全身垂直振动对去卵巢骨质疏松大鼠骨髓细胞分化的调节作用

目的:观察全身垂直振动对去卵巢骨质疏松大鼠骨髓细胞分化的调节作用。方法:将36只3月龄雌性未孕SD大鼠按体重分层后随机分为假手术、去卵巢静止和去卵巢振动3个组。去卵巢手术后10周,去卵巢振动组大鼠经过1周适应振动治疗后,开始接受正式全身垂直振动治疗,每次振动15 min、频率为90 Hz,每天振动2次,每周7次。治疗7周时,用721分光光度计检测血清碱性磷酸酶和甘油三酯水平;常规HE染色观察左侧胫骨组织形态学;收集右侧股骨和胫骨骨髓细胞,一部分细胞用作碱性磷酸酶和油红O染色,另一部分细胞用作尼罗红(Nile Red)染色。结果:与假手术组比较,去卵巢静止组骨髓细胞碱性磷酸酶阳性染色细胞数目显著降低,而骨髓细胞Nile Red阳性染色细胞百分比和胫骨骨髓脂肪空泡数目显著增加;与去卵巢静止组比较,去卵巢振动组骨髓细胞碱性磷酸酶阳性染色细胞数目显著增加,而骨髓细胞Nile Red阳性染色细胞百分比和胫骨骨髓脂肪空泡数目显著下降。结论:全身垂直振动不仅增加去卵巢骨质疏松大鼠骨髓细胞的成骨分化能力,而且降低去卵巢骨质疏松大鼠骨髓细胞的成脂分化能力。

冻存骨髓基质细胞体外成骨潜能的实验研究

目的观察冻存骨髓基质细胞(BMSC)的体外生长特点及诱导与非诱导条件下的成骨能力。方法取冻存11个月犬BMSC复苏后进行体外扩增培养,传代培养中加入10-8mol/L地塞米松、50 mg/L Vit C和10mmol/Lβ-甘油磷酸钠进行诱导分化,观察细胞形态、细胞诱导前后增殖情况、细胞碱性磷酸酶(ALP)含量以及形成矿化结节能力的改变。结果冻存BMSC复苏后呈成纤维样表现,增殖力强。在诱导液的作用下冻存BMSC增殖性能降低,ALP活性增加,诱导5 d后可见矿化结节。结论冻存BMSC增殖能力强,经诱导后表现出明显的成骨活性,可作为骨组织工程的种子细胞。

Passage of bone-marrow-derived liver stem cells in a proliferating culture system

AIM： To explore the feasibility of passage of bone- marrow-derived liver stem cells （BDLSCs） in culture systems that contain cholestatic serum.METHODS： Whole bone marrow cells of rats were purified with conditioning selection media that contained 50 mL/L cholestatic serum. The selected BDLSCs were grown in a proliferating culture system and a differentiating culture system. The culture systems contained factors that stimulated the proliferation and differentiation of BDLSCs. Each passage of the proliferated stem cells was subjected to flow cytometry to detect stem cell markers. The morphology and phenotypic markers of BDLSCs were characterized using immunohistochemistry, reverse transcription polymerase chain reaction （RT-PCR） and electron microscopy. The metabolic functions of differentiated cells were also determined by glycogen staining and urea assay.RESULTS： The conditioning selection medium isolated BDLSCs directly from cultured bone marrow cells. The selected BDLSCs could be proliferated for six passages and maintained stable markers in our proliferating system. When the culture system was changed to a differentiating system, hepatocyte-like colony-forming units （H-CFUs） were formed. H-CFUs expressed markers of embryonic hepatocytes （alpha-fetoprotein, albumin and cytokeratin 8/18）, biliary cells （cytokeratin 19）, hepatocyte functional proteins （transthyretin and cytochrome P450-261）, and hepatocyte nuclear factors 1α and -3β）. They also had glycogen storage and urea synthesis functions, two of the critical features of hepatocytes.CONCLUSION： BDLSCs can be selected directly from bone marrow cells, and pure BDLSCs can be proliferated for six passages. The differentiated cells have hepatocyte-like phenotypes and functions. BDLSCs represent a new method to provide a readily available alternate source of cells for clinical hepatocyte therapy.

