用消减杂交技术克隆人成骨样细胞机械牵张力敏感基因的研究  

Cloning Genes Sensitive to Mechanical Stretch in Osteoblasts through Substractive Hybridization Technique

作　　者：冯雪[1] 丁寅[1] 段银钟[1] 林珠[1] 欧阳为明[2] 蒲勤[3] 

机构地区：[1]第四军医大学秦都口腔医院正畸科 [2]第四军医大学免疫学教研室,陕西西安710032 [3]第四军医大学生物化学教研室,陕西西安710032

出　　处：《华西口腔医学杂志》2004年第4期278-280,共3页West China Journal of Stomatology

基　　金：国家自然科学基金资助项目 (30 1 70 2 4 5 ;30 370 374)

摘　　要：目的　对人成骨样细胞机械牵张力敏感基因进行克隆研究。方法　对人成骨样细胞Saos_2进行二维方向上的加载 ,变形幅度为 12 % ,牵拉频率为 6周期 分钟 ,加载 12h后分别提取牵张力作用后Saos_2细胞及未作处理的正常Saos_2对照细胞的mRNA ,反转录制备cDNA ,将cDNA进行消减杂交 ,构建加载细胞与未加载细胞的差异表达基因的扣除cDNA文库 ,克隆后进行序列测定。结果　克隆到的基因片段中 ,功能已知基因片段 15个 ,功能未知基因片段 2个 ,其中牵张力敏感基因 1(SSG1)位于 9号染色体上 ,功能未知 ;牵张力敏感基因 2 (SSG2 )位于 18号染色体上 ,与转录因子 2 ,4高度同源。结论　用消减杂交技术可以对人成骨细胞样机械牵张力敏感基因进行有效的克隆研究。Objective In this experiment, genes sensitive to mechanical stretch in osteoblast like cells were cloned through substractive hybridization technique.Methods Two dimensional mechanical stretch with deformation of 12% and frequency of 6 cycles was loaded on human osteoblastic like cell line Saos-2. Complementary deoxyribornucleic acid (cDNA) library of cells was constructed 12 h after loading, acting as tester. cDNA library of cells without loading was constructed, acting as driver. A subtractive cDNA library osteoblastic like cell stimulated with mechanical stretch was constructed through substractive hybridization technique.Results Of clones randomly selected from this library, fifteen genes were identified to be the differentially expressed genes. Comparing with the sequences published in GeneBank via Internet, two sequences located in chromosome 9 and 18 respectively were identified to be novel, which were named as stretch sensitive gene 1 and stretch sensitive gene 2.Conclusion It is an efficient approach to clone and study genes relative to mechanical stretch through substractive hybridization technique.

关 键 词：消减杂交 基因克隆 成骨细胞 机械牵张力 敏感基因 

分 类 号：R785[医药卫生—口腔医学]