Objective To construct inducible lentiviral vector containing human bone morphogenetic protein 2 (hBMP-2) gene and to study its expression in human umbil ical cord blood mesenchymal stem cells (HUMSCs). Methods hBMP-2 gene was ampl ified by PCR from a plasmid and was cloned into pDown by BP reaction. pLV/EXPN2-Neo-TRE-hBMP-2 and pLV/EXPN2-Puro-EF1A-reverse transactivator (rtTA) were obtained with GATEWAY technology, and then were sequenced and analyzed by PCR. The recombinant vectors were transfected into 293FT cells respectively through l ipofectamine, and the lentiviral viruses were harvested from 293FT cells, then the titer was determined. Viruses were used to infect HUMSCs in tandem. In order to research the influence of induction time and concentration, one group of HUMSCs was induced by different doxycl ine concentrations (0, 10, 100 ng/mL, and 1, 10, 100 μg/mL) in the same induction time (48 hours), and the other by the same concentration (10 μg/mL) in different time points (12, 24, 48, and 72 hours). The expression of target gene hBMP-2 was indentified by ELISA method. After 2-week osteogenic induction of transfected HUMSCs, the mineral ization nodes were detected with Al izarin bordeaux staining method. Results Therecombinant inducible lentiviral vectors (pLV/EXPN2-Neo-TRE-hBMP-2 and pLV/EXPN2-Puro-EF1A-rtTA) were successfully constructed. The lentiviruses were also obtained and mediated by 293FT cells, and the virus titers were 3.5 × 108 TU/mL and 9.5 × 107 TU/mL respectively. HUMSCs could expression hBMP-2 by induction of doxycycl ine. The expression of hBMP-2 reached the peak at 10 μg/mL doxycl ine at 48 hours of induction. After 2-week osteogenic induction, a lot of mineral ization nodes were observed. Conclusion The recombinant inducible lentiviral vectors containing hBMP-2 gene can be successfully constructed, which provide an effective and simple method for the further study of stem cells and animal experiment in vivo.
【 Abstract】 Objective To construct a lentiviral expression vector carrying Nogo extra cellular peptide residues 1-40(NEP1-40) and to obtain NEP1-40 efficient and stable expression in mammalian cells. Methods The DNA fragment ofNEP1-40 coding sequence was ampl ified by PCR with designed primer from the cDNA l ibrary including NEP1-40 gene, and then subcloned into pGC-FU vector with in-fusion technique to generate the lentiviral expression vector, pGC-FU-NEP1-40. The positive clones were screened by PCR and the correct NEP1-40 was confirmed by sequencing. Recombinant lentiviruses were produced in 293T cells after the cotransfection of pGC-FU-NEP1-40， and packaging plasmids of pHelper 1.0 and pHelper 2.0. Green fluorescent protein (GFP) expression of infected 293T cells was observed to evaluate gene del ivery efficiency. NEP1-40 protein expression in 293T cells was detected by Western blot. Results The lentiviral expression vector carrying NEP1-40 was successfully constructed by GFP observation， and NEP1-40 protein expression was detected in 293T cells by Western blot. Conclusion The recombinant lentivirus pGC-FU-NEP1-40 is successfully constructed and it lays a foundation for further molecular function study of NEP1-40.
Abstract： Objective To construct a nesprin-siRNA lentiviral vector（LV-siNesprin）, transfect it into bone marrow mesenchymal stem cells （MSCs）, and observe morphology changes of MSCs. Methods According to the target gene sequence of nesprin, we designed and synthesized four pairs of miRNA oligo, which were then annealed into double-strand DNA and identified by sequencing. MiRNA interference with the four kinds of plasmids （SR-1,SR-2,SR-3, andSR-4） were transfected into rat vascular smooth muscle cells, and reverse transcriptase chain reaction(RT-PCR) and Western blotting were performed to detect the interference effects and filter out the most effective interference sequence. We used the best interference sequence carriers and pDONR221 to react together to get the entry vectors with interference sequence. Then the objective carrier pLenti6/V5-DEST expressing both entry vectors and lentiviral vectors was restructured to get lentiviral expression vector containing interference sequence （LV-siNesprin＋green fluoresent protein （GFP））, which was packaged and the virus titer was determined. LV-siNesprin＋GFP was transfected to MSCs, and the expression of nesprin protein（LV-siNesprin＋GFP group，GFP control group and normal cell group）was detected by Western blotting. The morphology of MSCs nuclear was observed by 4’,6-diamidino-2-phenylindole （DAPI） stain. The proliferation of MSCs （LV-siNesprin＋GFP group，GFP control group and normal group） was detected by 3-(4,5-dimethylthia- zol-2-yl)-2,5-diphenyltetrazolium bromide（MTT） after lentivirus transfected to MSCs at 24, 48, 72, and 96 hours. Results The four pairs of miRNA oligo were confirmed by sequencing. Successful construction of LV-siNesprin was confirmed by sequencing. The best interference with miRNA plasmid selected by RT-PCR and Western blotting was SR-3. Lentiviral was packaged, and the activity of the virus titer of the concentrated suspension was 1×106 ifu/ml. After MSCs were transfected with LV-siNesprin, nesprin protein expression significantly decreased, and the nuclear morphology also changed including fusion and fragmentation. The proliferation rate of MSCs in the LV-siNesprin＋GFP group was significantly slower than that of the GFP control and normal cell groups by MTT. Conclusion Nesprin protein plays an important role in stabilizing MSCs nuclear membrane, maintaining spatial structure of MSCs nuclear membrane，and facilitating MSCs proliferation.
