天津医药

• 实验研究 •    

OCIL shRNA表达载体的构建及其对成骨细胞RANKL/OPG基因表达的调节

王君1,郑纺2,王宝利3   

  1. 1. 天津医科大学内分泌研究所
    2. 天津中医药大学
    3. 天津医科大学代谢病医院内分泌研究所、卫生部激素与发育重点实验室
  • 收稿日期:2011-12-29 修回日期:2012-06-28 出版日期:2012-12-15 发布日期:2012-12-15
  • 通讯作者: 王宝利

Construction of OCIL shRNA Expression construct and its Effect on RANKL/OPG Expression in Osteoblasts

1,ZHENG Fang 1   

  • Received:2011-12-29 Revised:2012-06-28 Published:2012-12-15 Online:2012-12-15

摘要： 目的：构建OCIL shRNA表达载体，转染UMR106成骨细胞，观察OPG和RANKL mRNA的表达及转染后的成骨细胞对破骨细胞分化的影响。方法：设计针对大鼠OCIL mRNA的shRNA编码序列，克隆于pRNAT-U6.1/Neo 载体；使用lipofectamine2000将OCIL shRNA重组表达载体转染UMR106成骨细胞系，实时荧光定量PCR检测各组UMR106细胞中OCIL mRNA的表达及OPG和RANKL mRNA的表达。用OCIL shRNA转染的成骨细胞条件培养基干预PTH诱导骨髓细胞，观察破骨细胞样细胞生成情况。结果：1、成功构建了针对大鼠OCIL shRNA重组表达载体。转染OCIL shRNA表达载体的UMR106细胞中OCIL mRNA 表达受到明显抑制。2、OCIL shRNA转染UMR106成骨细胞后，RANKL mRNA表达水平明显升高；而OPG mRNA表达水平明显降低。3、OCIL shRNA转染的成骨细胞条件培养基促进骨髓细胞向破骨细胞样细胞分化。结论：RANKL/OPG系统参与了OCIL对破骨细胞分化的调节作用。

关键词: 破骨细胞抑制性凝集素, 小干扰, 核因子-κB受体激活物配基, 护骨素, 成骨细胞

Abstract: Objective：To construct OCIL shRNA expression construct and to study its effects on RANKL/OPG expression in osteoblasts and on osteoclastogenesis. Methods：Small hairpin RNA (shRNA) coding sequences targeting rat OCIL mRNA were designed, synthesized and cloned into pRNAT-U6.1/Neo vector. The constructs were transfected into UMR106 cell line with lipofectamine 2000. The expression level of OCIL mRNA in the cells was determined by real-time quantitative RT-PCR. The expression levels of RANKL and OPG were also studied. The culture medium collected from the transfectant was used to treat rat bone marrow cells in the presence of PTH and the number of osteoclast like cells formed from marrow cells were scored. Results：The shRNA expression constructs significantly reduced OCIL mRNA in UMR106 cells after transfection. Moreover, the overexpression of OCIL shRNA resulted in a significant increase of RANKL mRNA and decrease of OPG mRNA. The culture medium collected from the transfectant promoted osteoclast-like cell formation from marrow cells in the presence of PTH. Conclusion：The inhibition of osteoclastogenesis by OCIL may involve the regulation of RANKL/OPG system.

Key words: osteoclast inhibitory lectin(OCIL), shRNA, receptor activator of nuclear factor κB ligand (RANKL), osteopmtegerin(OPG), osteoblast