AIM:To identify the genes induced and regulated by the MYC protein in generating tumors from liver stem cells.METHODS:In this study,we have used an immortal porcine liver stem cell line,PICM-19,to study the role of c-...AIM:To identify the genes induced and regulated by the MYC protein in generating tumors from liver stem cells.METHODS:In this study,we have used an immortal porcine liver stem cell line,PICM-19,to study the role of c-MYC in hepatocarcinogenesis.PICM-19 cells were converted into cancer cells(PICM-19-CSCs)by overexpressing human MYC.To identify MYC-driven differential gene expression,transcriptome sequencing was carried out by RNA sequencing,and genes identified by this method were validated using real-time PCR.In vivo tumorigenicity studies were then conducted by injecting PICM-19-CSCs into the flanks of immunodeficient mice.RESULTS:Our results showed that MYC-overexpressing PICM-19 stem cells formed tumors in immunodeficient mice demonstrating that a single oncogene was sufficient to convert them into cancer cells(PICM-19-CSCs).By using comparative bioinformatics analyses,we have determined that>1000 genes were differentially expressed between PICM-19 and PICM-19-CSCs.Gene ontology analysis further showed that the MYCinduced,altered gene expression was primarily associated with various cellular processes,such as metabolism,cell adhesion,growth and proliferation,cell cycle,inflammation and tumorigenesis.Interestingly,six genes expressed by PICM-19 cells(CDO1,C22orf39,DKK2,ENPEP,GPX6,SRPX2)were completely silenced after MYC-induction in PICM-19-CSCs,suggesting that the absence of these genes may be critical for inducingtumorigenesis.CONCLUSION:MYC-driven genes may serve as promising candidates for the development of hepatocellular carcinoma therapeutics that would not have deleterious effects on other cell types in the liver.展开更多
Objective To observe the expression profiles of osteoblast-related genes in human mesenchymal stem cells(MSCs) derived from bone marrow during osteogenic differentiation. Methods MSCs were induced to differentiate wit...Objective To observe the expression profiles of osteoblast-related genes in human mesenchymal stem cells(MSCs) derived from bone marrow during osteogenic differentiation. Methods MSCs were induced to differentiate with MSC osteogenic differentiation medium for 7, 14, 21 and 28 days respectively. Alizarin Red staining was used to detect matrix mineralization. Expression of osteoblast-related genes, including osteocalcin, osteopontin, Runt-related transcription factor 2(Runx2), alkaline phosphatase and collagen type Ⅰ, was assessed with quantitative reverse transcription-polymerase chain reaction. Results On day 14 after induction of differentiation, cells were stained positively with Alizarin Red. The expression levels of these genes exhibited an upward trend as induction time was prolonged. Exposure to osteogenic differentiation medium less than 21 days did not significantly induce osteocalcin expression; osteocalcin expression levels in the differentiated cells induced for 21 and 28 days were 1.63 and 2.46 times as high as the undifferentiated cells respectively(all P<0.05). Stimulation with MSC osteogenic differentiation medium over 14 days significantly enhanced bone marrow-derived MSCs to express osteopontin and Runx2 genes(all P<0.05). Osteogenic differentiation medium could significantly induce the expressions of alkaline phosphatase and collagen type Ⅰgenes(all P<0.05). Their expressions reached the peak levels on day 21, which were increased more than 4- and 3-fold respectively. Conclusion Human bone marrow-derived MSCs could exhibit the sequential expression pattern of osteoblast marker genes during osteogenic differentiation in vitro.展开更多
Retinal degenerative diseases pose a serious threat to eye health, but there is currently no effective treatment available. Recent years have witnessed rapid development of several cutting-edge technologies, such as g...Retinal degenerative diseases pose a serious threat to eye health, but there is currently no effective treatment available. Recent years have witnessed rapid development of several cutting-edge technologies, such as gene therapy, stem cell therapy, and tissue engineering. Due to the special features of ocular structure, some of these technologies have been translated into ophthalmological clinic practice with fruitful achievements, setting a good example for other fields. This paper reviews the development of the gene and stem cell therapies in ophthalmology.展开更多
Brachytic stem is a major trait in plant type .of soybean and its yield potential may be higher under high population when compared with normal stem. In the present investigation, 152 recombinant inbred line (RIL) f...Brachytic stem is a major trait in plant type .of soybean and its yield potential may be higher under high population when compared with normal stem. In the present investigation, 152 recombinant inbred line (RIL) families derived from the cross of Bogao (normal stem) and Nannong 94-156 (brachytic stem) were used to map genes and QTLs of three plant type traits and to identify the effects of brachytic stem on agronomic traits such as yield. The primary results indicated that brachytic stem (sb) and determinate growth habit (drl) were mapped on linkage groups B2 and L, three major QTLs related to plant height were detected and mapped on linkage group L near drl, another minor QTL was mapped near sb on linkage group B2-1. Lines with brachytic stem had shorter plant height, lower biomass, yield, harvest index and pods per plant, and essentially no differences in days to maturity and 100-seed weight when compared with normal stem lines. It was obvious that the effect of brachytic stem on yield was due to the decreased height, biomass and harvest index.展开更多
BACKGROUND: Transplantation of Akt-over-expressing mesenchymal stem ceils (Akt-MSCs) has been shown to repair infarcted myocardium and improve cardiac function. However, little is known about the therapeutic effect...BACKGROUND: Transplantation of Akt-over-expressing mesenchymal stem ceils (Akt-MSCs) has been shown to repair infarcted myocardium and improve cardiac function. However, little is known about the therapeutic effects of Akt-MSCs on cardiac autonomic neuropathy in chronic heart failure (CHF). OBJECTIVE: The present study used adriamycin-induced CHF rat models to observe the effect of Akt-MSCs on cardiac autonomic nervous regeneration and the factors mediating this effect. DESIGN, TIME AND SETTING: A randomized, controlled animal experiment was performed at the Central Laboratory of Basic Medical College, China Medical University, between September 2008 and April 2009. MATERIALS: Rabbit anti-choline acetyltransferase (CHAT), growth associated protein-43 (GAP-43) synaptophysin (SYN) polyclonal antibodies and the secondary antibody (goat anti-rabbit IgG) were purchased from Boster, China. Cat-A-Kit assay system was provided by Amersham, USA. METHODS: (1) Adult rat MSCs were isolated and cultured for the preparation of Akt-MSCs. (2) Forty male Wistar rats were intramyocardially