【摘要】：目的:青光眼是一類因?yàn)椴±硇匝蹓荷邔?dǎo)致視盤凹陷性萎縮以及視野缺損、縮小為主要特征的眼部疾病。小梁網(wǎng)房水流出阻力增加是青光眼視神經(jīng)損害的主要原因,而原發(fā)性開角型青光眼(Primary Open Angle Glaucoma,POAG)異常沉積的細(xì)胞外基質(zhì)(extracellular matrix,ECM)是房水自小梁網(wǎng)流出阻力增加、眼壓升高的主要影響因素。本實(shí)驗(yàn)主要研究人眼小梁網(wǎng)細(xì)胞(Human Trabecular Meshwork Cells,HTMCs)中,micro RNA-1(mi RNA-1)對(duì)HTMCs細(xì)胞功能及纖維連接蛋白(Fibronectin,FN)的調(diào)控作用,為青光眼診治開發(fā)新的靶點(diǎn)。方法:300μM過氧化氫(H2O2)刺激HTMCs 2h后,Real-time PCR檢測(cè)細(xì)胞中mi RNA-1基因的表達(dá)變化。Lipofectamine 2000轉(zhuǎn)染mi RNA-1過表達(dá)質(zhì)粒,應(yīng)用CCK-8實(shí)驗(yàn),檢測(cè)過表達(dá)mi RNA-1后對(duì)HTMCs增殖活性的影響,Transwell遷移實(shí)驗(yàn)檢測(cè)HTMCs的遷移能力。Real-time PCR,western blot和免疫熒光檢測(cè)過表達(dá)mi RNA-1后HTMCs的纖維連接蛋白(Fibronectin,FN)基因和蛋白的表達(dá)變化。雙熒光素酶報(bào)告基因和生物信息學(xué)預(yù)測(cè)靶基因的方法來驗(yàn)證mi RNA-1對(duì)FN的靶定作用。結(jié)果:1)300u M H2O2刺激HTMCs 2h建立氧化應(yīng)激模型后,相對(duì)于無血清處理組,mi RNA-1基因表達(dá)水平下調(diào);FN的m RNA表達(dá)水平上調(diào);2)而轉(zhuǎn)染mi RNA-1質(zhì)粒后,相對(duì)于對(duì)照組,q PCR結(jié)果顯示mi RNA-1表達(dá)水平上調(diào);CCK-8法檢測(cè)轉(zhuǎn)染質(zhì)粒后HTMCs的增殖活性的變化,pc DNA3對(duì)照組相對(duì)表達(dá)量是0.84+0.01,pc DNA3/pri-1實(shí)驗(yàn)組相對(duì)表達(dá)量是0.93+0.03,兩組相比差異具有顯著性意義(p0.05);Transwell法檢測(cè)轉(zhuǎn)染質(zhì)粒后HTMCs的遷移能力的變化,pc DNA3-對(duì)照組相對(duì)表達(dá)量是111+1.86,pc DNA3/pri-1實(shí)驗(yàn)組相對(duì)表達(dá)量是210+4.9,兩組相比差異具有顯著性意義(p0.05)。提示轉(zhuǎn)染pc DNA3/pri-1質(zhì)粒可以促進(jìn)HTMCs的增殖活性和遷移能力;3)q PCR顯示過表達(dá)mi RNA-1會(huì)抑制FNm RNA的表達(dá)水平,轉(zhuǎn)染c DNA3/pri-mi RNA-1實(shí)驗(yàn)組相對(duì)表達(dá)量是0.66±0.12,;western blot顯示過表達(dá)mi RNA-1會(huì)抑制FN蛋白水平的表達(dá),轉(zhuǎn)染c DNA3/pri-mi RNA-1實(shí)驗(yàn)組相對(duì)表達(dá)量是0.62±0.11;免疫熒光的結(jié)果進(jìn)一步驗(yàn)證了過表達(dá)mi RNA-1對(duì)FN蛋白水平的影響;4)三種mi RNA靶基因預(yù)測(cè)軟件和雙熒光素酶報(bào)告基因?qū)嶒?yàn)說明mi RNA-1可以靶定FN結(jié)論:在HTMCs氧化應(yīng)激模型中,mi RNA-1表達(dá)水平下調(diào)和FN的m RNA表達(dá)水平上調(diào);而過表達(dá)的mi RNA-1可以上調(diào)HTMCs的增殖活性和遷移能力。mi RNA-1可以直接靶定FN并抑制FN的表達(dá),這可能為青光眼的治療提供新途徑。
[Abstract]:Objective: glaucoma is a kind of ocular disease which is characterized by concave atrophy of optic disc and defect of visual field due to pathological elevation of intraocular pressure. The increase of aqueous outflow resistance of trabecular meshwork was the main cause of optic nerve damage in glaucoma, while the abnormal deposition of extracellular matrix (extracellular matrix,ECM) in primary open-angle glaucoma (Primary Open Angle Glaucoma,POAG) was the increase of outflow resistance of aqueous humor. The main influencing factors of intraocular pressure elevation. The aim of this study was to investigate the regulatory effects of micro RNA-1 (mi RNA-1) on the function of HTMCs cells and fibronectin (Fibronectin,FN) in human trabecular meshwork (Human Trabecular Meshwork Cells,HTMCs) cells, and to develop a new target for the diagnosis and treatment of glaucoma. Methods the expression of mi RNA-1 gene was detected by real-time PCR 2 hours after HTMCs was stimulated by hydrogen peroxide (H2O2). Lipofectamine 2000 was transfected into mi RNA-1 