摘要
目的通过基因芯片筛选同真皮间充质干细胞相关的创伤修复差异表达基因。方法利用贴壁生长的特性分离大鼠真皮间充质干细胞,用Tripure提取伤口液刺激前后dMSCs和创伤前后大鼠皮肤组织中总RNA。PCR扩增已构建抑制消减杂交差异表达文库中485个克隆的插入片断,送北京博奥制作基因芯片。提取的RNA反转录后分别同芯片杂交,找出在细胞水平和组织水平同时高表达的克隆。将这些克隆测序,进行生物信息学分析。结果分离的大鼠真皮间充质干细胞呈纺锤形,在体外显示多向分化潜能。抽提伤口液刺激前后dMSCs和创伤前后大鼠皮肤组织中总RNA,反转录后和基因芯片杂交,发现可能同创伤愈合相关的13条基因,其中热休克蛋白70(HSP70),基质金属蛋白酶3(MMP3),白细胞蛋白酶抑制因子(SLPI)等可能在创伤愈合中有重要意义。结论抑制消减杂交和基因芯片结合是筛选差异表达基因的一种有效方法。伤口液刺激前后dMSCs中差异表达基因的发现能为从基因水平探讨dMSCs参与创伤愈合的分子机制提供新的研究靶点。
Objective Find changed genes in dermal mesenchymal stem cells （dMSCs） associated with wound repair. Methotis Isolated stem cells from the rat skin tissue. We had obtained the cDNA library by suppression subtractive hybridization （SSH）. The inserted fragments of 485 clones in the library were expanded by polymerase chain reaction （PCR） method, and the PCR products were fixed on the cDNA mcroarry after the purification. The RNA was isolated from two groups： ①dMSCs before and after the stimulation by wound fluid; ②skin tissue before and after trauma. After reverse transcription, cDNA of these two groups was hybridized with the cDNA mcroarry respectively. The clones which both high expressed in two groups were selected, and the DNA clones were sequenced and analyzed by bioinformatics. Results Most of the neonatal DMCs were spindleshaped cells and exhibited muhilineage potential in vitro. Thirteen genes related to wound repair were found in our experiment. Some of the genes such as HSP70,MMP and SLPI might play the key roles in the startup and regulation of wound repair. Conclusion Combining with SSH and the cDNA mcroarry is an effective method to find changed genes. The discovery of the changed genes in dMSCs is helpful to explore the molecule mechanism of wound repair.
出处
《中国急救医学》
CAS
CSCD
北大核心
2006年第4期271-273,共3页
Chinese Journal of Critical Care Medicine
基金
国家自然科学基金项目资助(30370562)
国家重点基础研究发展规划(973)项目(2005CB522605)
关键词
基因芯片
干细胞
基因
cDNA mcroarry
Stem cells
Gene