administered adriamycin at 2 mg/kg over 3 days for a total of five times and once a week for additional five times thereafter to establish CHF models. At 2 weeks after final adriamycin treatment, 34 successful CHF rat models were randomized to three groups: Akt-MSCs (n = 11), simple MSCs (s-MSCs, n =11), and control (n = 12). Each group was intravenously administered Akt-MSCs (2x106 cells in 100 IJL PBS), s-MSCs (2×10^6 cells in 100 μL PBS) or equal volume of phosphate buffered saline, once a day for a total of three times. MAIN OUTCOME MEASURES: At 4 weeks after final adriamycin treatment, myocardial norepinephrine (NE) content was detected using a Cat-A-Kit assay system. Myocardial CHAT, SYN and GAP-43 were performed by immunohistochemistry and Western blot analysis. Prior to, 2 and 4 weeks after adriamycin treatment, echocardiographic examination was performed and left ventricular ejection fraction (LVEF) was determined. RESULTS: Myocardial NE content, as well as SYN-positive and GAP-43-positive nerve fiber density and expression, and LVEF, was the greatest in the Akt-MSCs group, followed by the s-MSCs group, and lastly the control group (P 〈 0.05 or P 〈 0.01). ChAT expression was similar between Akt-MSCs and s-MSCs groups, but it was higher compared with the control group (P 〈 0.05). NE contents were negatively correlated to LVEF (r = -0.64, P = 0.015). CONCLUSION: Transplantation of MSCs, in particular Akt-MSCs, promotes cardiac nervous regeneration in failing heart, which might be mediated by GAP-43.展开更多
The possibility of rats mesenchymal stem cells (MSCs) modified with murine hyperpolarization-activated cyclic nucleotide-gated 2 (mHCN2) gene as biological pacemakers in vitro was studied. The cultured MSCs were trans...The possibility of rats mesenchymal stem cells (MSCs) modified with murine hyperpolarization-activated cyclic nucleotide-gated 2 (mHCN2) gene as biological pacemakers in vitro was studied. The cultured MSCs were transfected with pIRES2-EGFP plasmid carrying enhanced green fluorescent protein (EGFP) gene and mHCN2 gene. The identification using restriction enzyme and sequencing indicated that the mHCN2 gene was inserted to the pIRES2-EGFP. Green fluorescence could be seen in MSCs after transfection for 24–48 h. The expression of mHCN2 mRNA and protein in the transfected cells was detected by RT-PCR and Western blot, and the quantity of mHCN2 mRNA and protein expression in transfected MSCs was 5.31 times and 7.55 times higher than that of the non-transfected MSCs respectively (P<0.05, P<0.05). IHCN2 was recorded by whole-cell patch clamp method. The effect of Cs+, a specific blocker of pacemaker current, was measured after perfusion by patch clamp. The results of inward current indicated that there was no inward current recording in non-transfected MSCs and a large voltage-dependent inward and Cs+-sensitive current activated on hyperpolarizations presented in the transfected MSCs. IHCN2 was fully activated around–140 mV with an activation threshold of –60 mV. The midpoint (V50) was –95.1±0.9 mV (n=9). The study demonstrates that mHCN2 mRNA and protein can be expressed and the currents of HCN2 channels can be detected in genetically modified MSCs. The gene-modified MSCs present a novel method for pacemaker genes into the heart or other electrical syncytia.展开更多
Inhibition of neurite growth, which is mediated by the Nogo-66 receptor (NgR), affects nerve regeneration following neural stem cell (NSC) transplantation. The present study utilized RNA interference to silence Ng...Inhibition of neurite growth, which is mediated by the Nogo-66 receptor (NgR), affects nerve regeneration following neural stem cell (NSC) transplantation. The present study utilized RNA interference to silence NgR gene expression in NSCs, which were subsequently transplanted into rats with traumatic brain injury. Following transplantation of NSCs transfected with small interfering RNA, typical neural cell-like morphology was detected in injured brain tissues, and was accompanied by absence of brain tissue cavity, increased growth-associated protein 43 mRNA and protein expression, and improved neurological function compared with NSC transplantation alone. Results demonstrated that NSC transplantation with silenced NgR gene promoted functional recovery following brain injury.展开更多
The emergence of a huge volume of "omics" data enables a computational approach to the investigation of the biology of cancer. The cancer informatics approach is a useful supplement to the traditional experi...The emergence of a huge volume of "omics" data enables a computational approach to the investigation of the biology of cancer. The cancer informatics approach is a useful supplement to the traditional experimental approach. I reviewed several reports that used a bioinformatics approach to analyze the associations among aging, stem cells, and cancer by microarray gene expression profiling. The high expression of aging- or human embryonic stem cell-related molecules in cancer suggests that certain important mechanisms are commonly underlying aging, stem cells, and cancer. These mechanisms are involved in cell cycle regulation, metabolic process, DNA damage response, apoptosis, p53 signaling pathway, immune/inflammatory response, and other processes, suggesting that cancer is a developmental and evolutional disease that is strongly related to aging. Moreover, these mechanisms demonstrate that the initiation, proliferation, and metastasis of cancer are associated with the deregulation of stem cells. These findings provide insights into the biology of cancer. Certainly, the findings that are obtained by the informatics approach should be justified by experimental validation. This review also noted that next-generation sequencing data provide enriched sources for cancer informatics study.展开更多
AIM: To investigate the genes regulated in mesenchymal stem cells(MSCs) and diffuse-type gastric cancer(GC),gene expression was analyzed. METHODS: Gene expression of MSCs and diffuse-type GC cells were analyzed by mic...AIM: To investigate the genes regulated in mesenchymal stem cells(MSCs) and diffuse-type gastric cancer(GC),gene expression was analyzed. METHODS: Gene expression of MSCs and diffuse-type GC cells were analyzed by microarray. Genes related to stem cells, cancer and the epithelial-mesenchymal transition(EMT) were extracted from human gene lists using Gene Ontology and reference information. Gene panels were generated, and messenger RNA gene expression in MSCs and diffuse-type GC cells was analyzed. Cluster analysis was performed using the NCSS software.RESULTS: The gene expression of regulator of G-protein signaling 1(RGS1) was up-regulated in diffuse-type GC cells compared with MSCs. A panel of stem-cell related genes and genes involved in cancer or the EMT were examined. Stem-cell related genes, such as growth arrest-specific 6, musashi RNA-binding protein 2 and hairy and enhancer of split 1(Drosophila), NOTCH family genes and Notch ligands, such as delta-like 1(Drosophila) and Jagged 2, were regulated.CONCLUSION: Expression of RGS1 is up-regulated, and genes related to stem cells and NOTCH signaling are altered in diffuse-type GC compared with MSCs.展开更多
