C-fos反义基因转染对缺血缺氧心肌细胞的保护作用

Protective effect of c-fos antisense gene recombinant transfection on ischemic hypoxic cardiomyocytes

目的：探讨c-fos反义基因重组体转染对缺氧复合烧伤血清处理心肌细胞的保护作用。 方法：实验于2002年在第三军医大学西南医院烧伤研究所完成。Wistar大鼠125只，正常对照组15只：未经缺血缺氧处理；非转染组45只：10％烧伤大鼠血清+缺氧(体积分数为0．01的氧)；转染组45只：先转染，然后[10％烧伤大鼠血清+缺氧(体积分数为0．01的氧)]处理。转染组和非转染组均按照时相点不同又分为1，3，7h三个时相点组，每组15只。烧伤血清采自30％全身体表面积Ⅲ度烫伤Wistar大鼠(20只)。采用含体积分数为0．01氧的混和气体作为缺氧模型，采用基因重组技术构建c-fos反义基因重组体，由脂质体胺试剂介导心肌细胞转染，缺氧复合烧伤血清处理后1，3，7h不同时相点采用反转录-多聚酶联反应检测c-fos mRNA的表达变化，检测c-fos蛋白、肌钙蛋白-T和β-微管蛋白的表达变化采用蛋白免疫印迹法。 结果：105只大鼠进入结果分析。对照组、转染组1，3，7h三个时相点及非转染组1，3，7h三个时相点各15只，另外20只用于制备烧伤血清。①c-fos蛋白表达：非转染组显著表达，3h达到最高峰，非转染组高于转染组(46．3±3．06，34．3±2．06，P<0．01)。②肌钙蛋白-T和β-微管蛋白的表达：与正常对照组比较，非转染组肌钙蛋白-T和β-微管蛋白表达显著下降，7h降至最低水平，非转染组低于转染组(肌钙蛋白-T：25．1±3．06，34．3±2．06；β-微管蛋白：44．8±3．17，66．5±3．14，P<0．01)。 结论：缺氧复合烧伤血清处理产生的c-fos会引起心肌细胞损伤，当在缺氧复合烧伤血清处理前转染c-fos反义基因后，心肌细胞肌钙蛋白-T和β-微管蛋白表达较单纯缺氧复合烧伤处理心肌细胞显著回升，表明c-fos反义基因转染能有效抑制心肌细胞肌钙蛋白-T和β-微管蛋白的表达下降，从而对缺血缺氧心肌细胞起保护作用。

AIM： To investigate the protective effect of c-fos antisense gene recombinant transfection on the cardiomyocytes following hypoxia and burn serum treatment. METHODS： The experiment was carried out in the Burn Institute of West China Hospital, the Third Military Medical University of Chinese PLA in 2002. Totally 125 Wistar rats were used. The rats were divided into normal control group （n=15, no ischemia hypoxia treatment）, non-transfected group [n=45, 10% burn serum＋hypoxia （oxygen with volume fraction of 0.01）], transfected group [n=45, the rats were transfected, and then treated with 10% burn serum＋hypoxia （oxygen with volume fraction of 0.01）]. The transfection group and non-transfection group were subdivided into 1, 3 and 7-hour groups respectively with 15 rats in each group. Burn serum was collected from Wistar rats （n=20） with 30% total body surface areaⅢ degree bum injury. The gas mixture containing oxygen with the volume fraction of 0.01 was used as hypoxia model. The c-fos antisense gene recombinant was constructed with the technique of gene recombination, the transfection of the cardiomyecytes was mediated by liposomes amine reagent. At 1, 3 and 7 hours after the hypoxia and bum serum treatment, the changes of c-fos mRNA expression were detected with reverse transcription-polymerase chain reaction （RT-PCR）, and the changes of c-fos proteins, troponin-T, β-Tubulin expressions in cardiomyecytes were assayed with Westem blot in the transfected and non-transfected groups. RESULTS： 105 rats were involved in the analysis of results, with 15 rats in the control group and the 1, 3 and 7-hour subgroups of the transfected and non-transfected groups respectively. Another 20 rats were used for the preparation of bum serum. ① The expression of c-fos protein： It expressed significantly in the non-transfected group, and reached the peak value at 3 hours, it was higher in the non-transfected group than in the transfected group （46.3±3.06, 34.3±2.06, P 〈 0.01）. ② The expressions of troponin-T and β-Tubulin： Compared with the normal control group, the expressions of troponin-T and β-Tubulin were significantly decreased, and decreased to the lowest level at 7 hours, and were lower in the non-transfected group than in the transfected group [troponin-T： 25.1±3.06, 34.3±2.06; β-Tubulin： 44.8±3.17, 66.5±3.14, P 〈 0.01]. CONCLUSION： The hypoxia and bum serum treated c-los can cause the myocardial injury, transfection of the c-fos antisense gene recombinant may reduce the myocardial injury. The results suggest that the c-fos antisense gene recombinant transfection can effectively inhibit the decreased expressions of troponin-T and β-Tubulin in the cardiomyocytes, and has the protective effect on cardiomyocytes exposed to burn sera and hypoxia.