The Study Of Hepatic Stellate Cells Regulated By Paracrine HGF From Bone Marrow Mesenchymal Stem Cells In Vitro

Object:Using transwell upper and lower bilayer co-culture system to investigate the effects of paracrine hepatocyte growth factor(HGF)paracrine from rat bone marrow mesenchymal stem cells(BMSCs)on the proliferation,apoptosis and activation of hepatic stellate cells(HSCs).Methods: The whole bone marrow adherent method was used to isolate,culture and purify rat BMSCs,and HSCs was cultured.The semi-permeable membrane of six-well plate was used to established a two-layer cell co-culture system with indirect contact.The study consisted of experimental group(BMSCs and HSCs Transwell co-culture),blank The control group(co-cultured with low sugar medium and HSCs),the negative control group(co-cultured with HSCs and HSCs),and the c-met inhibitor group(co-culture of BMSCs and HSCs Transwell and pretreatment with c-met inhibitor).The morphological changes of cells were observed under inverted phase contrast microscope.BMSCs were identified by flow cytometry.The activity of HSCs was identified by immunofluorescence confocal microscopy.After 48 hours of co-culture,the apoptosis rate of HSCs was detected by flow cytometry.The proliferation of HSCs was detected by MTT assay.Immunofluorescence confocal microscopy was used to quantitatively detect the expression of α-SMA in HSCs.The concentration of HGF in the supernatant of each co-culture system was detected by enzyme-linked immunosorbent assay(ELISA).Results: 1.BMSCs was observed to be fibroid,triangular or irregular under inverted phase contrast microscope.Flow cytometry(FCM)was used to find that MSCs overexpressed positive surface molecules CD29 and CD90,and low expression of hematopoietic cell surface marker CD45;Activated HSCs was fusiform and expressed α-SMA;2.The MTT results showed that the proliferation of HSCs in the experimental group was significantly lower than that in the blank control group,the negative control group and the c-met inhibitor group(P<0.01);3.Detection of apoptosis rate of HSCs by flow cytometry:The apoptosis rate of HSCs in the experimental group was significantly higher than that in the other groups(p < 0.05),but there was no significant difference between the blank control group,the negative control group and the c-met inhibitor group(p > 0.05);4.Quantitative analysis of immunofluorescence confocal results: Compared with the blank control group,the negative control group and the c-met inhibitor group,the expression of α-SMA in hscs in the experimental group was significantly lower than that in other group(p < 0.01).There was no significant difference in the expression of α-SMA among the other three groups(p > 0.05);5.ELISA results showed that the concentration of HGF in the supernatant of the experimental group was significantly higher than that in the other groups(p < 0.01)。 Conclusion: BMSCs can promote the apoptosis of HSCs,inhibit the proliferation and activation of HSCs through paracrine HGF.