Rat bone marrow mesenchymal stem cells expressing brain-derived neurotrophic factor were successfully obtained using a gene transfection method, then intravenously transplanted into rats with spinal cord injury. At 1,...Rat bone marrow mesenchymal stem cells expressing brain-derived neurotrophic factor were successfully obtained using a gene transfection method, then intravenously transplanted into rats with spinal cord injury. At 1,3, and 5 weeks after transplantation, the expression of brain-derived neurotrophic factor and neurofilament-200 was upregulated in the injured spinal cord, spinal cord injury was alleviated, and Basso-Beattie-Bresnahan scores of hindlimb motor function were significantly increased. This evidence suggested that intravenous transplantation of adenovirus- mediated brain-derived neurotrophic factor gene-modified rat bone marrow mesenchymal stem cells could play a dual role, simultaneously providing neural stem cells and neurotrophic factors.展开更多
Enhanced stem nematode resistance of transgenic sweetpotato (cv. Lizixiang) was achieved using Oryzacystatin-I (OCI) gene with Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain EHA105 harbor...Enhanced stem nematode resistance of transgenic sweetpotato (cv. Lizixiang) was achieved using Oryzacystatin-I (OCI) gene with Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain EHA105 harbors a binary vector pCAMBIA1301 with OCI gene, gusA gene and hptII gene. Selection culture was conducted using 25 mg L-1 hygromycin. A total of 1 715 plants were produced from the inoculated 1 450 cell aggregates of Lizixiang via somatic embryogenesis. GUS assay and PCR analysis of the putative transgenic plants randomly sampled showed that 90.54% of them were transgenic plants. Transgenic plants exhibited significantly enhanced resistance to stem nematodes compared to the untransformed control plants by the field evaluation with stem nematodes. Stable integration of the OCI gene into the genome of resistant transgenic plants was confirmed by Southern blot analysis, and the copy number of integrated OCI gene ranged from 1 to 4. Transgene overexpression in stem nematode-resistant plants was demonstrated by quantitative real-time PCR analysis. This study provides a way for improving stem nematode resistance in sweetpotato.展开更多
Hox genes are an evolutionary highly conserved gene family. They determine the anterior-posterior body axis in bilateral organisms and influence the developmental fate of cells. Embryonic stem cells are usually devoid...Hox genes are an evolutionary highly conserved gene family. They determine the anterior-posterior body axis in bilateral organisms and influence the developmental fate of cells. Embryonic stem cells are usually devoidof any Hox gene expression, but these transcription factors are activated in varying spatial and temporal patterns defining the development of various body regions. In the adult body, Hox genes are among others responsible for driving the differentiation of tissue stem cells towards their respective lineages in order to repair and maintain the correct function of tissues and organs. Due to their involvement in the embryonic and adult body, they have been suggested to be useable for improving stem cell differentiations in vitro and in vivo. In many studies Hox genes have been found as driving factors in stem cell differentiation towards adipogenesis, in lineages involved in bone and joint formation, mainly chondrogenesis and osteogenesis, in cardiovascular lineages including endothelial and smooth muscle cell differentiations, and in neurogenesis. As life expectancy is rising, the demand for tissue reconstruction continues to increase. Stem cells have become an increasingly popular choice for creating therapies in regenerative medicine due to their self-renewal and differentiation potential. Especially mesenchymal stem cells are used more and more frequently due to their easy handling and accessibility, combined with a low tumorgenicity and little ethical concerns. This review therefore intends to summarize to date known correlations between natural Hox gene expression patterns in body tissues and during the differentiation of various stem cells towards their respective lineages with a major focus on mesenchymal stem cell differentiations. This overview shall help to understand the complex interactions of Hox genes and differentiation processes all over the body as well as in vitro for further improvement of stem cell treatments in future regenerative medicine approaches.展开更多
To investigate the characteristics of multidrugresistance and transplantation of modified stem/ progenitor cells by multidrugresistant gene (mdr1 gene), we established PA317/MDR1 cell line which producing retroviruse...To investigate the characteristics of multidrugresistance and transplantation of modified stem/ progenitor cells by multidrugresistant gene (mdr1 gene), we established PA317/MDR1 cell line which producing retroviruses by transfecting the retroviral vector PHaMDR1/A into packging cell line PA317 by Lipofectin. The virus titer of the supernatants was 1.2×105 cfu/ml. We transfected the murine hematopietic cells collected from 5FU pretreated mice and they showed the ability to reconstitute the longterm hematopoiesis of preirradiated mice. After 4 months, both of bone marrow cells and peripheral blood cells of transplanted mice still contained mdr1 gene. We also transfered mdr1 gene into human bone marrow CD34+ cells selected by using magnetic cell sorting system. PCR analysis showed that transduced CD34+ cells maintained the mdr1 cDNA. A fraction of CFUGM originated from transfected CD34+ cells had the charactor of resistance to Taxol. It is indicated that mdr1 gene can be transduced into murine and human stem/proginitor cells through retroviral mediated gene transfer and it protects the transfected cells from cytotoxic drugs.展开更多
Stem lignin content(SLC) in common wheat(Triticum aestivum L.) contributes to lodging resistance. Caffeic acid 3-O-methyltransferase(COMT) is a key enzyme involved in lignin biosynthesis. Characterization of TaCOMT ge...Stem lignin content(SLC) in common wheat(Triticum aestivum L.) contributes to lodging resistance. Caffeic acid 3-O-methyltransferase(COMT) is a key enzyme involved in lignin biosynthesis. Characterization of TaCOMT genes and development of gene-specific markers could enable marker-assisted selection in wheat breeding. In the present study, the full-length genomic DNA(gDNA) sequences of TaCOMT genes located on chromosomes 3 A, 3 B, and 3 D were cloned by homologous cloning. Two allelic variants, TaCOMT-3 Ba and TaCOMT-3 Bb, were identified and differed by a 222-bp insertion/deletion(InDel) in the 3′-untranslated region(3′-UTR). A co-dominant gene-specific marker based on this InDel was developed and designated as Ta COMT-3 BM. A total of 157 wheat cultivars and advanced lines grown in four environments were used to validate the associations between allelic patterns and SLC. The SLC of cultivars with TaCOMT-3 Ba was significantly(P<0.01) higher than that of those with TaCOMT-3 Bb, and the marker TaCOMT-3 BM could be effectively used in wheat breeding.展开更多
