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摘要：
目的 研究新型融合蛋白——蛋白转导结构域（protein transduction domain, PTD）4‑超氧化物歧化酶（superoxide dismutase, SOD）1对心肌缺血/再灌注损伤（myocardial ischemia/reperfusion injury, MI/RI）大鼠肌酸激酶（creatine kinase, CK）、乳酸脱氢酶（lactic dehydrogenase, LDH）及丙二醛（malondialdehyde, MDA）的影响，探讨PTD4‑SOD1对再灌注心肌的保护作用。 方法 40只SPF级雄性SD大鼠，体重（330[±]20） g，按随机数字表法分为4组：假手术组（S组）、MI/RI组、SOD1蛋白组（SOD1组）、PTD4‑SOD1融合蛋白组（PTD4‑SOD1组），每组10只。S组只穿线不结扎左冠状动脉前降支（left anterior descending, LAD），其余3组均通过结扎与松解LAD的方法制备大鼠MI/RI模型，缺血30 min，再灌注120 min。S组、MI/RI组在再灌注的同时通过尾静脉分别注射0.9%氯化钠注射液（1 ml）， SOD1组在再灌注的同时通过尾静脉注射SOD1蛋白500 μg（1 ml），PTD4‑SOD1组在再灌注的同时通过尾静脉注射PTD4‑SOD1 500 μg（1 ml）。再灌注结束时通过左心室采血5 ml，后快速摘取心脏。分别使用免疫荧光检测PTD4‑SOD1在大鼠MI/RI模型中的穿膜能力，比色法检测CK、LDH的含量，硫代巴比妥酸（thiobarbituric acid, TBA）染色法检测MDA的含量。 结果 免疫荧光结果显示，S组、MI/RI组、SOD1组均没有代表融合蛋白的荧光出现，PTD4‑SOD1组出现荧光。与S组比较，其余3组CK、LDH和MDA含量均明显升高，差异有统计学意义（P<0.05）；MI/RI组CK、LDH、MDA含量与SOD1组差异无统计学意义（P>0.05），PTD4‑SOD1组CK、LDH、MDA含量较MI/RI组和SOD1组明显降低，差异有统计学意义（P<0.05）。 结论 PTD4与SOD1重组后表达纯化的PTD4‑SOD1能显著降低血清CK、LDH和MDA值，抑制脂质过氧化反应，实现了对心肌细胞的保护作用。
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Abstract: Objective To investigate the protection effects of the novel recombinant protein transduction domain (PTD) 4‑superoxide dismutase (SOD) 1 fusion protein on cardiocytes during the process of myocardial ischemia/reperfusion injury (MI/RI). This article explored the influence of PTD4‑SOD1 fusion protein on cardiocytes creatine kinase (CK), lactate dehydrogenase (LDH) and malondialdehyde (MDA) productions and activities. Methods Forty SPF male SD rats (330[±]20) g, according to the random number table method, were randomly divided into 4 groups (n=10): the sham group (S group), MI/RI group, the SOD1 group and the PTD4‑SOD1 group. In the MI/RI, SOD1 and PTD4‑SOD1 groups, this article used the method of ligaturing and then releasing the left anterior descending branch of coronary artery of for preparation the rat models of the myocardial ischemia reperfusion injury: ligaturing for 30 min, and then reperfusion for 120 min. While in the sham group, the thread was placed but the left anterior descending (LAD) branch of coronary artery of the mice were not ligatured and released. When reperfusion, the mice were injected 1 ml 0.9% NaCl, 1 ml pure SOD1 protein (500 μg) solution and 1 ml PTD4‑SOD1 fusion protein (500 μg) solution in the model mice, SOD1 and PTD4‑SOD1 groups respectively. After reperfusion for 120 min, 5 ml blood were collected from the left ventricular, and then the hearts of mice were removed immediately. In final examinations, immunofluorescence was used to analyze the penetrability of PTD4‑SOD1 fusion protein passing through the cell membranes, and also the CK, LDH and MDA volumes were examined. Results Immunofluorescence analysis indicated that, positive red fluorescence were existed in the mice of PTD4‑SOD1 group, while negative results were obtained in the other 3 groups. The volumes of CK，LDH and MDA in MI/RI, SOD1, PTD4‑SOD1 groups were significantly higher than those values of in S group (P<0.05); and the volumes in PTD4‑SOD1 group were significantly lower than that of in MI/RI and SOD1 groups (P<0.05). However, they were not significantly different between MI/RI and SOD1 groups (P>0.05). Conclusions The novel recombinant fusion protein of PTD4 and SOD1 can significantly decrease the volumes of CK, LDH and MDA; inhibit the lipid peroxidation that are caused by reactive oxygen species (ROS) and finally protect the cardiocytes.
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