激肽释放酶结合蛋白抑制高糖诱导的人视网膜毛细血管内皮细胞增殖作用的研究

发布时间：2018-06-12 00:22

本文选题：激肽释放酶结合蛋白 + 人视网膜毛细血管内皮细胞　；参考：《苏州大学》2016年博士论文

【摘要】：糖尿病视网膜病变(Diabetic retinopathy,DR)是糖尿病常见的微血管并发症,是世界性的重要致盲性眼病,它的非增殖期表现包括微血管瘤、视网膜内出血、视网膜内微血管异常,增殖期表现为新生血管形成、玻璃体积血或视网膜前出血。眼内新生血管生成是增殖性糖尿病视网膜病变(Proliferative diabeticretinopathy,PDR)恶化和视力丧失的主要原因。新生血管导致的出血、渗出和增生牵引导致眼内结构和功能破坏,终至视力丧失。所以,寻找有效的方法来防止和治疗DR迫在眉睫。内源性血管生成抑制因子激肽释放酶结合蛋白(Kallistatin,Kallikrein-Binding Protein,KBP)是丝氨酸蛋白酶抑制剂家族中的一员,有多种生物功能,包括调节血压、抗炎、血管松弛和刺激内膜增生等。研究发现KBP是一种血管生成抑制剂,能够抑制肿瘤组织的新生血管反应,也可抑制大鼠后肢缺血模型的自发性血管生成,但是KBP对于人眼部视网膜新生血管的作用研究不多。本研究在人眼部组织标本实验及离体细胞培养实验中探讨了KBP与糖尿病视网膜病变新生血管形成的关系。第一部分KBP在增殖性糖尿病视网膜病变患者玻璃体中表达水平的研究目的探讨KBP是否参与糖尿病视网膜病变新生血管的形成。方法临床因特发性黄斑裂孔、特发性黄斑前膜和PDR需行玻璃体切割手术的患者,糖尿病患者根据1999年WHO诊断标准确诊为2型糖尿病。分为2组:无糖尿病病史的特发性黄斑裂孔、特发性黄斑前膜患者作为对照组;因PDR手术的患者,为PDR组。25G玻璃体切割手术标准三通道切口完成后,将1ml注射器接玻切头的抽吸通道侧,玻切头进入前部玻璃体腔,未开灌注前,高速切割并抽吸玻璃体保存。采用酶联免疫吸附测定法(ELISA)检测玻璃体中KBP的表达。结果ELISA检测结果显示对照组玻璃体KBP含量为38.48±1.97μg/ml,PDR组玻璃体KBP含量为18.35±2.74μg/ml,两组比较差异有统计学意义(t=16.59,P0.05)。结论糖尿病视网膜病变PDR期患者玻璃体中KBP表达下降,可能与PDR的形成相关,KBP可能对糖尿病视网膜新生血管的形成有抑制作用。第二部分KBP抑制高糖诱导的人视网膜毛细血管内皮细胞增殖作用的研究目的观察KBP对高糖诱导的人视网膜毛细血管内皮细胞增殖是否有抑制作用。方法1.将人视网膜毛细血管内皮细胞(Human retinal endothelial cells,HRECs)分成两组,分别加入5m M和30m M浓度糖溶液干预24h后提取细胞RNA,检测不同糖浓度对KBP及血管内皮生长因子(VEGF)表达的影响。2.将细胞分成4组:加5m M浓度葡萄糖,加30m M浓度葡萄糖,加30m M浓度葡萄糖及100n M KBP或1000n M KBP。将细胞加入96孔板中,每孔约1000细胞,待细胞贴壁,饥饿处理24h,加入不同浓度葡萄糖及KBP干预24h,再加入CCK-8 10μl,37℃培养箱放置2-4h,酶联仪检测每孔内450nm波长的吸光度(OD值),记录分析。收集细胞,进行western blot检测VEGF的蛋白表达。3.将细胞分成3组:加5m M浓度葡萄糖,加30m M浓度葡萄糖,加30m M浓度葡萄糖及1000n M KBP。细胞迁移用24孔板,Transwell小室装置,细胞饥饿过夜加入膜上层,下层加入不同浓度葡萄糖和KBP,24h后,记录膜下层的细胞数。4.将细胞分成3组:加5m M浓度葡萄糖,加30m M浓度葡萄糖,加30m M浓度葡萄糖及1000n M KBP。将HRECs以及不同浓度的葡萄糖及KBP加入到含Matrigel基质胶的96孔板内,培养箱培养8h后拍照,管腔数目统计分析。5.将细胞分成两组:一组导入干扰KBP基因表达的si RNA,一组导入空载si RNA。在5m M葡萄糖环境下进行CCK-8实验、细胞迁移实验、管腔形成实验。分别记录结果统计。结果1.RT-PCR结果显示,与5m M糖浓度相比,30m M糖浓度刺激HRECs中KBP及VEGF的表达(P0.05)。2.与5m M糖浓度相比,30m M糖浓度促进细胞的增殖迁移及管腔形成,加入了100n M KBP或1000n M KBP后,这些作用被抑制(P0.05)。3.VEGF的蛋白表达在30m M葡萄糖+1000nmol/L KBP组低于30m M葡萄糖组。4.在5m M糖浓度环境下,用KBP si RNA干扰KBP基因表达可以提高细胞增殖迁移管腔形成的能力(P0.05)。5.在5m M糖浓度环境下,用KBP si RNA干扰细胞KBP基因,VEGF蛋白表达增加。结论1.高糖促进HRECs KBP及VEGF基因表达。2.100n M KBP或1000n M KBP抑制高糖刺激诱导的HRECs增殖。3.1000n M KBP抑制高糖刺激诱导的HRECs中VEGF的表达。4.抑制KBP基因表达可以提高HRECs的增殖作用。5.抑制KBP基因提高HRECs中VEGF的表达。
[Abstract]:Diabetic retinopathy (DR) is a common microvascular complication of diabetes. It is an important blind eye disease in the world. Its non proliferative stages include microangioma, intraretinal hemorrhage, and microvascular abnormalities in the retina. The proliferative stage is neovascularization, vitreous blood or retinal hemorrhage. Angiogenesis is the main cause of Proliferative diabeticretinopathy (PDR) deterioration