钙离子在蓝光诱导人视网膜色素上皮细胞凋亡中的作用机制研究

发布时间：2018-06-18 00:28

本文选题：视网膜色素上皮细胞 + 凋亡　；参考：《遵义医学院》2017年硕士论文

【摘要】：目的:探讨蓝光诱导人视网膜色素上皮(retinal pigment epithelial cells,RPE)细胞凋亡过程中,胞内钙离子(Ca~(2+))在Ca~(2+)-PKC信号传导通路及与线粒体损伤的凋亡途径的相关作用机制。方法:原代培养健康人RPE细胞,传至第3-6代用于本实验。采用蓝光(470nm-490nm),光照强度为(2,000±500)lx,经蓝光照射时间6h后,继续培养细胞24h,建立蓝光损伤人RPE细胞模型。采用免疫组化(IHC)染色光学显微镜下鉴定凋亡细胞,通过透射电镜(TEM)观察RPE的核型变和线粒体形态学改变。将体外培养的人RPE细胞随机分成4组:1A组:无光照组;1B组:光照组;1C组:光照+硝苯地平(Nifedipine)组;1D组:光照+(-)Bay K8644组。采用RT-PCR技术测定细胞膜L-型钙通道(L-type calcium channel)α1C、α1D、α1F亚型的m RNA表达量。将体外培养的人RPE细胞随机分成5组:2A组:无光照组;2B组:光照组;2C组:光照+硝苯地平组;2D组:光照+钙磷酸结合蛋白(Calphostin C)组;2E组:光照+佛波酯(PMA)组。激光扫描共聚焦显微镜(LSCM)用于检测各组RPE细胞内游离Ca~(2+)浓度,分析荧光强度。采用非放射性核素法检测RPE胞内PKC活性。采用Western blot法测定各组RPE胞内Caspase-9蛋白的表达。将体外培养的人RPE细胞随机分为3组,3A组:(500±100)lx、(2,000±500)lx、(3,000±500)lx光照强度,光照时间6h,光照后细胞终止培养时间24h;3B组:(2,000±500)lx光照强度,光照时间3h、6h、12h、24h,,光照后细胞终止培养时间24h;3C组:(2,000±500)lx光照强度,光照时间6h,光照后细胞终止培养时间6h、12h、24h、36h和48h;对照组:无光照组,流式细胞术用于检测细胞线粒体跨膜电位(ΔψM)。将体外培养的人RPE细胞随机分为2组:4A组:光照强度(2,000±500)lx,光照时间6h,光照后细胞终止培养时间6、12、24、36h;4B组:光照强度(2,000±500)lx,光照时间12h,光照后细胞终止培养时间6h、12h、24h和36h,酶联免疫吸附试验(ELISA)测定细胞色素C(Cyt C)浓度。将体外培养的人RPE细胞分成2组:5A组:无光照组;5B组:单纯光照组,Western blot法测定Bax、Bcl-2、Bcl-xl蛋白表达量。采用SPSS 20.0统计学软件对实验数据进行统计学分析。结果:体外培养的人RPE细胞在蓝光(470-490nm),光照强度(2000±500)lx,光照时间6h,光照后细胞终止培养时间24h后出现细胞凋亡,IHC染色表现为细胞核浓缩、染色质边聚成新月形、细胞核碎裂成数个;透射电子显微镜下观察可见线粒体膜破碎,嵴完全消失,染色质聚核膜边并浓集,呈现典型的细胞凋亡核型变。RT-PCR测定L-型膜型钙通道的α1C、α1D、α1F亚基m RNA表达量比较,差异均有统计学意义(F=45.92,P0.05;F=50.32,P0.05;F=18.50,P0.05)。LSCM检测RPE细胞内游离Ca~(2+)浓度结果显示,光照组(2E组2B组2D组2C组,P0.05)均高于无光照组(2A组),差异具有统计学意义(F=26764.22,P0.05)。非放射性核素法检测RPE细胞内PKC活性结果显示,光照组(2B组)IA值明显高于对照组(2A组)IA值(t=-9.869,P0.05)。胞内Ca~(2+)浓度与PKC活性通过线性回归分析显示:胞内Ca~(2+)与PKC呈正相关关系(Y=53.032X+127.565,其中:r=0.990,t=13.931,P0.05)。Western blot法测定各组RPE细胞内caspase-9蛋白相对表达量显示,2B、2D、2E组与2A组比较,caspase-9蛋白相对表达量均明显升高(P0.05)。流式细胞术检测细胞线粒体膜电位(ΔψM)显示:3A组随着光照强度增加,RPE线粒体膜电位(ΔψM)开始下降(P0.05);3B组中光照6h、12h组与对照组比较,RPE线粒体膜电位(ΔψM)明显下降(P0.05);3C组中的光照6h、12h、24h、36h、48h与对照组比较,RPE线粒体膜电位(ΔψM)明显下降(P0.05)。ELISA法测定Cyt C结果显示:4A组:光照6h处理继续培养的24h、36h组与对照组、6h、12h组比较,Cyt C浓度明显增加(P0.05)。4B组:光照12h处理继续培养12h、24h、36h组与对照组比较,Cyt C浓度明显增加(P0.05)。Western blot法测定Bax、Bcl-2、Bcl-xl蛋白表达量,结果显示:光照组(5B组)RPE细胞与无光照组(5A组)比较,Bax表达量增加,而Bcl-2、Bcl-xl表达量下降,差异均有统计学意义(P0.05),且Bax/Bcl-2值、Bax/Bcl-xl值与无光照组比较,明显高于无光照组,差异均有统计学意义(P0.05)。结论:蓝光可诱导人RPE细胞凋亡,出现典型的细胞凋亡核型变和胞内线粒体损伤。蓝光照射后可引起RPE细胞膜L-型钙通道α1C、α1D、α1F亚基的m RNA表达增高,细胞内Ca~(2+)荧光强度及PKC活性不同程度增强,且胞内Ca~(2+)浓度及PKC活性呈正相关关系。与此同时,蓝光照射后RPE细胞线粒体膜电位(ΔψM)明显下降,Cyt C大量释放,促凋亡因子Bax与抑凋亡因子Bcl-2、Bcl-xl比值下调,触发caspase-9蛋白相对表达量增加,最终诱导细胞凋亡。钙离子同时参与Ca~(2+)-PKC信号传导通路促凋亡过程及细胞线粒体凋亡途径,并起着启动、调控、促进细胞凋亡的关键作用。
