本文關(guān)鍵詞：miR-204對年齡相關(guān)性白內(nèi)障晶狀體上皮細(xì)胞氧化損傷的作用及機制　出處：《青島大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： miR-204 TP53INP1 p53 年齡相關(guān)性白內(nèi)障 氧化損傷

【摘要】：目的:探討mi R-204對年齡相關(guān)性白內(nèi)障氧化損傷的作用及機制。方法:收集20例皮質(zhì)性年齡相關(guān)性白內(nèi)障患者的晶狀體前囊膜和20例眼庫正常供體眼的透明晶狀體前囊膜,采用基因芯片技術(shù)檢測年齡相關(guān)性白內(nèi)障及透明晶狀體前囊膜中差異表達的mi RNAs,實時熒光定量PCR法驗證兩組晶狀體前囊膜中部分mi RNAs的表達;生物信息學(xué)分析并預(yù)測差異mi RNAs的靶基因,并采用熒光素酶報告基因驗證。將人晶狀體上皮細(xì)胞系(HLECs-B3)接種于六孔板進行培養(yǎng),細(xì)胞培養(yǎng)液中添加200μM的H2O2作用6h,模擬LECs氧化損傷,分別在細(xì)胞培養(yǎng)液中分別添加相應(yīng)的轉(zhuǎn)染劑:將細(xì)胞分為模型對照組、mi R-204擬似物組、擬似物對照組、mi R-204拮抗物組、拮抗物對照組,未用H2O2處理的LECs作為正常對照組,轉(zhuǎn)染24h后采用實時定量-PCR法測定各組LECs中mi R-204 m RNA的表達以驗證轉(zhuǎn)染率;采用流式細(xì)胞儀測定體外培養(yǎng)的各組細(xì)胞的凋亡率;采用實時熒光定量PCR和Western blot法檢測體外培養(yǎng)的各組LECs中TP53INP1及p53 m RNA和蛋白的相對表達量。結(jié)果:基因芯片結(jié)果顯示年齡相關(guān)性白內(nèi)障患者的晶狀體前囊膜中mi R-204表達顯著下降;熒光定量PCR驗證結(jié)果與基因芯片結(jié)果一致,年齡相關(guān)性白內(nèi)障患者晶狀體前囊膜組mi R-204 m RNA相對表達量明顯低于正常對照組,差異有統(tǒng)計學(xué)意義(t=14.21,P0.05)。生物信息學(xué)及熒光素酶報告基因?qū)嶒灧治霾Ⅱ炞CTP53INP1為mi R-204靶基因之一;細(xì)胞實驗中,實時熒光定量PCR和Western blot法結(jié)果顯示模型對照組細(xì)胞中TP53INP1、p53 m RNA及蛋白的相對表達量明顯高于正常組對照,差異有統(tǒng)計學(xué)意義(m RNA:t=6.44、10.72,均P0.01;蛋白:t=11.71、t=19.4,均P0.01);mi R-204擬似物組細(xì)胞中TP53INP1、p53 m RNA和蛋白的相對表達量低于擬似物對照組,差異均有統(tǒng)計學(xué)意義(m RNA:t=-19.28、-6.50,均P0.05;蛋白:t=-10.58、-6.36,均P0.05);mi R-204拮抗物組TP53INP1、p53m RNA的相對表達量高于拮抗物對照組,差異均有統(tǒng)計學(xué)意義(m RNA:t=4.07、7.18,均P0.05;蛋白:t=3.74、10.58,均P0.05),正常對照組、模型對照組、mi R-204擬似物組、擬似物對照組、mi R-204拮抗物組、拮抗物對照組細(xì)胞凋亡率分別為(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)%、模型對照組細(xì)胞凋亡率明顯高于正常對照組,差異有統(tǒng)計學(xué)意義(t=-20.69,P0.01);mi R-204擬似物組細(xì)胞凋亡率明顯低于擬似物對照組,而mi R-204拮抗物組細(xì)胞凋亡率明顯高于拮抗物對照組,差異均有統(tǒng)計學(xué)意義(t=-12.20,P0.001;t=3.79,P0.05)。結(jié)論:mi R-204通過作用于靶基因TP53INP1在LECs中的表達而調(diào)控LECs的凋亡,從而對年齡相關(guān)性白內(nèi)障LECs發(fā)揮抗氧化損傷作用,該作用可能是通過影響TP53INP1-p53通路實現(xiàn)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 鹵0.12 and 4.90 鹵0.28 respectively. The percentage of cell apoptosis in the antagonist group was 2.60 鹵0.15 and 4.39 鹵0.20% respectively. The apoptotic rate in the model control group was significantly higher than that in the normal control group (5.74 鹵0.13% and 4.34 鹵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.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R776.1

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