Objective To observe the effect of shRNA interference lentivirus vector targeting rat Sirt1 gene on the expression of Sirt1 in retinal ganglion cell (RGC). Methods Four short hairpin (sh) RNA interference sequences targeting rat Sirt1 gene were designed. The target sequences of Oligo DNA were synthesized and annealed to double strand DNA, which was subsequently connected with pGLV3 lentivirus vector to build the lentiviral vector. The positive clones were identified by polymerase chain reaction (PCR) and DNA sequencing. The lentiviral vector construct and lentiviral packaging plasmids were co-transfected into 293T cells, then the titer of lentivirus were determined. The RGC were divided into 6 groups including blank group, negative control group and si-Sirt1-1, si-Sirt1-2, si-Sirt1-3, si-Sirt1-4 groups. Real-time PCR and Western blotting were used to detect the expression of Sirt1 mRNA and protein in the RGC cells. Results PCR and DNA sequencing analysis confirmed that the shRNA sequence was successfully inserted into the lentivirus vector. The concentrated titer of virus suspension was 8×108 TU/ml after the recombinant lentiviral vector successfully transfected and harvested in 293T cells. Comparing with NC group, the expression of Sirt1 mRNA and protein were significantly decreased in the si-Sirt1-1, si-Sirt1-2, si-Sirt1-3 and si-Sirt1-4 groups (F=27.682, 1 185.206; P=0.000, 0.000). The si-Sirt1-2 group had the strongest effect in reducing the expression of Sirt1 mRNA and protein. Conclusion The 4 lentiviral vectors harboring RNAi targeting rat Sirt1 gene can effectively down regulate the expression of Sirt1 mRNA and protein in RGC cells.
Objective To investigate a change in the differentiation and biological function of the cultured rat fibroblast (FB) transfected by the myoblast determining gene (MyoD) and the connexin 43 (Cx43) gene and to explore the possible mechanism of the MyoD and Cx43 genes on treatment of ischemic heart disease (IHD). Methods The gene cloning technology was used to construct the eukaryotic expressed plasmid vector pLenti6/V5-DEST-MyoD and pLenti6/V5DEST-Cx43 in which MyoD cDNA or Cx43 cDNA was inserted. The RFL-6 FB cells were transfected with exogenetic MyoD cDNA or Cx43 cDNA via lipofectamine, followed by the Blasticidin (50 μg/ml) selection, according to the lentiviral expression system (ViraPower) protocol. The expression and the biological functions of MyoD and Cx43 in the transfectants were testified by RT-PCR, Western blot, and molecular and immunocytochemical methods. The mophological structure changes of the cells were observed under microscope before and after the transfection. Results The expression of MyoD and Cx43 was detected in the MyoD and Cx43 genes transfected FB with RT-PCR and Western blot. The immunocytochemical methods indicated the expressionsof the MyoD and Cx43 genes, while desmin and αactin were found in these cells. The myotubes were found from the cultures incubated a week in the differentiation medium, in which the transfected cells had a characteristic of the filamentsin their cytoplasm and showed a myoblast morphology. Conclusion MyoD cDNA can induce the cultured FB to differentiate into the myoblasts and Cx43 cDNA can enhance the gap junctional intercellular communication between the cell and the cell. Thus, a further experimental foundation for the therapy of IHD can be provided.
