ABSTRACT
ObjectiveTo investigate the role of transforming growth factor (TGF-β1) in hepatic fibrosis induced by echinococcus multilocular infection and its possible mechanisms in this process.Methods Forty-five C57BL/6 mice were randomly divided into the model group（30）and the control group (15). Protoscolece suspension of echinococcus multilocular was infused through portal vein in the model group (4000/each). Mice in the control group was injected the same volume of normal saline solution. Six mice in the model group and 3 mice in the control group were sacrificed at 1, 2, 4, 8 and 12 weeks after infection. The liver tissues were observed the histopathological changes by using hematoxylin-eosin (H&E) staining. The fibrosis degree and glycogen synthesis function of liver tissue were observed by Sirius-red staining and Periodic acid schiff (PAS), respectively. The expression levels of TGF-β1 and a-smooth muscle actin (α-SMA) were measured by immunohistochemical staining.ResultsThe obvious abnormal changes were not observed in 1 week after model setup. The diffuse distribution of multiple white spots began to appear at 2 weeks, but the amount of white plaques decreased after 8 weeks. Meanwhile, forming small lesions were not obviously observed the boundary with the surrounding normal liver tissue. Clear echinococcal vesicles were seen at week 12. H&E staining showed that hepatic tissue structure of control group was normal. In the model group, the number of lesions with worms decreased gradually and amount of granulomas were increased. The inflammatory lesions did not change significantly. Sirius-red staining demonstrated that collagen deposition in the control group was mainly around the bile duct and blood vessels. However, the deposition in the model group was mainly around the lesion and the degree of fibrosis became more serious with time. PAS staining displayed that the content of glycogen in the liver tissues of the control group was rich, evenly distributed and stained uniformly. However, the glycogen staining positive area decreased with the time of infection and the staining became lighter in the model group. Immunohistochemical staining indicated that the positive expression of α-SMA and TGF- β1 in the control group were mainly found in the bile ducts and perivascular areas. The positive areas in the model group were mostly granulomatous areas around the metacercariae and fibroblasts. Expression of α-SMA and TGF- β1 increased over time after infection with the expression peak at 12 weeks(16.80±2.09、4.10±2.14).ConclusionThe degree of fibrosis in liver tissues at different time points was consistent with the expression trend of TGF- β1 and α-SMA. TGF-β1 may promote collagen deposition and lead to fibrosis by activating hepatic stellate cells.
ABSTRACT
BACKGROUND: Difficulty in long-time survival and continuous proliferation is the main problem for transplanted fetal hepatic progenitor cells and co-transplantation with transforming growth factor beta 1 (TGF-β1)-induced hepatic stellate cells may be a promising way to solve this scientific obstacle. OBJECTIVE: To explore the therapeutic effects of co-transplantation of fetal hepatic progenitor cells and TGF-β1-induced hepatic stellate cells on acute liver injury in mice. METHODS: Over-expression vector pHBLV-CMVIE-TGF-β1 was infected to mouse hepatic stellate cells and transfection efficiency was detected by immunocytochemistory, western blot and qRT-PCR. Hepatic progenitor cells, mHPCs-E14.5, were cultured and identified by immunofluorescence in vitro.The mouse model of acute liver injury was established by intraperitoneal injection of CCl4in combination with 2/3 partial hepatectomy, followed by mHPCs-E14.5transplantation, co-transplantation of mHPCs-E14.5and mHSCs-pHBLV-CMVIE-TGF-β1 (experimental co-transplantation group) or co-transplantation of mHPCs-E14.5and mHSCs-pHBLV-CMVIE-GFP (control co-transplantation group) for cell transplantation assay. Confocal immunofluorescence staining against CK19, ALB, a-SMA was performed to analyze the engraftment and differentiation of transplanted cells in the splenic parenchyma 14 days post-transplantation; serum alanine transferase and aspartate transferase levels were monitored using an automatic biochemistry analyzer. RESULTS AND CONCLUSION: (1) A hepatic stellate cell line that over-expressing TGF-β1 was successfully established and expression levels of TGF-β1 and α-smooth muscle actin were efficiently up-regulated in the over-expression group (P < 0.01). (2) mHPCs-E14.5expressed massive AFP and low levels of ALB and CK19,confirming that this cell line was in complete conformity with fetal hepatic progenitor cells in vitro.(3)CK19 and ALB positive cells existed in the splenic parenchyma in mHPCs-E14.5transplantation group.Highly expressed ALB but less expressed α-SMA and CK19 were observed in the control co-transplantation group, while massive CK19 and a-SMA positive cells as well as less level of ALB positive cells existed in the experimental co-transplantation group. Serum alanine transferase and aspartate transferase levels were decreased remarkably after cell transplantation, and moreover, the decrease was more obvious in the experimental co-transplantation group (P < 0.05). Overall, transplanted fetal hepatic progenitor cells engraft and differentiate into hepatocytes and cholangiocytes in the splenic parechyma successfully in vivo.Importantly,hepatic stellate cells induced by TGF-β1 promote the differentiation of fetal hepatic progenitor cells into cholangiocytes and accelerate recovery from CCl4/partial hepatectomy induced acute liver injury.