This research aims to construct a lentiviral expression vector carrying the extracelluar domain (ED) of human hepatocyte growth factor receptor (C-Met), and to express it in transfected 293T cells. The extracellular domain of C-Met was amplified by RT-PCR, ligated with lentiviral expression vector p RRL-CMV-ED, and then expressed in 293T cell line. The expressed protein was purified and identified by RT-PCR and Western blot. The enzyme digestion and sequence analysis showed that the lentiviral expression vector p RRL-CMV-ED was constructed correctly. The size of amplified genes was about 2 700 bp. The purified protein with Ni-affinity column was about 105 kD analyzed by SDS-PAGE. The Western blot and ELISA results showed that the expressed protein which could bind to HGF specifically was the extracelluar domain of human hepatocyte growth factor receptor. This research may lay a foundation for further study of anti-C-MET monoclonal antibody and neutralizing antibody.
ObjectiveTo discuss the possibility of constructing injectable tissue engineered adipose tissue, and to provide a new approach for repairing soft tissue defects.MethodsHuman adipose-derived stem cells (hADSCs) were extracted from the lipid part of human liposuction aspirate by enzymatic digestion and identified by morphological observation, flow cytometry, and adipogenic induction. The hADSCs underwent transfection by lentivirus vector expressing hepatocyte growth factor and green fluorescent protein (HGF-GFP-LVs) of different multiplicity of infection (MOI, 10, 30, 50, and 100), the transfection efficiency was calculated to determine the optimum MOI. The hADSCs transfected by HGF-GFP-LVs of optimal MOI and being adipogenic inducted were combined with injectable fibrin glue scaffold, and were injected subcutaneously into the right side of the low back of 10 T-cell deficiency BALB/c female nude mice (transfected group); non-HGF-GFP-LVs transfected hADSCs (being adipogenic inducted) combined with injectable fibrin glue scaffold were injected subcutaneously into the left side of the low back (untransfected group); and injectable fibrin glue scaffold were injected subcutaneously into the middle part of the neck (blank control group); 0.4 mL at each point. Twelve weeks later the mice were killed and the implants were taken out. Gross observation, wet weight measurement, HE staining, GFP fluorescence labeling, and immunofluorescence staining were performed to assess the in vivo adipogenic ability of the seed cells and the neovascularization of the grafts.ResultsThe cultured cells were identified as hADSCs. Poor transfection efficiency was observed in MOI of 10 and 30, the transfection efficiency of MOI of 50 and 100 was more than 80%, so the optimum MOI was 50. Adipose tissue-like new-born tissues were found in the injection sites of the transfected and untransfected groups after 12 weeks of injection, and no new-born tissues was found in the blank control group. The wet-weight of new-born tissue in the transfected group [(32.30±4.06) mg] was significantly heavier than that of the untransfected group [(25.27±3.94) mg] (t=3.929, P=0.001). The mature adipose cells in the transfected group [(126.93±5.36) cells/field] were significantly more than that in the untransfected group [(71.36±4.52) cells/field] (t=30.700, P=0.000). Under fluorescence microscopy, some of the single cell adipocytes showed a network of green fluorescence, indicating the presence of GFP labeled exogenous hADSCs in the tissue. The vascular density of new-born tissue of the transfected group [(16.37±2.76)/field] was significantly higher than that of the untransfected group [(9.13±1.68)/field] (t=8.678, P=0.000).ConclusionThe hADSCs extracted from the lipid part after liposuction can be used as seed cells. After HGF-GFP-LVs transfection and adipose induction, the hADSCs combined with injectable fibrin glue scaffold can construct mature adipose tissue in vivo, which may stimulate angiogenesis, and improve retention rate of new-born tissue.
Objective To investigate effect of hepatocyte growth factor (HGF) after lentivirus-mediated RNA interference (RNAi) targeting c-Met on invasion of colonic carcinoma cell line SW480. Methods The experiment was assigned into 3 groups: NC group, the normal cells were infected by the shRNA negative control virus (the NC-20 andNC-40 represented the negative group which were added 20 ng/mL and 40 ng/mL respectively HGF after being infected); KD group, the normal cells were infected by the shRNA-c-Met target virus (the KD-20 and KD-40 represented the interfered group which were added 20 ng/mL and 40 ng/mL HGF respectively after being infected; KD1, KD2, KD3, and KD4 represented the different RNAi targets for the purpose gene); CON group, the normal cells were not infected by any virus. The lentiviral vector shRNA-c-Met was constructed and verified by polymerase chain reaction (PCR) and DNA sequencing. The SW480 cells were infected with the shRNA-c-Met after packed with lentivirus plasmid. Fourty-eight hours transfection later, the c-Met mRNA of the transfected SW480 cell was detected by real time PCR and the c-Met protein was examined by Western blot. Seventy-two hours after transfection, the cell apoptosis was detected by flow cytometry and the invasions in the different cells with stable transfection were detected by Transwell test. Results The RNAi sequence targeting c-Met gene was successfully inserted into the lentiviral vector. The shRNA-c-Met transfection resulted in an obviously reduced expression of c-Met mRNA in the SW480 cells. The efficency of gene knock down of the KD4 (the cells with No.4 target spot knocked down) was 81.4%. The shRNA-c-Met tansfection resulted in an obviously reduced expression of c-Met protein in the SW480 cells. After transfection, the apoptosis rate of the KD group was significantly higher than that in the NC group (P<0.001) or the CON group (P<0.001). The invasion ratios in the NC group, NC-20 group, and NC-40 group were significantly higher than those in the KD group (P<0.001), KD-20 group (P=0.015), and KD-40 group (P=0.017), respectively; which in the NC-20 group and NC-40 group were increased as compared with the NC group (P<0.001,P<0.001), and in the NC-40 group was increased as compared with the NC-20 group (P=0.005). The invasion ratios in the KD-20 group and KD-40 group were increased as compared with the KD group (P<0.001,P<0.001), and in the KD-40 group was increased as compared with the KD-20 group (P=0.014). Conclusion Lentivirus-mediated RNAi targeting c-Met could effectively suppress expression of c-Met in SW480 cells and could reduce invasion of HGF on SW480 cells with knocked down c-Met.