中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (45): 8365-8368.doi: 10.3969/j.issn.1673-8225.2010.45.002

• 干细胞培养与分化 stem cell culture and differentiation • 上一篇    下一篇

小分子干扰RNA介导RhoA沉默优化骨髓间充质干细胞的培养　

吕  刚1，姚  鑫2   

  1. 1天津医科大学研究生院，天津市 300000；2天津市环湖医院神经外科，天津市 300000
  • 出版日期:2010-11-05 发布日期:2010-11-05
  • 通讯作者: 姚鑫，硕士，主任医师，硕士生导师，天津市环湖医院神经外科中心，天津市300000 phoenix6a6@163.com
  • 作者简介:吕刚★，男，1978年生，天津市人，满族，天津医科大学在读硕士，主治医师，主要从事脑外伤，脊髓损伤的相关研究。 lgemail@eyou.com

Optimization of small interfering RNA-mediated RhoA silencing in bone marrow mesenchymal stem cell cultures

Lü Gang1, Yao Xin2   

  1. 1 Postgraduate College, Tianjin Medical University, Tianjin  300000, China; 2 Department of Neurosurgery, Huanhu Hospital of Tianjin, Tianjin  300000, China
  • Online:2010-11-05 Published:2010-11-05
  • Contact: Yao Xin, Master, Chief physician, Master’s supervisor, Department of Neurosurgery, Huanhu Hospital of Tianjin, Tianjin 300000, China phoenix6a6@163.com
  • About author:Lü Gang★, Studying for master’s degree, Attending physician, Postgraduate College, Tianjin Medical University, Tianjin 300000, China lgemail@eyou.com

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摘要：

背景：以往骨髓间充质干细胞的培养方法存在衰老和分化率低等问题。
目的：检测是否可以通过沉默RhoA基因的方法优化骨髓间充质干细胞培养。
方法：体外培养SD大鼠骨髓间充质干细胞，经小分子干扰RNA转染以沉默RhoA基因表达，分为3组培养：干细胞组(未转染小分子干扰RNA)、经随机打乱顺序的小分子干扰RNA 转染干细胞组、经小分子干扰RNA 转染的干细胞组。用RT-PCR，Western blot检测骨髓间充质干细胞在转染前后RhoA基因和蛋白的表达。应用细胞生长曲线、MTT比色法观察细胞生长的优化作用，采用流式细胞术测定细胞周期分布的变化。
结果与结论：与干细胞组、经随机打乱顺序的小分子干扰RNA 转染干细胞组比较，经小分子干扰RNA 转染的干细胞组RhoA基因和蛋白表达量明显降低(P < 0.05)，细胞的生长速度明显增快，细胞周期G0/G1期减少，S期细胞数增多(P < 0.05)。说明通过沉默RhoA基因的方法可以促进骨髓间充质干细胞增殖，优化培养方法。

关键词: 细胞培养, 骨髓间充质干细胞, RhoA, RNA干扰, 干细胞培养

Abstract:

BACKGROUND: Previous culture method of bone marrow mesenchymal stem cells (BMSCs) has problems such as senescence and low differentiation rate.
OBJECTIVE: To determine whether the RhoA gene silencing can optimize BMSC cultures.
METHODS: BMSCs from Sprague Dawley rats were cultured in vitro, and were transfected by small interfering RNA (siRNA) to knock down the expression of RhoA. BMSCs were divided into three groups: stem cell group (non-transfection of siRNA), disrupted by random sequence siRNA transfection stem cell group, and siRNA transfection of stem cell group. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot assay were used to assess RhoA gene and protein expression before and after transfection. Cellular proliferation optimization was determined by the cell growth curve and MTT assay. The cell cycle was analyzed by flow cytometry.
RESULTS AND CONCLUSION: Expression of RhoA gene and protein was significantly reduced following transfection of siRNA compared with stem cell group (P < 0.05). The cell growth speed was significantly increased, the cell cycle of G0/G1 phase was decreased and cell number in S phase was increased (P < 0.05). These indicated that the RhoA gene silencing can promote BMSC proliferation, and optimize culture methods. 

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