[Objectives] To optimize the culture medium for head and neck squamous cell carcinoma patient-derived organoid and screen suitable cytokines;compare the transfection efficiency of direct transfection and short-term su...[Objectives] To optimize the culture medium for head and neck squamous cell carcinoma patient-derived organoid and screen suitable cytokines;compare the transfection efficiency of direct transfection and short-term suspension transfection for organoid in matrigel. [Methods] Advanced DMEM/F12 medium, GlutaMax and HEPES buffer, nicotinamide, N-acetylcysteine, B27, A83-01, EGF, Y-27632 and Primocin primary cell antibiotics were prepared. On this basis, fibroblast growth factor 10(FGF10), Neuregulin 1, Noggin and R-spondin-1 were added in turn to prepare the selection medium, and the organoid diameter was used as the evaluation index to evaluate the effect of organoid medium. Using lentivirus, mCherry red fluorescent protein was transfected into HNSCC—PDO in different ways, and the transfection effect was evaluated by the fluorescence intensity of organoid sphere. [Results] Nrg1 Noggin and R-Spondin-1 promoted the growth of head and neck squamous cell carcinoma sphere(P<0.05) while FGF10 did not significantly promote the growth of head and neck squamous cell carcinoma sphere(P>0.05). Compared with direct transfection, short-term suspension transfection had higher transfection efficiency for HNSCC—PDO in matrigel. [Conclusions] R-Spondin-1 Nrg1 and Noggin may be the key cytokines in culture of HNSCC—PDO whereas FGF10 played an insignificant role in this study. Short-term suspension transfection could improve the transfection efficiency of lentivirus to HNSCC—PDO.展开更多
Glial cell line-derived neurotrophic factor recombinant adenovirus vector-transfected bone marrow mesenchymal stem cells were induced to differentiate into neuron-like cells using inductive medium containing retinoic ...Glial cell line-derived neurotrophic factor recombinant adenovirus vector-transfected bone marrow mesenchymal stem cells were induced to differentiate into neuron-like cells using inductive medium containing retinoic acid and epidermal growth factor. Cell viability, micro- tubule-associated protein 2-positive cell ratio, and the expression levels of glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43 protein in the su- pernatant were significantly higher in glial cell line-derived neurotrophic factor/bone marrow mesenchymal stem cells compared with empty virus plasmid-transfected bone marrow mes- enchymal stem cells. Furthermore, microtubule-associated protein 2, glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein743 mRNA levels in cell pellets were statistically higher in glial cell line-derived neurotrophic factor/bone marrow mesen- chymal stem cells compared with empty virus plasmid-transfected bone marrow mesenchymal stem cells. These results suggest that glial cell line-derived neurotrophic factor/bone marrow mesenchymal stem cells have a higher rate of induction into neuron-like cells, and this enhanced differentiation into neuron-like cells may be associated with up-regulated expression of glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43.展开更多
Toll-like receptor 3 protein expression has been shown to be upregulated during cerebral ische- mia/reperfusion injury in rats. In this study, rat primary cortical neurons were subjected to oxy- gen-glucose deprivatio...Toll-like receptor 3 protein expression has been shown to be upregulated during cerebral ische- mia/reperfusion injury in rats. In this study, rat primary cortical neurons were subjected to oxy- gen-glucose deprivation to simulate cerebral ischemia/reperfusion injury. Chemically synthesized small interfedng RNA (siRNA)-1280, -1724 and -418 specific to toll-like receptor 3 were transfected into oxygen-glucose deprived cortical neurons to suppress the upregulation of toll-like receptor 3 protein expression. Western blotting demonstrated that after transfection with siRNA, toll-like re- ceptor 3 protein expression reduced, especially in the toll-like receptor 3-1724 group. These results suggested that siRNA-1724 is an optimal sequence for inhibiting toll-like receptor 3 expression in cortical neurons following oxygen-glucose deprivation.展开更多
Objective: To investigate the inhibitory effect and IC50, (rAdp53) in colorectal cancer cells in vitro and to guide (50% inhibiting concentration) of the recombinant adenoviral p53 gene clinical practice. Methods...Objective: To investigate the inhibitory effect and IC50, (rAdp53) in colorectal cancer cells in vitro and to guide (50% inhibiting concentration) of the recombinant adenoviral p53 gene clinical practice. Methods: We evaluated the efficiency (IC50)of the rAdp53 and six kinds of anti-cancer drugs(5-fluorouracil, tegafur, mitomycin c, cisplatin, oxaliplatin, paclitaxel) in human colorectal cancer cell line-174 through the cell culture and MTT chemosensitivity assay to make sure the anti-cancer capability of rAdp53. Expression of p53 protein in transfection cells of colorectal cancer line-174 with rAdp53 was evaluated by immunohistochemical staining. Results: The rAdp53 is a dose-and time-dependent anti-cancer drug, its IC50 is 5.73×10^11 VP/ml, but its effect was not obvious when compared with other anti-cancer drugs. In control group, the immunohistochemistry stain was negative. However, rAd-p53 of five different concentrations were all positive in infected colorectal cancer cells with rAd-p53 and the earliest positive result would present 24 hours after infection. Conclusion: The rAdp53 has good anti-cancer efficacy is colorectal cancer cell line-174 in vitro. But its anti-cancer efficacy was less than those of the classical chemical medicine mitomycin c, 5-fluorouracil and cisplatin etc., when it was used alone.展开更多
This study sought to assess the potential of brain-derived neurotrophic factor (BDNF) to promote neuronal repair and regeneration in rats with diffuse axonal injury, and to examine the accompanying neurobiological c...This study sought to assess the potential of brain-derived neurotrophic factor (BDNF) to promote neuronal repair and regeneration in rats with diffuse axonal injury, and to examine the accompanying neurobiological changes. BDNF gene transfection reduced the severity of the pathological changes associated with diffuse axonal injury in cortical neurons of the frontal lobe and increased neurofilament protein expression. These findings demonstrate that BDNF can effectively promote neuronal repair and neurite regeneration after diffuse axonal injury.展开更多
