加压培养对视网膜神经节细胞存活状态的影响

发布时间：2018-02-04 17:41

本文关键词： 视网膜神经节细胞压力凋亡 OPTN基因青光眼　出处：《中国医科大学》2010年硕士论文　论文类型：学位论文

【摘要】： 青光眼是世界第二大致盲性眼病,视网膜神经节细胞(Retinal ganglion cells, RGCs)的凋亡是青光眼发病的基本病理机制,眼内压升高是最重要的危险因素,但是眼内压引起RGCs凋亡的确切机制还不清楚。有研究表明遗传因素在青光眼的发病中起着重要的作用。OPTN基因是POAG确定的两个致病基因之一,研究证实其在细胞存活和凋亡中起到调控作用。本实验对RGC-5 (Retinal ganglion cell-5)进行加压培养,通过对不同压力培养条件下视网膜神经节细胞的生长存活率、凋亡情况及OPTN基因表达等指标的观察,分析不同压力培养条件下RGC-5细胞的活性和总体存活状态,从而初步探讨压力对视网膜神经节细胞的直接作用及影响。方法对传代培养的RGC-5分别给予0、20、40、60、80mmHg压力48小时,应用MTT方法和流式细胞学检测方法检测压力对RGC-5细胞存活率和凋亡的影响。应用免疫组织化学方法、Q-Real-time PCR和Western-Blot的方法检测压力作用下培养的RGC-5细胞OPTN基因的表达量。统计结果均以均数±标准差表示,各组间的比较用配对t检验分析方法,均用SPSS13.0软件进行统计分析。结果 1、培养的RGC-5细胞呈梭型,平均分裂时间为2-3天左右,细胞生长旺盛。 2、MTT和流式检测结果显示在加压处理48小时后RGC-5存活率随着压力的增高逐渐下降,各组间差异有显著的统计学意义(P0.01)；RGC-5细胞凋亡率不断增高,在40mmHg时以早期凋亡为主,60mmHg时以晚期凋亡为主,在80mmHg时细胞以死亡为主。 3、免疫组织化学检测结果显示各加压组平均积分光密度(Integrated optical density average,IODA)值为20mmHg组：(5.32±0.42)；40mmHg组：(3.1±0.85)；60mmHg组：(5.8±1.13)：80mmHg组：(8.9±1.26)；对照组：(10.58±1.45)。各加压组IODA值与对照组比较,均有统计学差异(P0.05)；采用Q-Realtime-PCR定量分析,Gapdh作为内参,得出在20、40、60、80mmHg压力作用下RGC-5细胞内OPTN基因mRNA的表达量分别为对照组的0.12倍,0.04倍,0.35倍,0.48倍,各加压组细胞mRNA表达量与对照组比较均有统计学差异(P0.05)；采用Western blot方法分析,Gapdh作为内参,得出在20、40、60、80mmHg压力作用下RGC-5细胞Optineurin蛋白表达量分别为对照组的0.91倍,0.79倍,0.86倍,0.96倍,20mmHg和80mmHg加压组与对照组比较无统计学差异(P0.05),40mmHg和60mmHg加压组与对照组比较有显著的统计学差异(P0.01) 结论 1、压力直接影响培养RGC-5的存活状态； 2、随绝对压力的不断增高,培养RGC-5细胞存活率不断降低,细胞凋亡和死亡趋见明显。 3、随绝对压力的不断增高,RGC-5细胞OPTN基因表达水平的V字形波动提示在不同压力条件下OPTN基因对培养视网膜神经节细胞的存活的调控作用可能不同,存在着细胞受损晚期的保护性作用可能。
[Abstract]:Glaucoma is the world second big blind eye disease, retinal ganglion cells (Retinal ganglion cells, RGCs) apoptosis is the basic pathogenesis of glaucoma, increased intraocular pressure is the most important risk factor, but the exact mechanism of RGCs intraocular pressure caused by apoptosis is unclear. Studies have shown that plays in the pathogenesis of genetic factors in glaucoma the important role of.OPTN gene is one of the two genes identified by POAG, proved that it plays a regulatory role in cell survival and apoptosis. The experiments of RGC-5 (Retinal ganglion cell-5) the pressure training, according to the different pressure conditions of retinal ganglion cells survival rate, apoptosis and expression of OPTN gene other indicators, analysis of the cultured RGC-5 cell activity and survival status of the different pressure, so as to investigate the pressure on retinal ganglion cells The direct effect and influence of the cell.
Method
The cultured RGC-5 were given 0,20,40,60,80mmHg pressure for 48 hours, and affect the rate of apoptosis and flow cytometry using MTT method detection method of pressure on the survival of RGC-5 cells. Immunohistochemical method, Q-Real-time PCR method and Western-Blot detection for pressure expression of RGC-5 OPTN gene in cultured cells. The statistical results are the mean and standard deviation, paired t test analysis method comparison between two groups, were analyzed by using SPSS13.0 software.
Result
1, the cultured RGC-5 cells were spindle shaped, with an average split time of about 2-3 days, and the cells grew exuberant.
2, MTT and flow cytometry showed in the pressure after 48 hours of treatment RGC-5 survival rate decreased gradually with the increasing pressure, and there were significant differences between the groups (P0.01); the apoptosis rate of RGC-5 cells increased, mainly at the early stage of apoptosis of 40mmHg, 60mmHg in late apoptosis in 80mmHg cells by. Death.
3. Immunohistochemical detection results showed that the compression group average integral optical density (Integrated optical density average, IODA) = 20mmHg group: (5.32 + 0.42); group 40mmHg (3.1 + 0.85); group 60mmHg (5.8 + 1.13):80mmHg group: (8.9 + 1.26); control group: (10.58 + 1.45). The IODA value of compression group compared with the control group, were statistically significant (P0.05); the quantitative analysis of Q-Realtime-PCR, Gapdh as reference, the expression of RGC-5 OPTN gene in mRNA cells under pressure of 20,40,60,80mmHg was 0.12 times, 0.04 times that of the control group, 0.35 times, 0.48 times each. The expression of mRNA in cell compression group and control group were statistically significant (P0.05); Western analysis by using blot method, Gapdh as reference, obtained under pressure of 20,40,60,80mmHg RGC-5 cells Optineurin protein expression were 0.91 times, 0.79 times that of the control group, 0.86 times 0.96 times, there was no significant difference between the 20mmHg and 80mmHg compression group and the control group (P0.05). There was a significant difference between the 40mmHg and 60mmHg compression group and the control group (P0.01).
conclusion
1, the pressure directly affected the survival of RGC-5.
2, with the increase of absolute pressure, the survival rate of RGC-5 cells was decreased, and apoptosis and death were obvious.
3, with the increase of absolute pressure, the V shape fluctuation of OPTN gene expression level in RGC-5 cells suggests that the regulation of OPTN gene on the survival of cultured retinal ganglion cells may be different under different pressure conditions, and there may be a protective effect of late stage of cell damage.

【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R775.9

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