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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,尤其是从采用这种方法获得的差减文库中
BACKGROUND: Traditional suppressionsubtractive hybridization (SSH) is primarily used to compare gene clones that are expressed in only one set in two mRNA samples. In the process of cloning and screening of scar-free healing-related genes in rabbit skin tissue, it is necessary to consider both the difference between the trauma and the control and the difference between the fetus and the adult rabbit. OBJECTIVE: To obtain credible scar-free healing-related genes and to provide a very realistic pre-mat for understanding the underlying mechanisms of scarless healing. Design: Set up a controlled experimental study. Unit: Daping Hospital of PLA Third Military Medical University Field Surgery Research Institute. MATERIALS: Forty-two healthy white-eared white rabbits weighing 2.5 ~ 3kg were selected from Experimental Animal Center of Daping Hospital Field Surgery Institute, Third Military Medical University. Intervention: In order to reduce the interference caused by the genetic background, three groups were established: Fetal trauma (FT), Fetal control (FC) and adult rabbit trauma (Adult trau- ma, AT) Tester corresponds to two driver Modified multiple SSH technology. The main outcome measures: ① RNA extraction results. ② SSH results. Results: Tester was performed on embryonic trauma, adult rabbit and embryo respectively, and the other two were Drivers. Three groups of positive and negative hybridization products were obtained. The total RNA extracted from full-thickness skin tissue had good integrity. PCR validation analysis, G3PDH reduction efficiency is satisfactory. CONCLUSION: Multiple-suppression subtractive hybridization with self-improvement is feasible both in theory and in practice, especially in the subtractive library obtained by this method