Objective: To study the effects on human glioma cell line CHG-5 by ultrasonic microbubble intensifier with survivin antisense oligonucleotides (ASODN) transfection. Methods: Antisense oligonucleotides targeting su...Objective: To study the effects on human glioma cell line CHG-5 by ultrasonic microbubble intensifier with survivin antisense oligonucleotides (ASODN) transfection. Methods: Antisense oligonucleotides targeting survivin mRNA was designed and synthesized. Four regimen groups were designed, group A: survivin antisense oligonucleotides transfected with ultrasonic microbubble intensifier combined with ultrasound irradiation, group B: survivin antisense oligonucleotides transfected with lipofectamine combined with ultrasound irradiation, group C: survivin antisense oligonucelotides with lipofectamine transfection, group D: blank control. The expression changes of surviving protein were measured by immunohistochemical staining and Western blotting, and MTT assay was used to measure the changes of proliferation. Results: Survivin protein expression in group A was decreased significantly in human glioma cell line CHG-5 than other groups(P〈0.05), and the proliferating rate of CHG-5 in group A was also significantly inhibited(P〈0.05). Conclusion: Ultrasonic microbubble intensifier transfection combined with ultrasound irradiation is a promising method in gene transfection effectively and noninvasively.展开更多
We studied the regulatory effects of the estragen receptorβ(ERβ)gene on the downstream estrogen signal transfection pathway in colon cancer cells and the possible mechanisms involved.A human ERβ gene recombinant ex...We studied the regulatory effects of the estragen receptorβ(ERβ)gene on the downstream estrogen signal transfection pathway in colon cancer cells and the possible mechanisms involved.A human ERβ gene recombinant expression plasmid,pEGFP-C1-ERβ,was constructed and transfected into the Caco-2 colon cancer cell line,a line with low ERβ gene expression.The expression of ERβ mRNA and protein was detected 72h after transfection.RT-PCR was used to examine the expression levels of the progesterone recepror(PR)gene ...展开更多
Objective: To investigate the effect of Heme oxygenase-1 (HO-1) gene transfection on the viability of cultured rat islets, and to explore the potential value of HO-1 gene in islet transplantation. Methods:Recombin...Objective: To investigate the effect of Heme oxygenase-1 (HO-1) gene transfection on the viability of cultured rat islets, and to explore the potential value of HO-1 gene in islet transplantation. Methods:Recombinant adenovirus vector containing human HO-1 gene(Ad-HO-1 ) or enhanced green fluorescent protein gene(Ad-EGFP) was generated by using AdEasy system respectively. The rat islets were transfected with Ad-HO-1, Ad-EGFP or blank vector and then cultured for 7 days. Transfection was confirmed by expression of EGFP and human HO-1 protein detected by fluorescence photographs and western blot, respectively. The insulin release upon different concentration of glucose stimulation was detected using insulin radioimmunoassay kit, and stimulation index(SI) was calculated. Glucose-stimulated insulin release was used 'to assess islet viability. Results:Adenovirus vector successfully transferred HO-1 gene to rat islet cells in vitro, and the insulin release upon high level of glucose stimulation and stimulation index (SI) of Ad-HO-1-infected islets were significantly higher than those of Ad-EGFP-infected islets and control islets (P 〈 0.05). Conclusion: Adenovirus-mediated HO-1 gene transfection is a feasible strategy to confer cytoprotection and therefore protect the viability of cultured rat islets.展开更多
AIM:To investigate whether ultrasound-targeted cationic microbubbles(CMBs)destruction could deliver endostatingreen fluorescent protein(GFP)plasmids efficiently to the human retinal endothelial cells(HRECs)and inhibit...AIM:To investigate whether ultrasound-targeted cationic microbubbles(CMBs)destruction could deliver endostatingreen fluorescent protein(GFP)plasmids efficiently to the human retinal endothelial cells(HRECs)and inhibit retinal neovascularization in mice.METHODS:CMBs were prepared and the presentation of GFP reporter was confirmed by flow cytometry and laser confocal microscopy.Experiments assessing HRECs migration and vascular formation were per formed to evaluate gene therapy’s efficiency in vitro.A mouse model of oxygen-induced retinopathy was employed and the expression of Bcl-xl,Bcl-2,vascular endothelial growth factor(VEGF)and endostatin in the retina of mice were determined by Western blotting and quantitative polymerase chain reaction(q PCR).The expression of endostatin-GFP in the retina was examined by laser confocal microscopy at 5,14,and 28 d after treatment.RESULTS:The gene expression of endostatin was the highest in the group of the CMBs.Besides,the inhibition and antiangiogenesis effect of the migration and development of HRECs were improved following treatment with CMBs compared with the other groups in vitro.In vivo,retinal neovascularization was significantly inhibited and the fluorescence intensity of endostatin-GFP in the mouse retina was importantly higher in the group of CMBs than that in other groups.CONCLUSION:The research illustrates ultrasoundtargeted CMBs destruction possessed distinct effect on the inhibition of the vascular formation and the development of retinal neovascularization both in vitro and in vivo.展开更多
Three transfection reagents, Lipofectamine® 2000, TransIT-PRO® and linear 25 kDa polyethylenimine were evaluated for transient expression of enhanced green fluorescent protein in Chinese hamster ovary ce...Three transfection reagents, Lipofectamine® 2000, TransIT-PRO® and linear 25 kDa polyethylenimine were evaluated for transient expression of enhanced green fluorescent protein in Chinese hamster ovary cells. TransIT-PRO® was found to be more efficient under the examined conditions, but comes at an increased cost compared to the widely used PEI.展开更多