The size of the blind population in 2015 was estimated to be approximately 36 million(Bourne et al.,2017).According to the predictions by Bourne and co-workers,the number of the visually impaired is expected to reac...The size of the blind population in 2015 was estimated to be approximately 36 million(Bourne et al.,2017).According to the predictions by Bourne and co-workers,the number of the visually impaired is expected to reach nearly 100 million by 2050.展开更多
Objective :To investigate the influence of GST πgenetransferintohumancord blood hematopoie ticstem cellson their drugres is tanceagains tanti tumordrugs invivo .Methods: GST πgenetran sfectionin to human cord blood ...Objective :To investigate the influence of GST πgenetransferintohumancord blood hematopoie ticstem cellson their drugres is tanceagains tanti tumordrugs invivo .Methods: GST πgenetran sfectionin to human cord blood CD 34+cells was carried outusing are trovirusvec torPLJ GST πwith the aido f fibronect in .Succes sfulgenetransfer was confirmed by invitrocolony assay and RT PCR .GST πgenetrans duced human cord blood CD34+cells were the nengrafted in to 4 week old total body irradiated NOD/Scid mice and carboplatin was intraperitoneally admin is tered sequentially at 4 weeks interval 4 week saftereng raftment.Results :Peripheral blood (PB) WBC was significantly higher in GST πmice than control mice after 2 course of car boplat in .Retroviral GST π expression in bone marrow hematopoietic progenitor cells of recipient mice was detected by RT PCR 16 weeks after Xenotransplantation .Conclusion :The transfection of GST π gene could confer ,to some extent ,resistance to cord blood stem cells against carboplatin in vivo .展开更多
Neural stem cells transplantation plays an important role in repair and cell replacement therapy for nervous system degenerative diseases. However, the self-repair effects are minimal because of a limited absolute num...Neural stem cells transplantation plays an important role in repair and cell replacement therapy for nervous system degenerative diseases. However, the self-repair effects are minimal because of a limited absolute number, as well as the local microenvironment, post-transplantation. A combined treatment utilizing stem cell transplantation and gone therapy can exert a dual effect involving stem cells and neurotrophic factors. The adenovirus carrier is展开更多
Summary: A new type of TGF-β3 fusion protein with targeted therapy function was constructed, and its feasibility and target specificity of inducing chondrogenesis were investigated by transfecting LAP-MMP-mTGF-β3 g...Summary: A new type of TGF-β3 fusion protein with targeted therapy function was constructed, and its feasibility and target specificity of inducing chondrogenesis were investigated by transfecting LAP-MMP-mTGF-β3 gene into adipose-derived stem cells (ADSCs). The recombinant pIRES- EGFP-MMP was constructed by inserting the sense and antisense DNA of encoding the amino acid of the synthetic MMP enzyme cutting site into the eukaryotic expression vector pIRES-EGFE LAP and mTGF-β3 fragments were obtained by using RT-PCR and inserted into the upstream and downstream of MMP from pIRES-EGFP-MMP respectively, and the recombinant plasmid of pIRES-EGFP- LAP-MMP-mTGF-β3 was constructed, which was transferred to ADSCs. The ADSCs were cultured and divided in three groups: experimental group (MMP group), negative control group (no MMP) and non-transfection group. The morphological changes were observed microscopically, and the expression of proteoglycan and type II collagen (Col II) was detected by using Alcian blue staining and immuno- histochemistry staining at 7th, 14th and 21st day after culture. The recombinant plasmid of pIRES-EGFP-LAP-MMP-mTGF-β3 was correctly constructed by methods of enzyme cutting and se- quencing analysis. The mTGF-β3 fusion protein was successfully expressed after transfection, and in the presence of the MMP, active protein mTGF-β3 was generated, which significantly promoted differ- entiation of ADSCs into chondrocytes and the expression of cartilage matrix. The novel fusion protein LAP-MMP-mTGF-β3 can targetedly induce differentiation of ADSCs into chondrocytes, which would open up prospects for target therapy of cartilage damage repair in future.展开更多
Objective: To explore the survival and migration of bone mesenchymal stem cells transplantated in intervertebral disc of rabbits and expression of the exogenic genes. Methods. Thirty-two rabbits were used, A randomiz...Objective: To explore the survival and migration of bone mesenchymal stem cells transplantated in intervertebral disc of rabbits and expression of the exogenic genes. Methods. Thirty-two rabbits were used, A randomized block design was used and discs in the same rabbit were one block,the lumbar discs from L2-3 to L5-6 were randomly divided into blank group, saline group, cell transplantation group Ⅰand cell transplantation group Ⅱ. The fluorescence microscopy was used to determine the fluorescence of the maker protein GFP and DNA-PCR was used to analyze the copies of DNA of neomycin-resistant gene at 1, 3, 6, months after transplantation. Results: There was fluorescence in cell transplantation group Ⅰ and Ⅱ and none in blank group, saline group at 1, 3, 6 months after transplantation. In cell transplantation groups,the fluorescent distribution was more scatter with time, but no significant difference between cell groups Ⅰ and Ⅱ. The test of neomycin resistant gene expressed in cell transplantation group Ⅰ and Ⅱ and quantitative analysis showed that there was no significant difference between the cell groups Ⅰ and Ⅱ (P〉0.05). Conclusion: The transplanted bone mesenchymal stem cells can survive, migrate and the transfer genes can express efficiently, it suggests that the BMSC therapy may be effective to prevent and treat intervertebral disc degeneration.展开更多
