胎儿骨髓MSCs中Bmi-1和p16基因表达与其复制老化的关系  

Correlation between Bmi-1,p16 gene expressions and replicative senescence of fetal bone marrow-derived mesenchymal stem cells

作　　者：李晓波 顾建娟 关云谦[2] 陈蓓蕾 张愚[2] 

机构地区：[1]扬州大学临床医学院神经内科,江苏扬州225001 [2]首都医科大学宣武医院细胞生物室,北京100053

出　　处：《吉林大学学报（医学版）》2013年第4期715-719,I0002,共6页Journal of Jilin University：Medicine Edition

基　　金：江苏省卫生厅科研基金资助课题(H201049)

摘　　要：目的:探讨胎儿骨髓间充质干细胞(MSCs)中Bmi-1和p16基因表达随传代次数增加的变化情况,阐明其与MSCs复制老化之间的关系。方法:取孕16、17和25周流产胎儿的股骨,利用贴壁培养法分离、培养、扩增骨髓MSCs。采用流式细胞术检测第5代MSCs中CD13、CD34、CD44、CD45、CD73、CD90、CD105、CD133和HLA-DR的表达;连续传代30代,诱导培养MSCs老化;用BrdU掺入率检测第5(P5)、15(P15)和30代(P30)骨髓MSCs的增殖能力;采用Real-time PCR法检测上述细胞中Bmi-1和p16基因mRNA的表达量。结果:从孕16、17和25周的流产胎儿股骨骨髓中均能分离、培养、扩增出MSCs,均表达CD13、CD44、CD73、CD90和CD105,不表达CD34、CD45、CD133和HLA-DR;随着MSCs的不断传代,细胞生长速度逐渐减慢,细胞变成扁平状,折光性下降。P5与P15MSCs的BrdU掺入率比较差异无统计学意义(P>0.05),P30与P5和P15MSCs的BrdU掺入率比较均明显降低(P<0.05)。P5MSCs中Bmi-1mRNA表达水平高于P15和P30MSCs(P<0.05),P15MSCs中Bmi-1mRNA表达水平高于P30MSCs(P<0.05)。P5与P15 MSCs中p16mRNA表达水平比较差异无统计学意义(P>0.05),但P30 MSCs中p16mRNA表达水平明显低于P5和P15MSCs(P<0.05)。结论:胎儿股骨骨髓能培养出MSCs,其复制老化与Bmi-1的表达有关联,但其信号通路可能不通过p16INK4a-Rb介导。Objective To study the changes of Bmi-1and p16gene expressions in human fetal bone marrow-derived mesenchymal stem cells（MSCs）with the increasing of passage number,and to clarify the relationship between Bmi-1,p16gene expressions and replicative senescence of MSCs.Methods By using the adherence culture method,MSCs were isolated from the femoral bone marrow of three abortuses：16-week-old,17-week-old and 25-week-old,respectively;and then the MSCs were isolated,cultured and amplified.The expressions of CD13,CD34,CD44,CD45,CD73,CD90,CD105,CD133and HLA-DR in the fifth generation of MSCs were detected with flow cytometry.After successively subcultured,the fetal bone marrow-derived MSCs were induced to replicative senescence.The proliferation abilities of MSCs at passage 5（P5）,passage 15（P15）and passage 30（P30）were detected with BrdU incorporation assays.The expression levels of Bmi-1and p16gene mRNA in MSCs at P5,P15 and P30were determined with Real-time PCR.Results The MSCs were isolated from the femoral bone marrow of the three abortuses and then cultured successfully,with positive expressions for CD13,CD44,CD73,CD90,CD105and almost no expressions for CD34,CD45,CD133and HLA-DR;With the increasing of passage number,the cell growth rate was gradually slowed down,and the cells became flat,and the refractivity was decreased.The BrdU incorporation rates of MSCs between P5and P15had no significant difference（P0.05）,but the BrdU incorporation rate of MSCs at P30was lower than those of MSCs at P5and P15（P0.05）.The expression level of Bmi-1gene mRNA of MSCs at P5was higher than those of MSCs at P15and P30（P0.05）,and the expression level of Bmi-1gene mRNA of MSCs at P15was higher than that of MSCs at P30（P0.05）.The expression levels of p16gene mRNA of MSCs between P5and P15had no significant difference（P0.05）,but the expression level of p16gene mRNA of MSCs at P30was lower than those of MSCs at P5and P15（P0.05）.Conclusion MSCs can be successfully isolated from the femoral bone marrow o

关 键 词：BMI-1基因 P16基因 骨髓间充质干细胞 老化 

分 类 号：R329.1[医药卫生—人体解剖和组织胚胎学]