胎兔皮肤创伤消减文库构建与瘢痕愈合基因的初步筛选  

Construction of the subtractive cDNA library from injured fetal rabbit skins and rudnment screening of scar realing gene

作　　者：张波[1] 王正国[1] 朱佩芳[1] 

机构地区：[1]解放军第三军医大学野战外科研究所四室,重庆市400042

出　　处：《中国临床康复》2003年第4期574-575,T003,共3页Chinese Journal of Clinical Rehabilitation

基　　金：973项目(G1999054204)

摘　　要：目的:许多哺乳动物的胎儿在妊娠期前2/3内皮肤创伤为无瘢痕愈合,其发生机制至今尚不完全清楚。应用抑制性消减杂交获得差异表达的片段并构建消减文库,为差异基因的筛选及可能出现的未知基因的功能研究提供前期铺垫。方法:选择孕20d的胎兔及孕兔分别在腰部做全层皮肤切口,12h后取材作为胎兔创伤(fetaltrauma,FT)、胎兔对照(fetalcontrol,FC)和成兔创伤(adulttrauma,AT)。提取总RNA,合成dscDNA,经RsaⅠ酶切、Adaptor连接,两次杂交及两次抑制性PCR可获得差异性表达的片段。将扩增产物插入T载体并转染工程菌,培养后挑选阳性克隆。结果:经逐步验证获得61个较为可靠的阳性克隆。结论:设计并应用改良的SSH(2Drivers)替代传统的SSH,经实验证实在理论及实际操作上均可行且合理。消减文库的构建对无瘢痕愈合相关基因及其功能的后续研究提供了可靠的材料。AIM:Early gestation mammalian fetuses possess the amazing ability to heal cut aneous wounds in scarless fashion.Over the past years, scientists have been work ing to decipher the mechanisms underlying this regenerative repair.The remarkabl e phenotypic differences between fetal and adult healing may lead us want to kno w their characteristics in genetics,which represent potentially important mechan isms to explain the differences in the quality of wound repair observed in fetal versus adult tissues.It is reasonable to construct subtractive cDNA library for future research.METHODS:Middle laparotomy and hysterotomy were performed on pr egnant rabbits on 20-day gestation to expose the fetal back, longitudinal incis ion which penetrated full skin was made on the back of fetus.The trauma fetus sk in was harvested at 12 hours post-operation (FT), the fetus control (FC) and tr auma adult skin(AT) were taken at the same time. dscDNA was synthesized from tot al RNA of skin samples with SMART technology.Taking one of the three samples as Tester respectively, the other two as Drivers,we got 1 forward and 2 reverse hyb ridization products. Having had amplified by selective PCR, the products were in serted into vector,then transferred into E.coli HB101, the colonies were screene d afterwards.RESULTS:The wounded fetuses kept alive even after birth. Every dete rminant step like RNA isolation, cDNA synthesis, RsaⅠ digestion, adaptor ligat ion and hybridization was well-operated. Subtractive efficiency identification demonstrated that the suppression subtractive hybridization(SSH) was successful. Inserting into vector and transferring to E.coli were satisfactory.CONCLUSION:I nstead of classic SSH, an improved SSH with 2 Drivers was applied for the experi ment. Results confirm that the improved program be reasonable and correct in bot h theory and practice. The subtractive cDNA library we have got is helpful for f uture researches of scarless healing related gene(s) and its (their) function(s) .

关 键 词：胎兔 皮肤创伤 消减文库 构建 瘢痕愈合 基因筛选 

分 类 号：R619.6[医药卫生—外科学] R751.05[医药卫生—临床医学]