BACKGROUND: Gene therapy is an effective expression of genes within target cells after transferring exogenous target genes. Both vector selection and transfection method are important factors for gene transfection. A...BACKGROUND: Gene therapy is an effective expression of genes within target cells after transferring exogenous target genes. Both vector selection and transfection method are important factors for gene transfection. An ideal gene vector is required for a high transfusion of target gene and an exact introduction of target gene into specific target cells so as to express gene products. OBJECTIVE: To study the expression of mRNA and protein after transfecting rat bone marrow mesenchymal stem cells (BMSCs) with brain-derived neurotrophic factor (BDNF) genes based on cationic polymer vector. DESIGN, TIME AND SETTING: A randomized, controlled in vitro study using gene engineering, performed at the Neurobiology Laboratory, Xuzhou Medical College between October 2007 and April 2008. MATERIALS: PcDNA3.1 BDNF was obtained from Youbiai Biotechnological Company, Beijing and cationic polymer vector used was the SofastTM gene transfection reagent that was made by Taiyangma Biotechnological Co., Ltd., Xiamen. METHODS: BMSCs extracted from six Sprague Dawley (SD) rats aged 1 month were isolated and cultured in vitro. Third passage BMSCs were inoculated on a 6-well culture plate at the density of 1×106 cells/L. At about 80% confluence, BMSCs were transfected with PcDNA3.1-BDNF (2 μg) combined with SofastTM gene transfection reagent (6 μg) (BDNF group) or with PcDNA3.1 (2 μg) combined with SofastTM gene transfection reagent (6 μg) (blank vector group). Cells that were not transfected with any reagents but still cultured under primary culture conditions were used as a non-transfection group. MAIN OUTCOME MEASURES: Enzyme linked immunosorbent assay was used to measure time efficiency of BMSC-secreted BDNF protein. Twenty-four hours after gene transfection, RT-PCR was used to detect expression of BDNF mRNA in the BMSCs. Immunohistochemistry was used to determine expression of BDNF protein in the BMSCs. RESULTS: BDNF protein expression was detected at day 1 after gene transfection, rapidly increased after 5–9 days and gradually increased after 11–15 days in the BDNF group; moreover, BDNF protein expression was higher than that in the non-transfection group and the blank vector group at different time points (P 〈 0.01). Additionally, BDNF mRNA expression in the BDNF group was higher than that in the blank vector group and the non-transfection group (P 〈 0.01). CONCLUSION: A cationic polymer vector can effectively mediate the BDNF gene to transfect BMSCs; genetically modified BMSCs can express BDNF protein effectively for a long term.展开更多
基金Supported by Departmental funds to Dr.Aravalli RN
文摘AIM:To identify the genes induced and regulated by the MYC protein in generating tumors from liver stem cells.METHODS:In this study,we have used an immortal porcine liver stem cell line,PICM-19,to study the role of c-MYC in hepatocarcinogenesis.PICM-19 cells were converted into cancer cells(PICM-19-CSCs)by overexpressing human MYC.To identify MYC-driven differential gene expression,transcriptome sequencing was carried out by RNA sequencing,and genes identified by this method were validated using real-time PCR.In vivo tumorigenicity studies were then conducted by injecting PICM-19-CSCs into the flanks of immunodeficient mice.RESULTS:Our results showed that MYC-overexpressing PICM-19 stem cells formed tumors in immunodeficient mice demonstrating that a single oncogene was sufficient to convert them into cancer cells(PICM-19-CSCs).By using comparative bioinformatics analyses,we have determined that>1000 genes were differentially expressed between PICM-19 and PICM-19-CSCs.Gene ontology analysis further showed that the MYCinduced,altered gene expression was primarily associated with various cellular processes,such as metabolism,cell adhesion,growth and proliferation,cell cycle,inflammation and tumorigenesis.Interestingly,six genes expressed by PICM-19 cells(CDO1,C22orf39,DKK2,ENPEP,GPX6,SRPX2)were completely silenced after MYC-induction in PICM-19-CSCs,suggesting that the absence of these genes may be critical for inducingtumorigenesis.CONCLUSION:MYC-driven genes may serve as promising candidates for the development of hepatocellular carcinoma therapeutics that would not have deleterious effects on other cell types in the liver.
基金Supported by the National Natural Science Foundation of China(81372007)
文摘Objective To observe the expression profiles of osteoblast-related genes in human mesenchymal stem cells(MSCs) derived from bone marrow during osteogenic differentiation. Methods MSCs were induced to differentiate with MSC osteogenic differentiation medium for 7, 14, 21 and 28 days respectively. Alizarin Red staining was used to detect matrix mineralization. Expression of osteoblast-related genes, including osteocalcin, osteopontin, Runt-related transcription factor 2(Runx2), alkaline phosphatase and collagen type Ⅰ, was assessed with quantitative reverse transcription-polymerase chain reaction. Results On day 14 after induction of differentiation, cells were stained positively with Alizarin Red. The expression levels of these genes exhibited an upward trend as induction time was prolonged. Exposure to osteogenic differentiation medium less than 21 days did not significantly induce osteocalcin expression; osteocalcin expression levels in the differentiated cells induced for 21 and 28 days were 1.63 and 2.46 times as high as the undifferentiated cells respectively(all P<0.05). Stimulation with MSC osteogenic differentiation medium over 14 days significantly enhanced bone marrow-derived MSCs to express osteopontin and Runx2 genes(all P<0.05). Osteogenic differentiation medium could significantly induce the expressions of alkaline phosphatase and collagen type Ⅰgenes(all P<0.05). Their expressions reached the peak levels on day 21, which were increased more than 4- and 3-fold respectively. Conclusion Human bone marrow-derived MSCs could exhibit the sequential expression pattern of osteoblast marker genes during osteogenic differentiation in vitro.
基金Supported by the Beijing Commission of Education Science Technology Development Key Project (No. KZ201010025019) the Institute of Ophthalmology, Capital Medical Uni Project (No. 201205)
文摘Retinal degenerative diseases pose a serious threat to eye health, but there is currently no effective treatment available. Recent years have witnessed rapid development of several cutting-edge technologies, such as gene therapy, stem cell therapy, and tissue engineering. Due to the special features of ocular structure, some of these technologies have been translated into ophthalmological clinic practice with fruitful achievements, setting a good example for other fields. This paper reviews the development of the gene and stem cell therapies in ophthalmology.
基金This work was supported by the National 973 Program(2004CB7206)National 863 Program of China(2002AA211052)Jiangsu Provincial Natural Science Foundation(BK2004045).
文摘Brachytic stem is a major trait in plant type .of soybean and its yield potential may be higher under high population when compared with normal stem. In the present investigation, 152 recombinant inbred line (RIL) families derived from the cross of Bogao (normal stem) and Nannong 94-156 (brachytic stem) were used to map genes and QTLs of three plant type traits and to identify the effects of brachytic stem on agronomic traits such as yield. The primary results indicated that brachytic stem (sb) and determinate growth habit (drl) were mapped on linkage groups B2 and L, three major QTLs related to plant height were detected and mapped on linkage group L near drl, another minor QTL was mapped near sb on linkage group B2-1. Lines with brachytic stem had shorter plant height, lower biomass, yield, harvest index and pods per plant, and essentially no differences in days to maturity and 100-seed weight when compared with normal stem lines. It was obvious that the effect of brachytic stem on yield was due to the decreased height, biomass and harvest index.