Objective To construct human recombinant lentiviral expression vector of microRNA-210 (miR-210)and to explore the over-expression of miR-210 on the capillary formation in human umbilical vein endothelial cells 12 (HUVE-12). Methods The recombinant lentiviral expression vector of pGCSIL-green fluorescent protein (GFP)-pre-miR-210 wasconstructed by molecular cloning and transfected to HUVE-12 (LV-miR-210-GFP group), only pGCSIL-GFP was transfectedas control group (LV-GFP group). The miR-210 expression activity was evaluated by GFP reporter through fluorescencedetection and real-time fluorescent quantitative PCR. The ephrinA3 protein expression was measured by flow cytometry. Theconcentration of vascular endothelial growth factor (VEGF) in culture supernatant was determined by ELISA. The cells werecultured in 96-well culture plate coated with Matrigel to assess the abil ity of capillary formation. Results The recombinantplasmid pGCSIL-GFP-pre-miR-210 was confirmed by restriction endonuclease analysis and DNA sequencing. Fluorescencedetection showed that the fluorescence intensity of GFP was highest between 48 and 72 hours after transfection. Real-timefluorescent quantitative PCR showed that the miR-210 expression of LV-miR-210-GFP group was 9.72 times higher than thatin LV-GFP group (t= —11.10，P=0.00). Flow cytometry analysis showed that the positive cell rate of enphrinA3 in LV-miR-210-GFP group (12.52% ± 0.67%) was significantly lower than that in LV-GFP group (73.22% ± 1.45%) (t= —66.12，P=0.00).The concentration of VEGF in supernatant in LV-miR-210-GFP group was significantly higher than that in LV-GFP group[(305.29 ± 16.52) pg/mL vs. (42.52 ± 3.11) pg/mL, t= —27.06，P=0.00]. In vitro capillary-l ike formation assay showed that thenumber of capillaries was significantly larger in LV-miR-210-GFP group than in LV-GFP group (17.33 ± 6.33 vs. 6.33 ± 2.33,t= —2.83，P=0.04). Conclusion The recombinant lentiviral expression vector of miR-210 is constructed successfully andHUVE-12 over-expressing miR-210 can significantly increase the capillary formation, which facil itates further study on themolecular functions of miR-210 in angiogenesis.
Objective To construct gene-modified hepatic stem cells (WB-F344 cells), which have rat IL-13 gene and can secrete the recombinant rat IL-13 cytokine in the cells. Methods Firstly, the rat IL-13 sequences were synthesized. Then the sequences were amplificated in bacterium coli after recombinated with pWPXL-MOD plasmid. After PCR and sequence identification, the positive clones were packaged into lentivirus. After detecting the virus titer, the WB-F344 cells with constructed lentivirus vector with rat IL-13 gene were cultured, then the valid targets (expression level of the IL-13) were detected by real time-PCR and Western blot in cultured WB-F344 cells on 5 days. Results The valid DNA of rat IL-13 was recombinated and packaged in lentivirus vector. The recombinant gene sequence was correct by checking with gene sequence test. Then the recombinant was introducted into the WB-F344 cells cultures. The best multiplicity of infection (MOI) value for effective transfection was 5. IL-13 had been detected on day 5 after transfection by checking with real-time PCR and Western blot. Conclusion The recombinant rat IL-13 gene with lentivirus vector is constructed and gene-modified WB-F344 cells are cultured successfully, which can be used in next animal experiment.
Objective To construct the lentiviral vector containing homo sapiens forkhead box C2 (Foxc2) gene and to detect its expression in bone marrow mesenchymal stem cells (BMSCs) of rabbits. Methods Human Foxc2 gene coding region fragment was obtained by RT-PCR and then cloned into the plasmid of LV-green fluorescent protein (GFP) to prepare Foxc2 lentiviral plasmid. Foxc2 lentiviral plasmid, pGC-LV, pHelper1.0, and pHelper2.0 were co-transfected into 293T cells to obtain recombinant virus containing Foxc2 gene. The lentiviral titer was detected. BMSCs were isolated from bone marrow of rabbit and infected with Foxc2 recombined lentiviral, then the optimum multiplicity of infection (MOI) was determined by detecting the intensity of fluorescence expression. The expression of Foxc2 in the infected BMSCs was determined at 1, 3, and 7 days after transfection by inverted fluorescence microscope and Western blot. After osteogenic induction, Alizarin red staining was done to observe the formation of mineralized nodule. Results The Foxc2 recombinant lentiviral vector was constructed and was confirmed by restriction enzyme digestion and sequencing analysis. It could efficiently transfect 293T cells and express in 293T cells. The lentiviral titer was 2 × 108 TU/mL. The optimum MOI was 200. The inverted fluorescence microscope observation showed that the Foxc2 gene expressed in 84.5% ± 4.8% of infected BMSCs at 3 days after transfection. The expression of Foxc2 in infected BMSCs was stable and high, and increased gradually within 7 days after transfection by Western blot. At 2 weeks after osteogenic induction, Alizarin red staining showed that there were a large number of red calcified matrix deposition in the cytoplasm. Conclusion Foxc2 recombined lentivirus with high viral titer is successfully constructed and packaged, and the Foxc2 gene can be transfected into BMSCs with stable and high expression of Foxc2 in infected cells, and these cells may be applied for gene therapy of avascular necrosis of the femoral head.
