人脐带间质干细胞体外条件下调控人肝细胞生长及功能的研究

A study on human umbilical cord mesenchymal stem cell regulated the growth and function of human liver cell in vitro

目的 探讨人脐带间质干细胞体外条件下调控人原代肝细胞增殖及功能的作用及其机制.方法 实验组将人脐带间质干细胞/原代肝细胞按1 ＆#215;105/1 ＆#215;105（个/孔）的比例接种于Transwell共培养板上下层共培养;对照组为原代肝细胞单独培养;共培养1、3、7d后应用流式细胞仪分析细胞周期;酶联免疫吸附试验（ELISA）法检测白蛋白（ALB）和尿素的分泌;逆转录-聚合酶链反应（RT-PCR）检测ALB、细胞角蛋白-18（CK18）、肝细胞生长因子（HGF）及c-met mRNA的表达.结果 共培养1、3、7d后原代肝细胞处于S期细胞比例与对照组比较明显增多,且增殖作用随共培养时间的增多而加强,差异有统计学意义（55.71％ ＞44.53％ ＞36.02％ ＞29.13％,P＜0.05）;共培养组ALB分泌量（μg/L）明显增加,差异有统计学意义（21.18＞ 17.12,30.90＞ 13.46,15.14＞8.80,P ＜0.05）,尿素分泌量（mg/L）得出同样的结果（211.18＞ 190.40,280.84＞ 75.64,152.06＞ 33.90,P ＜0.05）,在共培养各组别中,共培养3d时,尿素及白蛋白达到峰值,差异有统计学意义（P＜0.05）,而且共培养7d时仍保持较高水平的尿素及白蛋白分泌水平,与共培养1d比较,差异无统计学意义（P＞0.05）;与对照组比较,共培养各组别（1、3、7d）均可上调细胞内ALB、CK18、HGF及c-met mRNA的表达,共培养3d时原代肝细胞（PHHs）细胞内ALB mRNA上调最显著,而CK18、HGF及c-met mRNA的表达上调则在共培养7d时最为显著.结论 人脐带间质干细胞在体外条件下可通过旁分泌机制促进人原代肝细胞的生长增殖,保护其细胞功能,上调人原代肝细胞内HGF及其受体的表达可能是其中的机制之一.

Objective To investigate the mechanism of human liver cell＇s function and growth regulated by human umbilical cord mesenchymal stem cells in vitro.Methods The experimental group,human umbilical cord mesenchymal stem cells / primary hepatocytes by 1 ＆#215; 105/1 ＆#215; 105（cells / well） were seeded in the Transwell co-culture plates; the control group,liver cells （1 ＆#215; 105 cells / well） were cultured alone ; then cell cycle analysis by flowcytometry after cultured for 1,3,7 d.enzyme linked immunosorbent assay （ELISA） assay was to detect Albumin （ALB） secretion and urea; and reverse transcription-polymerase chain reaction （RT-PCR） was to analyse ALB,cytokeratin 18 （CK18）,hepatocyte growth factor （HGF） and c-met in primary hepatocytes.Results Cells in s phase in co-cultured group were more than control group and the proliferative effect was more and more as time goes by,the difference was statistically significant （55.71% 〉44.53% 〉36.02 % 〉29.13%,P 〈0.05）.The secretion of ALB and urea in the co-cultured cells were more than control group （μg/L）.In the same manner with the result （21.18 〉 17.12,30.90〉13.46,15.14〉8.80,P 〈0.05; 211.18〉190.40,280.84〉75.64,152.06〉33.90,P〈0.05）.In the co-culture groups,when co-cultured 3d,urea and albumin reached its peak,the difference was statistically significant （P 〈 0.05）,and co-cultured primary human hepatocytes （PHHs） still maintain a high level of urea and albumin secretion in d7 ; compared with the control group,the RNA levels of ALB,CK18,HGF and c-met increased CK18.Conclusion Human umbilical cord mesenchymal stem cells can promote the proliferation of primary human hepatocytes through paracrine mechanisms.HGF and its receptor were up-regulated in the human primary hepatocytes may be one of the mechanisms.