Intermedin对缺氧复氧所致H9c2心肌细胞氧化应激损伤的保护作用
发布时间：2018-08-05 12:14
【摘要】：目的通过培养Intermedin(IMD)质粒转染后的大鼠H9c2心肌细胞建立缺氧/复氧(H/R)模型,证实IMD对心肌细胞的H/R氧化应激损伤具有保护作用,为研究IMD基因对心血管疾病的作用及分子生物学机制奠定基础。 方法实验分为四组：1)正常对照组：用DMEM培养基培养H9c2心肌细胞48h；2)H/R组：常规培养H9c2心肌细胞48h后,用5mmol/1的连二亚硫酸钠使其缺氧2h,后用DMEM培养基复氧1h；3)H/R+空质粒组(空质粒组)：pIRES2-EGFP质粒(即空质粒)转染后稳定培养48h,之后给予缺氧/复氧处理；4)H/R+IMD质粒组(IMD质粒组)：PIRES2-EGFP/IMD质粒(即IMD质粒)转染后稳定培养48h,之后给予缺氧/复氧处理。实验终止后,在倒置相差显微镜下观察H9c2心肌细胞形态；质粒转染后48h,在荧光显微镜下观察心肌细胞形态；通过流式细胞仪检测细胞转染效率以确定质粒的最佳转染量；采用化学比色法测定细胞上清液中乳酸脱氢酶(LDH)的释放；用硫代巴比妥酸显色法测定细胞上清液中丙二醛(MDA)含量；用黄嘌呤氧化酶法测定细胞上清液中超氧化物歧化酶(SOD)活性；运用流式细胞术检测心肌细胞凋亡率 结果(1)倒置相差显微镜下观察H9c2心肌细胞为长梭形,呈鱼群状或漩涡状排列,贴壁紧密； (2)荧光显微镜下观察心肌细胞胞浆内呈现绿色荧光,呈长梭形,贴壁生长； (3)终浓度为.1ug/ml的质粒细胞转染效率最高； (4)与正常对照组相比,H/R组细胞上清液中LDH含量增加,MDA含量增加,SOD活性下降(p均0.05)；IMD质粒的转染显著逆转了上述这些指标的变化,与H/R组相比具有显著差异(p0.05)；空质粒组与H/R组相较,各指标测定值无统计学差异(p0.05)； (5)与正常对照组相比,H/R组细胞凋亡率(中晚期)显著增加(p0.05),IMD质粒组细胞凋亡率(中晚期)较H/R组有所下降(p0.05)；空质粒组与H/R组相较则无统计学差异(p0.05); 结论(1)H/R可诱导心肌细胞氧化应激损伤和凋亡； (2)IMD基因可通过质粒转染的方法进入细胞发挥作用； (3)IMD可通过减轻氧化应激,抑制细胞凋亡,对心肌细胞H/R损伤起到保护作用；
[Abstract]:Objective to establish the model of hypoxia / reoxygenation (H / R) by cultured rat H9c2 cardiomyocytes transfected with Intermedin (IMD) plasmid, and to prove that IMD has protective effect on H / R oxidative stress injury of cardiomyocytes. It provides a basis for the study of the role of IMD gene in cardiovascular disease and the molecular biological mechanism. Methods the experiment was divided into four groups: 1) normal control group: H9c2 cardiomyocytes were cultured in DMEM medium for 48 h) H / R group: after 48 hours of conventional culture, H9c2 cardiomyocytes were exposed to anoxia for 2 h with 5mmol/1 sodium disulfite, and then reoxygenated on DMEM medium for 1 h. 3) the empty H / R plasmid group (empty plasmid group): pIRES2-EGFP plasmid (empty plasmid) was transfected and cultured steadily for 48h, then treated with anoxia / reoxygenation. 4) the H / R IMD plasmid group (IMD plasmid group): PIRES2-EGFP / IMD plasmid (IMD plasmid) was transfected and cultured steadily for 48h, then treated with anoxia / reoxygenation. At the end of the experiment, the morphology of H9c2 cardiomyocytes was observed under inverted phase contrast microscope, the morphology of cardiomyocytes was observed under fluorescence microscope at 48h after transfection, the transfection efficiency was detected by flow cytometry to determine the optimal amount of plasmid transfection. The release of lactate dehydrogenase (LDH) in cell supernatant was determined by chemical colorimetry, malondialdehyde (MDA) content in cell supernatant by thiobarbituric acid colorimetry, superoxide dismutase (SOD) activity in cell supernatant by xanthine oxidase method. The results of flow cytometry were as follows: (1) under inverted phase contrast microscope, H9c2 cardiomyocytes were long fusiform, arranged in fish-like or whirlpool shape and closely adhered to the wall; (2) under fluorescence microscope, the cytoplasm of cardiomyocytes showed green fluorescence with long fusiform and adherent growth. (3) the transfection efficiency of plasmid cells with final concentration of .1ugrml was the highest. (4) compared with the normal control group, the LDH content in the supernatant of the H / R group increased and the activity of LDH decreased (p0.05). The transfection of LDH plasmid significantly reversed the changes of these indexes, and there was a significant difference compared with the H / R group (p0.05). There was no significant difference between the empty plasmid group and the H / R group (p0. 05); (5). Compared with the normal control group, the apoptotic rate (p0. 05) in the H- / R group was significantly increased (p0. 05). The apoptosis rate (p0. 05) in the middle and late stage of the plasmid group was lower than that in the H- / R group (p0. 05). Conclusion (1) H / R can induce oxidative stress injury and apoptosis of cardiomyocytes, (2) IMD gene can be transfected into the cells by plasmid transfection. (3) IMD can reduce oxidative stress, inhibit apoptosis and protect cardiomyocytes from H / R injury.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363