Differentiation of hepatocytoid cell induced from whole-bone-marrow method isolated rat myeloid mesenchymal stem cells

AIM： To explore the expansion and differentiation of hepatocytoid cell induced from myeloid mesenchymal stem cell （MSC） in vib＇o, in order to find suitable resource of hepatocytes for bioartiflcial liver or liver transplantation. METHODS： The rat myeloid MSC was isolated and divided into three groups which were cultured by Friedensteion method, and then were induced by culture fluid, culture fluid plus cholestatic serum and culture fluid plus hepatocyte growth factor （HGF）, respectively. Hepatocytoid cell as well as expression of CK18 and AFP was observed by immunohistochemistry. RESULTS： After the induction for 21 d, hepatocytoid cell was observed, and its expression of CK18 and AFP was detected by immunohistochemistry in MSC cultured with cholestatic serum. Furthermore, on the 35^th d, albumin mRNA was expressed in the cell, suggesting the inducing effect was similar to that by HGF.CONCLUSION： Rat myeloid MSC can differentiate into hepatocyte lineage under appropriate condition. This method is easy to operate.

Differentiation of rat bone marrow stem cells in liver after partial hepatectomy

AIM： To investigate the differentiation of rat bone marrow stem cells in liver after partial hepatectomy. METHODS： Bone marrow cells were collected from the tibia of rat with partial hepatectomy, the medial and left hepatic lobes were excised. The bone marrow stem cells （Thy^＋CD3^-CD45RA^- cells） were enriched from the bone marrow cells by depleting red cells and fluorescence-activated cell sorting. The sorted bone marrow stem cells were labeled by PKH26-GL in vitro and autotransplanted by portal vein injection. After 2 wk, the transplanted bone marrow stem cells in liver were examined by the immunohistochemistry of albumin （hepatocyte-specific marker）. RESULTS： The bone marrow stem cells （Thy^＋CD3^-CD45RA^- cells） accounted for 2.8% of bone marrow cells without red cells. The labeling rate of 10 μM PKH26- GL on sorted bone marrow stem cells was about 95%. There were sporadic PKH26-GL-labeled cells among hepatocytes in liver tissue section, and some of the cells expressed albumin. CONCLUSION： Rat bone marrow stem cells can differentiate into hepatocytes in regenerative environment and may participate in liver regeneration after partial hepatectomy.

Chondrogenic differentiation of rat bone marrow mesenchymal stem cells induced by puerarin and tetrandrine

Objective: This study aims to clarify the effect of the active components puerarin and tetrandrine on the chondrogenic differentiation of bone marrow mesenchymal stem cells(BMSCs).Methods: Using network pharmacology, protein targets of puerarin and tetrandrine were predicted, and a database of cartilage formation targets was established. The protein target information related to disease was then collected, and the drug-targeting network was constructed by analyzing the protein–protein interactions. Genes related to chondrogenesis induced by puerarin and tetrandrine and chondroblast differentiation signaling pathways were searched. Finally, potential drug-and disease-related genes,as well as proteins, were screened and verified using real-time RT-PCR and western blotting.Results: Network pharmacological studies have shown that puerarin and tetrandrine are involved in BMSCs cartilage differentiation. The experimental results showed that puerarin and tetrandrine could regulate the expression of cartilage differentiation-related genes and proteins. Puerarin increased the protein expression of COL2 A1, COL10 A1, MMP13, and SOX-9,as well as the gene expression of Col2 a1, Mmp13, Tgfb1, and Sox-9. Tetrandrine increased the protein expression of COL2 A1,COL10 A1, MMP13, and SOX-9, as well as the gene expression of Col10 a1, Tgfb1, Sox-9, and Acan. The combination of puerarin and tetrandrine increased the protein expression of COL2 A1, COL10 A1, MMP13, and SOX-9 and the gene expression of Col2 a1,Col10 a1, Sox-9, and Acan.Conclusions: Puerarin, tetrandrine, and their combination can promote the proliferation of BMSCs and induce their differentiation into chondrocytes, and they are thus expected to be inducers of chondrogenic differentiation. These results suggest that puerarin and tetrandrine have potential therapeutic effects on osteoarthritis.

