人IκBα真核表达载体的构建及其在大鼠肝移植缺血-再灌注损伤中的作用

Construction of human IκBα mutant gene eukaryotic expression vectors and their effects on the ischmia-reperfusion injury of rat after orthotopic liver transplantation

目的:构建人IκBα真核表达质粒,探讨对大鼠肝移植缺血-再灌注损伤的保护作用。方法:应用RT-PCR和重叠延伸PCR技术,得到点突变的人IκBα,定向克隆至PcDNA3.0真核表达载体。将SD大鼠原位肝移植模型分为三组:Ⅰ组为空白对照组,Ⅱ组为PcDNA3.0空载体转染组,Ⅲ组为PcDNA3.0-IκBα转染组。分别于术后2、12、24和72 h取材。用RT-PCR测定肝组织中核转录因子(NF-κB)p65、肿瘤坏死因子α(TNF-α)、细胞间黏附分子1(ICAM-1)mRNA的表达及肝功能变化。结果:经酶切和DNA序列测定鉴定,证实重组质粒构建正确;Ⅲ组与Ⅰ组、Ⅱ组相比:NF-κBp65、TNFα、ICAM-1 mRNA的表达水平及肝酶学指标均明显降低(P<0.05)。结论:真核表达质粒PcDNA3.0-IκBα构建成功,IκBα通过抑制NF-κBp65 mRNA的表达及其核移位减轻大鼠肝移植缺血-再灌注损伤。

Objective： To construct a recombinant eukaryotice expression plasmid inserted human IkBα utant gene and study the protective effects on ischmia-reperfusion injury in rat after orthotopic liver transplantation. Methods： RT-PCR and overlap extension PCR were used to get site-mutant human IkBα. The RCR product was cloned into plasmid PcDNA 3.0. After orthotopic liver transplantation was constructed, the rats were divided into three groups： group Ⅰ was the control, all rats in group Ⅱ were per- fused with empty PcDNA3.0 and all those in group Ⅲ were perfused with PcDNA3.0-IkBαM. NF-kB p65 mRNA, TNF-α mRNA and intercellular adhesive molecular （ICAM） -1 mRNA were measured by RTPCR. And the serum levels of AST, ALT, and LAH were measured. Results： Restriction enzyme digestion and DNA sequencing confirmed that the recombinant eukaryotic expression plasmid inserted IkBα gene （PcDNA3.0-IkBα） had been constructed correctly. Comparison with group Ⅰ and group Ⅱ , the mRNA expression of p65,TNF-α, ICAM-1 , and the serum levels of AST, ALT, and LAH were decreased significantly in group Ⅲ（P 〈 0. 05 ）. Conclusion： An eukaryotice expression plasmid PcDNA3.0-IkBα has been constructed successfully. The IkBα can protect against orthotopic liver transplantation ischemia-reperfusion injury in rats by inhibiting the mRNA expression of the NF-kB and transporting to the nucleus.