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.
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 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.
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.
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 explore the effects on osteogenic differentiation of adipose derived stem cells (ADSCs) by simultaneously down-regulating Noggin combined with up-regulating bone morphogenetic protein 14 (BMP-14) in vitro.
MethodsPrimary ADSCs were isolated and expanded in vitro from 5 Sprague Dawley rats (weighing, 250-300 g). ADSCs were transfected with lentiviral (Lv)-enhanced green fluorescent protein in group A (control group), with Lv-BMP-14 in group B, and with Lv-BMP-14 and Lv-Noggin shRNA in group C. BMP-14 and osteogenesis-related genes[collagen type I, alkaline phosphatase (ALP), and osteocalcin (OCN)] mRNA expression levels were detected by real time fluorescence quantitative PCR at 3, 7, and 14 days after transfection. Alizarin red staining for calcium nodules was also employed to assess the osteogenic ability of co-transfected ADSCs.
ResultsAt 3 days after transfection, no significant difference was found in BMP-14 mRNA expression among groups P>0.05). At 7 and 14 days after transfection, BMP-14 mRNA expression was significantly higher in group C than groups A and B, and in group B than group A (P<0.05). At 3 days after transfection, collagen type I, ALP, and OCN mRNA expressions of group C were significantly higher than those of groups A and B (P<0.05), but no significant difference was shown between groups A and B P>0.05). At 7 and 14 days, collagen type I, ALP, and OCN mRNA expressions were higher in group C than groups A and B, and in group B than group A, showing significant difference (P<0.05) except collagen type I mRNA expression at 7 days between groups A and B P>0.05). The results of alizarin red staining showed that the amount of calcium nodules presented an increased tendency in the order of group A, group B, and group C.
ConclusionBMP-14 is capable of enhancing osteogenic differentiation of ADSCs. A combination of inhibiting Noggin gene expression and enhancing BMP-14 gene expression in ADSCs can significantly strengthen osteogenic differentiation capability, showing significant synergistic effect.
ObjectiveTo observe the effect of lentivirus-mediated cyclooxygenase 2 (COX-2) and Aggrecanase-1 silencing and insulin-like growth factor 1 (IGF-1) in BMSCs after injecting into the knee joint cavity in cynomolgus monkeys with knee osteoarthritis (OA).
MethodsBMSCs were isolated from the bone marrow of 10 donors. The lentivirus vector expressing genes of COX-2, Aggrecanase-1, and IGF-1 were constructed, and transfected into the third generation human BMSCs at 40 multiplicity of infection (virus group); BMSCs transfected with lentivirus-empty vector served as blank-virus group. The growth status and number of BMSCs were observed under inverted phase contrast microscope, and normal BMSCs were used as normal control group. At 1 week after transfected, the mRNA expressions of COX-2, Aggrecanase-1, and IGF-1 were detected with RT-PCR. Nine 3-year-old cynomolgus monkeys were selected to establish the OA model according to Hulth modeling method, and were randomly divided into 3 groups (n=3). At 6 weeks after remodeling, the right knee joint cavity was injected accordingly with 1 mL BMSCs (about 1×107 cells) in virus group and blank-virus group, with 1 mL of normal saline in the blank control group; the left knee served as normal controls. The general condition was observed after injection; at 1, 4, and 6 weeks, the concentrations of prostaglandin E2 (PGE2), IL-1, Aggrecanase-1, and IGF-1 of double knee liquid were detected with ELISA; at 6 weeks, MRI, general observation, histology method, and immunohistochemistry method were used to detect the knee cartilage changes and the expressions of COX-2, Aggrecanase-1, and IGF-1 were measured with RT-PCR.
ResultsNo significant difference was found in cell morphology and growth curve between 2 groups after transfection. By RT-PCR, COX-2, and Aggrecanase-1 expressions were significantly reduced, IGF-1 expression was significantly increased in virus group when compared with normal control group and the blank-virus group (P < 0.05). All monkeys survived to the end of the experiment after injection. When compared with blank-virus group and blank control group, the concentrations of PGE2, Aggrecanase-1, and IL-1 significantly decreased and the concentration of IGF-1 significantly increased in the virus group (P < 0.05), but the indicators in 3 groups were significantly higher than those in the normal control group (P < 0.05). MRI showed that abnormal articular surface with high density could be found in virus group, blank-virus group, and blank control group, while the virus group had the minimum area. Gross observation and histological observation showed that the cartilage morphology of virus group, blank-virus group, and blank control group was accordance with early OA articular cartilage changes, but virus group was better than blank-virus group and blank control group in repair degree, whose improved Pineda score was significantly lower (P < 0.05). Immunohistochemical staining showed that the virus group had deeper dyeing with occasional brown particles and more chondrocytes than blank-virus group and blank control group. By RT-PCR, COX-2 and Aggrecanase-1 mRNA expressions of cartilage in virus group were significantly decreased, and IGF-1 expression was significantly increased when compared with blank control group and the blank-virus group (P < 0.05).
ConclusionLentivirus-mediated multi-genes co-transfection in BMSCs can inhibit the expressions of COX-2 mRNA and Aggrecanase-1 mRNA, and enhance the IGF-1 mRNA expression, which decreases the concentration of inflammatory factors, and protects the joint cartilage effectively.
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.
