The Mechanism Of Hepatocyte Growth Factor To Promote The Apoptosis Of Hepatic Stellate Cells

Objective To investigate rat bone marrow mesenchymal stem cells (BMSCs) in vitro isolation, culture, mass generation, identification and ability of osteoblasts and adipocyte. It would provide stable function cells for BMSCs research. Methods BMSCs were isolated from SD rats, cultured and purified in vitro by the whole bone marrow adherence method. Living cells dynamic morphous changes were observed under an inverted phase contrast microscope. The grow curve of BMSCs was drawn by MTT assay. Phenotypic characterization of BMSCs was analyzed by flow cytometry. Osteogenesis and adipoegenesis induced liquid were used to induce osteogenic and adipoegenic differentiation of BMSCs.Results (1) BMSCs isolated by the whole bone marrow adherence method were uniform spindle shaped and typical swirl morphology.(2) Its growth curve resembled S shape and had three periods:latent period, rapid growth period and slow growth period.(3) Flow cytometry showed that CD29+99.45%, CD34+1.45%, CD44+99.52%, CD45+1.41%.(4) BMSCs have multiple differentiation potential which could be induced into osteoblasts and adipocytes. Osteoblasts were stained orange by alizarin red and adipocytes were stained red by oil red o.Conclusions BMSCs isolated by the whole bone marrow adherence method are high purity cells and have multiple differentiational potential. Objective To investigate the role of exogenous hepatocyte growth factor (HGF) on apoptosis of primary hepatic stellate cells (HSCs) induced by tumor necrosis factor-related apoptosis inducing-ligand (TRAIL) promoting and its possible mechanisms.Methods HSCs were recovered and mass genernated. The cells were divided into the following groups:(1) HSCs blank group;(2) HGF group;(3) TRAIL group;(4) HGF+TRAIL group. The morphology of HSCs was observed by the inverted phase contrast microscope. HSCs were characterized by the expression of activation protein markers alpha-smooth muscle actin (a-SMA) using immunocytochemisty. OD value of HSCs was measured by MTT assay. The apoptosis rate and DR5fluorescence intensity of HSCs were measured by Annexin-V-FITC/PI double staining and immunofluorescence respectively. The expression of DR5protein was determined by Western blot.Results (1) The expression of a-SMA protein in HSCs was positive.(2) MTT assay indicated that HGF and TRAIL had no effect on HSCs proliferation under the concentration of50-200ng/mL and0.5-1.5ug/mL respectively. TRAIL at2ug/ml could inhibit HSCs proliferation. OD value of HSCs at24h and48h were:(0.22±0.02)%and (0.25±0.08)%(P<0.01).(3) Apoptosis of HSCs in HGF+TRAIL group was higher than that of HSCs blank group and HGF group (P<0.01).(4) HSCs immunofluorescence results showed that DR5fluorescence intensity in HGF+TRAIL group was higher than that of HSCs blank group HGF group and TRAIL group (P<0.01).(5) The expression of DR5protein in HGF+TRAIL group was higher than that of HSCs blank group, HGF group and TRAIL group (P<0.01).Conclusions HGF can promote the apoptosis of HSCs induced by TRAIL The possible machanism is that HGF can increase the expression of DR5protein on HSCs surface. Objective:To observe the effect of downregulation of hepatocyte growth factor (HGF) mediated by lentivirus in bone marrow mesenchymal stem cells (BMSCs) and hepatic stellate cells (HSCs) on the apoptosis of primary HSCs. To explore the role of BMSCs and HSCs derived HGF on promoting apoptosis of HSCs in co-culture system. It would provide molecular basis for BMSCs to liver fibrosis treatment.Methods:BMSCs were isolated from SD rats, cultured and purified in vitro. Primary HSCs were recovered. BMSCs were cultured in the transwell insert (1.3×105cells/well) and the HSCs (1.0×105cells/well) were cultured in the plastic plates (6-wells) to establish the upper and lower double-cell co-culture system. Cells were cultured24h,48h and72h and divided into five groups:(1) HSCs blank group:HSCs cultured alone;(2) BMSCs+HSCs co-cultured group: BMSCs co-cultured with HSCs;(3) HSCs transfection co-cultured group:HSCs were transfected lenti-HGF-ShRNA and then co-cultured with BMSCs;(4) BMSCs transfection co-cultured group:BMSCs were transfected lenti-HGF-ShRNA and then co-cultured with HSCs;(5) TNF-a pretreatment group:BMSCs were stimulated by TNF-a for six hours and then co-cultured with HSCs with fresh medium. To observe cells morphology under the inverted phase contrast microscope. The concentration of HGF in co-culture group and transfection group was determined by ELISA. Apoptosis of HSCs was evaluated by Annexin-V-FITC/PI double staining; the expression of DR5mRNA, Caspase-8mRNA and protein in HSCs were tested by FQ-PCR and Western blot respectively.Results:(1) Plasmid sequence results were consistent with building sequence.(2) The expression of green fluorescent in two cells after transfection72h were about eighty percent under inverted phase contrast fluorescent microscope. The levels of HGF protein in BMSCs transfection group and HSCs transfection group were down-regulated (60.2±2.5)%and (63.3±4.3)%respectively. The expression of green fluorescent in BMSCs transfection group was seventy percent.(3) The concentration of HGF in BMSCs blank control group and HSCs blank control group was higher than that in BMSCs transfection group and HSCs transfection group respectively (P<0.01).(4) The concentration of HGF in TNF-a pretreatment group obviously higher than that of in BMSCs group, BMSCs+HSCs co-culture group and HSCs blank control group (P<0.01); The oncentration of TRAIL in the BMSCs group was higher than that of in HSCs blank control group, BMSCs+HSCs co-culture group and TNF-α pretreament group (P<0.01).(5) Apoptosis rate of HSCs in TNF-α pretreatment group was obviously higher than that of the other four groups and had time-dependent (P<0.01); Apoptosis rate of BMSCs transfection co-culture group was lower than that of BMSCs+HSCs co-culture group (P<0.01).(6) The expressions of DR5、Caspase-8protein and mRNA in TNF-α pretreatment group was higher than that of in HSCs group and the other three groups (P<0.01). The expressions of DR5、Caspase-8protein and mRNA in co-culture group was significantly higher than that of in BMSCs transfection co-culture group (P<0.01).Conclusions:BMSCs co-cultured with HSCs can promote apoptosis of HSCs which may be associated with BMSCs derived HGF can up-regulate the expression of DR5and Caspase-8protein. TNF-α can enhance the effect. HSCs derived HGF play less role in promoting the apoptosis of HSCs in the system.