【作者】 李扣华；

【导师】 文珠；

【作者基本信息】 江西医学院 ， 细胞生物学， 2004， 硕士

【摘要】 目的：通过对参皂甙Rb1、黄芪对体外培养的新生大鼠大脑皮层神经元缺氧性凋亡保护作用及其机制的研究，为临床提供理论依据。方法：1. 应用 “Neurobasa加B27 Supplement 培养液”进行无血清神经细胞体外培养。2.采用Trypan blue拒染法判断缺氧时间与培养的神经细胞活性之间的关系。3. 神经细胞缺氧培养及药物干预实验: 取培养4天的新生大鼠大脑皮层神经细胞进行缺氧实验。分组如下：(1) 正常对照组：不缺氧常规培养，即37℃、5%CO2、饱和湿度的培养箱中培养；(2) 凋亡诱导组（阳性对照组）：常规培养4天后缺氧复氧培养；(3) 人参皂甙Rb1组1(10μg/ml)；(4) 人参皂甙Rb1组2(50μg/ml)；(5) 人参皂甙Rb1组3(100μg/ml)；(6) 黄芪注射液组1(10mg/ml)；(7) 黄芪注射液组2(50mg/ml)；(8) 黄芪注射液组3(100mg/ml)。(3)-(8)组等药物干预组，常规培养4天后于缺氧前0小时加入指定浓度的药物，然后与凋亡诱导组的培养板一道置缺氧罐中通入5% CO2＋95%N2的混合气在37℃条件下缺氧培养12小时。在缺氧12小时后，再置37℃、5%CO2培养箱中复氧培养48小时。复氧培养48小时后，应用DNA琼脂糖凝胶电泳法、流式细胞仪PI染色法、Hoechst 33342荧光染色法检测人参皂甙Rb1和黄芪对体外培养的大鼠皮层神经元缺氧诱导的神经细胞凋亡的影响。4．采用免疫细胞化学染色法测定缺氧12小时复氧48小时后药物组、阳性对照组及正常对照组的神经细胞Bcl-2和Bax蛋白表达的变化。结果：1.缺氧6-12小时复氧48小时神经细胞存活率均大于80%,缺氧24小时复氧48小时<WP=4>神经细胞活率小于50%,缺氧组细胞活率与阴性对照组相比有显著性差异（p<0.05）。2．阴性对照组细胞经Hoechst 33342荧光染色表现为核形态正常，核内染色质呈均匀蓝绿色荧光，流式细胞仪检测为正常二倍体峰，DNA琼脂糖凝胶电泳无凋亡梯带。在凋亡诱导组中部分细胞表现为核染色质凝集致密浓染，荧光增强，有的核固缩碎裂呈颗粒状和块状荧光，流式细胞仪检测可见凋亡的亚二倍体峰，DNA琼脂糖凝胶电泳可见典型的凋亡梯带。通过Hoechst 33342荧光染色及流式细胞仪检测细胞凋亡率发现不同浓度的人参皂甙Rb1组和黄芪注射液组与凋亡诱导组相比细胞凋亡率呈不同程度降低，且呈剂量依赖关系。在人参皂甙Rb1组2和黄芪注射液组2DNA琼脂糖凝胶电泳亦未见典型的凋亡梯带。3. 缺氧12小时复氧48小时使神经细胞Bcl-2的表达上调,而使Bax的表达降低; 一定浓度的人参皂甙Rb1,黄芪上调缺氧神经细胞Bcl-2的表达,同时下调缺氧神经细胞Bax的表达。结论：1. 人参皂甙Rb1,黄芪能在一定浓度范围內抑制缺氧诱导的神经细胞凋亡。2. 人参皂甙Rb1,黄芪在一定浓度范围內可上调缺氧神经细胞Bcl-2的表达,同时下调缺氧神经细胞Bax的表达。3. 体外无血清原代新生SD大鼠大脑皮层神经元是一种研究神经细胞凋亡和药物干预抗神经细胞凋亡的作用机理好的模型。更多还原

