miR-204对年龄相关性白内障晶状体上皮细胞氧化损伤的作用及机制

发布时间：2018-01-03 16:00

本文关键词：miR-204对年龄相关性白内障晶状体上皮细胞氧化损伤的作用及机制　出处：《青岛大学》2016年硕士论文　论文类型：学位论文

【摘要】：目的:探讨mi R-204对年龄相关性白内障氧化损伤的作用及机制。方法:收集20例皮质性年龄相关性白内障患者的晶状体前囊膜和20例眼库正常供体眼的透明晶状体前囊膜,采用基因芯片技术检测年龄相关性白内障及透明晶状体前囊膜中差异表达的mi RNAs,实时荧光定量PCR法验证两组晶状体前囊膜中部分mi RNAs的表达;生物信息学分析并预测差异mi RNAs的靶基因,并采用荧光素酶报告基因验证。将人晶状体上皮细胞系(HLECs-B3)接种于六孔板进行培养,细胞培养液中添加200μM的H2O2作用6h,模拟LECs氧化损伤,分别在细胞培养液中分别添加相应的转染剂:将细胞分为模型对照组、mi R-204拟似物组、拟似物对照组、mi R-204拮抗物组、拮抗物对照组,未用H2O2处理的LECs作为正常对照组,转染24h后采用实时定量-PCR法测定各组LECs中mi R-204 m RNA的表达以验证转染率;采用流式细胞仪测定体外培养的各组细胞的凋亡率;采用实时荧光定量PCR和Western blot法检测体外培养的各组LECs中TP53INP1及p53 m RNA和蛋白的相对表达量。结果:基因芯片结果显示年龄相关性白内障患者的晶状体前囊膜中mi R-204表达显著下降;荧光定量PCR验证结果与基因芯片结果一致,年龄相关性白内障患者晶状体前囊膜组mi R-204 m RNA相对表达量明显低于正常对照组,差异有统计学意义(t=14.21,P0.05)。生物信息学及荧光素酶报告基因实验分析并验证TP53INP1为mi R-204靶基因之一;细胞实验中,实时荧光定量PCR和Western blot法结果显示模型对照组细胞中TP53INP1、p53 m RNA及蛋白的相对表达量明显高于正常组对照,差异有统计学意义(m RNA:t=6.44、10.72,均P0.01;蛋白:t=11.71、t=19.4,均P0.01);mi R-204拟似物组细胞中TP53INP1、p53 m RNA和蛋白的相对表达量低于拟似物对照组,差异均有统计学意义(m RNA:t=-19.28、-6.50,均P0.05;蛋白:t=-10.58、-6.36,均P0.05);mi R-204拮抗物组TP53INP1、p53m RNA的相对表达量高于拮抗物对照组,差异均有统计学意义(m RNA:t=4.07、7.18,均P0.05;蛋白:t=3.74、10.58,均P0.05),正常对照组、模型对照组、mi R-204拟似物组、拟似物对照组、mi R-204拮抗物组、拮抗物对照组细胞凋亡率分别为(1.31±0.12)%、(4.90±0.28)%、(2.60±0.15)%、(4.39±0.20)%、(5.74±0.13)%和(4.34±0.63)%、模型对照组细胞凋亡率明显高于正常对照组,差异有统计学意义(t=-20.69,P0.01);mi R-204拟似物组细胞凋亡率明显低于拟似物对照组,而mi R-204拮抗物组细胞凋亡率明显高于拮抗物对照组,差异均有统计学意义(t=-12.20,P0.001;t=3.79,P0.05)。结论:mi R-204通过作用于靶基因TP53INP1在LECs中的表达而调控LECs的凋亡,从而对年龄相关性白内障LECs发挥抗氧化损伤作用,该作用可能是通过影响TP53INP1-p53通路实现的。
[Abstract]:Objective: to investigate the effect and mechanism of mi R-204 on oxidative injury of age-related cataract. The anterior capsule of lens was collected in 20 cases of cortical age-related cataract and 20 cases of normal donor eyes. The differential expression of mi RNAs in anterior capsule of age-related cataract and transparent lens was detected by gene chip technique. The expression of partial mi RNAs in anterior capsule of lens was detected by real-time fluorescence quantitative PCR. The target genes of the differential mi RNAs were analyzed and predicted by bioinformatics and verified by luciferase reporter gene. The human lens epithelial cell line (HL-ECs-B3) was cultured on a six-hole plate. The cells were treated with 200 渭 M H2O2 for 6 h to simulate the oxidative damage of LECs. The cells were divided into two groups: model control group. Mi R-204 mimic group, mimic control group, antagonist group, antagonist control group, LECs without H _ 2O _ 2 treatment as normal control group. After 24 hours of transfection, the expression of mi R-204 m RNA in LECs was measured by real-time quantitative PCR to verify the transfection rate. The apoptosis rate of each group was measured by flow cytometry. Real-time fluorescence quantitative PCR and Western blot were used to detect the relative expression of TP53INP1 and p53 m RNA and protein in LECs cultured in vitro. The results of gene chip showed that the expression of miR-204 in the anterior capsule of the patients with age-related cataract was significantly lower than that in the patients with age-related cataract. The results of fluorescence quantitative PCR were consistent with those of gene chip. The relative expression of mi R-204m RNA in patients with age-related cataract was significantly lower than that in normal controls. The difference was statistically significant (P 0.05). Bioinformatics and luciferase reporter gene were analyzed and verified that TP53INP1 was one of the target genes of mi R-204. In cell experiment, the results of real-time fluorescence quantitative PCR and Western blot showed TP53INP1 in the model control group. The relative expression of p53 m RNA and protein was significantly higher than that of normal control group, and the difference was statistically significant (P 0.01). The protein was 11.71 and 19.4, all of which were P0.01C; The relative expression of p53 m RNA and protein in the mimic group of miR-204 was lower than that in the control group. The differences were statistically significant (P 0.05). The protein content was 10. 58%-6. 36%, respectively (P0. 05%). The relative expression of TP53INP1p53m RNA in MIR-204 antagonist group was higher than that in antagonist control group, and the difference was statistically significant. 7.18, P 0.05; Protein: t0. 3. 74n 10.58, P0.05, normal control group, model control group, model control group, mimic group, mimic control group, mimic control group, Mi R-204 antagonist group. The rate of apoptosis in the antagonist control group was 1.31 卤0.12 and 4.90 卤0.28 respectively. The percentage of cell apoptosis in the antagonist group was 2.60 卤0.15 and 4.39 卤0.20% respectively. The apoptotic rate in the model control group was significantly higher than that in the normal control group (5.74 卤0.13% and 4.34 卤0.63%), and the difference was statistically significant (P < 0.05). P0.01; The apoptotic rate of the mimicry group was significantly lower than that of the control group, while the apoptosis rate of the antagonist group was significantly higher than that of the antagonist group. The difference was statistically significant (P 0.001). Conclusion: MiR-204 regulates the apoptosis of LECs by acting on the expression of target gene TP53INP1 in LECs. Thus, the anti-oxidative damage of LECs in age-related cataract may be achieved by affecting the TP53INP1-p53 pathway.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R776.1

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