【摘要】：目的：通過(guò)氧化損傷標(biāo)記物8-羥基脫氧鳥(niǎo)苷（8-OHDG）及細(xì)胞凋亡標(biāo)記物半胱氨酸天冬氨酸蛋白酶-3（caspase-3）的檢測(cè)，分析心肌缺血缺氧、心肌缺血再灌注、心肌細(xì)胞氧化應(yīng)激的病理變化及相關(guān)機(jī)制，在此基礎(chǔ)上研究氧化樟腦對(duì)氧化應(yīng)激損傷后心肌細(xì)胞的保護(hù)作用。 方法：采用H9c2大鼠心肌細(xì)胞復(fù)制缺氧模型，隨機(jī)分為4組：正常組、氧化應(yīng)激組、氧化應(yīng)激+氧化樟腦A組、氧化應(yīng)激+氧化樟腦B組。正常對(duì)照組：不作任何特殊處理；氧化應(yīng)激組：將心肌細(xì)胞更換為無(wú)氧培養(yǎng)液，放入缺氧裝置內(nèi)，繼續(xù)培養(yǎng)4h后，更換復(fù)氧液，再進(jìn)行有氧培養(yǎng)4h，完成缺氧/復(fù)氧模型；氧化應(yīng)激+氧化樟腦組：在心肌細(xì)胞缺氧/復(fù)氧完成后分別加入10ul/50ml（A組）、20ul/50ml（B組）濃度的氧化樟腦繼續(xù)培養(yǎng)6h。通過(guò)噻唑藍(lán)微量自動(dòng)比色法（MTT）檢測(cè)各組心肌細(xì)胞活力。通過(guò)免疫化學(xué)染色觀察各組細(xì)胞氧化損傷標(biāo)記物8-羥基脫氧鳥(niǎo)苷（8-OHDG）及細(xì)胞凋亡標(biāo)記物半胱氨酸天冬氨酸蛋白酶-3（caspase-3）水平。統(tǒng)計(jì)學(xué)處理以Sigma Stat3.5軟件進(jìn)行統(tǒng)計(jì)學(xué)處理，應(yīng)用t-Test或one-wayANOVA分析，結(jié)果以Mean±SD表示，P0.05為有統(tǒng)計(jì)學(xué)差異。 結(jié)果： 1.氧化應(yīng)激組H9c2心肌細(xì)胞繼續(xù)培養(yǎng)6h后進(jìn)行MTT檢測(cè)。結(jié)果表明：氧化應(yīng)激組吸光度值（A值）低于對(duì)照組（P0.05）。 2.氧化應(yīng)激+氧化樟腦組H9c2心肌細(xì)胞繼續(xù)培養(yǎng)6h后進(jìn)行MTT檢測(cè)。結(jié)果表明：氧化應(yīng)激組吸光度值（A值）低于氧化應(yīng)激+氧化樟腦組，氧化應(yīng)激+氧化樟腦組吸光度值（A值）低于對(duì)照組（P0.05），氧化應(yīng)激+氧化樟腦組較氧化應(yīng)激組心肌細(xì)胞活力明顯升高。氧化應(yīng)激+氧化樟腦B組心肌細(xì)胞活力高于氧化應(yīng)激+氧化樟腦A組。 3.8-OhdG表達(dá)在對(duì)照組最低，氧化應(yīng)激組最高，而氧化應(yīng)激+氧化樟腦組較氧化應(yīng)激組明顯降低，，氧化應(yīng)激+氧化樟腦B組8-OhdG表達(dá)低于氧化應(yīng)激+氧化樟腦A組。 4. caspase-3的蛋白表達(dá)在對(duì)照組最低，氧化應(yīng)激組最高，而氧化應(yīng)激+氧化樟腦組較氧化應(yīng)激組明顯降低，氧化應(yīng)激+氧化樟腦B組的caspase-3蛋白表達(dá)低于氧化應(yīng)激+氧化樟腦A組。 結(jié)論：氧化樟腦對(duì)氧化應(yīng)激損傷后心肌細(xì)胞具有保護(hù)作用，它可減輕氧化應(yīng)激后心肌細(xì)胞的損傷，抑制氧化應(yīng)激后心肌細(xì)胞的凋亡。
[Abstract]:Objective: to detect the oxidative damage marker 8-hydroxydeoxyguanosine (8-OHDG) and the apoptotic marker cysteine aspartate protease-3 (caspase-3) to analyze myocardial ischemia and hypoxia and myocardial ischemia-reperfusion. On the basis of pathological changes and related mechanisms of oxidative stress in cardiomyocytes, the protective effect of camphor on myocardial cells after oxidative stress was studied. Methods: hypoxia model was induced by H9c2 rat cardiomyocytes. The rats were randomly divided into 4 groups: normal group, oxidative stress oxidative camphor A group and oxidative stress oxidative camphor B group. Normal control group: without any special treatment, oxidative stress group: the cardiomyocytes were replaced with anaerobic culture medium, put into anoxic equipment, then cultured for 4 hours, then reoxygenated solution was changed, then aerobic culture was carried out for 4 hours to complete the hypoxia / reoxygenation model. Oxidative stress Oxycamphor group: after hypoxia / reoxygenation of cardiomyocytes were completed, 10ul/50ml (group A) 20 ulr / 50ml (group B) was added respectively to culture camphor for 6 h. The activity of myocardial cells in each group was detected by (MTT) with thiazolyl blue microcolorimetry. The levels of 8-hydroxydeoxyguanosine (8-OHDG) and cysteine aspartate protease -3 (caspase-3) were detected by immunocytochemical staining. Statistical processing was performed by Sigma Stat3.5 software, and t-Test or one-wayANOVA analysis was used. The result was that Mean 鹵SD was used to show statistical difference (P0.05). Results: 1. In oxidative stress group, H9c2 cardiomyocytes were cultured for 6 h and MTT was detected. The results showed that the absorbance (A value) of oxidative stress group was lower than that of control group (P0.05). H9c2 cardiomyocytes in oxidative stress group were cultured for 6 h and MTT was detected. The results showed that the absorbance value (A value) of oxidative stress group was lower than that of oxidative stress oxidative camphor group. The absorbance (A value) of oxidative stress camphor group was lower than that of control group (P0.05). The activity of myocardial cells in oxidative stress camphor group was significantly higher than that in oxidative stress group. The activity of cardiomyocytes in oxidative stress camphor B group was higher than that in oxidative stress camphor A group. The expression of 3.8-OhdG was the lowest in the control group and the highest in the oxidative stress group, while the oxidative stress oxidative camphor group was significantly lower than that in the oxidative stress group. The expression of 8-OhdG in oxidative stress camphor B group was lower than that in oxidative stress camphor A group. 4. The protein expression of caspase-3 was the lowest in the control group and the highest in the oxidative stress group, while the oxidative stress oxidative camphor group was significantly lower than that in the oxidative stress group. The expression of caspase-3 protein in oxidative stress camphor B group was lower than that in oxidative stress camphor A group. Conclusion: Oxycamphor has protective effect on cardiomyocytes after oxidative stress. It can reduce the injury of cardiomyocytes after oxidative stress and inhibit the apoptosis of cardiomyocytes after oxidative stress.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R363

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