In order to compare the transfection efficiency of lncRNA malat1 plasmid and lentivirus vectors in primary cultured rat DRG-derived satelite glial cells(SGCs).FISH was employed to detect the subcellular localization I...In order to compare the transfection efficiency of lncRNA malat1 plasmid and lentivirus vectors in primary cultured rat DRG-derived satelite glial cells(SGCs).FISH was employed to detect the subcellular localization IncRNA-malat1l,qPCR was used to detect the expression of IncRNA-malat1 before and after transfection of the interference vector.It was found that malat1 was not silenced by IncRNA Smart silencers plasmid.qPCR results showed no significant difference in expression level of malat1 mRNA before and after transfection smart silencers plasmid of malat1 at 24 h,48h,3 d and7 d.The subcellular localization analysis found that malat1 was mainly located in the nucleus.It is speculated that it is difficult to achieve silencing of lncRNA located in the nucleus by applying interference technology of siRNA plasmid vector.qPCR results showed that the expression level of malat1 mRNA before and after malat1 transfection was significantly decreased at 24 h and 3 d compared with the normal group and the no-load group(P<0.05).In conclusion,it was suggested that the SGCs of DRG belonged to primary cells.展开更多
Nucleic acids-based therapies have recently developed as next-generation agents for treating and preventing viral infection, cancer, and genetic disorders, but their use is still limited due to its relatively poor del...Nucleic acids-based therapies have recently developed as next-generation agents for treating and preventing viral infection, cancer, and genetic disorders, but their use is still limited due to its relatively poor delivery into targeted cells. We designed and synthesized new amphiphilic amino acid derivatives (cysteine-based) of low molecular weight, formed by the same pentapeptide (AG2: WWCOO) N-acylated, with different hydrophobic chains containing from 12 to 18 carbons, named AG2-Cn (N), which dimerize by oxidation in the presence of pLenti-CMV-GFP Puro plasmid (P) in the respective gemini. We determined transfection efficiency, critical micelle concentration, particle size, ζ-potential and cytotoxicity for the derivatives obtained. We found that all the synthesized compounds were active for DNA delivery and had greater ability to transfect CHO-K1 cells. In particular, AG2-C18 is a promising carrier for gene delivery because it showed no cytotoxicity and its activity was greater than or equal to the commercial actives currently used.展开更多
Objective:To obtain the bone marrow mesenchymal stem cells(BMSCs).complete phenotypic identification and successfully transfecl rat BMSCs by recombinant plasmid pF.GFP/Ang-1.Methods:BMSCs were isolated from bone marro...Objective:To obtain the bone marrow mesenchymal stem cells(BMSCs).complete phenotypic identification and successfully transfecl rat BMSCs by recombinant plasmid pF.GFP/Ang-1.Methods:BMSCs were isolated from bone marrow using density gradient centrifugation method and adherence screening method,and purified.Then the recombinant plasmid pEGFP/Ang-1was used to transfect BMSCs and the positive clones were obtained by the screen of C418 and observed under light microscopy inversely.Green fluorescent exhibited by protein was enhanced to measure the change time of the expression amount of Ang-1.Results:BMSCs cell lines were obtained successfully by adherence screening method and density gradient ccntrifugation.Ang-1 recombinant plasmid was transfected smoothly into rat BMSCs,which can express Ang-1 for 3 d and decreased after 7 d.Conclusions:Adherence screening method und density gradient ceiilrifugation can be effective methods lo obtain BMSCs with high purity and rapid proliferation.Besides,the expression of transfected recombinant plasmid pEGFP/Ang-1 in rat BMSCs is satisfactory.展开更多
In order to investigate the possible relationshipbetween the transfection of IL-2 gene and the changes intumor cells biological characteristics, we used retroviralvector to transduce human mammary carcinoma MCF-7cell ...In order to investigate the possible relationshipbetween the transfection of IL-2 gene and the changes intumor cells biological characteristics, we used retroviralvector to transduce human mammary carcinoma MCF-7cell line with the IL-2 cDNA and explored the changes incell membrane components and function. The genemodified cells releasing 70 - 169 U/106 cells of IL-2 in 24hrs. showed apparent morphological conversion includingthe appearance change from epitheloid form展开更多
Since the beginning of gene therapy, most of genetransfection were focused on the tumor cells or effectorcells. We selected macrophages as the target cells of genetransfection because they are not only antitumor effec...Since the beginning of gene therapy, most of genetransfection were focused on the tumor cells or effectorcells. We selected macrophages as the target cells of genetransfection because they are not only antitumor effectorcells but also antigen-presenting cells.They act as abridge connecting tumor cells with immune effector cells.Two cytokines we chosen are closely linkcd with thefunctions of macrophage. IFN-γis a principle factor toactivate macrophages and it incrcases MHC expression ofthem which can improve their antigen presenting ability.M-CSF is an important cytokine to keep theproliferation, differentiation and maturation ofmacrophage progenitor cells. In this study, we used展开更多
The elimination of the tumor is closely relatedwith the sensitivity of tumor cells to the cytotoxicityof immune effector cells. We supposed that cytokinegenetransfection may increase the cytotoxicitysusceptibility of ...The elimination of the tumor is closely relatedwith the sensitivity of tumor cells to the cytotoxicityof immune effector cells. We supposed that cytokinegenetransfection may increase the cytotoxicitysusceptibility of tumor cells to effector cells, and as aconsequence, the tumorigenicity decreased. Beforekilling tumor cells, effector cells required first torecognize non-specific surface adhesion molecules展开更多
