Intermedin對缺氧復氧所致H9c2心肌細胞氧化應激損傷的保護作用
發(fā)布時間：2018-08-05 12:14
【摘要】：目的通過培養(yǎng)Intermedin(IMD)質粒轉染后的大鼠H9c2心肌細胞建立缺氧/復氧(H/R)模型,證實IMD對心肌細胞的H/R氧化應激損傷具有保護作用,為研究IMD基因對心血管疾病的作用及分子生物學機制奠定基礎。 方法實驗分為四組：1)正常對照組：用DMEM培養(yǎng)基培養(yǎng)H9c2心肌細胞48h；2)H/R組：常規(guī)培養(yǎng)H9c2心肌細胞48h后,用5mmol/1的連二亞硫酸鈉使其缺氧2h,后用DMEM培養(yǎng)基復氧1h；3)H/R+空質粒組(空質粒組)：pIRES2-EGFP質粒(即空質粒)轉染后穩(wěn)定培養(yǎng)48h,之后給予缺氧/復氧處理；4)H/R+IMD質粒組(IMD質粒組)：PIRES2-EGFP/IMD質粒(即IMD質粒)轉染后穩(wěn)定培養(yǎng)48h,之后給予缺氧/復氧處理。實驗終止后,在倒置相差顯微鏡下觀察H9c2心肌細胞形態(tài)；質粒轉染后48h,在熒光顯微鏡下觀察心肌細胞形態(tài)；通過流式細胞儀檢測細胞轉染效率以確定質粒的最佳轉染量；采用化學比色法測定細胞上清液中乳酸脫氫酶(LDH)的釋放；用硫代巴比妥酸顯色法測定細胞上清液中丙二醛(MDA)含量；用黃嘌呤氧化酶法測定細胞上清液中超氧化物歧化酶(SOD)活性；運用流式細胞術檢測心肌細胞凋亡率 結果(1)倒置相差顯微鏡下觀察H9c2心肌細胞為長梭形,呈魚群狀或漩渦狀排列,貼壁緊密； (2)熒光顯微鏡下觀察心肌細胞胞漿內呈現綠色熒光,呈長梭形,貼壁生長； (3)終濃度為.1ug/ml的質粒細胞轉染效率最高； (4)與正常對照組相比,H/R組細胞上清液中LDH含量增加,MDA含量增加,SOD活性下降(p均0.05)；IMD質粒的轉染顯著逆轉了上述這些指標的變化,與H/R組相比具有顯著差異(p0.05)；空質粒組與H/R組相較,各指標測定值無統(tǒng)計學差異(p0.05)； (5)與正常對照組相比,H/R組細胞凋亡率(中晚期)顯著增加(p0.05),IMD質粒組細胞凋亡率(中晚期)較H/R組有所下降(p0.05)；空質粒組與H/R組相較則無統(tǒng)計學差異(p0.05); 結論(1)H/R可誘導心肌細胞氧化應激損傷和凋亡； (2)IMD基因可通過質粒轉染的方法進入細胞發(fā)揮作用； (3)IMD可通過減輕氧化應激,抑制細胞凋亡,對心肌細胞H/R損傷起到保護作用；
[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.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R363
本文編號：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.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R363
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