Comparison of human amniotic fluid-derived and umbilical cord Wharton＇s Jelly-derived mesenchymal stromal cells： Characterization and myocardial differentiation capacity

Objective To compare the characterization and myocardial differentiation capacity of arnniotic fluid-derived mesenchymal stromal cells （AF MSCs） and umbilical cord Wharton＇s Jelly-derived mesenchymal stromal cells （WJ MSCs）. Methods The human AF MSCs were cultured from amniotic fluid samples obtained by amniocentesis. The umbilical cord WJ MSCs were obtained from Wharton＇s Jelly of umbilical cords of infants delivered full-term by normal labor. The morphology, growth curves, and analyses by flow cytometry of cell surface markers were compared between the two types of cells. Myocardial genes （GATA-4, c-TnT, a-actin, and Cx43） were detected by real-time PCR and the corresponding protein expressions were detected by Western blot analysis after myocardial induced in AF MSCs and WJ MSCs. Results Our findings revealed AF MSCs and WJ MSCs shared similar morphological characteristics of the fibroblastoid shape. The AF MSCs were easily obtained than the WJ MSCs and had a shorter time to reach adherence of 2.7 ± 1.6 days to WJ MSCs of 6.5 ± 1.8 days. The growth curves by MTT cytotoxic assay showed the AF MSCs had a similar proliferative capacity at passage 5 and passage 10. However, the proliferative capacities ofWJ MSCs were decreased at 5 passage relative to 10 passage. Both AF stem cells and WJ stem cells had the characteristics of mesenchymal stromal cells with some characteristics of embryonic stem cells. They express CD29 and CD105, but not CD34. They were positive for Class I major histocompatibility （MHC I） antigens （HLA-ABC）, and were negative, or mildly positive, for MHC Class II （HLA-DR） antigen. Oct-4 was positive in all the two cells types. Both AF MSCs and WJ MSCs could differentiate along myocardium. The differentiation capacities were detected by the expression of GATA-4, c-TnT, a-actin, Cx43 after myocardial induction. Conclusions Both AF MSCs and WJ MSCs have the potential clinical application for myogenesis in cardiac regenerative therapy.

Experimental study on the induction of bone marrow stromal cells differentiating into cardiomyocyte-like cells with cardiomyocytes in vitro

Objective：To investigate the feasibility of bone marrow stromal cells （BMSCs） differenti ating into cardiomyocyte like cells in heterogeneous cardiomyocytes microenvironment in vitro. Methods： Mouse GFP-BMSCs were isolated by centrifugation through a Ficoll step gradient and purified by plating culture and depletion of the non-adherent cells. Neonatal rat cardiomyocytes （CMs） were isolated by enzymatic dissociation from hearts of 1-to 2-day old Sprague-Dawley （SD） rats and differentially plated to remove fibroblasts. Mouse GFP-BMSCs were cocuhured with neonatal rat CMs through direct and indirect contact, respectively. Cardiomyogenic differentiation of BMSCs was evaluated by immunostaining with an- ti-a-actin monoclonal antibody and observing synchronous contraction with adjacent CMs by phase contrast microphotography. Results： On day 7 of cocuhure, GFP-BMSCs （CMs ： BMSCs：4 ： 1）attached to nonfluorescent contracting cells （rat-derived CMs） showed myotube-like formation and started to contract synchronously with adjacent cardiomyocytes. About 10% of the fluorescent GFP-BMSCs were cardiomyocyte-like cells as determined by cell morphology and positive actin staining. Conclusion;Direct cell to-cell interaction with CMs is crucial for cardiomyogenic differentiation of BMSCs in heterogeneous CMs microenvironment in vitro. This provides a novel inducing pathway for directional differentiation of cardiovascular tissue engineering seed cells.

Experimental study on MyoD gene induced differentiation of bone marrow mesen-chymal stem cells into myoblasts in vitro

Objective: To explore the possibility of the transfection of MyoD gene induced bone marrow mesenchymal stem cells ( MSCs) to differentiate into myoblasts in vitro. Methods: The eukaryotic expression plasmid vector pIRES2-EGFP-MyoD was transfected into MSCs with lipotransfection method, and the positive cells were selected by G418; The expression of MyoD was detected in the transfected MSCs with RT-PCR and the amplified, purified product was identified by sequencing; The reporter gene enhanced green fluorescence protein ( EFGP) was observed in the transfected cells under a fluorescent and a laser confocal microscopes; Immunohistochemical methods was used to examine the expressions of MyoD, myogenin, myosin, myoglobin and desmin in the differentiated cells. The ultrastructure changes of the cells before and after transfection were observed with electron microscopy. Results: The expression of MyoD was detected in the transfected MSCs with RT-PCR and the amplified, purified product was as same in sequence as that from Genbank; Green fluorescence was observed in the transfected cells under a fluorescent and a laser confocal microscopes; Immunohistochemical methods indicated that MyoD, myogenin, myosin, myoglobin and desmin were expressed in the transfected cells; The transfected cells showed the morphological characteristics of mature cells with filaments in their cytoplasm. Conclusion: MyoD gene can induce cultured MSCs to successfully differentiate into myoblasts , probably providing an experimental foundation for trauma repair.