BACKGROUND: At present, a universal method and vector for transfecting enhanced green fluorescent protein (EGFP) into neural stem cells does not exist. The traditional use of liposome to transfect GFP shows low lab...BACKGROUND: At present, a universal method and vector for transfecting enhanced green fluorescent protein (EGFP) into neural stem cells does not exist. The traditional use of liposome to transfect GFP shows low labeling efficiency and short labeling time. However, there is an increasing number of reports in recent years utilizing adeno-associated virus (AAV) transfection of neural stem cells. OBJECTIVE: To compare differences of neural stem cell transfection via rAAV-2-EGFP or liposome, with regard to transfection efficiency, stability, and safety. DESIGN, TIME AND SETTING: A parallel, controlled experiment at a cellular molecular level was performed in the Central Laboratory, Clinical Neuromedicine Research Center, Tongji Medical College, Huazhong University of Science and Technology, between June 2007 and March 2008. MATERIALS: Liposome 2000 was purchased from Invitrogen, USA; rAAV-2-EGFP was offered from Beijing AGTC Gene Technology, China. METHODS: Cerebral cortical cells from embryonic day 12 C57BL/6 mouse embryo were isolated and cultivated, and the logarithmically growing neural stem cells were divided into three groups. Liposome transfection: neural stem cells were transfected with liposome/EGFP plasmid mixture comprising 2 pg pcDNA-3.0-EGFP plasmid and 12 μg Liposome 2000 in complete culture solution. AAV transfection: neural stem cells were transfected with virus transfection solution comprising rAAV-2-EGFP and complete culture solution at multiplicity of infection = 10^5. Negative control: physiological saline was used instead of virus transfection solution. MAIN OUTCOME MEASURES: At different time points after transfection (36 hours, 1 week, 2 weeks, 1 month, and 6 months), the proportion of green fluorescent cells was quantified under fluorescent microscopy. Transfection efficiency and proliferative activity of the transfected neural stem cells were detected with flow cytometry and 3-(4,5)-dimethylthiahiazo (-z-yl)-3,5-di- phenytetrazoliumremide, respectively. RESULTS: The neural stem cells began to express green fluorescence 36 hours after transfection with rAAV-2-EGFP. Transfection efficiency reached a peak (61.2%) at 1 week, and was higher than the liposome transfection group (38.7%; P 〈 0.05). Green fluorescence was detectable for 6 months, with no weakness of expression, and rAAV-2-EGFP transfection showed no obvious effects on the proliferation activity of neural stem cells. In the liposome transfection group, green fluorescence was observed after 24 hours and reached a peak at 3 days. Fluorescence expression and proliferation activity disappeared at 2 weeks. CONCLUSION: rAAV-2-EGFP transfection of neural stem cells was superior to liposome transfection.展开更多
Human-induced pluripotent stem cells(iPSCs)are an accessible source of adult-derived,patient-specific pluripotent stem cells for use in basic research,drug discovery,disease modeling,and stem cell therapy.Improving th...Human-induced pluripotent stem cells(iPSCs)are an accessible source of adult-derived,patient-specific pluripotent stem cells for use in basic research,drug discovery,disease modeling,and stem cell therapy.Improving the accessibility of methods to obtain iPSCs regardless of the cell source can enhance their clinical application.Therefore,our purpose is to report a simple protocol to obtain iPS-like cells from urine-derived renal epithelial cells(RECs)using different extracellular matrices and transfection reagents.In this study,we began by culturing urine-derived cells from healthy donors to establish a primary culture of renal epithelial cells,followed by their characterization.Subsequently,we generated iPS-like cells by transfecting renal epithelial cells(RECs)with vectors expressing Oct4,Sox2,L-Myc,Lin-28,and Klf4,and we compared the efficacy of different extracellular matrices and transfection reagents.The resultant iPS-like cells showed a human embryonic stem cell-like morphology and expressed the specific pluripotency markers Oct3/4,Nanog,Lin28,and Klf4.We concluded that Lipofectamine Stem Cell transfection reagent is more effective than FuGENE in obtaining iPS-like cells under the conditions tested.Moreover,the three matrices are similar in their efficiency of obtaining iPS-like cells.This report provides an experimental protocol for obtaining and generating iPS-like cells from urine samples for further cell therapy research on different human diseases.展开更多
Objective: To investigate the effect of different therapeutic ultrasound (TUS) parameters and culture conditions on the cell viability and transfection efficiency of human cervical cancer cells (HeLa). Methods: ...Objective: To investigate the effect of different therapeutic ultrasound (TUS) parameters and culture conditions on the cell viability and transfection efficiency of human cervical cancer cells (HeLa). Methods: HeLa cells were cultured using two different protocols (in suspension or in monolayer). Subsequently, cells were exposed to different TUS intensity (0.4 W/cm^2, 1.0 W/cm^2, 1.6 W/cm^2, 2.2 W/cm^2), duty cycle (DC)(10%, 20%, 50%), exposure time (1 min or 3 min). Cell viability was analyzed by flow cytometry. Gene transfection of red fluorescent protein (DsRED) was detected. Results: TUS intensity and duty cycle had a great impact on the overall results (P〈0.01). Cell injury were found to increase progressively with intensity (1.6 W/cm^2, 2.2 W/cm^2) and duty cycle (50%) and cell detachment was accompanied by ultrasound exposure in adherent cells Results of factorial design showed that the fashion of cell culture and the TUS parameters had interaction (P〈0.01). The ideal conditions that cell viability above 80% producing maximum efficiency were noted to be at 1.0 W/cm^2 irradiated 3 min with a duty cycle of 20% in cell suspension. Conclusion: TUS parameters and transfection conditions have a great impact on the gene transfection and cell viability. Optimal parameters could enhance cell inembrane permeability, which facilitate to delivering the macromolecules into cells.展开更多
