【摘要】：目的通過(guò)培養(yǎng)Intermedin(IMD)質(zhì)粒轉(zhuǎn)染后的大鼠H9c2心肌細(xì)胞建立缺氧/復(fù)氧(H/R)模型,證實(shí)IMD對(duì)心肌細(xì)胞的H/R氧化應(yīng)激損傷具有保護(hù)作用,為研究IMD基因?qū)π难芗膊〉淖饔眉胺肿由飳W(xué)機(jī)制奠定基礎(chǔ)。 方法實(shí)驗(yàn)分為四組：1)正常對(duì)照組：用DMEM培養(yǎng)基培養(yǎng)H9c2心肌細(xì)胞48h；2)H/R組：常規(guī)培養(yǎng)H9c2心肌細(xì)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(fù)氧1h；3)H/R+空質(zhì)粒組(空質(zhì)粒組)：pIRES2-EGFP質(zhì)粒(即空質(zhì)粒)轉(zhuǎn)染后穩(wěn)定培養(yǎng)48h,之后給予缺氧/復(fù)氧處理；4)H/R+IMD質(zhì)粒組(IMD質(zhì)粒組)：PIRES2-EGFP/IMD質(zhì)粒(即IMD質(zhì)粒)轉(zhuǎn)染后穩(wěn)定培養(yǎng)48h,之后給予缺氧/復(fù)氧處理。實(shí)驗(yàn)終止后,在倒置相差顯微鏡下觀察H9c2心肌細(xì)胞形態(tài)；質(zhì)粒轉(zhuǎn)染后48h,在熒光顯微鏡下觀察心肌細(xì)胞形態(tài)；通過(guò)流式細(xì)胞儀檢測(cè)細(xì)胞轉(zhuǎn)染效率以確定質(zhì)粒的最佳轉(zhuǎn)染量；采用化學(xué)比色法測(cè)定細(xì)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(cè)定細(xì)胞上清液中丙二醛(MDA)含量；用黃嘌呤氧化酶法測(cè)定細(xì)胞上清液中超氧化物歧化酶(SOD)活性；運(yùn)用流式細(xì)胞術(shù)檢測(cè)心肌細(xì)胞凋亡率 結(jié)果(1)倒置相差顯微鏡下觀察H9c2心肌細(xì)胞為長(zhǎng)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(xì)胞胞漿內(nèi)呈現(xiàn)綠色熒光,呈長(zhǎng)梭形,貼壁生長(zhǎng)； (3)終濃度為.1ug/ml的質(zhì)粒細(xì)胞轉(zhuǎn)染效率最高； (4)與正常對(duì)照組相比,H/R組細(xì)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(zhì)粒的轉(zhuǎn)染顯著逆轉(zhuǎn)了上述這些指標(biāo)的變化,與H/R組相比具有顯著差異(p0.05)；空質(zhì)粒組與H/R組相較,各指標(biāo)測(cè)定值無(wú)統(tǒng)計(jì)學(xué)差異(p0.05)； (5)與正常對(duì)照組相比,H/R組細(xì)胞凋亡率(中晚期)顯著增加(p0.05),IMD質(zhì)粒組細(xì)胞凋亡率(中晚期)較H/R組有所下降(p0.05)；空質(zhì)粒組與H/R組相較則無(wú)統(tǒng)計(jì)學(xué)差異(p0.05); 結(jié)論(1)H/R可誘導(dǎo)心肌細(xì)胞氧化應(yīng)激損傷和凋亡； (2)IMD基因可通過(guò)質(zhì)粒轉(zhuǎn)染的方法進(jìn)入細(xì)胞發(fā)揮作用； (3)IMD可通過(guò)減輕氧化應(yīng)激,抑制細(xì)胞凋亡,對(duì)心肌細(xì)胞H/R損傷起到保護(hù)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(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R363

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