体外共培养大鼠骨髓间充质干细胞对肝星状细胞增殖的影响:CyclinD1与P27表达调控  

Influence of bone marrow mesenchymal stem cells on hepatic stellate cells proliferation:Regulation of Cyclin D1 and P27 expression

作　　者：王东旭[1] 姜海行[1] 苏思标[1] 覃山羽[1] 梁梓宇[1] 

机构地区：[1]广西医科大学第一附属医院消化内科,广西壮族自治区南宁市530021

出　　处：《中国组织工程研究与临床康复》2010年第10期1764-1768,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：广西自然科学基金资助项目(0640133);广西自然科学基金资助项目(0897008);广西"新世纪十百千人才工程"专项资金资助项目(2006206);广西卫生厅青年基金(桂卫Z2009102)~~

摘　　要：背景:在肝纤维化发生发展过程中,肝星状细胞起着关键作用。研究证实骨髓间充质干细胞移植可作为治疗肝纤维化的方法,但其逆转肝纤维化的机制不明。目的:探讨体外共培养条件下,大鼠骨髓间充质干细胞对肝星状细胞增殖的调控机制。方法:实验组在半透膜上接种大鼠骨髓间充质干细胞,在6孔塑料培养板上接种肝星状细胞,建立上下双层细胞共培养体系;对照组将大鼠骨髓间充质干细胞更换为成纤维细胞;空白组仅行肝星状细胞单独培养。WST-8法检测肝星状细胞生长抑制率,流式细胞仪分析细胞周期,RT-PCR检测肝星状细胞CyclinD1和P27mRNA的表达,Westernblot检测肝星状细胞CyclinD1和P27蛋白的表达。结果与结论:与空白组、对照组比较,实验组共培养24,48,72h肝星状细胞生长抑制率均显著升高(P<0.01);共培养72hG0/G1期细胞显著增加(P<0.01),S期细胞显著减少(P<0.01),可阻滞肝星状细胞由G0/G1期向S期转换。共培养24h,实验组CyclinD1mRNA及蛋白表达开始下调,至72h时表达量显著低于对照组、空白组(P<0.01);共培养全过程中各组p27mRNA的表达无明显差异(P>0.05),共培养24h时实验组p27蛋白表达较对照组、空白组均显著上调(P<0.01)。结果证实大鼠骨髓间充质干细胞能够抑制肝星状细胞的增殖,其机制可能是通过下调CyclinD1表达、上调P27蛋白表达,使细胞周期停滞于G0/G1期实现的。BACKGROUND： The hepatic stellate cells （HSCs） plays a key role in the development of liver fibrosis. Studies have shown that bone marrow-derived mesenchymal stem cell （BMSCs） transplantation can be used to treat liver fibrosis, but the mechanism for reversal of liver fibrosis remains unknown. OBJECTIVE： To explore the mechanism of bone marrow mesenchymal stem cells to regulate the proliferation of HSCs under co-culture in vitro. METHODS： Rat BMSCs and HSCs in the experimental group were cultured in the plastic culture plate （6 holes） to establish the upper and lower double-cell co-culture system. Rat normal fibroblast cell lines were seeded as control group; HSCs were cultured alone as blank group. Cell proliferation was determined by WST8 and cell cycle was determined by flow cytometry. The Cyclin D1 and P27 mRNA expression in HSC was determined by reverse transcription-polymerase chain reaction （RT-PCR） and the level of Cyclin D1 and P27 protein by Western blot. RESULTS AND CONCLUSION： HSCs co-cultured with BMSCs significantly inhibited HSC proliferation compared with the blank and control groups at 24, 48, and 72 hours （P 0.01）; Flow cytometry showed that the percentage of G0/G1 phase cells of co-culture group was increased but the S phase cells reduced （P 0.01） compared with the other groups at 72 hours, and BMSCs blocked HSC to convert from G0/G1 period to S phase. After HSCs co-cultured with BMSCs for 24 hours, the expression of CyclinD1 mRNA and protein was reduced, and significantly less than the blank and control groups at 72 hours （P 0.01）; no differences were detected in P27 mRNA expression in each group during the co-culture （P 0.05）. After co-culture of 24 hours, the p27 protein expression was significantly increased compared with the blank and control groups （P 0.01）. BMSCs inhibited the proliferation of HSCs, possibly through inhibiting CyclinD1 expression, increasing the p27 protein expression to cause cell cycle arresting in G0/G1 phase.

关 键 词：细胞周期 CYCLIND1 P27 共培养 肝星状细胞 骨髓间充质干细胞 

分 类 号：R394.2[医药卫生—医学遗传学]