Three cationic lipids with lysylated(1), histidylated(2), and arginylated(3) headgroups and cholesterol hydrophobic moiety were synthesized. The average sizes of liposomes and lipoplexes were around 100 and 160 ...Three cationic lipids with lysylated(1), histidylated(2), and arginylated(3) headgroups and cholesterol hydrophobic moiety were synthesized. The average sizes of liposomes and lipoplexes were around 100 and 160 nm, respectively. The gene transfection efficiency of the three lipoplexes loaded with pGL3 or pORF-LacZ was compared on 293T cells in the presence or the absence of serum. The transfection efficiency of the three lipoplexes in a serum-free medium was 2 to 3-fold higher than that of dioleoyl-trimethylammonium propane(DOTAP). In the presence of serum, however, most of the lipoplexes showed lower transfection activities; only lipoplex 3 retained its high transfection efficiency.展开更多
基金Supported by Natural Science Foundation of China(82160386)Guangxi Natural Science Foundation(2023GXNSFAA0261892021GXNSFAA075042)。
文摘[Objectives] To optimize the culture medium for head and neck squamous cell carcinoma patient-derived organoid and screen suitable cytokines;compare the transfection efficiency of direct transfection and short-term suspension transfection for organoid in matrigel. [Methods] Advanced DMEM/F12 medium, GlutaMax and HEPES buffer, nicotinamide, N-acetylcysteine, B27, A83-01, EGF, Y-27632 and Primocin primary cell antibiotics were prepared. On this basis, fibroblast growth factor 10(FGF10), Neuregulin 1, Noggin and R-spondin-1 were added in turn to prepare the selection medium, and the organoid diameter was used as the evaluation index to evaluate the effect of organoid medium. Using lentivirus, mCherry red fluorescent protein was transfected into HNSCC—PDO in different ways, and the transfection effect was evaluated by the fluorescence intensity of organoid sphere. [Results] Nrg1 Noggin and R-Spondin-1 promoted the growth of head and neck squamous cell carcinoma sphere(P<0.05) while FGF10 did not significantly promote the growth of head and neck squamous cell carcinoma sphere(P>0.05). Compared with direct transfection, short-term suspension transfection had higher transfection efficiency for HNSCC—PDO in matrigel. [Conclusions] R-Spondin-1 Nrg1 and Noggin may be the key cytokines in culture of HNSCC—PDO whereas FGF10 played an insignificant role in this study. Short-term suspension transfection could improve the transfection efficiency of lentivirus to HNSCC—PDO.
文摘Glial cell line-derived neurotrophic factor recombinant adenovirus vector-transfected bone marrow mesenchymal stem cells were induced to differentiate into neuron-like cells using inductive medium containing retinoic acid and epidermal growth factor. Cell viability, micro- tubule-associated protein 2-positive cell ratio, and the expression levels of glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43 protein in the su- pernatant were significantly higher in glial cell line-derived neurotrophic factor/bone marrow mesenchymal stem cells compared with empty virus plasmid-transfected bone marrow mes- enchymal stem cells. Furthermore, microtubule-associated protein 2, glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein743 mRNA levels in cell pellets were statistically higher in glial cell line-derived neurotrophic factor/bone marrow mesen- chymal stem cells compared with empty virus plasmid-transfected bone marrow mesenchymal stem cells. These results suggest that glial cell line-derived neurotrophic factor/bone marrow mesenchymal stem cells have a higher rate of induction into neuron-like cells, and this enhanced differentiation into neuron-like cells may be associated with up-regulated expression of glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43.
基金supported by the National Natural Science Foundation of China,No.30970995the Postgraduate Science Research Innovation Project of Higher Learning University of Jiangsu Province in China,No.CXLX11_0735
文摘Toll-like receptor 3 protein expression has been shown to be upregulated during cerebral ische- mia/reperfusion injury in rats. In this study, rat primary cortical neurons were subjected to oxy- gen-glucose deprivation to simulate cerebral ischemia/reperfusion injury. Chemically synthesized small interfedng RNA (siRNA)-1280, -1724 and -418 specific to toll-like receptor 3 were transfected into oxygen-glucose deprived cortical neurons to suppress the upregulation of toll-like receptor 3 protein expression. Western blotting demonstrated that after transfection with siRNA, toll-like re- ceptor 3 protein expression reduced, especially in the toll-like receptor 3-1724 group. These results suggested that siRNA-1724 is an optimal sequence for inhibiting toll-like receptor 3 expression in cortical neurons following oxygen-glucose deprivation.
文摘Objective: To investigate the inhibitory effect and IC50, (rAdp53) in colorectal cancer cells in vitro and to guide (50% inhibiting concentration) of the recombinant adenoviral p53 gene clinical practice. Methods: We evaluated the efficiency (IC50)of the rAdp53 and six kinds of anti-cancer drugs(5-fluorouracil, tegafur, mitomycin c, cisplatin, oxaliplatin, paclitaxel) in human colorectal cancer cell line-174 through the cell culture and MTT chemosensitivity assay to make sure the anti-cancer capability of rAdp53. Expression of p53 protein in transfection cells of colorectal cancer line-174 with rAdp53 was evaluated by immunohistochemical staining. Results: The rAdp53 is a dose-and time-dependent anti-cancer drug, its IC50 is 5.73×10^11 VP/ml, but its effect was not obvious when compared with other anti-cancer drugs. In control group, the immunohistochemistry stain was negative. However, rAd-p53 of five different concentrations were all positive in infected colorectal cancer cells with rAd-p53 and the earliest positive result would present 24 hours after infection. Conclusion: The rAdp53 has good anti-cancer efficacy is colorectal cancer cell line-174 in vitro. But its anti-cancer efficacy was less than those of the classical chemical medicine mitomycin c, 5-fluorouracil and cisplatin etc., when it was used alone.