基金Scientific Research Program of Higher Education Institute in Liaoning Province, No. 2008S248
文摘BACKGROUND: Transplantation of Akt-over-expressing mesenchymal stem ceils (Akt-MSCs) has been shown to repair infarcted myocardium and improve cardiac function. However, little is known about the therapeutic effects of Akt-MSCs on cardiac autonomic neuropathy in chronic heart failure (CHF). OBJECTIVE: The present study used adriamycin-induced CHF rat models to observe the effect of Akt-MSCs on cardiac autonomic nervous regeneration and the factors mediating this effect. DESIGN, TIME AND SETTING: A randomized, controlled animal experiment was performed at the Central Laboratory of Basic Medical College, China Medical University, between September 2008 and April 2009. MATERIALS: Rabbit anti-choline acetyltransferase (CHAT), growth associated protein-43 (GAP-43) synaptophysin (SYN) polyclonal antibodies and the secondary antibody (goat anti-rabbit IgG) were purchased from Boster, China. Cat-A-Kit assay system was provided by Amersham, USA. METHODS: (1) Adult rat MSCs were isolated and cultured for the preparation of Akt-MSCs. (2) Forty male Wistar rats were intramyocardially administered adriamycin at 2 mg/kg over 3 days for a total of five times and once a week for additional five times thereafter to establish CHF models. At 2 weeks after final adriamycin treatment, 34 successful CHF rat models were randomized to three groups: Akt-MSCs (n = 11), simple MSCs (s-MSCs, n =11), and control (n = 12). Each group was intravenously administered Akt-MSCs (2x106 cells in 100 IJL PBS), s-MSCs (2×10^6 cells in 100 μL PBS) or equal volume of phosphate buffered saline, once a day for a total of three times. MAIN OUTCOME MEASURES: At 4 weeks after final adriamycin treatment, myocardial norepinephrine (NE) content was detected using a Cat-A-Kit assay system. Myocardial CHAT, SYN and GAP-43 were performed by immunohistochemistry and Western blot analysis. Prior to, 2 and 4 weeks after adriamycin treatment, echocardiographic examination was performed and left ventricular ejection fraction (LVEF) was determined. RESULTS: Myocardial NE content, as well as SYN-positive and GAP-43-positive nerve fiber density and expression, and LVEF, was the greatest in the Akt-MSCs group, followed by the s-MSCs group, and lastly the control group (P 〈 0.05 or P 〈 0.01). ChAT expression was similar between Akt-MSCs and s-MSCs groups, but it was higher compared with the control group (P 〈 0.05). NE contents were negatively correlated to LVEF (r = -0.64, P = 0.015). CONCLUSION: Transplantation of MSCs, in particular Akt-MSCs, promotes cardiac nervous regeneration in failing heart, which might be mediated by GAP-43.
文摘The possibility of rats mesenchymal stem cells (MSCs) modified with murine hyperpolarization-activated cyclic nucleotide-gated 2 (mHCN2) gene as biological pacemakers in vitro was studied. The cultured MSCs were transfected with pIRES2-EGFP plasmid carrying enhanced green fluorescent protein (EGFP) gene and mHCN2 gene. The identification using restriction enzyme and sequencing indicated that the mHCN2 gene was inserted to the pIRES2-EGFP. Green fluorescence could be seen in MSCs after transfection for 24–48 h. The expression of mHCN2 mRNA and protein in the transfected cells was detected by RT-PCR and Western blot, and the quantity of mHCN2 mRNA and protein expression in transfected MSCs was 5.31 times and 7.55 times higher than that of the non-transfected MSCs respectively (P<0.05, P<0.05). IHCN2 was recorded by whole-cell patch clamp method. The effect of Cs+, a specific blocker of pacemaker current, was measured after perfusion by patch clamp. The results of inward current indicated that there was no inward current recording in non-transfected MSCs and a large voltage-dependent inward and Cs+-sensitive current activated on hyperpolarizations presented in the transfected MSCs. IHCN2 was fully activated around–140 mV with an activation threshold of –60 mV. The midpoint (V50) was –95.1±0.9 mV (n=9). The study demonstrates that mHCN2 mRNA and protein can be expressed and the currents of HCN2 channels can be detected in genetically modified MSCs. The gene-modified MSCs present a novel method for pacemaker genes into the heart or other electrical syncytia.
基金the Science and Technology Foundation of Tianjin Health Bureau,No.2010KY04
文摘Inhibition of neurite growth, which is mediated by the Nogo-66 receptor (NgR), affects nerve regeneration following neural stem cell (NSC) transplantation. The present study utilized RNA interference to silence NgR gene expression in NSCs, which were subsequently transplanted into rats with traumatic brain injury. Following transplantation of NSCs transfected with small interfering RNA, typical neural cell-like morphology was detected in injured brain tissues, and was accompanied by absence of brain tissue cavity, increased growth-associated protein 43 mRNA and protein expression, and improved neurological function compared with NSC transplantation alone. Results demonstrated that NSC transplantation with silenced NgR gene promoted functional recovery following brain injury.
文摘The emergence of a huge volume of "omics" data enables a computational approach to the investigation of the biology of cancer. The cancer informatics approach is a useful supplement to the traditional experimental approach. I reviewed several reports that used a bioinformatics approach to analyze the associations among aging, stem cells, and cancer by microarray gene expression profiling. The high expression of aging- or human embryonic stem cell-related molecules in cancer suggests that certain important mechanisms are commonly underlying aging, stem cells, and cancer. These mechanisms are involved in cell cycle regulation, metabolic process, DNA damage response, apoptosis, p53 signaling pathway, immune/inflammatory response, and other processes, suggesting that cancer is a developmental and evolutional disease that is strongly related to aging. Moreover, these mechanisms demonstrate that the initiation, proliferation, and metastasis of cancer are associated with the deregulation of stem cells. These findings provide insights into the biology of cancer. Certainly, the findings that are obtained by the informatics approach should be justified by experimental validation. This review also noted that next-generation sequencing data provide enriched sources for cancer informatics study.
基金Cancer Research from the Ministry of Health,Labour and Welfare
文摘AIM: To investigate the genes regulated in mesenchymal stem cells(MSCs) and diffuse-type gastric cancer(GC),gene expression was analyzed. METHODS: Gene expression of MSCs and diffuse-type GC cells were analyzed by microarray. Genes related to stem cells, cancer and the epithelial-mesenchymal transition(EMT) were extracted from human gene lists using Gene Ontology and reference information. Gene panels were generated, and messenger RNA gene expression in MSCs and diffuse-type GC cells was analyzed. Cluster analysis was performed using the NCSS software.RESULTS: The gene expression of regulator of G-protein signaling 1(RGS1) was up-regulated in diffuse-type GC cells compared with MSCs. A panel of stem-cell related genes and genes involved in cancer or the EMT were examined. Stem-cell related genes, such as growth arrest-specific 6, musashi RNA-binding protein 2 and hairy and enhancer of split 1(Drosophila), NOTCH family genes and Notch ligands, such as delta-like 1(Drosophila) and Jagged 2, were regulated.CONCLUSION: Expression of RGS1 is up-regulated, and genes related to stem cells and NOTCH signaling are altered in diffuse-type GC compared with MSCs.
基金Research Fund for the Doctoral Program of Higher Education of China, No. 20060392003
文摘Rat bone marrow mesenchymal stem cells expressing brain-derived neurotrophic factor were successfully obtained using a gene transfection method, then intravenously transplanted into rats with spinal cord injury. At 1,3, and 5 weeks after transplantation, the expression of brain-derived neurotrophic factor and neurofilament-200 was upregulated in the injured spinal cord, spinal cord injury was alleviated, and Basso-Beattie-Bresnahan scores of hindlimb motor function were significantly increased. This evidence suggested that intravenous transplantation of adenovirus- mediated brain-derived neurotrophic factor gene-modified rat bone marrow mesenchymal stem cells could play a dual role, simultaneously providing neural stem cells and neurotrophic factors.
