用DDRT-PCR分析烧伤创面愈合相关基因的表达

Analysis of the expression of burn wound healing related genes by DDRT-PCR

目的：比较烫伤后第５天创面组织与正常皮肤组织间基因表达的差异。方法：用差异显示逆转录ＰＣＲ技术（ＤＤＲＴ－ＰＣＲ）分析基因表达差异，将所有差异表达基因割胶回收并再扩增，用点杂交和Ｎｏｒｔｈｅｒｎ杂交进一步证实基因表达差异，克隆并测序部分差异表达片段，然后将ＤＮＡ序列与Ｇｅｎｂａｎｋ作同源性比较。结果：在显示出的约１２０００条ＤＮＡ条带中获取差异表达基因片段２８９个。用点杂交对其中１２６个片段进一步分析，用Ｎｏｒｔｈｅｒｎ杂交证实了３个创面愈合中高表达基因片段，经序列测定和同源性分析证实，其中一个为纤溶酶原激活因子抑制因子２Ａ，其余２个为新基因片段。

Objective: To compare the gene expression differences between normal skin and the 5th postburn wound tissue in rats. Methods: Differential display reverse transcript PCR(DDRT-PCR) was used to reveal the gene expression differences. All the differentially expressed gene bands were recovered from the gel slices and were submitted to reamplification. Dot blot hybridization followed by Northern blot hybridizations were performed to confirm the gene expression differences. Cloning and sequencing were done followed by alignment of the obtained sequences with Genbank database. Results: A sum of 289 differential gene bands among 12 000 revealed bands were screened out. There were 126 bands whose expression differences were confirmed by Dot blot hybridization. There were 3 bands with increased gene expression level in wound healing confirmed by Northern blot hybridization. Sequence analysis showed that one of the bands was the mRNA for plasminogen activator inhibitor 2 type A, while the other two bands might be novel gene candidates. Conclusion: The plasminogen activator inhibitor 2 type A plays an important role in wound healing. 