[Abstract]:Objective: To investigate the mechanism of intracellular calcium (Ca~ (2+)) in Ca~ (2+) -PKC signal transduction pathway and the apoptosis pathway of mitochondrial damage during apoptosis of retinal pigment epithelial cells (RPE) cells. Methods: primary culture of healthy human RPE cells was used for the 3-6 generation to be used in this experiment. Blue light (470nm-490nm), light intensity (2000 + 500) LX, after blue light irradiation time 6h, continue to culture 24h, establish a human RPE cell model with blue light damage. The apoptotic cells were identified by immunohistochemical (IHC) staining optical microscope, and the morphological changes of RPE and mitochondrial morphology were observed by transmission electron microscopy (TEM). The human RPE was cultured in vitro. The cells were randomly divided into 4 groups: group 1A: no light group; group 1B: light group; group 1C: Light + nifedipine (Nifedipine) group; 1D group: Light + (-) Bay K8644 group. RT-PCR technique was used to determine the L- type calcium channel of cell membrane (L-type calcium channel). Group 2B: light group; group 2C: Light + nifedipine group; group 2D: Light + calcium phosphate binding protein (Calphostin C) group; 2E group: Light + phorbol ester (PMA) group. Laser scanning confocal microscopy (LSCM) was used to detect the concentration of free Ca~ (2+) in RPE cells in each group and analyze the fluorescence intensity. Ern blot method was used to determine the expression of Caspase-9 protein in RPE cells. The RPE cells in vitro were randomly divided into 3 groups, 3A group: (500 + 100) LX, (2000 + 500) LX, (3000 + 500) LX light intensity, light time 6h, and cell termination culture time 24h, 3B group: (2000 + 500) light intensity, illumination time, lighting time, cell termination Culture time 24h; group 3C: (2000 + 500) LX light intensity, light time 6h, cell termination culture time after light 6h, 12h, 24h, 36h and 48h; control group: no light group, flow cytometry was used to detect cell mitochondrial transmembrane potential (delta M). The cultured human RPE cells were randomly divided into 2 groups: 4A group: illumination intensity (2000 + 500), illumination time, Cell terminating culture time after light was 6,12,24,36h; 4B group: light intensity (2000 + 500) LX, light time 12h, cell termination culture time 6h, 12h, 24h and 36h, enzyme linked immunosorbent assay (ELISA) determination of cytochrome C (Cyt C) concentration. The cultured human cells were divided into 2 groups: no light group; simple light group Lot method was used to determine the expression of Bax, Bcl-2, Bcl-xl protein. The experimental data were statistically analyzed by SPSS 20 statistical software. Results: the human RPE cells in vitro were in blue light (470-490nm), light intensity (2000 + 500) LX, light time 6h, cell apoptosis after the cell termination culture time 24h, IHC staining showed nuclear concentration, The chromatin was clustered into crescent shaped crescent shape, and the nucleus fragmentation was several. Under transmission electron microscope, the mitochondrial membrane breakage was observed, the crista completely disappeared, the chromatin polynuclear membrane edge was concentrated, and the expression of alpha 1C, alpha 1D, and alpha 1F subunit m RNA in a typical apoptotic karyotype change was statistically significant (F=4) (F=4). 