中国组织工程研究

中国组织工程研究 ›› 2011, Vol. 15 ›› Issue (7): 1199-1204.doi: 10.3969/j.issn.1673-8225.2011.07.013

• 组织构建细胞学实验 cytology experiments in tissue construction • 上一篇    下一篇

反转录病毒介导靶向瞬时感受器电位M7基因siRNA抑制RBL-2H3细胞的活化

吕志强，吴毅梅，江山平，张  蔚，黄林洁   

  1. 中山大学附属第二医院呼吸内科，广东省广州市510120
  • 收稿日期:2010-09-23 修回日期:2010-12-08 出版日期:2011-02-12 发布日期:2011-02-12
  • 通讯作者: 江山平，教授，博士生导师，中山大学附属第二医院呼吸内科，广东省广州市 510120 shanpingjiang@126.com
  • 作者简介:吕志强，男，1964年生，广东省海丰县人，汉族，1988年中山大学毕业，副教授，主要从事支气管哮喘的临床与基础研究。 gzmeggiewoo@hotmail.com
  • 基金资助:

    课题国家自然基金项目(81070027)，广东省自然基金项目(10151008901000078)，广东省科技计划项目(2009B030801093)及广东省医学科研基金项目(B2009071)资助。

Retrovirus-mediated siRNA targeting transient receptor potential melastatin 7 gene suppresses activation of RBL-2H3 cells  

Lü Zhi-qiang, Wu Yi-mei, Jiang Shan-ping, Zhang Wei, Huang Lin-jie   

  1. Department of Respiratory Medicine, the Second Affiliated Hospital, Sun Yat-sen University, Guangzhou  510120, Guangdong Province, China
  • Received:2010-09-23 Revised:2010-12-08 Online:2011-02-12 Published:2011-02-12
  • Contact: Jiang Shan-ping, Professor, Doctoral supervisor, Department of Respiratory Medicine, the Second Affiliated Hospital, Sun Yat-sen University, Guangzhou 510120, Guangdong Province, China shanpingjiang @126.com
  • About author:Lü Zhi-qiang, Associate professor, Department of Respiratory Medicine, the Second Affiliated Hospital, Sun Yat-sen University, Guangzhou 510120, Guangdong Province, China gzmeggiewoo@hotmail.com
  • Supported by:

    the National Natural Science Foundation of China, No. 81070017*; the Natural Science Foundation of Guangdong Province, No. 10151008901000078*; the Science and Technology Planning Project of Guangdong Province, No. 2009B030801093*; Medical Scientific Research Foundation of Guangdong Province, No. B2009071*

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被引次数

1

摘要：

背景：钙离子在肥大细胞活化后的脱颗粒反应起重要作用。瞬时感受器电位M7(transient receptor potential melastatin 7，TRPM7)是肥大细胞重要的候选通道。
目的：构建携带大鼠靶向TRPM7-siRNA的反转录病毒载体并检测其对大鼠RBL-2H3细胞抗原活化的影响。
方法：实验设计3个TRPM7-siRNA序列和1个无关对照序列，克隆到酶切的pSuper-retro-neo-GFP反转录病毒载体，用重组质粒pSuper-retro-neo-GFP-shTRPM7-(1, 2, 3) 采用脂质体Lipofectamine 2000转染RBL-2H3细胞，采用Western blot检测干扰效率。筛选出最有效的pSuper-retro-neo-GFP-siTRMP7与包装质粒共转染293FT细胞生成反转录病毒并感染RBL-2H3细胞，荧光实时定量PCR及Western blot检测TRPM7-siRNA的沉默效果。检测β-氨基已糖苷酶活性探讨RBL-2H3细胞抗原活化程度的改变。
结果与结论：转染后的各组细胞中siTRPM7-3转染组的沉默效率最高(P < 0.05)。pSuper-retro-neo-GFP-siTRMP7-3干扰组的TRPM7基因的mRNA水平和蛋白水平显著下调，致敏后其β-氨基已糖苷酶活性明显降低(P  < 0.05)。结果提示，降低TRPM7基因的表达可抑制RBL-2H3细胞的抗原活化。

关键词: 反转录病毒, RNA干扰, RBL-2H3细胞, 瞬时感受器电位M7通道, 心脏, 组织工程

Abstract:

BACKGROUND: Calcium ion plays an important role in the degranulation process for activated mast cells. Transient receptor potential melastatin 7 (TRPM7) is an important candidate channel for mast cells.
OBJECTIVE: To construct a recombinant retrovirus vector siRNA targeting rat TRPM7 gene and explore its influence on antigen-induced activation of RBL-2H3 cells.
METHODS: Three TRPM7-siRNA sequences and a negative sequence were designed and cloned into linearized pSuper-retro-neo-GFP vector. The above recombinants were transfected by lipofectamine 2000 into RBL-2H3 cells. The gene silencing efficacy of the 3 targets was evaluated by Western blot. The optimized pSuper-retro-neo-GFP-siTRMP7 and packaging plasmid were co-transfected into 293FT cells to produce retrovirus, which was applied to infect RBL-2H3 cells. The RNAi efficiency was confirmed by real-time PCR and Western blot. Measurement of β-hexosaminidase was performed before and after TRPM7-siRNA transfection to explore the changes on activation of RBL-2H3 cells.
RESULTS AND CONCLUSION: The gene silencing efficacy of siTRPM7-3 transfected group was highest among all Lipofectamine 2000 transfected groups (P < 0.05). Compared to the normal control group, TRPM7 expression of pSuper-retro-neo-GFP-siTRMP7-3 transfected group was significantly reduced both at mRNA and protein levels (P < 0.05). The results revealed that, down regulation of TRPM7 channel can suppress the antigen-induced activation of RBL-2H3 cells.

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