| PART 1 EXPERIMENT in vivo: THE EFFECT OF MSCs TRANSPLANTATION ON THE ANTI-HEPATIC FIBROSIS INDUECED BY CCL4 IN RATSOBJECTIVE: To study the effect of MSCs transplantation against hepatic fibrosis and the effect on expression of Col-I, RhoA,Cdc42,Rac1 in rats with CCl4-induced hepatic fibrosis. fibrosis.METHODS: For MSC isolation, male Sprague-Dawley (SD) rats were killed by cervical dislocation and the limbs removed. MSC were flushed with Dulbecco's Modified Eagle medium(DMEM)culture medium from the medullary cavities of femurs using a 25-G needle.MSC were separated, grown, and propagated in culture for 4 pasagges and were characterized morphologically. A part of the 4th pasagge cells were treated with HGF for 2 week. The 4th pasagge MSCs and HGF-induced MSCs were then infused into the tail vein of female rats with CCl4–induced liver fibrosis.Hepatic fibrosis model of SD rats were induced by subcutaneous injection (s.c.) of CCl4 on the dorsum at dose of 0.2 m1 of 40% CCl4/100 g (40% CCl4=4ml CCl4 : 6ml peanut oil), twice a week. For rats in the normal control group, normal saline ( NS ) were given, s.c., 0.2 m1 of NS/100g , twice a week. Sixty femail SD hepatic fibrosis rats were divided into 3 groups randomly:①model control group(C, n=20): treated by infusion of normal saline.②MSC group(M, n=20): treated by injection of MSCs suspension via tail vein.③HGF-induced MSC group(HM, n=20): treated by injection of HGF-induced MSCs suspension via tail vein . All fibrosis model rats were treated with infusion of MSC suspension or sham via tail vein once time at the beginning of 7th week, on the different pointed time after treatment(on weeks of 1,2,3 and 4), rats were anesthetized with the sodium pentobarbitol, the blood of the rats was taken from eyeball, and separated serum was finally frozen at -20℃until assay. About 50-100 mg hepatic tissue was treated with the liquid nitrogen rapidly, then being frozen in -80℃freezer for extracting total RNA and protein. Two pieces of hepatic tissue in the identical spot of liver, size 1 cm3×1 cm3×l cm3, were taken, one was soaked in 10% neutral formalin solution for the light microscope observation and immunohistochemistry.The serological indexes, e.g. hyaluronic acid (HA), laminin (LN), collagen IV (Col-IV) were assayed by enzyme linked immunoabsorbent assay (ELISA). The degree of fibrosis in hepatic tissue was observed under light microscope by hematoxylin and eosin(HE). The collagen expression degree was evaluated by Masson staining method. mRNA expression of Col-I, RhoA,Cdc42,Rac1 in hepatic tissues were detected by RT- PCR, whereas protein expression of Col-I,RhoA,Cdc42,Rac1 in hepatic tissues were detected by Western blot.RESULTS:①The influence of MSCs on serological indexes in hepatic fibrosis rats: Compared with the model control group, the serological concentrations of HA, LN, and Col-IV in HGF-induced MSC treated group or MSC treated group were decreased significantly at different pointed time (P<0.01, P<0.05), furthermore, the serological concentrations of HA, LN, and Col-IV in HGF-induced MSC treated group were significantly lower than MSC treated group (P<0.01 , P<0.05).②The influence of MSCs on histomorphology in hepatic fibrosis rats: We analyzed liver histology in rats with MSCs and HGF-induced MSCs in CCL4 modals by HE staining and MT staining.HE staining showed that architecture of liver got better in MSCs and HGF-induced MSCs. Inflammation reduced and pseudolobules were resolved.Collagen accumulation and fatty degeneration were significantly lower in MSCs and HGF- induced MSCs compared to the model control. The necrosis of hepatocyte was replaced by regenerate.The thickened septal fibrosis become thinner or disappeared. Histopathological examination of liver tissue showed that MSCs had a significant antifibrotic effect as evidence by the decrease in liver collagen stained with masson trichrome compared to the model group that exhibited marked periportal fibrosis in hepatic lobules.③The influence of MSCs transplantation on Col-I, RhoA,Cdc42,Rac1 mRNA expression in hepatic fibrosis rats. Compared with the model control group, MSCs group and HGF-induced MSCs group could down regulate significantly Col-I, RhoA,Cdc42,Rac1 mRNA expression (P<0.01, P<0.05) on week 3 and 4 after treatment.