5.92, P0.05, F=50.32, P0.05, F=18.50, P0.05).LSCM detection of intracellular free Ca~ (2+) concentration in RPE cells showed that the light group (2E group 2B group 2D group) was higher than the non light group, and the difference was statistically significant. The IA value of the control group (group 2A) (t=-9.869, P0.05). The intracellular Ca~ (2+) concentration and PKC activity by linear regression analysis showed that the intracellular Ca~ (2+) was positively correlated with PKC. The relative expression of -9 protein increased significantly (P0.05). The cell mitochondrial membrane potential (delta M) detected by flow cytometry showed that the mitochondrial membrane potential (delta M) of RPE began to decrease with the increase of light intensity (P0.05) in the 3A group, and 6h in 3B group, and the RPE mitochondrial membrane potential (delta M) decreased significantly in the 3B group, and the RPE mitochondrial membrane potential (delta M) was significantly decreased. 24 H, 36h, 48h compared with the control group, RPE mitochondrial membrane potential (P0.05) significantly decreased (P0.05).ELISA method for the determination of Cyt C results showed: 4A group: 6h treatment of 24h, compared with the control group. The expression of Bax, Bcl-2, Bcl-xl protein was measured with the addition of (P0.05).Western blot. The results showed that the expression of RPE cells in the light group (5B group) was higher than that in the non light group (5A group), and the expression of Bax increased, while Bcl-2, the Bcl-xl expression decreased, and the difference was significantly higher than that in the non light group, and the difference was significantly higher than that in the non light group. Statistical significance (P0.05). Conclusion: blue light can induce apoptosis of human RPE cells, and there are typical apoptotic karyotype changes and intracellular mitochondrial damage. Blue light irradiation can cause the L- calcium channel alpha 1C, alpha 1D, the m RNA expression of the alpha 1F subunit in the RPE cell membrane, the enhancement of the Ca~ (2+) fluorescence intensity and the PKC activity in the cell, and the intracellular relaxation. At the same time, there was a positive correlation between the concentration and PKC activity. At the same time, the mitochondrial membrane potential (delta M) of RPE cells decreased significantly, Cyt C was released, the apoptosis factor Bax and the anti apoptotic factor Bcl-2, Bcl-xl ratio down regulated, triggered the increase of the relative expression of caspase-9 protein, and the ultimate induction of apoptosis. Calcium ions participated in Ca~ (2+) -PKC signal at the same time. Conduction pathway promotes apoptosis and cell mitochondria apoptosis pathway, and plays a key role in initiating, regulating and promoting cell apoptosis.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R774

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