雌激素对星形胶质细胞缺氧、缺糖损伤的影响及其机制的探讨

发布时间：2018-06-30 03:09

本文选题：星形胶质细胞 + 雌激素　；参考：《中南大学》2008年硕士论文

【摘要】： 目的研究17-β雌二醇对大鼠大脑皮层星形胶质细胞(AS)缺氧、缺糖损伤的影响,并对其可能的机制进行初步探讨。方法原代培养Sprague-Dawley大鼠大脑皮层AS,建立缺氧、缺糖损伤模型,以吉姆萨染色观察细胞形态学变化;以透射电镜观察细胞超微结构尤其是凋亡小体的变化;以四甲基偶氮唑盐(MTT)还原实验检测细胞活力;以乳酸脱氢酶(LDH)漏出率作为细胞损伤指标;以流式细胞术检测细胞凋亡率,研究氧糖剥夺(OGD)对AS的影响。在此基础上,通过钙离子敏感的荧光探针Fura-2/AM负载细胞,荧光双波长分光光度计检测细胞内钙离子浓度的变化;以黄嘌呤氧化酶法、硫代巴比妥酸法检测细胞超氧化物歧化酶(SOD)活性及丙二醛(MDA)含量的变化;免疫细胞化学染色和RT-PCR方法观测凋亡相关分子Caspase-3和Caspase-12在OGD前后的变化。结果①17-β雌二醇增强细胞活力的效应10nmol/L开始出现,在20nmol/L达到最高峰;降低LDH漏出率和抗凋亡效应均从1nmol/L开始,也是在20nmol/L达到最高峰。②缺氧缺糖处理即刻、缺氧缺糖处理前6h、12h、24h、36h,加入终浓度为20nmol/L的17-β雌二醇,均使缺氧缺糖损伤AS细胞活力增高,LDH漏出率降低,细胞凋亡率降低。其中缺氧缺糖处理前24h加入17-β雌二醇在上述5个时间点中,增强细胞活力的效应最强。③缺氧缺糖损伤可使细胞内游离钙离子浓度显著增高,17-β雌二醇预处理后可明显降低钙离子浓度,调节钙稳态失衡。④缺氧缺糖损伤可致AS内SOD活性降低,MDA含量升高,17-β雌二醇预处理后可显著升高SOD活性,降低MDA的含量。⑤RT-PCR和免疫细胞化学染色结果显示,缺氧缺糖可使AS内凋亡相关分子Caspase-3、Caspase-12的表达增加,17-β雌二醇预处理后可显著抑制两者的表达。结论①17-β雌二醇对体外培养AS缺氧、缺糖损伤具有直接的保护作用,并呈一定的量效、时效关系。②17-β雌二醇可能通过调节钙离子浓度、Caspase-3和Caspase-12的表达对缺氧、缺糖损伤AS发挥保护作用。③17-β雌二醇对缺氧、缺糖损伤皮层AS的保护作用可能与提高SOD活性或降低其消耗率,从而抑制缺氧、复氧过程中大量生成的氧自由基对DNA和细胞器膜等的损伤有关;可能与降低脂质过氧化反应有关。
[Abstract]:Objective to study the effects of 17- 尾 estradiol on cerebral cortical astrocyte (as) injury induced by hypoxia and glucose deficiency, and to explore its possible mechanism. Methods the primary cultured Sprague-Dawley rat cerebral cortex ASS was used to establish the model of hypoxia and glucose deficiency, and the morphological changes of the cells were observed by Gimsa staining, and the ultrastructure of the cells, especially the apoptotic corpuscles, was observed by transmission electron microscope (TEM). MTT assay was used to detect cell viability, lactate dehydrogenase (LDH) leakage rate was used as the index of cell damage, and apoptosis rate was detected by flow cytometry to study the effect of oxygen glucose deprivation (OGD) on as. On this basis, the changes of intracellular calcium concentration were detected by the calcium sensitive fluorescent probe Fura-2 / AM loaded cells, and the changes of intracellular calcium concentration were detected by xanthine oxidase method. The activity of superoxide dismutase (SOD) and the content of malondialdehyde (MDA) were measured by thiobarbituric acid method, and the changes of apoptosis related molecules Caspase-3 and Caspase-12 before and after OGD were observed by immunocytochemical staining and RT-PCR. Results the effect of 117- 尾 estradiol on enhancing cell viability began to appear at 10nmol / L and reached its peak at 20nmol / L, and the decrease of LDH leakage rate and anti-apoptotic effect began at 1nmol / L, and immediately after 20nmol / L hypoxia and glucose deprivation treatment. Before hypoxia and glucose deprivation treatment, the LDH leakage rate and apoptosis rate of as cells increased and apoptosis rate decreased 6 hours before hypoxia and glucose deprivation treatment, and 17- 尾 estradiol at the final concentration of 20 nmol / L increased the activity of as cells after hypoxia and glucose deprivation treatment for 24 hours and 36 hours before hypoxia and glucose deprivation treatment, and the final concentration of 17- 尾 estradiol was 20 nmol / L. Among them, 17- 尾 estradiol was added 24 hours before hypoxia and glucose deprivation treatment, and 17 尾 estradiol was added at the above 5 time points. The effect of enhancing cell viability was strongest. 3 hypoxia and glucose deprivation injury could significantly increase intracellular free calcium concentration and decrease calcium concentration after pretreatment with 17- 尾 estradiol. Regulation of calcium homeostasis. 4 hypoxia and glucose deficiency injury could reduce the activity of SOD and increase the content of MDA. After pretreatment with 17- 尾 estradiol, SOD activity could be significantly increased, and the content of MDA could be decreased by RT-PCR and immunocytochemical staining. Hypoxia and glucose deprivation could significantly inhibit the expression of apoptosis related molecule Caspase-3 and Caspase-12 after pretreatment with 17- 尾 estradiol. Conclusion 117- 尾 estradiol has direct protective effect on anoxia and glucose deficiency injury of as cultured in vitro, and has a dose-dependent effect. The aging relationship of estradiol. 217- 尾 estradiol may regulate the expression of Caspase-3 and Caspase-12 in Ca ~ (2 +) concentration. The protective effect of estradiol on hypoxia may be related to increasing SOD activity or decreasing consumption rate, thus inhibiting hypoxia. The large amount of oxygen free radicals produced during reoxygenation may be related to the damage of DNA and organelle membrane, and may be related to the reduction of lipid peroxidation.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R363

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