Objective To investigate the feasibility of recombinant lentivirus (LVs) mediated hyperpolarization- activated cyclic nucleotide-gated cation channel 4 (HCN4) gene transfecting rat bone mesenchymal stem cells (BMSCs) so as to construct the biological pacemaker cells. Methods Sprague Dawley rats at the age of 3-5 weeks were selected to isolate and culture BMSCs using modified whole bone marrow adherent culture method. LVs was used as carrier, and enhanced green fluorescent protein (EGFP) as marker to build LVs-HCN4-EGFP virus liquid. The BMSCs at passage 3 were transfected with LVs-HCN4-EGFP virus liquid (experimental group) and LVs-EGFP null virus liquid (control group). Fluorescence microscope was used to observe the green fluorescent protein expression after 24, 48, and 72 hours of transfection; Western blot method was used to detect the HCN4 protein expression. The electrophysiology was used to detect the pacemaker current in the experimental group. Results After transfection, BMSCs in the experimental group showed normal morphology and good growth; scattered green fluorescence could be seen at 48 hours under fluorescence microscope, with a transfection efficiency of about 10%; the fluorescence expression increased slightly, with the transfection efficiency of 20% to 25% at 72 hours. While no expression of green fluorescence was seen in the control group. Western blot results showed that the same band expression as a relative molecular mass of HCN4 protein were found at 72 hours after transfection in the experimental group, only weak expression of protein band was seen in the control group; the gray value of the experimental group (33.75 ± 0.41) was significantly higher than that of the control group (23.39 ± 0.33) (t=17.524, P=0.013). In the experimental group, the pacemaker current was recorded, and it could be blocked by CsCl, in accordance with the characteristics of pacemaker current. Conclusion The recombinant LVs mediated HCN4 gene is successfully transfected into rat BMSCs, and the expression of HCN4 protein and the pacemaker current can be detected.
ObjectiveTo investigate the inhibitory effect of lentivirus-mediated polypyrimidine bundle binding protein-associated splicing factor (PSF) on retinal neovascularization (RNV) in mice model of oxygen-induced retinopathy (OIR).MethodsOne hundred and twelve 5-day-old C57BL/6J mice were randomly divided into normal control group, simple OIR model group, OIR model + lentivirus empty vector treatment group (Vec group) and OIR model + PSF lentivirus treatment group (PSF group), with 16, 32, 32 and 32 mice, respectively. When the mice were 7 days old, the mice in the normal control group were fed in a routine environment, and the mice in the OIR model group, Vec group and PSF group were established OIR model. The mice in the Vec group and PSF group were given an intravitreal injection of 1 μl of lentiviral vector and PSF lentivirus (titer 1×1011 TU/ml) at the age of 12 days. No injection was performed in the normal control group and simple OIR group. RNV was evaluated by counting the number of pre-retinal neovascular cells and analysis of non-perfusion area by immunofluorescent staining of the mouse retina. Real-time quantitative PCR was applied to detect the mRNA expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and hemeoxygenase-1 (HO-1). Western blot analysis was applied to detect the protein expression of Nrf2, HO-1 and PSF. Results Of the normal control group, simple OIR model group, Vec group and PSF group, the number of pre-retinal neovascular cell nuclei were 0.00, 14.36±5.50, 15.67±4.96, 8.13±2.09, the non-perfusion area were 0.00%, (35.71±2.81)%, (36.57±4.53)%, (15.33±4.75)%, respectively. The differences of the number of pre-retinal neovascular cell nuclei and non-perfusion area among 4 groups were significant (F=24.87, 165.70; P＜0.05). Compared with the normal control group, there were more pre-retinal neovascular cell nucleis and larger non-perfusion area in the simple OIR model group and Vec group (P＜0.05). Compared with the simple OIR model group and Vec group, there were lower pre-retinal neovascular cell nucleis and smaller non-perfusion area in the PSF group (P＜0.05). Real-time quantitative PCR and Western blot showed that the mRNA expression of Nrf2, HO-1 (F=53.66, 83.54) and protein expression of Nrf2, HO-1 and PSF (F=58.38, 52.69, 24.79) among 4 groups were significant (P＜0.05). The mRNA expression of Nrf2, HO-1 and protein expression of Nrf2, HO-1 and PSF in the simple OIR model group and Vec group decreased significantly than those in the normal control group (P＜0.05). The mRNA expression of Nrf2, HO-1 and protein expression of Nrf2, HO-1 and PSF in the PSF group were increased significantly than those in the simple OIR model group and Vec group (P＜0.05). model group and Vec group (P＜0.05).ConclusionIntravitreal injection of lentivirus-mediated PSF inhibits RNV in mice model of OIR possibly through up-regulating the expression of Nrf2 and HO-1.
