【作者】 曾志成；

【导师】 彭清华； 武正清；

【作者基本信息】 湖南中医药大学 ， 中西医结合临床， 2009， 硕士

【摘要】 目的1.制备不同浓度青光安颗粒含药血清。2.培养、传代人小梁细胞并建立体外高压模型。3.初步探讨青光安颗粒含药血清对体外加压后人小梁细胞凋亡的影响及其与Caspase-3、Caspase-8蛋白酶表达的关系。方法1.随机将60只SD大鼠分组,进行灌胃,以获取青光安2.5倍、5倍、10倍、20倍含药血清,益脉康5倍含药血清以及不含药大鼠血清。2.建立人小梁细胞体外高压模型。3.MTT法检测经不同含药血清作用后加压人小梁细胞的活性。取加压人小梁细胞接种到培养板中,加入不同含药血清,分别检测不同药物与浓度含药血清组不同时间段细胞的OD值。4. TUNEL技术检测不同含药血清作用后加压人小梁细胞的凋亡指数,免疫组化检测不同含药血清作用后加压人小梁细胞Caspase-3、Caspase-8蛋白酶的表达指数。5.流式细胞仪检测不同含药血清作用后加压人小梁细胞的凋亡率和Caspase-3、Caspase-8蛋白表达率。结果1.成功获取含药血清,并将其作用于体外加压的人眼小梁细胞。2.成功制备体外加压的人眼小梁细胞。3.MTT法发现,OD值与不同药物含药血清有关,还与作用时间有关。青光安5倍、10倍、20倍组和益脉康5倍组都能够明显提高细胞OD值,与空白组比较差异有显著统计学意义(P<0.01)；青光安10倍、20倍组与益脉康5倍组组比较差异有显著统计学意义(P<0.01),但是青光安5倍组与益脉康5倍组组之间比较差异没有统计学意义(P>0.05)；青光安10倍组与20倍组之间比较差异没有统计学意义(P>0.05)；培养24h、36h的OD值明显高于培养12h的OD值,比较差异有显著统计学意义(P<0.01),但是培养24h与培养36h时间段之间比较差异无统计学意义(P>0.05)。4.TUNEL技术、免疫组化检测、流式细胞仪检测共同发现青光安5倍、10倍、20倍组和益脉康5倍组都可以明显抑制加压人小梁细胞的凋亡和Caspase-3、Caspase-8蛋白酶的表达,与空白组比较差异有显著统计学意义(P<0.01)；与益脉康5倍组比较,青光安5倍组差异无统计学意义(P>0.05),青光安10倍、20倍组差异有统计学意义(P<0.05)；与青光安10倍组比较,青光安5倍组、益脉康5倍组的凋亡和Caspase-3、Caspase-8蛋白酶表达都较高,比较差异有统计学意义(P<0.05),青光安20倍组差异无统计学意义(P>0.05)。5.根据流式细胞仪检测结果统计发现各组加压后人小梁细胞Caspase-3蛋白表达率与凋亡率的决定系数(R2)为0.784；Caspase-8蛋白表达率与凋亡率的决定系数(R2)为0.813。结论1.青光安颗粒含药血清能提高加压人小梁细胞活性、抑制其凋亡。2.青光安颗粒含药血清能抑制加压人眼小梁细胞Caspase-3、Caspase-8蛋白酶的激活与表达。3.青光安颗粒含药血清抑制加压人小梁细胞凋亡可能主要是通过抑制细胞Caspase-3、Caspase-8蛋白酶激活的途径而起作用。更多还原

【Abstract】 Objective 1. prepared serum containing Qing-guang-an grains of different concentrations.2. cultivated and Subcultured human trabecular cells and established high-pressure model in vitro.3. After intervention of serum containing different concentrations Qing-guang-an grains, preliminary studied apoptosis of pressurized human trabecular cells in vitro and its relation with Caspase-3、Caspase-8 protein expression.METHODS 1. Randomly grouped 60 SD rats, intragastric administration, obtained serums containing 2.5times、5 times、10 times、20 times Qing-guang-an grains、5 times yi-Mai-kang Tablet and free medicine.2. Established high-pressure model of human trabecular cells in vitro.3. MTT checked activity of pressurized Human trabecular cells after intervention of different serums. Inoculated pressurized human trabecular cells to the culture plate, added serums containing differents drugs and concentrations to the culture plate, detected optical density value (OD value) of pressurized human trabecular cells after intervention different serums and different periods.4. TUNEL detected apoptosis index (AI) of pressurized human trabecular cells after intervention of different serums; Histochemistry detected Caspase-3、Caspase-8 protease expression index (PEI) of pressurized human trabecular cells after intervention of different serums.5. Flow cytometry detected the apoptosis rate and the Caspase-3、Caspase-8 protease expression of pressurized human trabecular cells after intervention of different serums.Results 1. successfully gained serums containing different drug, and be utilized to pressurized human trabecular cells in vitro.2. successfully established high-pressure model of human trabecular cells in vitro.3. Found by MTT, OD values relate with serums containing different drugs, but also the time.5 times、10 times、20 times Qing-guang-an Grains groups and 5 times yi-Mai-kang Tablets group could enhance the OD value of Cells Obviously, were higher than blank group(P<0.01),10 times and 20 times Qing-guang-an Grains groups were higher than 5 times Yi-Mai-Kang Tablets group(P<0.01), but no different between 5 times Qing-guang-an Grains groups and 5 times Yi-Mai-Kang Tablets group(P>0.05); There were no between 10 times Qing-guang-an Grains group and 20 times Qing-guang-an Grains group(P>0.05); 0D value of the cells cultured for 24 hours and 36 hours were conspicuouslly higher than that cultured for 12 hours(P<0.01), but no difference between 24 hours and 36 hours(P>0.05).4. Found by TUNEL、Histochemistry and Flow cytometry,5 times、10 times、20 times Qing-guang-an Grains groups and 5 times Yi-Mai-Kang Tablets group could inhibit the apoptosis and the expression of Caspase-3、Caspase-8 of pressurized Human Trabecular Cells obviously. As compared with the blank group, the apoptosis and the expressions of Caspase-3 and Caspase-8 were conspicuouslly lower(P< 0.01); As compared with the 5 times Yi-Mai-Kang Tablets group,there were no difference between 5 times Qing-guang-an Grains group and it(P>0.05), but the apoptosis and the expressions of Caspase-3 and Caspase-8 of 10 times、20 times Qing-guang-an Grains groups were lower(P<0.05); As compared with the 10 times-Qing-guang-an Grains group, the apoptosis and the expressions of Caspase-3 and Caspase-8 of 5 times Qing-guang-an Grains and 5 times Yi-Mai-Kang Tablets groups were higher(P<0.05); But there were no difference between 10 times Qing-guang-an Grains group and 20 times Qing-guang-an Grains group(P>0.05).5. Found by Statistics of flow cytometry, each group pressurized human trabecular cells Caspase-3 protein expression rate and the apoptosis rate coefficient of determination (R2) is 0.784; Caspase-8 protein expression rate and the apoptosis rate coefficient of determination (R2) is 0.813.Conclusion 1. Serums containing Qing-guang-an Grains can enhance pressurized human trabecular cells posterity activity, inhibit apoptosis.2. Serums containing Qing-guarig-an Grains can inhibit the pressurized human trabecular cells Caspase-3、Caspase-8 activation and expression of protease.3. Serums containing Qing-guang-an Grains inhibit apoptosis of pressurized human trabecular cells possible mainly through the inhibition of the channels of Caspase-3、Caspase-8 protease activation.更多还原