吉林大学学报(医学版)

• 基础研究 • 上一篇    下一篇

胎儿骨髓MSCs中Bmi-1和p16基因表达与其复制老化的关系

李晓波1, 顾建娟1, 关云谦2, 陈蓓蕾1, 张愚2   

  • 收稿日期:2013-02-01 出版日期:2013-07-28 发布日期:2013-08-17
  • 通讯作者: 李晓波(Tel:0514-87373835,E-mail:lxbljy@163.com) E-mail:lxbljy@163.com
  • 作者简介:李晓波(1972-),男,江苏省泰兴市人, 副主任医师,医学博士,主要从事干细胞的基础和临床应用研究。
  • 基金资助:

    江苏省卫生厅科研基金资助课题(H201049)

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

LI Xiao-bo1,GU Jian-juan1,GUAN Yun-qian2,CHEN Bei-lei1,ZHANG Yu2   

  1. 1. Department of Neurology,Clinical Medical School, Yangzhou University, Yangzhou  225001,China;2. Department of Cell Biology,Xuanwu Hospital,Captial Medical University,Beijing 100053,China
  • Received:2013-02-01 Online:2013-07-28 Published:2013-08-17

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P15 MSCs的 BrdU掺入率比较差异无统计学意义(P>0.05),P30 与P5和P15 MSCs的 BrdU掺入率比较均明显降低(P<0.05)。P5 MSCs中Bmi-1 mRNA表达水平高于P15和P30 MSCs(P<0.05),P15 MSCs中Bmi-1 mRNA表达水平高于P30 MSCs(P<0.05)。P5与P15 MSCs中p16 mRNA表达水平比较差异无统计学意义(P>0.05),但P30 MSCs中p16 mRNA表达水平明显低于P5和P15 MSCs(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。

关键词: Bmi-1基因, p16基因, 骨髓间充质干细胞, 老化

Abstract:

Abstract:Objective To study the changes of Bmi-1 and p16 gene 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,p16 gene 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,CD133 and 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-1 and  p16 gene mRNA in MSCs at P5,P15 and P30 were 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,CD105 and almost no expressions for CD34,CD45,CD133 and 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 P5 and P15 had no significant difference(P>0.05),but the BrdU incorporation rate of  MSCs at P30 was lower than those of MSCs at P5 and P15(P<0.05). The expression level of Bmi-1 gene mRNA of MSCs at P5 was higher than those of MSCs at P15 and P30 (P<0.05),and the expression level of Bmi-1 gene mRNA of MSCs at P15 was higher than that of MSCs at P30 (P<0.05). The expression levels of p16 gene mRNA of MSCs between P5 and P15 had no significant difference(P>0.05),but the expression level of p16 gene mRNA of MSCs at P30 was lower than those of MSCs at P5 and P15 (P<0.05). Conclusion MSCs can be successfully isolated from the femoral bone marrow of the human fetus. The replicative senescence of fetal bone marrow-derived MSCs has correlation with the mRNA expression level of Bmi-1,but the signal pathway may not mediate through p16INK4a-Rb pathway .

Key words: Bmi-1 gene, p16 gene, bone marrow-derived mesenchymal stem cells, senescence

中图分类号: 

  • R329.1