Objective Biliary epithelial cell (BEC) proliferated actively induced by ischemia-type biliary lesion (ITBL), which played an important role in the development of biliary complication after orthortopic liver transplantation (OLT). The aims of this study is to provide novel method to protect the liver endured cold preservation and reperfusion injury (CPRI) and reduce posttransplant biliary complication, and explore its possible mechanism.Methods Based on constructed OLT models for studying ITBL, the hepatic oval cell (HOC) or the IL-13 genemodified HOC to the portal vein of the recipient 〔OLT+HOC group and OLT+IL-13· HOC group〕 were-transfused, then the pathology change, the liver function and the expressions of the α-smooth muscle actin (αSMA) and Heme oxygenase-1 (HO-1) mRNA of the transplanted liver of CPRI were observed, the proliferation of BEC and survival rate of the recipients were also observed. Results The BEC injury was showed in grafts with prolonged ischemia time, characterized by induction of BEC proliferation, liver function injury and cholestasis sign reflecting the increase of serum ALT, AST and TBIL. The OLT+IL-13·HOC group had better results than OLT and OLT+HOC group, which indicated the OLT+IL-13·HOC group had low level of expression α-SMA (after operation 7 d, Plt;0.05) and proliferation of BEC (after operation 3 d, Plt;0.05). The expressions of HO-1 mRNA were higher in OLT+IL-13·HOC group than in other groups. The survival rate of OLT group was lower than that of the OLT+IL-13·HOC group and sham operation group (Plt;0.05).Conclusion High expression level of IL-13 in recipient rats could promote the expression of HO-1 mRNA in transplant liver, and profit to protection donor liver, and recover of the liver function after liver transplantation. It perhaps is the mechanism of protective effect of IL-13 on graft that stimulate the expression of HO-1 mRNA significantly.
ObjectiveTo investigate whether Akt1 gene transfection mediated by recombinant lentivirus (LVs) in the bone marrow mesenchymal stem cells (BMSCs) could enhance the ability of hypoxia tolerance so as to provide a theoretical basis for improving the effectiveness of stem cells transplantation. MethodLVs was used as transfection vector, enhanced green fluorescent protein (EGFP) was used as markers to construct the pLVX-EGFP-3FLAG virus vector carrying the Akt1 gene. The 3rd generation BMSCs from 3-5 weeks old Sprague Dawley rats were transfected with pLVX-EGFP virus solution as group B and with pLVX-EGFP-3FLAG virus solution as group C; and untransfected BMSCs served as control group (group A). At 2-3 days after transfection, the expression of green fluorescent was observed by fluorescence microscope; and at 48 hours after transfection, Western blot method was used to detect the expression of Akt1 protein in groups B and C. BMSCs of groups B and C were given hypoxia intervention with 94%N2, 1%O2, and 5%CO2 for 0, 3, 6, 9, and 12 hours (group B1 and group C1) . The flow cytometry was used to analyze the cell apoptosis rate and cell death rate, and the MTT method to analyze the cell proliferation, and Western blot to detect the expression of apoptosis related gene Caspase-3. ResultsAfter transfection, obvious green fluorescence was observed in BMSCs under fluorescence microscopy in groups B and C, the transfection efficiency was about 60%. Akt1 expression of group C was significantly higher than that of group B (t=17.525, P=0.013) . The apoptosis rate and cell death rate of group B1 increased gradually with time, and difference was significant (P<0.05) . In group C1, the apoptosis rate and cell death rate decreased temporarily at 3 hours after hypoxia intervention, then increased gradually, and difference was significant (P<0.05) . The apoptosis rate and cell death rate of group C1 were significantly lower than those of group B1 at each time point (P<0.05) except at 0 hour. MTT assay showed that absorbance (A) values of groups B and C were significantly higher than those of groups B1 and C1 at each time point (P<0.05) ; the A value of group B was significantly lower than that of group C at each time point (P<0.05) . The A value of group B1 was significantly lower than that of group C1 at 6, 9, and 12 hours after hypoxia intervention (P<0.05) . Western blot results showed that the Caspase-3 expression of group C1 significantly reduced when compared with group B1 at each time point (P<0.05) . ConclusionsAkt1 gene transfection mediated by recombinant LVs could significantly improve hypoxia tolerance of BMSCs by inhibiting the apoptosis, which could provide new ideas for improving the effectiveness of stem cells transplantation.