Identification of key genes responsible for cytokine-induced erythroid and myeloid differentiation and switching of hematopoietic stem cells by RAGE

We utilized a unique culture system to analyze the expression patterns of gene, protein, and cell surface antigen, and the biological process of the related genes in erythroid and myeloid differentiation and switching of hematopoietic stem cells （HSCs） in response to cytokine alterations. Gene-specific fragments （266） identified from five populations of cytokine-stimulated HSCs were categorized into three groups： （1） expressed specifically in a single cell population; （2） expressed in two cell populations, and （3） expressed in three or more populations. Of 145 defined cDNAs, three （2%） were novel genes. Protein two-dimensional gel electrophoresis and flow cytometry analyses showed overlapped and distinguished protein expression profiles in the cell populations studied. Biological process mapping of mRNAs expressed in erythroid and myeloid lineages indicated that mRNAs shared by both lineages attended ＇core processes,＇ whereas genes specifically expressed in either lineage alone were related to specific processes or cellular maturation. Data from this study support the hypothesis that committed HSCs （El4 or G14） cells can still be redirected to develop into myeloid or erythroid cells when erythropoietin （EPO） is replaced with granulocyte-colony stimulating factor （G-CSF） under erythroid-cultured condition or G-CSF with EPO in myeloid-cultured environment, respectively. Our results suggest that genes or proteins co-expressed in erythroid and myeloid lineages may be essential for the lineage maintenance and switching in hematopoiesis.

Neurotrophic Effect of Bone Proliferation and Committed Mesencephalic Precursors Marrow Stromal Cells on Differentiation of Ventral

OBJECTIVE To explore the potential neurotrophic effect of bone marrow stromal cells （BMSCs） on cell proliferation and committed neuronal differentiation of ventral mesencephalic precursors （VMPs） in vitro. METHODS Ventral mesencephalic precursors from Ell inbred rat embryos and BMSCs from adult rats were cultured both separately and in co-culture. After a 7-day incubation in vitro, three conditioned culture media were obtained, termed VMP or common medium, BMSC medium, and BMSC±VMP medium. Ventral mesen- cephalic precursors cells were cultured in each of these media and the effects on proliferation and VMP differentiation were assessed. The relative yield of TH± cells was calculated and compared by immunocytochemical staining. RESULTS After a 7-day culture and induction of VMPs, the total cell counts were increased by （44.13±4.75）-fold （common）, （60.63±5.25）-fold （BMSC）, and （64.00±7.63）-fold （BMSC±VMP）. The proportions of TH＋ cells were （18.76±5.20）%, （23.49±4.10）%, and （28.08± 5.42）%, respectively, with statistically significant differences among the treatment groups. CONCLUSION BMSCs release factors that promote the proliferation of VMPs and facilitate the committed differentiation of VMPs into dopaminergic neurons.

Mesenchymal Stem Cells Express Low Levels of Cardiomyogenic Genes and Show Limited Plasticity towards Cardiomyogenic Phenotype

Mesenchymal stem cell differentiation towards osteogenic, chondrogenic and adipogenic lineages have been extensively described and reproduced in the literature. In contrast, cardiomyogenic differentiation still remains largely controversial. In this study the authors aim to shed new light into this unclear phenomenon and test whether BMMSC （bone marrow mesenchymal stem cells） and ATMSC （adipose tissue derived mesenchymal stem cells） are able to differentiate into functional cardiomyocytes, investigating two differentiation protocols. AT and BMMSC behaved differently when cultured in differentiation media and presented lower levels of proliferation and alkaline phosphatase production, expression of cardiomyocyte-specific transcription factors such as GATA-4, Nkx2-5 and proteins such as ct and 13 Myosin Heavy Chains. Furthermore, MSC started to express higher levels of Connexin-43 and c~ sarcomeric actinin protein. Unfortunately, though, MSC did not present cardiomyocyte-like electrophysiological properties. In order to analyze a possible explanation for such limited plasticity, the authors decided to address the issue using a quantitative approach. Gene expression was quantified by Real time PCR, and, for the first time, the authors show that a possible explanation for limited plasticity of MSC is that even though differentiated cells presented differential gene expression, the levels of key cardiomyogenic genes did not reach expression levels presented by adult cardiomyocytes, nor were maintained along differentiation, reaching peaks at 4 days of stimulation, and decaying thereafter.

