Zhang Bo, Liu Da-wei, Wang Zheng-guo, Zhu Pei-fang, Zhou Ji-hong, Jiang Jian-xin
Research Institute of Surgery, Daping Hospital, Chongqing 400042, China.
Chin J Traumatol. 2004 Feb;7(1):13-7.
Early gestational mammalian fetuses possess the amazing ability to heal cutaneous wounds in a scarless fashion. Over the past years, scientists have been working to decipher the mechanisms underlying this regenerative repair. The remarkable phenotypic differences between fetal and adult healings behooves us to learn their characteristics in genetics, which represents potentially important mechanisms involved in wound repair observed in fetal versus adult tissues. In this sense, it is reasonable to construct subtractive cDNA library for future research.
Middle laparotomy and hysterotomy were performed on pregnant rabbits at 20-day gestation to expose the fetal back, and a longitudinal incision through the skin was made on the back of the fetus. The traumatized fetal skin was harvested 12 hours post-operation, the fetus control and traumatized adult skin specimens were taken at the same time. dscDNA was synthesized from total RNA of skin samples with SMART technology. Taking one of the three samples as Tester respectively and the other two as Drivers, we obtained 1 forward and 2 reverse hybridization products. After being amplified with selective polymerase chain reaction, the products were inserted into a vector, and then transferred into E. coli HB101. The colonies were screened afterwards.
The wounded fetuses were alive for a long time even after birth. Every determinant step, such as RNA isolation, cDNA synthesis, Rsa I digestion, adaptor ligation and hybridization, was well-operated. Subtractive efficiency identification demonstrated that the suppression subtractive hybridization (SSH) was successful. Insertion into vector and transferring to E. coli were satisfactory.
Instead of classic SSH, an improved SSH with 2 Drivers was applied for the experiment. Results confirmed that the improved program was reasonable and correct in both theory and practice. The subtractive cDNA library we have obtained is going to be used for future researches to reveal scarless healing related gene(s) and its (their) expression.
妊娠早期的哺乳动物胎儿具有以无瘢痕方式愈合皮肤伤口的惊人能力。在过去几年中，科学家们一直在努力破解这种再生修复的潜在机制。胎儿和成人愈合之间显著的表型差异促使我们了解它们在遗传学方面的特征，这代表了在胎儿与成人组织中观察到的伤口修复所涉及的潜在重要机制。从这个意义上说，构建消减cDNA文库以供未来研究是合理的。
对妊娠20天的孕兔进行中腹部剖腹术和子宫切开术以暴露胎儿背部，并在胎儿背部做一个贯穿皮肤的纵向切口。术后12小时采集受伤的胎儿皮肤，同时采集胎儿对照和受伤的成人皮肤标本。使用SMART技术从皮肤样本的总RNA合成双链cDNA。分别以三个样本中的一个作为测试样本，另外两个作为驱动样本，我们获得了1个正向和2个反向杂交产物。经过选择性聚合酶链反应扩增后，将产物插入载体，然后转入大肠杆菌HB101。随后对菌落进行筛选。
受伤的胎儿即使出生后也能存活很长时间。每个关键步骤，如RNA分离、cDNA合成、Rsa I消化、接头连接和杂交，都操作良好。消减效率鉴定表明抑制消减杂交（SSH）成功。载体插入和大肠杆菌转化效果良好。
本实验采用了改进的含2个驱动样本的SSH而非经典的SSH。结果证实改进后的方案在理论和实践上都是合理且正确的。我们获得的消减cDNA文库将用于未来研究，以揭示无瘢痕愈合相关基因及其表达。