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 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 observe the influences of uncoupling protein 2 (UCP-2) rs660339 variants transfection on cell proliferation and apoptosis of human umbilical vein endothelial cell (HUVEC). Methods Two UCP-2 green fluorescent protein (GFP) lentivirus constructs were created with the rs660339 locus carried C or T (UCP-2C or UCP-2T), respectively. HUVEC were cultured after lentiviral infection of UCP-2C or UCP-2T. The expression of UCP-2C or UCP-2T was detected with real time polymerase chain reaction. Cell proliferation and cell apoptosis were compared among negative control (NC) group, UCP-2T group and UCP-2C group using CCK-8 cell viability and flow cytometry. Western blot and immunostaining were employed to examine the expression of Bcl-2 gene. Results The lentivirus constructs were successfully created. ＞80% of the transfected cells were found to express GFP under fluorescent microscope. The mRNA levels of UCP-2 gene were significantly increased (F=29.183,P=0.001) in the UCP-2T group and UCP-2C group. The CCK-8 assay revealed that on day two (F=15.970,P=0.004), day three (F=16.738,P=0.004), day four (F=5.414,P=0.045) post-infection, UCP-2T and UCP-2C group showed significantly greater proliferation than the NC cells. The apoptotic rate in the UCP-2T and UCP-2C group was significantly lower than NC group (F=277.138,P=0.000), and the apoptotic rate of UCP-2T was significantly lower than that of UCP-2C (P=0.003). The protein levels of Bcl-2 in the UCP-2T and UCP-2C group were significantly greater than that in the NC group (F=425.679,P=0.000), and the Bcl-2 expression of UCP-2T was greater than that of UCP-2C (P=0.002). The Bcl-2 density in the UCP-2T and UCP-2C group were greater than that in the NC group (F=11.827,P=0.008), while there was no difference between UCP-2T and UCP-2C group (P=0.404). Conclusion The variants of UCP-2 rs660339 may influence HUVEC proliferation and apoptosis, and UCP-2T showed a stronger effect of inhibiting apoptosis than UCP-2C.
ObjectiveTo construct the connective tissue growth factor (CTGF) recombinant interference vector (shRNA) and observe its inhibitory effect on the expression of endogenous CTGF in retinal vascular endothelial cells. Methods The human CTGF shRNA was constructed and the high-titer CTGF shRNA lentivirus particles was acquired via three-plasmid lentivirus packaging system to infect retinal vascular endothelial cells. The optimal multiplicity and onset time of lentivirus infection were identified by tracing down the red florescent protein in interference vector. The cells were classified into three groups: blank control group, infection control group and CTGF knockdown group. The differences in cells migrating ability was observed through Transwell allay. The mRNA and protein expression of CTGF, fibronectin, α-smooth muscle actin (α-SMA) and collagen Ⅰ (Col Ⅰ) were quantified through real-time PCR testing and Western blot system. Data between the three groups were examined via one-way analysis of variance. ResultsThe result showed that an optimal multiplicity of 20 and onset time of 72 hours were the requirements to optimize lentivirus infection. Transwell allay result showed a contrast in the number of migrated cells in the CTGF knockdown group and that in the blank control group and infection control group (F=20.64, P=0.002). Real-time PCR testing showed a contrast in related gene expression (CTGF, fibronectin, α-SMA and Col Ⅰ) in the CTGF knocked-down group and that in the blank control group and infection control group (F=128.83, 124.44, 144.76, 1 374.44; P=0.000, 0.000, 0.000, 0.000). Western blot system showed the statistical significance of the contrasted number of related protein expression (CTGF, fibronectin, α-SMA and Col Ⅰ) in the knockdown group and that in the blank control group (F=22.55, 41.60, 25.73, 161.68; P=0.002, 0.000, 0.001, 0.000). ConclusionThe success in producing CTGF shRNA lentivirus particle suggests that CTGF shRNA lentivirus can effectively knock down CTGF expression.
