P21^(Cip/WAF1)及P27^(Kip1)与增殖细胞核抗原在人骨髓间充质干细胞向肝细胞分化过程中的表达

Expression of P21^(Cip/WAF1),P27^(Kip1) and proliferating cell nuclear antigen during differentiation of human bone marrow mesenchymal stem cells into hepatocytes

背景:P21Cip/WAF1、P27Kip1是两种重要的细胞周期的负调控因子,增殖细胞核抗原是反映干细胞增殖的指标。目的:观察体外诱导人骨髓间充质干细胞向肝细胞分化过程中P21Cip/WAF1,P27Kip1及增殖细胞核抗原的表达变化。设计、时间及地点:细胞学基因水平观察,于2006-12/2007-06在中山大学附属第二医院林百欣医学研究中心实验室完成。材料:骨髓来源于中山大学附属第二医院行骨髓穿刺的健康供者。方法:全骨髓贴壁筛选法体外分离培养人骨髓间充质干细胞,传至第5代后,加入含20μg/L肝细胞生长因子、10μg/L成纤维细胞生长因子4的opti-MEMI低血清培养基向肝细胞诱导分化。主要观察指标:诱导后RT-PCR检测细胞内P21Cip/WAF1,P27Kip1及增殖细胞核抗原基因mRNA的表达。结果:骨髓间充质干细胞向肝细胞定向诱导后0,1,3,5,7d,细胞内P21Cip/WAF1mRNA表达的相对量逐渐升高(P<0.01),P27Kip1mRNA表达的相对量无明显差异(P>0.05),增殖细胞核抗原mRNA表达的相对量除诱导后0,1d无明显差异(P>0.05)外,其余各时间点均逐渐降低(P<0.01)。结论:P21Cip/WAF1高表达抑制了骨髓间充质干细胞增殖,同时在其向肝细胞分化过程中起一定调控作用,P27Kip1可能并不起作用,增殖细胞核抗原的表达则与P21Cip/WAF1相反,呈逐步减少趋势。

BACKGROUND： P21^cip/WAF1 and P27^Kip1 are two important negative regulatory factors of cell cycle. Proliferating cell nuclear antigen （PCNA） is an index reflecting stem cell proliferation. OBJECTIVE： To study the expression of P21^cip/WAF1, P27^Kip1and PCNA gene during differentiation of human bone marrow mensenchymal stem cells （BMSCs） into hepatocytes in vitro. DESIGN, TIME AND SETTING： The cytology, gene level, observation experiment was conducted at the Laboratory of Linbaixin Medical Institute Center, Second Afftiated Hospital, Sun Yat-sen University from December 2006 to June 2007. MATERIALS： Bone marrow was collected from healthy donors undergoing bone marrow aspiration at the Second Affiliated Hospital, Sun Yat-sen University. METHODS： Human BMSCs were isolated by the whole bone marrow adherence method in vitro. At passage 5, BMSCs were incubated in opti-MEMI low-serum medium containing 20μg/L hepatocyte growth factor, 10 μ g/L flbroblast growth factor-4 to induce the differentiation into hepatocytes. MAIN OUTCOME MEASURES： After induction, the expression of P21^cip/WAF1, P27^Kip1and PCNA mRNA in 13MSCs was detected by RT-PCR. RESULTS： After BMSCs were induced for 0 day, 1 day, 3 days, 5 days, 7 days, the relative P21^cip/WAF1, P27^Kip1 mRNA value gradually increased （P 〈 0.01 ）, but no significant differences in relative P27Kip1 mRNA value （P 〉 0.05）. The relative PCNA mRNA value gradually decreased （P 〈 0.01 ）, except no significant differences were detected at 0 and 1 day （P 〉 0.05）. CONCLUSION： Overexpression of P21^cip/WAF1 inhibits BMSC proliferation, and has a regulatory effect during the differentiation of BMSCs into hepatocytes. P27Kip1 may has no effects. PCNA expression has an opposite effect compared to P21^cip/WAF1 , resulting in a gradually decreased tendency.