miRNA-1调控人小梁网细胞功能及纤维连接蛋白的作用研究
发布时间：2018-09-11 14:21
【摘要】：目的:青光眼是一类因为病理性眼压升高导致视盘凹陷性萎缩以及视野缺损、缩小为主要特征的眼部疾病。小梁网房水流出阻力增加是青光眼视神经损害的主要原因,而原发性开角型青光眼(Primary Open Angle Glaucoma,POAG)异常沉积的细胞外基质(extracellular matrix,ECM)是房水自小梁网流出阻力增加、眼压升高的主要影响因素。本实验主要研究人眼小梁网细胞(Human Trabecular Meshwork Cells,HTMCs)中,micro RNA-1(mi RNA-1)对HTMCs细胞功能及纤维连接蛋白(Fibronectin,FN)的调控作用,为青光眼诊治开发新的靶点。方法:300μM过氧化氢(H2O2)刺激HTMCs 2h后,Real-time PCR检测细胞中mi RNA-1基因的表达变化。Lipofectamine 2000转染mi RNA-1过表达质粒,应用CCK-8实验,检测过表达mi RNA-1后对HTMCs增殖活性的影响,Transwell迁移实验检测HTMCs的迁移能力。Real-time PCR,western blot和免疫荧光检测过表达mi RNA-1后HTMCs的纤维连接蛋白(Fibronectin,FN)基因和蛋白的表达变化。双荧光素酶报告基因和生物信息学预测靶基因的方法来验证mi RNA-1对FN的靶定作用。结果:1)300u M H2O2刺激HTMCs 2h建立氧化应激模型后,相对于无血清处理组,mi RNA-1基因表达水平下调;FN的m RNA表达水平上调;2)而转染mi RNA-1质粒后,相对于对照组,q PCR结果显示mi RNA-1表达水平上调;CCK-8法检测转染质粒后HTMCs的增殖活性的变化,pc DNA3对照组相对表达量是0.84+0.01,pc DNA3/pri-1实验组相对表达量是0.93+0.03,两组相比差异具有显著性意义(p0.05);Transwell法检测转染质粒后HTMCs的迁移能力的变化,pc DNA3-对照组相对表达量是111+1.86,pc DNA3/pri-1实验组相对表达量是210+4.9,两组相比差异具有显著性意义(p0.05)。提示转染pc DNA3/pri-1质粒可以促进HTMCs的增殖活性和迁移能力;3)q PCR显示过表达mi RNA-1会抑制FNm RNA的表达水平,转染c DNA3/pri-mi RNA-1实验组相对表达量是0.66±0.12,;western blot显示过表达mi RNA-1会抑制FN蛋白水平的表达,转染c DNA3/pri-mi RNA-1实验组相对表达量是0.62±0.11;免疫荧光的结果进一步验证了过表达mi RNA-1对FN蛋白水平的影响;4)三种mi RNA靶基因预测软件和双荧光素酶报告基因实验说明mi RNA-1可以靶定FN结论:在HTMCs氧化应激模型中,mi RNA-1表达水平下调和FN的m RNA表达水平上调;而过表达的mi RNA-1可以上调HTMCs的增殖活性和迁移能力。mi RNA-1可以直接靶定FN并抑制FN的表达,这可能为青光眼的治疗提供新途径。
[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 卤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 卤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.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R775
本文编号：2236939
[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 卤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 卤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.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R775
【参考文献】
相关期刊论文 前1条
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
，本文编号：2236939
本文链接：https://www.wllwen.com/yixuelunwen/wuguanyixuelunwen/2236939.html
最近更新
教材专著