【 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.
Objective To build the lentiviral vectors of pigment epithelial derived factor (PEDF) gene, and investigate their expression in human umbilical cord mesenchymal stem cells (hUCMSCs). Methods The PEDF lentiviral vectors (LV-PEDF) were built by DNA recombination and confirmed by DNA sequencing. hUCMSCs were transfected by LV-PEDF with MOI 10, 30, 50, respectively. The transfection efficiency was observed under fluorescence microscope. Cell immunofluorescence, immunocytochemistry and real-time PCR methods were used for detecting the expression of PEDF and VEGF. Results The PEDF cDNA was sub-cloned into pCDH-CMV-MCS-EF1-copGFP vector successfully. DNA sequencing analysis confirmed that PEDF gene sequence was exactly the same with that reported in GenBank. pCDH-PEDF infected cells could show green fluorescence under fluorescence microscope. The transfection efficiency was 72.1% in PEDF-MSCs. Immunofluorescence and immunochemical staining confirmed that PEDF protein was overexpressed in hUCMSCs. The relative expression of PEDF mRNA in experimental group and control group was (0.170±0.028) and (0.015±0.007) respectively by RT-PCR, the difference was statistically significant (P＜0.001). The relative expression levels of VEGF mRNA in the two groups were (0.265±0.022) and (0.285±0.049), respectively, with no significant difference (P＞0.05). Conclusions We successfully built a lentivirus vector carrying PEDF gene and obtained hUCMSCs with overexpressed PEDF.
ObjectiveTo construct a lentiviral vector carrying rat sirt1 gene and observe the expression of sirt1 in retinal ganglion cell (RGC) of rat. MethodsRat sirt1 cDNA was inserted into pLV5 vector. After identification by sequencing analysis and PCR, the recombinant sirt1expressinglentivirus vector was packaged by cotransfecting 293T cells with packaged plasmid.Then pLV5-sirt1 was used to infect the cultured Sprague-Dawley rat RGC cell in vitro.The expressions of sirt1 protein and mRNA in infected rat RGC were detected by quantitative real-time PCR and Western blot. ResultsThe sirt1 expression vector pLV5 was successful constructed and sequence was proved to be correct. The expression of sirt1 protein and mRNA in RGC was significantly increased than that in cells infected with control lentiviruses(P < 0.05). ConclusionWe have successful constructed a sirt1 expression lentivirus vector pLV5-sirt1 and it can increase the expression of sirt1 protein and mRNA in the rat retinal ganglion cells.
ObjectiveTo construct a lentiviral vector-mediated gene-targeted small interfering RNA (siRNA) vector to vascular endothelial growth factor (VEGF), and choose the RNAi with the highest silence efficiency to VEGFA gene. MethodsThree kinds of VEGFA gene-targeted hairpin siRNA was designed (KD1, KD2, KD3), then two complementary oligo nucleotide strand were synthesized and inserted into pGCSIL-GFP vector. After annealing, the recombined vector pGCSIL-GFP-siVEGFA was gotten, which was digested by restrictive enzyme and sequenced, and was co-transfected with the pHelper 1.0 and pHelper 2.0 into 293T cells by Lipofectamine 2000. After that, the new vector was transfected into human umbilical vein endothelial cells (HUVECs), and the mRNA expression level of VEGFA gene in cells was detected by RT-PCR. Then we compared the mRNA expression level of VEGFA gene of the 3 groups. ResultspGCSIL-GFP-siVEGFA was built successfully, and all the siRNA could silence the expression of VEGFA mRNA in the HUVECs, and the relative expressions of VEGFA mRNA to the control group were 0.614±0.043 (KD1), 0.334±0.030 (KD2), and 0.201±0.015 (KD3) respectively. ConclusionWe've successfully constructed the siRNA vector for VEGFA mRNA, which can obviously suppress the expression of VEGFA mRNA.