基金the National Natural Science Foundation of China (Key Program and General Program), No. 10832012 10872078
文摘This study sought to assess the potential of brain-derived neurotrophic factor (BDNF) to promote neuronal repair and regeneration in rats with diffuse axonal injury, and to examine the accompanying neurobiological changes. BDNF gene transfection reduced the severity of the pathological changes associated with diffuse axonal injury in cortical neurons of the frontal lobe and increased neurofilament protein expression. These findings demonstrate that BDNF can effectively promote neuronal repair and neurite regeneration after diffuse axonal injury.
基金the grants from the National 863 Scientific and Technological Research Projects[National Science Fortune Word No.(2006)501]the Highlight of National Natural Science Foundation of China(No.30430230)
文摘Objective: To study the effects on human glioma cell line CHG-5 by ultrasonic microbubble intensifier with survivin antisense oligonucleotides (ASODN) transfection. Methods: Antisense oligonucleotides targeting survivin mRNA was designed and synthesized. Four regimen groups were designed, group A: survivin antisense oligonucleotides transfected with ultrasonic microbubble intensifier combined with ultrasound irradiation, group B: survivin antisense oligonucleotides transfected with lipofectamine combined with ultrasound irradiation, group C: survivin antisense oligonucelotides with lipofectamine transfection, group D: blank control. The expression changes of surviving protein were measured by immunohistochemical staining and Western blotting, and MTT assay was used to measure the changes of proliferation. Results: Survivin protein expression in group A was decreased significantly in human glioma cell line CHG-5 than other groups(P〈0.05), and the proliferating rate of CHG-5 in group A was also significantly inhibited(P〈0.05). Conclusion: Ultrasonic microbubble intensifier transfection combined with ultrasound irradiation is a promising method in gene transfection effectively and noninvasively.
基金supported by a grant from the National Natural Sciences Foundation of China(No.30500488)
文摘We studied the regulatory effects of the estragen receptorβ(ERβ)gene on the downstream estrogen signal transfection pathway in colon cancer cells and the possible mechanisms involved.A human ERβ gene recombinant expression plasmid,pEGFP-C1-ERβ,was constructed and transfected into the Caco-2 colon cancer cell line,a line with low ERβ gene expression.The expression of ERβ mRNA and protein was detected 72h after transfection.RT-PCR was used to examine the expression levels of the progesterone recepror(PR)gene ...
基金This work was supported by National Natural Science Foundation of China, No 30571759
文摘Objective: To investigate the effect of Heme oxygenase-1 (HO-1) gene transfection on the viability of cultured rat islets, and to explore the potential value of HO-1 gene in islet transplantation. Methods:Recombinant adenovirus vector containing human HO-1 gene(Ad-HO-1 ) or enhanced green fluorescent protein gene(Ad-EGFP) was generated by using AdEasy system respectively. The rat islets were transfected with Ad-HO-1, Ad-EGFP or blank vector and then cultured for 7 days. Transfection was confirmed by expression of EGFP and human HO-1 protein detected by fluorescence photographs and western blot, respectively. The insulin release upon different concentration of glucose stimulation was detected using insulin radioimmunoassay kit, and stimulation index(SI) was calculated. Glucose-stimulated insulin release was used 'to assess islet viability. Results:Adenovirus vector successfully transferred HO-1 gene to rat islet cells in vitro, and the insulin release upon high level of glucose stimulation and stimulation index (SI) of Ad-HO-1-infected islets were significantly higher than those of Ad-EGFP-infected islets and control islets (P 〈 0.05). Conclusion: Adenovirus-mediated HO-1 gene transfection is a feasible strategy to confer cytoprotection and therefore protect the viability of cultured rat islets.
基金Supported by the National Natural Science Foundation for Youth of China(No.81901765)。
文摘AIM:To investigate whether ultrasound-targeted cationic microbubbles(CMBs)destruction could deliver endostatingreen fluorescent protein(GFP)plasmids efficiently to the human retinal endothelial cells(HRECs)and inhibit retinal neovascularization in mice.METHODS:CMBs were prepared and the presentation of GFP reporter was confirmed by flow cytometry and laser confocal microscopy.Experiments assessing HRECs migration and vascular formation were per formed to evaluate gene therapy’s efficiency in vitro.A mouse model of oxygen-induced retinopathy was employed and the expression of Bcl-xl,Bcl-2,vascular endothelial growth factor(VEGF)and endostatin in the retina of mice were determined by Western blotting and quantitative polymerase chain reaction(q PCR).The expression of endostatin-GFP in the retina was examined by laser confocal microscopy at 5,14,and 28 d after treatment.RESULTS:The gene expression of endostatin was the highest in the group of the CMBs.Besides,the inhibition and antiangiogenesis effect of the migration and development of HRECs were improved following treatment with CMBs compared with the other groups in vitro.In vivo,retinal neovascularization was significantly inhibited and the fluorescence intensity of endostatin-GFP in the mouse retina was importantly higher in the group of CMBs than that in other groups.CONCLUSION:The research illustrates ultrasoundtargeted CMBs destruction possessed distinct effect on the inhibition of the vascular formation and the development of retinal neovascularization both in vitro and in vivo.
