| Objective: The objective of this study was to analyze the effect of HGF-transfected hUCMSCs on rat hepatic stellate cells line(HSCs-T6),to explore the possible mechanism of HGF-transfected hUCMSCs in inhibiting proliferation of HSCs-T6,promoting their apoptosis,inhibiting their activation and reducing the expression of Collagen Ⅰ and Collagen Ⅲ by inhibiting the activation of TGF-β1/Smads signaling pathway,which would provide a research basis for treatment of liver fibrosis.Methods: HGF-transfected hUCMSCs and HSCs-T6 were co-cultured by Transwell indirect co-culture system,with hUCMSCs cultured in upper layer and HSCs-T6 in lower layer.The experiment was divided into four groups: control group: HSCs-T6+complete medium;HGF group: HSCs-T6+HGF-hUCMSCs;LV5-NC group: HSCs-T6+LV5-NC-hUCMSCs and hUCMSC group: HSCs-T6+hUCMSCs.After co-culturing for 1,2,and 3 days,cell morphology was observed by microscope and cell viability was measured by MTT assay;the apoptosis of HSCs-T6 was measured by Hoechst33342 staining and flow cytometry;the expression of α-SMA,Collagen Ⅰ,Collagen Ⅲ and TGF-β1,Smad2,Smad3 in HSCs-T6 was measured by immunocytochemistry,Western blotting and RT-PCR.Results: 1.Microscopic observation showed that HSCs-T6 were shrunk and sparsely arranged in HGF,LV5-NC,and hUCMSC group compared with control group;the changes were the most significant in HGF group whereas there were no significant differences between LV5-NC and hUCMSC group.2.MTT assay results showed that when co-cultured for 1 day,the OD value was decreased in HGF group compared with control group(P<0.05).When co-cultured for 2 days,the OD value was significantly decreased in HGF,LV5-NC and hUCMSC group compared with control group(P<0.01).When co-cultured for 3 days,the OD value was more significantly decreased in HGF,LV5-NC and hUCMSC group compared with control group(P<0.001);the OD value was significantly decreased in HGF group compared with LV5-NC and hUCMSC group(P<0.01)whereas there was no significant difference between LV5-NC and hUCMSC group(P>0.05).3.Hoechst33342 staining results showed that when co-cultured for 1,2 and 3 days,the number of HSCs-T6 apoptotic bodies was increased in HGF,LV5-NC and hUCMSC group compared with control group;the increase was the most significant in HGF group whereas there was no significant difference between LV5-NC and hUCMSC group.Moreover,flow cytometry showed that when co-cultured for 1,2 and 3 days,the proportion of HSCs-T6 apoptotic cells was increased in HGF,LV5-NC and hUCMSC group compared with control group(P<0.05);the apoptotic cells proportion in HGF group was increased compared with LV5-NC and hUCMSC group(P<0.05)whereas there was no significant difference between LV5-NC and hUCMSC group(P>0.05).4.Immunocytochemical results showed that when co-cultured for 1 day,the protein expression of α-SMA,Collagen Ⅰ,Collagen Ⅲ and TGF-β1,Smad2,Smad3 has no significant difference in HGF,LV5-NC and hUCMSC group compared with control group(P>0.05);when co-cultured for 2 days and 3 days,the above indicators were decreased in HGF,LV5-NC and hUCMSC group compared with control group(P<0.05);the above indicators were decreased in HGF group compared with LV5-NC and hUCMSC group(P<0.05)whereas there was no significant difference between LV5-NC and hUCMSC group(P>0.05).5.Western blotting and RT-PCR showed that when co-culture for 2 days and 3 days,either in protein or m RNA level,the expression of α-SMA,Collagen Ⅰ,Collagen Ⅲ and TGF-β1,Smad2,Smad3 in HGF,LV5-NC and hUCMSC group was decreased compared with control group(P<0.05);the above indicators were decreased in HGF group compared with LV5-NC and hUCMSC group(P<0.05)whereas there were no significant differences between LV5-NC and hUCMSC group(P>0.05).Conclusion: HGF-transfected hUCMSCs can decrease cell viability of HSCs-T6,promote apoptosis,and then inhibit growth;inhibit their activation and reduce the expression of Collagen Ⅰ and Collagen Ⅲ,which may be related to inhibiting the activation of TGF-β1/Smads signaling pathway. |
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