【作者】 林瑶；

【导师】 张召才；

【作者基本信息】 浙江大学 ， 重症医学， 2021， 硕士

【摘要】 背景:心肌功能障碍是严重脓毒症和脓毒症休克病人的主要并发症之一,称为脓毒症诱导的心肌功能障碍（Sepsis induced myocardial dysfunction,SIMD）。然而,针对SIMD的治疗,目前有效的策略依然十分有限。整合素β1是心肌细胞上主要的整合素亚单位,其主要功能是将机械信号转化为生物化学信号。我们此前发现整合素β1表达异常与大鼠脓毒症心肌功能障碍有关,本研究旨在探索整合素β1在脓毒症心肌功能障碍中的确切作用及其潜在细胞分子机制。方法:本研究采用心内慢病毒转染技术调节心脏整合素β1的表达,用以开展体内和体外实验。体内实验:44只雄性SD大鼠纳入研究,以脂多糖10mg/kg腹腔注射12小时诱导脓毒症模型,以缓冲液（PBS）腹腔注射为对照;以超声心动图结合心肌损伤生物标志物（CK-MB和c Tn I）检测确认脓毒症心肌功能障碍;动物随机分为6组:空白对照组（PBS）、脓毒症模型组（PBS+LPS）、整合素β1过表达+LPS组(lenti-Integrinβ1^Over+LPS),整合素β1干扰+LPS组(lenti-Integrinβ1^Si+LPS),慢病毒空载对照+LPS组（lenti-Mock+LPS）和慢病毒阴性对照+LPS组（lenti-Scramble+LPS）。体外实验:用100ng/m L LPS孵育乳大鼠心肌细胞（NRCM）处理12小时来诱导脓毒症体外模型,以培养基（DMEM）孵育的NRCM作为对照,将NRCM用慢病毒转染,并随机分为6组:DMEM组;DMEM+LPS组;lenti-Integrinβ1^Over+LPS组;lenti-Integrinβ1^Si+LPS组;lenti-Mock+LPS组;lenti-Scramble+LPS组。收集各组大鼠心肌组织和离体培养心肌细胞,分别检测心肌细胞凋亡、心肌线粒体损伤、心肌细胞骨架蛋白的表达和心肌细胞膜稳定性。结果:LPS腹腔注射12小时后,整合素β1过表达(lenti-Integrinβ1^Over+LPS)的大鼠心功能明显优于整合素β1干扰组(lenti-Integrinβ1^Si+LPS)和空载对照组（lenti-Mock+LPS）大鼠;整合素β1干扰组(lenti-Integrinβ1^Si+LPS)和慢病毒阴性对照组（lenti-Scramble+LPS）之间无统计学差异。体内体外实验均发现:上调整合素β1可致脓毒症大鼠心脏组织或LPS刺激的心肌细胞凋亡减少、心肌线粒体损伤减轻、心肌细胞骨架蛋白表达上调、心肌细胞膜稳定性提高。结论:心肌整合素β1对SIMD有保护作用,其保护机制包括:1)减少心肌细胞凋亡,2)改善心肌细胞线粒体损伤,3)增强心肌细胞骨架蛋白丰度,4)改善心肌细胞膜稳定性。本研究结果为靶向心脏整合素β1,以逆转和改善SIMD提供了一定的前期理论依据,为后续探索SIMD干预策略指明了新的方向。更多还原

【Abstract】 Background:Myocardial dysfunction is one of the main complications in patients with severe sepsis and septic shock,which is called sepsis induced myocardial dysfunction（SIMD）.However,the effective strategies for the treatment of SIMD are still very limited.Integrinβ1 is a major integrin subunit in cardiomyocytes,whose main function is to transform mechanical signals into biochemical signals.We previously found that the abnormal expression of integrinβ1 is related to SIMD in rats.This study aimed to explore the exact role of integrinβ1 in SIMD and its underlying cellular and molecular mechanisms.Methods:In the present study,the expression of cardiac integrinβ1 was regulated by lentiviral transfection for in vivo and in vitro experiments.In vivo experiment:44 male SD rats were included in the study.Sepsis model was induced by intraperitoneal injection of lipopolysaccharide（LPS）with a dose of 10 mg/kg for 12 hours,and buffer solution（PBS）intraperitoneal injection was used as control;sepsis induced myocardial dysfunction（SIMD）was confirmed by echocardiography combined with biomarkers for myocardial injury（CK-MB and c Tn I）.The animals were randomly divided into 6 groups:blank control group（PBS）,sepsis model group（PBS+LPS）,lenti-integrinβ1^over+LPS group,lenti-integrinβ1^si+LPS group,lenti-Mock+LPS group and lenti-Scramble+LPS group.In vitro experiment:neonatal rat cardiomyocytes（NRCM）were incubated with 100 ng/m L LPS for 12 hours to induce sepsis model in vitro.NRCM incubated with DMEM was used as control.NRCM transfected with lentivirus was randomly divided into 6 groups:DMEM group,DMEM+LPS group,lenti-integrinβ1^over+LPS group,lenti-integrinβ1^si+LPS group,lenti-Mock+LPS group and lenti-Scramble+LPS group.Myocardial tissue and cultured cardiomyocytes were collected from each group.Cardiomyocyte apoptosis,myocardial mitochondrial injury,myocardial cytoskeleton protein expression and myocardial cell membrane stability were detected.Results:After LPS intraperitoneal injection for 12 hours,the cardiac function of rats with overexpression of integrinβ1(lenti-Integrinβ1^Over+LPS group)was significantly better than that of rats in integrinβ1 interference group(lenti-Integrinβ1^Si+LPS)and empty control（lenti-Mock+LPS）group;there was no significant difference between integrinβ1interference group(lenti-Integrinβ1^Si+LPS)and lentivirus negative control group（lenti-Scramble+LPS）.In vivo and in vitro experiments showed that up-regulation of integrinβ1 could reduce the apoptosis in cardiac tissue or LPS stimulated cardiomyocytes,alleviate the damage of myocardial mitochondria,up-regulate the expression of myocardial cytoskeleton protein and improve the stability of myocardial cell membrane in septic rats.Conclusion:Myocardial integrinβ1 has a protective effect on SIMD,and its protective mechanisms include:1)reducing myocardial cell apoptosis,2)improving myocardial cell mitochondrial damage,3)enhancing myocardial cytoskeleton protein abundance,4)improving myocardial cell stability.The results of this study provide a theoretical basis for targeting cardiac integrinβ1 to reverse and improve SIMD,and point out a new direction for the subsequent exploration of intervention strategy against SIMD.更多还原