文摘Three transfection reagents, Lipofectamine® 2000, TransIT-PRO® and linear 25 kDa polyethylenimine were evaluated for transient expression of enhanced green fluorescent protein in Chinese hamster ovary cells. TransIT-PRO® was found to be more efficient under the examined conditions, but comes at an increased cost compared to the widely used PEI.
文摘In order to compare the transfection efficiency of lncRNA malat1 plasmid and lentivirus vectors in primary cultured rat DRG-derived satelite glial cells(SGCs).FISH was employed to detect the subcellular localization IncRNA-malat1l,qPCR was used to detect the expression of IncRNA-malat1 before and after transfection of the interference vector.It was found that malat1 was not silenced by IncRNA Smart silencers plasmid.qPCR results showed no significant difference in expression level of malat1 mRNA before and after transfection smart silencers plasmid of malat1 at 24 h,48h,3 d and7 d.The subcellular localization analysis found that malat1 was mainly located in the nucleus.It is speculated that it is difficult to achieve silencing of lncRNA located in the nucleus by applying interference technology of siRNA plasmid vector.qPCR results showed that the expression level of malat1 mRNA before and after malat1 transfection was significantly decreased at 24 h and 3 d compared with the normal group and the no-load group(P<0.05).In conclusion,it was suggested that the SGCs of DRG belonged to primary cells.
基金supported by grants from Universidad Nacional del Litoral(U.N.L)(CAI+D,2011),República Argentina.
文摘Nucleic acids-based therapies have recently developed as next-generation agents for treating and preventing viral infection, cancer, and genetic disorders, but their use is still limited due to its relatively poor delivery into targeted cells. We designed and synthesized new amphiphilic amino acid derivatives (cysteine-based) of low molecular weight, formed by the same pentapeptide (AG2: WWCOO) N-acylated, with different hydrophobic chains containing from 12 to 18 carbons, named AG2-Cn (N), which dimerize by oxidation in the presence of pLenti-CMV-GFP Puro plasmid (P) in the respective gemini. We determined transfection efficiency, critical micelle concentration, particle size, ζ-potential and cytotoxicity for the derivatives obtained. We found that all the synthesized compounds were active for DNA delivery and had greater ability to transfect CHO-K1 cells. In particular, AG2-C18 is a promising carrier for gene delivery because it showed no cytotoxicity and its activity was greater than or equal to the commercial actives currently used.
基金Supported by National Natural Science Foundation of China(Grant No.20114020)
文摘Objective:To obtain the bone marrow mesenchymal stem cells(BMSCs).complete phenotypic identification and successfully transfecl rat BMSCs by recombinant plasmid pF.GFP/Ang-1.Methods:BMSCs were isolated from bone marrow using density gradient centrifugation method and adherence screening method,and purified.Then the recombinant plasmid pEGFP/Ang-1was used to transfect BMSCs and the positive clones were obtained by the screen of C418 and observed under light microscopy inversely.Green fluorescent exhibited by protein was enhanced to measure the change time of the expression amount of Ang-1.Results:BMSCs cell lines were obtained successfully by adherence screening method and density gradient ccntrifugation.Ang-1 recombinant plasmid was transfected smoothly into rat BMSCs,which can express Ang-1 for 3 d and decreased after 7 d.Conclusions:Adherence screening method und density gradient ceiilrifugation can be effective methods lo obtain BMSCs with high purity and rapid proliferation.Besides,the expression of transfected recombinant plasmid pEGFP/Ang-1 in rat BMSCs is satisfactory.
文摘In order to investigate the possible relationshipbetween the transfection of IL-2 gene and the changes intumor cells biological characteristics, we used retroviralvector to transduce human mammary carcinoma MCF-7cell line with the IL-2 cDNA and explored the changes incell membrane components and function. The genemodified cells releasing 70 - 169 U/106 cells of IL-2 in 24hrs. showed apparent morphological conversion includingthe appearance change from epitheloid form
文摘Since the beginning of gene therapy, most of genetransfection were focused on the tumor cells or effectorcells. We selected macrophages as the target cells of genetransfection because they are not only antitumor effectorcells but also antigen-presenting cells.They act as abridge connecting tumor cells with immune effector cells.Two cytokines we chosen are closely linkcd with thefunctions of macrophage. IFN-γis a principle factor toactivate macrophages and it incrcases MHC expression ofthem which can improve their antigen presenting ability.M-CSF is an important cytokine to keep theproliferation, differentiation and maturation ofmacrophage progenitor cells. In this study, we used
文摘The elimination of the tumor is closely relatedwith the sensitivity of tumor cells to the cytotoxicityof immune effector cells. We supposed that cytokinegenetransfection may increase the cytotoxicitysusceptibility of tumor cells to effector cells, and as aconsequence, the tumorigenicity decreased. Beforekilling tumor cells, effector cells required first torecognize non-specific surface adhesion molecules
基金General Program of the National Natural Science Foundation of China,No.30500167863 Project of the National Science and Technology Ministry of China,No. 2007AA03Z312