基金supported by the Earmarked Fund for Modern Agro-Industry Technology Research System(Sweetpotato), Chinathe National High-Tech R&D Pro-gram of China (2009AA10Z102)+2 种基金the National Transgenic Plants Project of China (2009ZX08009-064B)the Natinal NaturalScience Foundation of China(30871570)the Scientific Fund to Graduate Re-search and Innovation Projects of China Agricultural University (15059201-kycx09018)
文摘Enhanced stem nematode resistance of transgenic sweetpotato (cv. Lizixiang) was achieved using Oryzacystatin-I (OCI) gene with Agrobacterium tumefaciens-mediated transformation. A. tumefaciens strain EHA105 harbors a binary vector pCAMBIA1301 with OCI gene, gusA gene and hptII gene. Selection culture was conducted using 25 mg L-1 hygromycin. A total of 1 715 plants were produced from the inoculated 1 450 cell aggregates of Lizixiang via somatic embryogenesis. GUS assay and PCR analysis of the putative transgenic plants randomly sampled showed that 90.54% of them were transgenic plants. Transgenic plants exhibited significantly enhanced resistance to stem nematodes compared to the untransformed control plants by the field evaluation with stem nematodes. Stable integration of the OCI gene into the genome of resistant transgenic plants was confirmed by Southern blot analysis, and the copy number of integrated OCI gene ranged from 1 to 4. Transgene overexpression in stem nematode-resistant plants was demonstrated by quantitative real-time PCR analysis. This study provides a way for improving stem nematode resistance in sweetpotato.
基金BMBF,Adi Pa D,1720X06,BMBF,FHprof Unt,FKZ:03FH012PB2FH-Extra,"Europischer Fonds für regionale Entwicklung","Europa-Investition in unsere Zukunft",FKZ:z1112fh012EFRE co-financed NRW Ziel 2:"Regionale Wettbewerbsfhigkeit und Beschftigung",DAAD,PPP Vigoni,FKZ:314-vigoni-dr and FKZ:54669218 for Edda Tobiasch
文摘Hox genes are an evolutionary highly conserved gene family. They determine the anterior-posterior body axis in bilateral organisms and influence the developmental fate of cells. Embryonic stem cells are usually devoidof any Hox gene expression, but these transcription factors are activated in varying spatial and temporal patterns defining the development of various body regions. In the adult body, Hox genes are among others responsible for driving the differentiation of tissue stem cells towards their respective lineages in order to repair and maintain the correct function of tissues and organs. Due to their involvement in the embryonic and adult body, they have been suggested to be useable for improving stem cell differentiations in vitro and in vivo. In many studies Hox genes have been found as driving factors in stem cell differentiation towards adipogenesis, in lineages involved in bone and joint formation, mainly chondrogenesis and osteogenesis, in cardiovascular lineages including endothelial and smooth muscle cell differentiations, and in neurogenesis. As life expectancy is rising, the demand for tissue reconstruction continues to increase. Stem cells have become an increasingly popular choice for creating therapies in regenerative medicine due to their self-renewal and differentiation potential. Especially mesenchymal stem cells are used more and more frequently due to their easy handling and accessibility, combined with a low tumorgenicity and little ethical concerns. This review therefore intends to summarize to date known correlations between natural Hox gene expression patterns in body tissues and during the differentiation of various stem cells towards their respective lineages with a major focus on mesenchymal stem cell differentiations. This overview shall help to understand the complex interactions of Hox genes and differentiation processes all over the body as well as in vitro for further improvement of stem cell treatments in future regenerative medicine approaches.
文摘To investigate the characteristics of multidrugresistance and transplantation of modified stem/ progenitor cells by multidrugresistant gene (mdr1 gene), we established PA317/MDR1 cell line which producing retroviruses by transfecting the retroviral vector PHaMDR1/A into packging cell line PA317 by Lipofectin. The virus titer of the supernatants was 1.2×105 cfu/ml. We transfected the murine hematopietic cells collected from 5FU pretreated mice and they showed the ability to reconstitute the longterm hematopoiesis of preirradiated mice. After 4 months, both of bone marrow cells and peripheral blood cells of transplanted mice still contained mdr1 gene. We also transfered mdr1 gene into human bone marrow CD34+ cells selected by using magnetic cell sorting system. PCR analysis showed that transduced CD34+ cells maintained the mdr1 cDNA. A fraction of CFUGM originated from transfected CD34+ cells had the charactor of resistance to Taxol. It is indicated that mdr1 gene can be transduced into murine and human stem/proginitor cells through retroviral mediated gene transfer and it protects the transfected cells from cytotoxic drugs.
基金supported by the National Natural Science Foundation of China(31161140346 and 31461143021)the Beijing Municipal Science and Technology Project,China(D151100004415003)+1 种基金the National Key Technology R&D Program of China(2014BAD01B05)the earmarked fund for China Agriculture Research System(CARS-3-1-3)
文摘Stem lignin content(SLC) in common wheat(Triticum aestivum L.) contributes to lodging resistance. Caffeic acid 3-O-methyltransferase(COMT) is a key enzyme involved in lignin biosynthesis. Characterization of TaCOMT genes and development of gene-specific markers could enable marker-assisted selection in wheat breeding. In the present study, the full-length genomic DNA(gDNA) sequences of TaCOMT genes located on chromosomes 3 A, 3 B, and 3 D were cloned by homologous cloning. Two allelic variants, TaCOMT-3 Ba and TaCOMT-3 Bb, were identified and differed by a 222-bp insertion/deletion(InDel) in the 3′-untranslated region(3′-UTR). A co-dominant gene-specific marker based on this InDel was developed and designated as Ta COMT-3 BM. A total of 157 wheat cultivars and advanced lines grown in four environments were used to validate the associations between allelic patterns and SLC. The SLC of cultivars with TaCOMT-3 Ba was significantly(P<0.01) higher than that of those with TaCOMT-3 Bb, and the marker TaCOMT-3 BM could be effectively used in wheat breeding.
基金funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 746526from the National Health and Medical Research Council(RG1063046)
文摘The size of the blind population in 2015 was estimated to be approximately 36 million(Bourne et al.,2017).According to the predictions by Bourne and co-workers,the number of the visually impaired is expected to reach nearly 100 million by 2050.
