创伤早期伤口液作用真皮多能干细胞差异表达cDNA文库的构建  

Construction of differentially expressed cDNA library of dermal pluripotent stem cells stimulated by early-phase wound transudate

作　　者：屈纪富[1] 史春梦[1] 郑怀恩[1] 郝利[1] 程天民[1] 

机构地区：[1]第三军医大学全军复合伤研究所创伤,烧伤与复合伤研究国家重点实验室,重庆400038

出　　处：《感染．炎症．修复》2005年第1期3-6,2,共5页Infection Inflammation Repair

基　　金：国家自然科学基金项目资助(30370562)

摘　　要：目的：研究创伤早期伤口液作用前后真皮多能干细胞基因表达的变化,探讨真皮多能干细胞参与创伤修复启动的分子机制。方法：取伤后1d伤口液作为创伤修复启动微环境,刺激真皮多能干细胞,24h后提取总RNA为实验方(tester),未予刺激的真皮多能干细胞总RNA为驱动方(driver)。实验方、驱动方总RNA由SMART cDNA方法合成cDNA,然后用抑制消减杂交(SSH)方法获得消减杂交产物。消减杂交产物克隆到T载体构建消减文库。对部分克隆进行杂交筛选及序列比对。结果：消减杂交文库挑取615个克隆进行PCR扩增,阳性率约为97%。取其中48个克隆进行反向Northern杂交,获得5个差异表达基因。经Genebank检索,这些差异表达基因与已知序列有较高同源性,其中大多为与创伤修复相关的基因。结论：我们成功构建了创伤早期伤口液刺激前后大鼠真皮多能干细胞差异表达cDNA文库,并筛选、克隆出部分与真皮多能干细胞参与创伤修复相关的差异表达基因。Objective:To investigate the differentially expressed genes in dermal pluripotent stem cells(DPSCs) stimulated by early-phase wound transudate so as to explore the molecular mechanisms of participation of DPSCs in the initiation of wound repair.Methods:Suppression subtractive hybridization(SSH)was performed to screen the differentially expressed genes in DPSCs stimulated by early—phase wound transudate.The subtractive products were cloned into T vector to construct cDNA library.To restrict the number of false positive clones,reverse Northern hybridization was used to further verify the differentially expressed cDNA clones,and the positive clones were sequenced.Results:There were 615 clones in the cDNA library.After the reverse Northern hybridization of 48 clones,5 differntially expressed genes were obtained,and most of them were associated with wound repair. Conclusions:Differentially expressed cDNA library of DPSCs were constructed by being stimulated by early-phase wound transudate and 5 differentially expressed genes in DPSCs associated with wound repair were screened.

关 键 词：真皮多能干细胞 创伤愈合 抑制消减杂交 

分 类 号：R64[医药卫生—外科学]