文摘BACKGROUND: At present, a universal method and vector for transfecting enhanced green fluorescent protein (EGFP) into neural stem cells does not exist. The traditional use of liposome to transfect GFP shows low labeling efficiency and short labeling time. However, there is an increasing number of reports in recent years utilizing adeno-associated virus (AAV) transfection of neural stem cells. OBJECTIVE: To compare differences of neural stem cell transfection via rAAV-2-EGFP or liposome, with regard to transfection efficiency, stability, and safety. DESIGN, TIME AND SETTING: A parallel, controlled experiment at a cellular molecular level was performed in the Central Laboratory, Clinical Neuromedicine Research Center, Tongji Medical College, Huazhong University of Science and Technology, between June 2007 and March 2008. MATERIALS: Liposome 2000 was purchased from Invitrogen, USA; rAAV-2-EGFP was offered from Beijing AGTC Gene Technology, China. METHODS: Cerebral cortical cells from embryonic day 12 C57BL/6 mouse embryo were isolated and cultivated, and the logarithmically growing neural stem cells were divided into three groups. Liposome transfection: neural stem cells were transfected with liposome/EGFP plasmid mixture comprising 2 pg pcDNA-3.0-EGFP plasmid and 12 μg Liposome 2000 in complete culture solution. AAV transfection: neural stem cells were transfected with virus transfection solution comprising rAAV-2-EGFP and complete culture solution at multiplicity of infection = 10^5. Negative control: physiological saline was used instead of virus transfection solution. MAIN OUTCOME MEASURES: At different time points after transfection (36 hours, 1 week, 2 weeks, 1 month, and 6 months), the proportion of green fluorescent cells was quantified under fluorescent microscopy. Transfection efficiency and proliferative activity of the transfected neural stem cells were detected with flow cytometry and 3-(4,5)-dimethylthiahiazo (-z-yl)-3,5-di- phenytetrazoliumremide, respectively. RESULTS: The neural stem cells began to express green fluorescence 36 hours after transfection with rAAV-2-EGFP. Transfection efficiency reached a peak (61.2%) at 1 week, and was higher than the liposome transfection group (38.7%; P 〈 0.05). Green fluorescence was detectable for 6 months, with no weakness of expression, and rAAV-2-EGFP transfection showed no obvious effects on the proliferation activity of neural stem cells. In the liposome transfection group, green fluorescence was observed after 24 hours and reached a peak at 3 days. Fluorescence expression and proliferation activity disappeared at 2 weeks. CONCLUSION: rAAV-2-EGFP transfection of neural stem cells was superior to liposome transfection.
基金the Programa de Apoyo a la Investigación Científica y Tecnológica(PAICyT,grant no.SA813-19)from the Universidad Autónoma de Nuevo Leónthe Consejo Nacional de Ciencia y Tecnología(CONACyT,grant no.CB-2015/255725)ORR thanks CONACyT for its support through a scholarship under register number 296354.
文摘Human-induced pluripotent stem cells(iPSCs)are an accessible source of adult-derived,patient-specific pluripotent stem cells for use in basic research,drug discovery,disease modeling,and stem cell therapy.Improving the accessibility of methods to obtain iPSCs regardless of the cell source can enhance their clinical application.Therefore,our purpose is to report a simple protocol to obtain iPS-like cells from urine-derived renal epithelial cells(RECs)using different extracellular matrices and transfection reagents.In this study,we began by culturing urine-derived cells from healthy donors to establish a primary culture of renal epithelial cells,followed by their characterization.Subsequently,we generated iPS-like cells by transfecting renal epithelial cells(RECs)with vectors expressing Oct4,Sox2,L-Myc,Lin-28,and Klf4,and we compared the efficacy of different extracellular matrices and transfection reagents.The resultant iPS-like cells showed a human embryonic stem cell-like morphology and expressed the specific pluripotency markers Oct3/4,Nanog,Lin28,and Klf4.We concluded that Lipofectamine Stem Cell transfection reagent is more effective than FuGENE in obtaining iPS-like cells under the conditions tested.Moreover,the three matrices are similar in their efficiency of obtaining iPS-like cells.This report provides an experimental protocol for obtaining and generating iPS-like cells from urine samples for further cell therapy research on different human diseases.
基金supported by a grant from the National Natural Science Foundation of China(No.30670548).
文摘Objective: To investigate the effect of different therapeutic ultrasound (TUS) parameters and culture conditions on the cell viability and transfection efficiency of human cervical cancer cells (HeLa). Methods: HeLa cells were cultured using two different protocols (in suspension or in monolayer). Subsequently, cells were exposed to different TUS intensity (0.4 W/cm^2, 1.0 W/cm^2, 1.6 W/cm^2, 2.2 W/cm^2), duty cycle (DC)(10%, 20%, 50%), exposure time (1 min or 3 min). Cell viability was analyzed by flow cytometry. Gene transfection of red fluorescent protein (DsRED) was detected. Results: TUS intensity and duty cycle had a great impact on the overall results (P〈0.01). Cell injury were found to increase progressively with intensity (1.6 W/cm^2, 2.2 W/cm^2) and duty cycle (50%) and cell detachment was accompanied by ultrasound exposure in adherent cells Results of factorial design showed that the fashion of cell culture and the TUS parameters had interaction (P〈0.01). The ideal conditions that cell viability above 80% producing maximum efficiency were noted to be at 1.0 W/cm^2 irradiated 3 min with a duty cycle of 20% in cell suspension. Conclusion: TUS parameters and transfection conditions have a great impact on the gene transfection and cell viability. Optimal parameters could enhance cell inembrane permeability, which facilitate to delivering the macromolecules into cells.
基金Supported by the National Basic Research Program of China(No.2005CB623903).
文摘Three cationic lipids with lysylated(1), histidylated(2), and arginylated(3) headgroups and cholesterol hydrophobic moiety were synthesized. The average sizes of liposomes and lipoplexes were around 100 and 160 nm, respectively. The gene transfection efficiency of the three lipoplexes loaded with pGL3 or pORF-LacZ was compared on 293T cells in the presence or the absence of serum. The transfection efficiency of the three lipoplexes in a serum-free medium was 2 to 3-fold higher than that of dioleoyl-trimethylammonium propane(DOTAP). In the presence of serum, however, most of the lipoplexes showed lower transfection activities; only lipoplex 3 retained its high transfection efficiency.