文摘Objective :To investigate the influence of GST πgenetransferintohumancord blood hematopoie ticstem cellson their drugres is tanceagains tanti tumordrugs invivo .Methods: GST πgenetran sfectionin to human cord blood CD 34+cells was carried outusing are trovirusvec torPLJ GST πwith the aido f fibronect in .Succes sfulgenetransfer was confirmed by invitrocolony assay and RT PCR .GST πgenetrans duced human cord blood CD34+cells were the nengrafted in to 4 week old total body irradiated NOD/Scid mice and carboplatin was intraperitoneally admin is tered sequentially at 4 weeks interval 4 week saftereng raftment.Results :Peripheral blood (PB) WBC was significantly higher in GST πmice than control mice after 2 course of car boplat in .Retroviral GST π expression in bone marrow hematopoietic progenitor cells of recipient mice was detected by RT PCR 16 weeks after Xenotransplantation .Conclusion :The transfection of GST π gene could confer ,to some extent ,resistance to cord blood stem cells against carboplatin in vivo .
文摘Neural stem cells transplantation plays an important role in repair and cell replacement therapy for nervous system degenerative diseases. However, the self-repair effects are minimal because of a limited absolute number, as well as the local microenvironment, post-transplantation. A combined treatment utilizing stem cell transplantation and gone therapy can exert a dual effect involving stem cells and neurotrophic factors. The adenovirus carrier is
基金supported by the National Natural Science Foundation of China(No.81101376)
文摘Summary: A new type of TGF-β3 fusion protein with targeted therapy function was constructed, and its feasibility and target specificity of inducing chondrogenesis were investigated by transfecting LAP-MMP-mTGF-β3 gene into adipose-derived stem cells (ADSCs). The recombinant pIRES- EGFP-MMP was constructed by inserting the sense and antisense DNA of encoding the amino acid of the synthetic MMP enzyme cutting site into the eukaryotic expression vector pIRES-EGFE LAP and mTGF-β3 fragments were obtained by using RT-PCR and inserted into the upstream and downstream of MMP from pIRES-EGFP-MMP respectively, and the recombinant plasmid of pIRES-EGFP- LAP-MMP-mTGF-β3 was constructed, which was transferred to ADSCs. The ADSCs were cultured and divided in three groups: experimental group (MMP group), negative control group (no MMP) and non-transfection group. The morphological changes were observed microscopically, and the expression of proteoglycan and type II collagen (Col II) was detected by using Alcian blue staining and immuno- histochemistry staining at 7th, 14th and 21st day after culture. The recombinant plasmid of pIRES-EGFP-LAP-MMP-mTGF-β3 was correctly constructed by methods of enzyme cutting and se- quencing analysis. The mTGF-β3 fusion protein was successfully expressed after transfection, and in the presence of the MMP, active protein mTGF-β3 was generated, which significantly promoted differ- entiation of ADSCs into chondrocytes and the expression of cartilage matrix. The novel fusion protein LAP-MMP-mTGF-β3 can targetedly induce differentiation of ADSCs into chondrocytes, which would open up prospects for target therapy of cartilage damage repair in future.
基金The Study of Differentiation of Bone Mesenchymal Stem Cells Transplanted in Intervertebral Disc and Expression of ExogenousGene(30400163)
文摘Objective: To explore the survival and migration of bone mesenchymal stem cells transplantated in intervertebral disc of rabbits and expression of the exogenic genes. Methods. Thirty-two rabbits were used, A randomized block design was used and discs in the same rabbit were one block,the lumbar discs from L2-3 to L5-6 were randomly divided into blank group, saline group, cell transplantation group Ⅰand cell transplantation group Ⅱ. The fluorescence microscopy was used to determine the fluorescence of the maker protein GFP and DNA-PCR was used to analyze the copies of DNA of neomycin-resistant gene at 1, 3, 6, months after transplantation. Results: There was fluorescence in cell transplantation group Ⅰ and Ⅱ and none in blank group, saline group at 1, 3, 6 months after transplantation. In cell transplantation groups,the fluorescent distribution was more scatter with time, but no significant difference between cell groups Ⅰ and Ⅱ. The test of neomycin resistant gene expressed in cell transplantation group Ⅰ and Ⅱ and quantitative analysis showed that there was no significant difference between the cell groups Ⅰ and Ⅱ (P〉0.05). Conclusion: The transplanted bone mesenchymal stem cells can survive, migrate and the transfer genes can express efficiently, it suggests that the BMSC therapy may be effective to prevent and treat intervertebral disc degeneration.
文摘BACKGROUND: Gene therapy is an effective expression of genes within target cells after transferring exogenous target genes. Both vector selection and transfection method are important factors for gene transfection. An ideal gene vector is required for a high transfusion of target gene and an exact introduction of target gene into specific target cells so as to express gene products. OBJECTIVE: To study the expression of mRNA and protein after transfecting rat bone marrow mesenchymal stem cells (BMSCs) with brain-derived neurotrophic factor (BDNF) genes based on cationic polymer vector. DESIGN, TIME AND SETTING: A randomized, controlled in vitro study using gene engineering, performed at the Neurobiology Laboratory, Xuzhou Medical College between October 2007 and April 2008. MATERIALS: PcDNA3.1 BDNF was obtained from Youbiai Biotechnological Company, Beijing and cationic polymer vector used was the SofastTM gene transfection reagent that was made by Taiyangma Biotechnological Co., Ltd., Xiamen. METHODS: BMSCs extracted from six Sprague Dawley (SD) rats aged 1 month were isolated and cultured in vitro. Third passage BMSCs were inoculated on a 6-well culture plate at the density of 1×106 cells/L. At about 80% confluence, BMSCs were transfected with PcDNA3.1-BDNF (2 μg) combined with SofastTM gene transfection reagent (6 μg) (BDNF group) or with PcDNA3.1 (2 μg) combined with SofastTM gene transfection reagent (6 μg) (blank vector group). Cells that were not transfected with any reagents but still cultured under primary culture conditions were used as a non-transfection group. MAIN OUTCOME MEASURES: Enzyme linked immunosorbent assay was used to measure time efficiency of BMSC-secreted BDNF protein. Twenty-four hours after gene transfection, RT-PCR was used to detect expression of BDNF mRNA in the BMSCs. Immunohistochemistry was used to determine expression of BDNF protein in the BMSCs. RESULTS: BDNF protein expression was detected at day 1 after gene transfection, rapidly increased after 5–9 days and gradually increased after 11–15 days in the BDNF group; moreover, BDNF protein expression was higher than that in the non-transfection group and the blank vector group at different time points (P 〈 0.01). Additionally, BDNF mRNA expression in the BDNF group was higher than that in the blank vector group and the non-transfection group (P 〈 0.01). CONCLUSION: A cationic polymer vector can effectively mediate the BDNF gene to transfect BMSCs; genetically modified BMSCs can express BDNF protein effectively for a long term.