人IκBαM真核表达载体的构建及其对大鼠肝移植缺血再灌注中ICAM-1的影响

Construction of human IκBα Mutant gene eukaryotic expression vectors and their effect on the expression of ICAM-1 in the ischmia-reperfusion injury of rat orthotopic liver transplantation

目的构建人IκBαM真核表达质粒,探讨人IκBαM对大鼠肝移植缺血再灌注中ICAM-1的影响。方法应用RT-PCR和重叠延伸PCR技术,得到点突变的人IκBαM,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4组:组Ⅰ为假手术组(行游离肝脏手术),组Ⅱ为空白对照组(行大鼠原位肝移植手术),组Ⅲ为PcDNA3.0空载体转染组(移植前两天行人PcDNA3.0转染供肝),组Ⅳ为PcDNA3.0-IκBαM转染组(移植前两天行人PcDNA3.0-IκBαM转染供肝)。分别于术后2h、12h、24h和3d取材。应用免疫组化、RT-PCR测定肝组织中ICAM-1的表达,同时检测各时点肝脏酶学的变化。结果经酶切和DNA序列测定鉴定,证实重组质粒构建正确;PcDNA3.0-IκBαM转染组与组Ⅱ、组Ⅲ相比:免疫组化示ICAM-1于移植后12h、24h的表达差异具有显著性;术后12hICAM-1mRNA的表达水平具有显著性差异;肝脏受损指标(ALT)在各时点差异具有显著性,尤其在术后12h最为显著(P<0.05)。各移植组与Ⅰ组差异有显著性,(P<0.05)。结论真核表达质粒PcDNA3.0-IκBαM构建成功,IκBαM通过抑制ICAM-1的表达减轻大鼠肝移植缺血再灌注损伤。

[Objective] To construct a recombinant eukaryotice expression plasmid inserted human IκBαMutant gene and study the effect on the expression of ICAM-1in ischmia-reperfusion injury of rat orthotopic liver transplantation.[Methods] RT-PCR and overlap extension PCR were used to get site-mutant human IκBα.The RCR product was cloned into plasmid PcDNA 3.0.After orthotopic liver transplantation was constructed,all the SD rats were divided into four groups：group I was the sham operation,group II was the blank control,the livers of all rats in group III were perfused with empty PcDNA3.0,and the livers of all rats in group were perfused with PcDNA3.0-IκBαM.The expression of ICAM-1 was measured by Immunohistochemistry and RT-PCR.And the serum level of ALT in every group was measured.[Results] Restriction enzyme digestion and DNA sequencing confirmed that the recombinant eukaryotic expression plasmid inserted IκBαM gene（PcDNA3.0-IκBαM）had been constructed correctly.Compared with group II and group III,the expression of ICAM-1 and the serum level of ALT decreased significantly in groupⅣ（P 〈0.05）.All liver transplantation group had significantly difference with group I.[Conclusion] An eukaryotice expression plasmid PcDNA3.0-IκBαM has been constructed successfully.The IκBαM can protect against orthotopic liver transplantation ischemia-reperfusion injury in rats by inhibiting the expression of the ICAM-1.