④The influence of MSCs on Col-I, RhoA,Cdc42,Rac1 protein expression in hepatic tissues of hepatic fibrosis rats: After treament with MSCs at week 3 and 4, the protein expression of Col-I , RhoA,Cdc42,Rac1 in hepatic tissues reduced significantly as compared with the model control group (P<0.05, P<0.01).Moreover, we observed that the antifibosis effect of MSCs in HGF-induced MSCs group was superior to MSCs group.CONCLUSIONS : MSCs transplantation may suppress collagenous fibre-proliferation in tissues of hepatic fibrosis rats, down-regulate the expression of Col-I, RhoA,Cdc42,Rac1 mRNA and its protein in tissues of hepatic fibrosis rats. The results indicate that MSCs could ameliorate liver fibrosis induced by CCl4 in SD rats, and reduce the collagen synthesis and the deposition, the underlying mechanism may be involvement of inhibition of Rho signaling pathway, which has been implied as an important new target for suppressing the deposition of ECM. PART 2 EXPREMENT in vitro: THE CONCOMITANT INHIBITORY EFFECTS OF MSCs ON THE EXPRESSION OF Col-I and RhoA,Cdc42,Rac1 IN HEPATIC STELLATE CELLSOBJECTIVE: To investigate the effects of mesenchymal stem cells (MSCs) on the mRNA and protein expression of Col-I, RhoA,Cdc42,Rac1 in hepatic stellate cells (HSCs) in vitro, and explore the underlying mechanism of anti- fiborsis of MSCs on the HSCs.METHODS : MSCs were isolated from BM in rats and grown, and propagated in culture flask. HSCs were recoveried and activated morphologically , a-SMA expression in HSCs was evaluated immuno- histochemically. An indirect coculture system between MSC and HSC or fiberblast cells was established using Transwell membranes (24mm diameter, 0.4μm pore size). Approximately 2.0×105 HSCs were placed in the lower chamber with either 2.0×105 MSCs or fiberoblast cells placed on the membrance insert. Cocultures were maintained in HSC medium for three days.Four groups were divided randomly:①HSC control group②Fiberoblast control group.③M SC group.④H GF- induced MSC group.At the different pointed time(on days of 1, 2 and 3), the HSCs in co-culture system were collected and the mRNA expressions of Col-I, RhoA,Cdc42,Rac1 in HSCs were detected by RT- PCR . The protein expressions of Col-I,RhoA,Cdc42,Rac1 in HSCs were evaluated by Western blot.RESULTS:①With the coculture time lasting, the inhibitory rate of HSCs proliferation with MSCs coculture were significant higher than HSC control group and Fiberoblast control group at different pointed time(P < 0.01).②The influence of MSCs on Col-I, RhoA,Cdc42,Rac1 mRNA expression in HSC of coculture system: Compared with the control group, the mRNA expression of RhoA,Cdc42,Rac1 in HGF-induced MSCs group and Cdc42 expression in MSCs group were significantly reduced on day 1(P<0.05, P<0.01), and all of the parameters in HGF-induced MSCs group and MSCs group were significantly decreased on day 2 and day 3(P<0.01, P<0.05, moreover, the expression of Cdc42 in HGF-induced MSC group was significant lower than MSC group on day 2( P<0.05) and the expression of Col-I, Cdc42, Rac1 in HGF-induced MSC group were significant lower than MSC group on day 3( P<0.05).③The influence of MSCs on Col-I, RhoA,Cdc42,Rac1 protein expression in HSC of coculture system: Compared with the control group, the protein expression of RhoA,Cdc42 in HGF-induced MSCs group were significantly reduced on day 1(P<0.05),and all of the parameters in HGF-induced MSCs group and MSCs group were significantly decreased on day 2 and day 3(P<0.01, P<0.05), furthermore, the expression of Cdc42 in HGF-induced MSCs group was significant lower than MSCs group on day 2( P<0.05) and the expression of Col-I, RhoA , Cdc42 in HGF-induced MSCs group were significantly lower than MSCs group on day 3( P<0.05或P<0.01).④The statistical analysis the relationship of the expession of Col-I and RhoA,Cdc42 ,Rac1 in HSC after treatment with MSC:There were positive relationship between the mRNA expression of Col-I and RhoA,Cdc42(r=0.711, P<0.05;r=0.812, P<0.05)on day 1,so as for the mRNA expression of Col-I and Rac1(r=0.751, P<0.01) on day 2, and the mRNA expression of Col-I and RhoA,Cdc42(r=0.826,P<0.05, r=0.646,P<0.05)on day 3.There were positive relationship between the protein expression of Col-I and RhoA,Cdc42 (r=0.865, P<0.05;r=0.733, P<0.05) on day 1;so as for Col-I and RhoA,Cdc42,Rac1(r=0.825, P<0.01;r=0.744, P<0.05;r=0.586, P<0.05) on day 2; as well as Col-I and RhoA,Cdc42(r=0.841, P<0.01;r=0.665, P<0.05) on day 3.CONCLUSIONS:①MSCs could inhibit the proliferation and activation of HSC and induce apoptosis of HSC in vitro, so that reducing the synthesis of ECM and alleviate the hepatic fibrosis.②MSCs could down-regulate the expression of Col I mRNA and its protein in HSC, finally reducing the synthesis of ECM, which may be one of the possible molecular mechanism againist hepatic fibrosis.③HGF-induced MSC may be superior to MSC for anti-fibrosis and inhibitory proliferation in HSCs. |
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