本文编号：2165765
[Abstract]:Objective to establish the model of hypoxia / reoxygenation (H / R) by cultured rat H9c2 cardiomyocytes transfected with Intermedin (IMD) plasmid, and to prove that IMD has protective effect on H / R oxidative stress injury of cardiomyocytes. It provides a basis for the study of the role of IMD gene in cardiovascular disease and the molecular biological mechanism. Methods the experiment was divided into four groups: 1) normal control group: H9c2 cardiomyocytes were cultured in DMEM medium for 48 h) H / R group: after 48 hours of conventional culture, H9c2 cardiomyocytes were exposed to anoxia for 2 h with 5mmol/1 sodium disulfite, and then reoxygenated on DMEM medium for 1 h. 3) the empty H / R plasmid group (empty plasmid group): pIRES2-EGFP plasmid (empty plasmid) was transfected and cultured steadily for 48h, then treated with anoxia / reoxygenation. 4) the H / R IMD plasmid group (IMD plasmid group): PIRES2-EGFP / IMD plasmid (IMD plasmid) was transfected and cultured steadily for 48h, then treated with anoxia / reoxygenation. At the end of the experiment, the morphology of H9c2 cardiomyocytes was observed under inverted phase contrast microscope, the morphology of cardiomyocytes was observed under fluorescence microscope at 48h after transfection, the transfection efficiency was detected by flow cytometry to determine the optimal amount of plasmid transfection. The release of lactate dehydrogenase (LDH) in cell supernatant was determined by chemical colorimetry, malondialdehyde (MDA) content in cell supernatant by thiobarbituric acid colorimetry, superoxide dismutase (SOD) activity in cell supernatant by xanthine oxidase method. The results of flow cytometry were as follows: (1) under inverted phase contrast microscope, H9c2 cardiomyocytes were long fusiform, arranged in fish-like or whirlpool shape and closely adhered to the wall; (2) under fluorescence microscope, the cytoplasm of cardiomyocytes showed green fluorescence with long fusiform and adherent growth. (3) the transfection efficiency of plasmid cells with final concentration of .1ugrml was the highest. (4) compared with the normal control group, the LDH content in the supernatant of the H / R group increased and the activity of LDH decreased (p0.05). The transfection of LDH plasmid significantly reversed the changes of these indexes, and there was a significant difference compared with the H / R group (p0.05). There was no significant difference between the empty plasmid group and the H / R group (p0. 05); (5). Compared with the normal control group, the apoptotic rate (p0. 05) in the H- / R group was significantly increased (p0. 05). The apoptosis rate (p0. 05) in the middle and late stage of the plasmid group was lower than that in the H- / R group (p0. 05). Conclusion (1) H / R can induce oxidative stress injury and apoptosis of cardiomyocytes, (2) IMD gene can be transfected into the cells by plasmid transfection. (3) IMD can reduce oxidative stress, inhibit apoptosis and protect cardiomyocytes from H / R injury.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363
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