摘要
目的利用抑制消减杂交(suppressionsubtractivehybridization,SSH)技术,比较瘢痕疙瘩与正常皮肤组织之间基因表达差异,筛选瘢痕疙瘩特有的差异表达基因片段,为揭示瘢痕疙瘩的病因提供实验依据。方法提取瘢痕疙瘩与正常皮肤组织细胞mRNA,逆转录成cDNA。以瘢痕疙瘩cDNA为检测子,正常皮肤组织细胞cDNA为驱动子,选择四碱基内切酶Rsa将cDNA酶切;连接特殊设计的接头进行消减杂交和PCR反应,得到消减混合物,与T载体连接,转化DH5α感受态细菌,建立差异消减文库。Southern杂交筛选鉴定差异基因,进行测序和同源分析。结果经SSH筛选、Southern杂交鉴定得到13个瘢痕疙瘩差异表达基因,其中已知功能的基因11个,如TA结合因子1、E4基因转录因子1、ephrinB2型受体基因、血小板生长因子受体基因等;有与肿瘤发生有关的基因,如G抗原7基因等;未知功能基因2个。结论筛选得到的瘢痕疙瘩差异表达基因,对了解瘢痕疙瘩发生发展的病因学基础和指导临床治疗具有重要意义。
Objective To compare gene express difference of keloid and normal skin tissues by using the suppression subtractive hybridization （SSH） so as to find the differential express gene in keloid. Methods mRNA extracted from keloid and normal skin tissues was used as the template to synthesis cDNA of keloid and normal skin. The cDNA of keloid served＇as a tester, the cDNA of normal skin as a driver, cDNA was digested with Rsa I . Adaptorligated tester cDNA was prepared. Then first hybridization, second hybridization and PCR amplification were done. Differentially expressed cDNA was selectively amplified during these reactions. After SSH, the PCR mixture was ligated with T-vector. The positive clones were selected and the insert gene fragments were analyzed. Southern hybridization identified the keloid differential express genes. The positive clones of Southern hybridization were selected, and these sequences were analyzed. The results were compared with that of GeneBank. Results Thirteen differential genes were found in keloid, of which 11 gene clones have been known their function, and 2 clones have not known their function. Conclusion Keloid differentially expressed gene was screened successfully by SSH.
出处
《中国修复重建外科杂志》
CAS
CSCD
北大核心
2006年第5期495-498,共4页
Chinese Journal of Reparative and Reconstructive Surgery
基金
国家自然科学基金资助项目(30170972)~~
关键词
瘢痕疙瘩
抑制消减杂交
差异表达基因
Keloid Suppression subtractive hybridization Differential express gene