【Abstract】 Objective：To investigated the effect of ginsenosideRb1 and huangqi on the apoptosis of cultured cerebral cortical neurons caused by hypoxia and the mechanism of the protctive effect in order to supply some experimental bases for clinical application.Methods：① The primary culure of cerebral neurons of postnatal rats was established with neurobasal medium suppled 2%B27 supplement .② Neuronal viability was investigated by trypan blue exclusion to determine the effect of hypxia of different time（6、12、24 hours） and reperfusion on cultured cerebral neurons．③ Neuronal hypoxic culture and drugs interventive experiment:At 4 Div ,the cultured neurons of postnatal rat cerebral cortex were used to hypoxic investigation .there were divided to such research groups: (1)the normal control group (the negtve control group):the neurons of which was cultured in 5%CO2/95%air atmosphere at 37℃without hypoxia; (2)the induced apoptotic group(the postive control group):the neurons were deprivated oxygen and then returned to normal culture condition;(3)GinsenosideRb1 experimental group1(10μg/ml)；(4)GinsenosideRb1experimental group2(50μg/ml)；(5)GinsenosideRb1 experimental group3(100μg/ml)；(6)huangqi injection experimental group 1(10mg/ml)；(7)huangqi injection experimental group 2(50mg/ml)；(8)huangqi experimental group 3(100mg/ml)；(3)-(8)groups were drugs interventive groups,the drugs of specific concentration were added to the medium of cultured neurons at 4 Div,then the neurons together with that of the postive control group were placed in a gas-tight <WP=6>tank flushed with 5%CO2/95%N2 at 37℃ for 12 hours.After 12h of hypoxia the neurons were further incubated up to 48h in 5%CO2/95%air atmosphere at 37℃.Then apoptotic incident of cultured neurons was evaluated by Hoechst 33342 staining ，flow cytometer , DNA agarose gel electrophoresis. ④The change of expression of apoptotic controlling genes bcl-2/bax were detected by immunocytochemistry among drugs intereventive groups,the postive control group and the negetive control group.RESULTS：①After cultured neurons were hypoxiaed for different times(6,12,24 hours) and reperfused for 48 hours, compared with the normal control group, there were less live cells. The cell viability of the normal control group , 6 hours hypoxia group and 12 hours hypoxia group was more than 80%,but that of 24 hours hypoxia group was less than 50%. ② Little apoptosis was detected by FACS、DNA agarose gel electrophoresis and Hoechst 33342 staining in negative control group. In the apoptosis induced group, many of the neurons had condensed chromatin(some of them broken into debris) which emitted brighter green fluorescence by using the the fluorescence dye Hoechst 33342; Sub-G1 peak was detected by flow cytometry and typical apoptotic DNA ladders were also detected by electrophoresis. The apoptotic rates of neurons in different concentration the ginsenoside Rb1 group and huangqi injection group were decreaser compared with that in the postive group by using the fluorescence dye Hoechst 33342 and flow cytometry,furthermore apoptotic rates of neurons were lower and lower with the increase of contreation of added drugs . typical apoptotic DNA ladders could hardly be detected by DNA agarose gel electrophoresis in the ginsenoside Rb1 group 2and huangqi injection group2.The expression of bcl-2 in cultured cerebral neurons was found to lower level in contrasts with that of bax measured by immunocytochemistry after hypoxia and reperfusion . Ginsenoside Rb1 and huangqi could up-regulate bcl-2 protein and down–regulate bax protein expression.CONCLUSION： <WP=7>Ginsenoside Rb1 and huangqi could prevent the apoptotic incidence trigger by hypoxia.Ginsenoside Rb1 and huangqi could up-regulate the expression of bcl-2 protein of hypoxic neurons and down–regulate that of bax.The primary culure of crebral neurons of postnatal rats with neurobasal medium suppled 2%B27 supple was a good model for investigation of the drugs that prevent from hypoxic apoptosis of neuron and the mechnism o更多还原