人金属蛋白酶1组织抑制剂基因表达对大鼠心肌梗死后炎症反应的影响及机制

Effects of overexpression of human tissue inhibitor of metalloproteinase-1 on the inflammatoryresponse in rats with myocardial infarction and related mechanisms

目的探讨携带人金属蛋白酶1组织抑制剂（hTIMP-1）的重组腺病毒（Ad-hTIMP-1）对大鼠心肌梗死后炎症反应的影响及机制。方法将Wistar雄性大鼠按照随机数字表法分为假手术组、生理盐水组、Ad-Track组和Ad-hTIMP-1组（每组各8只大鼠）。除假手术组外，其余各组大鼠均通过结扎冠状动脉左前降支建立心肌梗死模型。生理盐水组、Ad-Track组和Ad-hTIMP-1组分别在心肌梗死部位注射生理盐水、空载体重组腺病毒（Ad-Track）和Ad-hTIMP-1。4周后以超声心动图检测各组大鼠的心功能指标，然后处死大鼠并留取心脏组织。以苏木精-伊红（HE）和Masson染色法观察各组大鼠的心肌组织形态，实时荧光定量PCR检测各组心肌组织中肿瘤坏死因子-α（TNF-）、白细胞介素-6（IL-6）、白细胞介素-10（IL·10）和C反应蛋白（CRP）的mRNA表达水平，免疫组织化学法检测各组IL-6和CRP的蛋白表达水平。结果（1）超声心动图显示，假手术组、生理盐水组、Ad-Track组和Ad-hTIMP-1组的左心室收缩末期内径分别为（4．25±0．46）、（5．10±0．72）、（4．88±0．64）和（4．13±0．35）mm，生理盐水组和Ad-Track组均高于假手术组和Ad-hTIMP-1组（P均〈0．05）；假手术组、生理盐水组、Ad-Track组和Ad-hTIMP-1组的左心室射血分数分别为（72．46±5．74）％、（64．27±8．52）％、（64．65±3．90）％和（71．55±6．95）％，短轴缩短率分别为（36．90±4．97）％、（29．03±3．40）％、（30．95±2．51）％和（36．31±5．68）％，生理盐水组和Ad-Track组的左心室射血分数和短轴缩短率均低于假手术组和Ad-hTIMP-1组（P均〈0．05）。（2）HE和Masson染色显示，Ad-hTIMP-l组无心肌细胞坏死，有少量炎性细胞浸润和间质纤维化。（3）实时荧光定量PCR显示，Ad-hTIMP-t组的TNF-d、IL-6、IL-10和CRPmRNA表达水平均低于生理盐水组（P均〈0．05）；Ad·hTIMP-l组的TNF-α、IL-10和CRPmRNA表达水平均低于Ad-Track组（P均〈0．05）。（4）免疫组织化学法检测显示，生理盐水组和Ad-Track组的IL-6和CRP的蛋白表达水平均高于Ad-hTIMP-1组（P均〈0．05）。结论重组腺病毒Ad-hTIMP-1可改善大鼠心肌梗死后心功能，其机制可能与hTIMP-1抑制炎症反应，减少炎性细胞因子TNF-α、IL-6和CRP的表达有关。

Objective To observe the effects of recombinant adenovirus with human tissue inhibitor of metalloproteinase-1 （Ad-hTIMP-1） on the inflammatory response in rats with myocardial infarction （MI） and explore the related mechanisms. Methods The male Wistar rats were randomly divided into sham- operated group, saline group, Ad-Track group and Ad-hTIMP-1 group according to the random number table （n =8 each group）. MI was induced by ligation of the left anterior descending coronary artery and MI rats were injected with saline, Ad-Track and Ad-hTIMP-1, respectively. Sham-operated rats received similar surgical procedure without ligation of the left anterior descending coronary artery. After 4 weeks, the cardiac function was measured by echocardiography, then rats were sacrificed and hearts were removed for morphological and biological analysis. The morphology of myocardial tissue in each group was detected by HE staining and Masson staining. The mRNA expressions of tumor necrosis factor （TNF） -α, interleukin （IL）-6, IL-10 and C-reactive protein （CRP） were detected by real-time PCR. Immune histochemical staining was performed to observe the protein expression levels of IL-6 and CRP. Results （1） Left ventricular end systolic dimension derived from echocardiography was increased in saline group （ （5.10 ± 0. 72） ram） and Ad-Track group （ （4. 88±0. 64） mm） compared to sham-operated group （ （4. 25±0. 46） mm） , which was reduced in Ad-hTIMP-1 group （ （4. 13±0. 35） mm, all P 〈0. 05）. The left ventricular ejection fraction was （72.46 ± 5.74 ） %, （ 64. 27 ± 8.52 ） %, （ 64. 65 ± 3.90 ） %, and （ 71.55 ± 6.95 ） %, the fractional shortening was （ 36. 90 ± 4. 97 ） %, （29.03 ± 3.40） %, （ 30. 95 ± 2. 51 ） %, and （ 36. 31 ± 5.68） % in sham-operated group, saline group, Ad-Track group and Ad-hTIMP-1 group, respectively. The left ventricular ejection fraction and fractional shortening in saline group and Ad-Track group were lower than those in sham-operated group and Ad-hTIMP-1 group （all P 〈0.05）. （2） Necrosis of myocardial ceils was not found and a small amount of immune cell infiltration and interstitial fibrosis were observed on HE and Masson stained myocardial sections of Ad-hTIMP-1 group. （3） Real-time PCR showed that mRNA expressions of TNF-α, IL-6, IL-10 and CRP were lower in Ad-hTIMP-1 group than in saline group, mRNA expressions of TNF-α, IL-10 and CRP were lower in Ad-hTIMP-1 group than in Ad-Track group （ all P 〈 0. 05 ）. （4） Immune histochemical staining showed that protein expressions of IL-6 and CRP were higher in saline group and Ad-Track group than those in Ad-hTIMP-1 group （all P〈0.05）. Conclusion Recombinant adenovirus Ad-hTIMP-1 can improve cardiac function in rats with myocardial infarction via inhibiting the inflammatory response and downregulating the expression of TNF-α, IL-6 and CRP.