人脐带间充质干细胞上清液对大鼠肝星状细胞生长的影响

Effection of Human Umbilical Cord Mesenchymal Stem Cells Supernatant on Rat Hepatic Stellate Cells Growth

目的研究人脐带间充质干细胞(hUCMSCs)上清液对大鼠肝星状细胞(HSC-T6)生长的影响,探讨hUCMSCs上清液抑制HSC-T6生长的作用机制。方法采用组织块贴壁法获得hUCMSCs,并对其进行纯化、扩增、冻存和复苏。低温离心收集hUCMSCs上清液,实验分为对照组、25%hUCMSCs上清液组、50%hUCMSCs上清液组。于培养后不同时间点,观察HSC-T6细胞的形态学变化;MTT法检测HSC-T6细胞的增殖变化;采用Annexin V-FITC/PI检测HSC-T6细胞的凋亡变化;运用免疫组化方法检测HSC-T6细胞TGF-β1、α-SMA的表达变化。结果组织块贴壁法成功获得hUCMSCs。25%hUCMSCs上清液组、50%hUCMSCs上清液组与对照组相比,HSC-T6细胞数量明显减少,细胞形态更加不规则,突起减少;随着培养时间的增加,25%hUCMSCs上清液组、50%hUCMSCs上清液组与对照组相比,HSC-T6细胞增殖明显受到抑制;HSC-T6细胞凋亡数目明显增多;50%hUCMSCs上清液组较对照组相比,TGF-β1、α-SMA明显减少。结论 hUCMSCs上清液能够抑制大鼠肝星状细胞的增殖及活化;促进肝星状细胞的凋亡;与其抑制大鼠肝星状细胞TGF-β1的表达密切相关。

Objective To study the effection of Human Umbilical Cord Mesenchymal Stem Cells supernatant on the growth of rat hepatic stellate cells（HSC-T6）and to explore the mechanism of the inhibition of HSC-T6 growth.Methods HUCMSCs were obtained by tissue block adherence and purified,expanded,frozen and resuscitated.The hUCMSCs supernatants were collected by cryogenic centrifugation.The experiments were divided into control group,25%hUCMSCs supernatant group,and 50% hUCMSCs supernatant group.At different time points after culture,The morphology of HSC-T6 cells was observed;MTT assay for the proliferation of HSC-T6 cells;Annexin V-FITC/PI was used to detect the apoptosis of HSC-T6 cells.The expression of TGF-β1 andα-SMA in HSC-T6 cells was detected by immunohistochemistry.Results HUCMSCs cells were successfully obtained by tissue adherent method.In the supernatants of 25% hUCMSCs and 50% hUCMSCs supernatants compared with the control group,the number of HSC-T6 cells was significantly reduced,the cell morphology was more irregular,and the projections were reduced;As cultivation time increases,Compared with the control group,the proliferation of HSC-T6 cells was significantly reduced in 25%hUCMSCs group and 50% hUCMSCs group;the number of HSC-T6 cell apoptosis was significantly increased;TGF-β1 andα-SMA were significantly reduced in 50% hUCMSCs supernatant group compared with the control group.Conclusion The supernatant of hUCMSCs can inhibit the proliferation and activation of rat hepatic stellate cells and promote the apoptosis of hepatic stellate cells.It is closely related to the inhibition of the expression of TGF-β1 in rat hepatic stellate cells.