骨髓间质干细胞培养上清液调控肝细胞的体外研究

目的探讨骨髓间质干细胞(bone marrow mesenchymal stem cells,BMSC)培养上清液对肝细胞增殖、凋亡的影响,初步探讨BMSC治疗肝纤维化的旁分泌机制。方法采用密度梯度离心及贴壁细胞分离相结合提取BMSC。培养48h的BMSC细胞培养上清液按BMSC不同处理(培养)时间和含量两种方法建组。不同处理时间建组:取培养48hBMSC上清液处理肝细胞(完全培养),分为处理24h、48h、72h组,对照组为1640培养基处理24h的肝细胞。不同含量BMSC建组:实验1组为完全BMSC培养上清液(含肝细胞的6孔板中每孔加入BMSC上清液2ml)处理肝细胞;实验2组为部分BMSC培养上清液(每孔加BMSC上清液1ml+1640培养基1ml)处理肝细胞;对照组为完全细胞培养基(每孔加1640培养基2ml)处理肝细胞。用酶联免疫吸附试验(ELISA)试剂盒检测BMSC旁分泌肝细胞生长因子(hepatocyte growth factor,HGF)情况及培养时间对其的影响;用流式细胞仪观察肝细胞细胞分期和检测凋亡细胞数;用蛋白免疫印迹法(Western-blot)检测样本上清液中白蛋白的表达。结果 BMSC分泌HGF,其含量变化具有时间依赖性。加入BMSC培养上清液后,与对照组相比,实验1组中的培养上清液可以明显促进肝细胞增殖、抑制其凋亡,并随着处理时间的延长而增加(处理72h>处理48h>处理24h,均为P<0.05);实验组(1组、2组)的白蛋白分泌较对照组上调,且随着处理时间的延长白蛋白含量增加。结论 BMSC有可能通过分泌HGF促进肝细胞增殖、抑制凋亡,促进白蛋白分泌,从而抑制肝脏纤维化。

Extracorporeal research of culture supernatant of bone marrow mesenchymal stem cells on regulating hepatocyte

Objective To investigate the effect of culture supernatant of bone marrow mesenchymal stem cells(BMSC) on proliferation and apoptosis of hepatocyte,and to discuss the paracrine mechanism of BMSC on treating hepatic fibrosis preliminarily.Methods BMSC were extracted by density gradient centrifugation and adherent cell separation.BMSC were cultured for 48 h and the culture supernatant was divided into groups according to different processing(culturing) time and contentration.In groups of different processing time,hepatocytes(completely culture) were processed by 48 h BMSC culture supernatant for 24 h,48 h,72 h.In control group,hepatocyes were processed in 1640 culture medium for 24 h.In groups of different contentration,hepatocytes were divided into experimental group 1,experimental group 2 and control group.In experimental group 1,hepatocytes were processed by completely culture supernatant(2 ml BMSC supernatant was added into every orifice containing hepatocytes in six orifice plate).In experimental group 2,hepatocytes were processed by incomplete culture supernatant(1 ml BMSC supernatant and 1 ml 1640 culture medium were added into every orifice containing hepatocytes).In control group,hepatocytes were processed by completely culture medium(2 ml 1640 culture medium was added into every orifice containing hepatocytes).Hepatocyte growth factor(HGF) secretion by paracrine mechanism of BMSC and the impact of culture time were detected by enzyme-linked immune absorbent assay(ELISA).Cell staging and apoptosis cell counts of hepatocyte were detected by flow cytometry.The expression of albumin in supernatant was detected by Western-blot.Results The contentration of HGF secreted by BMSC was determined by culture time.After adding BMSC culture supernatant,the culture supernatant of experimental group 1 significantly promoted proliferation and inhibited apoptosis of hepatocytes compared with that in control group,and the effect improved the prolongation of processing time(processed 72 h > processed 48 h > processed 24 h,all in P<0.05).The secretion of albumin in experimental groups(group 1 and group 2) increased compared with that in control group,and increased as the prolongation of processing time.Conclusions BMSC may promote proliferation and inhibit apoptosis of hepatocytes through the secretion of HGF.It increases the secretion of albumin in order to inhibit hepatic fibrosis.