Osteogenic differentiation of mesenchymal stem cells promoted by overexpression of connective tissue growth factor

Objective:Large segmental bone defect repair remains a clinical and scientific challenge with increasing interest focusing on combining gene transfection with tissue engineering techniques.The aim of this study is to investigate the effect of connective tissue growth factor(CTGF) on the proliferation and osteogenic differentiation of the bone marrow mesenchymal stem cells(MSCs).Methods:A CTGF-expressing plasmid(pCTGF) was constructed and transfected into MSCs.Then expressions of bone morphogenesis-related genes,proliferation rate,alkaline phosphatase activity,and mineralization were examined to evaluate the osteogenic potential of the CTGF gene-modified MSCs.Results:Overexpression of CTGF was confirmed in pCTGF-MSCs.pCTGF transfection significantly enhanced the proliferation rates of pCTGF-MSCs(P<0.05).CTGF induced a 7.5-fold increase in cell migration over control(P<0.05).pCTGF transfection enhanced the expression of bone matrix proteins,such as bone sialo-protein,osteocalcin,and collagen type I in MSCs.The levels of alkaline phosphatase(ALP) activities of pCTGF-MSCs at the 1st and 2nd weeks were 4.0-and 3.0-fold higher than those of MSCs cultured in OS-medium,significantly higher than those of mock-MSCs and normal control MSCs(P<0.05).Overexpression of CTGF in MSCs enhanced the capability to form mineralized nodules.Conclusion:Overexpression of CTGF could improve the osteogenic differentiation ability of MSCs,and the CTGF gene-modified MSCs are potential as novel cell resources of bone tissue engineering.

Lack of telomerase activity in rabbit bone marrow stromal cells during differentiation along neural pathway

To investigate telomerase activity in rabbit bone marrow stromal cells （BMSCs） during their committed differentiation in vitro along neural pathway and the effect of glial cell line-derived neurotrophic factor （GDNF） on the expression of telomerase. Methods ： BMSCs were acquired from rabbit marrow and divided into control group, GDNF （10 ng/ml） group. Cytokine . NSCs medium （prepared by our lab, Patent No. ZL02134314. 4） supplemented with 10% fetal bovine serum （FBS） was used to induce BMSCs differentiation along neural pathway. Fluorescent was employed to identify the expressions of Nestin, neuronspecific endase （NSE）, and gial fibrillary acidic protein （GFAP）. The growth curves of the cells and the status of cell cycles were analyzed, respectively. During the differentiation, telomerase activitys were detected using the telomeric repeat amplification protocol-enzyme-linked immunosorbent assay （TRAP-ELISA）. Results： BMSCs were successfully induced to differentiate along neural pathway and expressed specific markers of fetal neural epithelium, mature neuron and glial cells. Telomeruse activities were undetectahle in BMSCs during differentiation along neural pathway. Similar changes of cell growth curves, cell cycle status and telomeruse expression were observed in the two groups. Conclusions ： Rabbit BMSCs do not display telomeruse activity during differentiation along neural pathway. GDNF shows little impact on proliferation and telomeruse activity of BMSCs.

Identification of neuron-related genes for cell therapy of neurological disorders by network analysis

Bone mesenchymal stem cells（BMSCs） differentiated into neurons have been widely proposed for use in cell therapy of many neurological disorders. It is therefore important to understand the molecular mechanisms underlying this differentiation. We screened differentially expressed genes between immature neural tissues and untreated BMSCs to identify the genes responsible for neuronal differentiation from BMSCs. GSE68243 gene microarray data of rat BMSCs and GSE18860 gene microarray data of rat neurons were received from the Gene Expression Omnibus database. Transcriptome Analysis Console software showed that 1248 genes were up-regulated and 1273 were down-regulated in neurons compared with BMSCs. Gene Ontology functional enrichment, protein-protein interaction networks, functional modules, and hub genes were analyzed using DAVID, STRING 10, BiN GO tool, and Network Analyzer software, revealing that nine hub genes, Nrcam, Sema3 a, Mapk8, Dlg4, Slit1, Creb1, Ntrk2, Cntn2, and Pax6, may play a pivotal role in neuronal differentiation from BMSCs. Seven genes, Dcx, Nrcam, Sema3 a, Cntn2, Slit1, Ephb1, and Pax6, were shown to be hub nodes within the neuronal development network, while six genes, Fgf2, Tgfβ1, Vegfa, Serpine1, Il6, and Stat1, appeared to play an important role in suppressing neuronal differentiation. However, additional studies are required to confirm these results.