Gpx1-Klk1转基因对肾缺血再灌注损伤保护作用的研究

目的 制备人谷胱甘肽过氧化物酶（Gpx1）、 组织激肽释放酶（Klk1）共转基因小鼠，在此基础上制作肾缺血再灌注损伤小鼠模型。观察转基因小鼠对缺血再灌注损伤的耐受能力。在体研究Gpx1-Klk1转基因对肾缺血再灌注的保护作用。 方法 应用基因工程技术制备pKsp-Gpx1-IRES-Klk1质粒，酶切后回收转基因片段并纯化，通过外源基因受精卵雄原核显微注射法制备Gpx1-Klk1共转基因小鼠。采用丝线悬吊控制法制备转基因小鼠肾缺血再灌注模型。设立C57BL野生型小鼠同法制备的肾缺血再灌注损伤为对照。在肾缺血再灌注实验前后，测定血尿素氮、血肌酐、肾组织中超氧化物歧化酶（SOD）、丙二醛（MDA）、总一氧化氮合酶（tNOS）及诱导型一氧化氮合酶（iNOS）。留取肾脏组织制备病理切片，观察Gpx1-Klk1转基因小鼠抗肾缺血再灌注损伤的能力。 结果 获得全长为6.585 kb的pKsp-Gpx1-IRES-Klk1重组质粒，并证明了该克隆的正确性。在转基因小鼠制备过程中，共获得525枚注射卵，移植成功率为81.0%，小鼠出生总数109只。经基因组DNA的PCR检测，确认10只为转基因阳性小鼠，阳性率为9.2%。Western印迹法检测证实了阳性转基因小鼠肾脏组织有Gpx1和Klk1蛋白强表达。转基因小鼠（实验组）和野生型小鼠（对照组）肾缺血再灌注损伤模型建立后，实验组血样中尿素氮及肌酐水平显著低于对照组（P < 0.01）；肾脏组织中SOD显著高于对照组 （P < 0.01），MDA显著低于对照组（P < 0.01）；两组肾组织中tNOS较缺血再灌注前均显著升高，且实验组显著高于对照组（P = 0.025），实验组肾组织中iNOS显著低于对照组（P < 0.01）。缺血再灌注后，实验组肾组织间质轻度水肿，肾小管上皮坏死细胞较少，对标本损伤程度采用半定量评分后显示，实验组损伤程度显著轻于对照组（1.58±1.05比3.95±0.80，P < 0.05）。 结论成功制备Gpx1-Klk1转基因小鼠。Gpx1-Klk1过表达对肾缺血再灌注损伤的保护作用。

Protective effect of Gpxl-Klkl transgene on the renal ischemia reperfusion injury

Objective To establish Gpx1-Klk1 combined transgenic mouse model, and to investigate the protective effect of Gpx1-Klk1 transgene on renal ischemia reperfusion injury in vivo. Methods pKsp-Gpx1-IRES-Klk1 recombinant plasmid was constructed and purified for microinjection into the male pronucleus of the zygote in order to prepare the Gpx1-Klk1 combined transgenic mice. A controlled thread hanging method was used to prepare the ischemia and reperfusion model in those successful transgenic mice. The C57BL wild-type mice were chosen in the control group. BUN and creatinine level in serum, SOD, MDA, tNOS and iNOS level in renal tissue, and the structure changes of renal tissue were all investigated before and after the experimental ischemia and reperfusion. Results A 6.585 kb-longed pKsp-Gpx1-IRES-Klk1 recombinant plasmid was obtained and identified. A total of 525 gene-injected ovums were transplanted into oviducts of 21 female mice, resulted in 17 pregnant (81.0%). Ten of those 109 born mice were accepted as transgenic positive by PCR assessment (9.2%). Gpx1-Klk1 combined transgenic mice were successfully established, and proved to have strong expression of Gpx1 and Klk1 protein confirmed by Western blotting. The difference between transgenic group and control group was examined. BUN and Scr of transgenic group were obviously lower than those of control (P<0.01). SOD of renal tissue in transgenic group was higher than that of control (P<0.01), while MDA was lower than control (P<0.01). tNOS of renal tissue in both groups increased significantly compared to that of pre-operation, and tNOS of the transgenic group was obviously higher than that of control (P=0.025), while the tissue iNOS of the transgenic group was obviously lower than that of control (P<0.01). After ischemia reperfusion, slight edema was found in renal interstitial tissue of the transgenic group, while relatively few necrosis was seen in renal tubular epithelial cells. The semiquantitative analysis of the tissue injury severity indicated that the injury of transgenic group was less than that of control group (1.58±1.05 vs 3.95±0.80, P<0.05). Conclusions A new type of renal ischemia and reperfusion mice model based on the Gpx1-Klk1 combined transgenic mice is successfully established. Gpx1-Klk1 gene over-expression has obviously protective effect on renal ischemia and reperfusion injury.