overexpression plasmid. CCK-8 assay was used. The effect of overexpression of mi RNA-1 on the proliferative activity of HTMCs was detected by Transwell migration assay. Real-time PCR,western blot of HTMCs and Fibronectin,FN gene and protein expression of HTMCs after mi RNA-1 expression were detected by immunofluorescence. Double luciferase reporter gene and bioinformatics method were used to predict target gene to verify the targeting effect of mi RNA-1 on FN. Results the oxidative stress model was established by stimulating HTMCs with 300U M H2O2 for 2 h. The expression level of RNA-1 gene was down-regulated and the m RNA expression level of FN was up-regulated in serum free treatment group (P < 0.05), and then transfected into mi RNA-1 plasmid. The expression level of mi RNA-1 was up-regulated compared with that of control group. CCK-8 method was used to detect the proliferative activity of HTMCs after transfection. The relative expression of PC DNA3 in the control group was 0.84. 01 and the relative expression of PC DNA3/pri-1 was 0. 93. 03, there was a significant difference between the two groups. There was significant difference between the two groups (p0.05). The relative expression of PC DNA3- in the control group was 2104.9. The difference between the two groups was significant (p0.05). The relative expression level of pc DNA3- in the control group was 2104.9, and the difference between the two groups was significant (p0.05). The relative expression of pc DNA3- in the control group was 2104.9, and there was a significant difference between the two groups (p0.05). It was suggested that transfection of pc DNA3/pri-1 plasmid could promote the proliferation and migration of HTMCs. The results showed that overexpression of mi RNA-1 could inhibit the expression of FNm RNA. The relative expression of c DNA3/pri-mi RNA-1 in the experimental group was 0.66 鹵0.12 blot. The overexpression of mi RNA-1 could inhibit the expression of FN protein. The relative expression of c DNA3/pri-mi RNA-1 was 0.62 鹵0.11.The results of immunofluorescence further verified the effect of overexpression of mi RNA-1 on the level of FN protein. 4) three mi RNA target gene prediction software and double-luciferase reporter gene experiment showed mi RNA-1. Conclusion: in the oxidative stress model of HTMCs, the expression of MMI RNA-1 was down-regulated and the expression of m RNA of FN was up-regulated. Overexpression of mi RNA-1 can up-regulate the proliferative activity and migration ability of HTMCs. Mi RNA-1 can directly target FN and inhibit the expression of FN, which may provide a new approach for the treatment of glaucoma.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R775

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 Chao Hu;Shi-Qiang Shen;Zhong-Hui Cui;Zu-Bing Chen;Wei Li;;Effect of microRNA-1 on hepatocellular carcinoma tumor endothelial cells[J];World Journal of Gastroenterology;2015年19期

，

本文編號(hào)：2236939