兔皮肤组织无瘢痕愈合相关基因克隆中应用改良多重抑制性消减杂交技术的可行性(英文)  

Feasibility of applying the improved multiple suppression subtractive hybridization technique in cloning of scarless healing related genes in rabbit skin

作　　者：张波[1] 刘大维[1] 王正国[1] 朱佩芳[1] 

机构地区：[1]解放军第三军医大学大坪医院野战外科研究所四室,重庆市400042

出　　处：《中国临床康复》2004年第32期7335-7337,共3页Chinese Journal of Clinical Rehabilitation

基　　金：国家重点基础研究发展规划资助(GB1999054204)~~

摘　　要：背景:传统的抑制性消减杂交(suppressionsubtractivehybridization,SSH)主要用来比较两组mRNA样品中仅在一组表达的基因克隆。在兔皮肤组织无瘢痕愈合相关基因的克隆与筛选过程中,既要同时考虑到创伤与对照间的差异,又要考虑到胎兔与成年兔之间的差异。目的:获得可信的无瘢痕愈合相关基因,为从根本上认识无瘢痕愈合的发生机制提供极具现实意义的前期铺垫。设计:设立对照的实验研究。单位:解放军第三军医大学大坪医院野战外科研究所。材料:选用42只体质量2.5～3kg健康的大耳白雌兔(第三军医大学大坪医院野战外科研究所实验动物中心提供)。干预:为了减少因遗传背景带来的干扰,实验设立了3组:胚胎创伤(Fetaltrauma,FT)、胚胎对照(Fetalcontrol,FC)和成年兔创伤(Adulttrau-ma,AT);使用自行改良的一个检测子(Tester)对应两个驱动子(Driver)改良的多重SSH技术。主要结局观察指标:①RNA抽提结果。②SSH结果。结果:分别以胚胎创伤、成年兔创伤及胚胎对照为Tester,其余两个共同为Drivers,共获得正反向杂交产物3组,由全层皮肤组织抽提的总RNA具有较好的完整性,经PCR验证分析,G3PDH的消减效率令人满意。结论:自行改良的多重抑制性消减杂交技术在理论与实际操作方面都是可行的。BACKGROUND:Traditional suppression subtractive hybridization(SSH) is a highly effective technique to compare two populations of mRNA so as to obtain clones of genes that are expressed in one population but not in the other.In the process screening and cloning of scarless healing related genes in rabbit skin,both the differences between normal group and trauma group and the differences between adult group and fetus group have to be considered. OBJECTIVE:To obtain the genuine scarless healing related genes so as to provide a practical basis for basically understanding the mechanism of scarless healing. DESIGN:A controlled experimental study. SETTING:The study was conducted at the Research Institute of Surgery,Daping Hospital,Third Military Medical University. MATERIALS: Forty two healthy flap eared female white rabbits with a body mass of 2.5-3 kg(provided by the Center for Experimental Animals,Research Institute of Surgery,Daping Hospital,Third Military Medical University) were selected for our study. INTERVENTIONS:To decrease the possible differences caused by hereditary backgrounds among the samples,it is reasonable to set up three groups of samples:fetal trauma(FT),fetal control(FC) and adult trauma(AT).An improved multiple SSH with one tester and two corresponding drivers was used. MAIN OUTCOME MEASURES:①Results of RNA extraction;②Results of SSH. RESULTS:FT,FC and AT were set as Tester separately,while the other two were both as Drivers.Then we got three groups of hybridization products.The total RNA extracted from full thickness skin tissue was intact.PCR analysis suggests that the subtractive efficiency of G3PDH was successful. CONCLUSION:The improved multiple SSH is feasible in theory and practice.Especially,those positive clones that were screened from subtractive library by this improved SSH were much closer to the target genes.

关 键 词：杂交 基因 克隆 分子 

分 类 号：R619[医药卫生—外科学]