and loss of vision in proliferative diabetic retinopathy. Hemorrhage, exudation and hyperplastic traction caused by neovascularization lead to structural and functional damage to the eye, and end to visual loss. Therefore, the search for effective methods to prevent and treat DR is imminent. Kallistatin (Kallikrein-Binding Protein, KBP), a member of the serine protease inhibitor family, is a member of the serine protease inhibitor family. It has a variety of biological functions, including regulating blood pressure, anti-inflammatory, vascular relaxation, and stimulating intima hyperplasia. The development of KBP is a angiogenesis inhibitor that inhibits tumor tissue. The neonatal vascular response can also inhibit spontaneous angiogenesis in the rat hind limb ischemia model, but the effect of KBP on human retinal neovascularization is not much studied. In this study, the relationship between KBP and the formation of neovascularization in diabetic retinopathy was investigated in human ocular tissue specimens and in vitro culture experiments. The first part K Study of the expression level of BP in the vitreous body of patients with proliferative diabetic retinopathy to investigate whether KBP participates in the formation of neovascularization of diabetic retinopathy. Methods patients with idiopathic macular holes, idiopathic macular macular membrane and PDR need vitrectomy, and diabetic patients are determined according to the 1999 WHO diagnostic criteria. Type 2 diabetes mellitus. It was divided into 2 groups: the idiopathic macular hole without the history of diabetes, the patients with idiopathic macular membrane as the control group; the 1ml syringe received the vitreous head of the 1ml syringe after the three channel incision of the.25G vitrectomy, and the vitreous head entered the anterior vitreous cavity after the operation of the.25G vitrectomy. The expression of KBP in vitreous body was detected by enzyme linked immunosorbent assay (ELISA). Results the results of ELISA detection showed that the KBP content in the vitreous body of the control group was 38.48 + 1.97 mu g/ml and the KBP content in the vitreous body of PDR group was 18.35 + 2.74 mu g/ml, and the two groups were statistically significant (t=16.59, P0.05). Conclusion diabetes mellitus (t=16.59, P0.05). Conclusion diabetes mellitus. The decrease of KBP expression in vitreous body of patients with retinopathy PDR may be related to the formation of PDR. KBP may inhibit the formation of retinal neovascularization in diabetic retina. Second part KBP inhibits the proliferation of human retinal capillary endothelial cells induced by high glucose, and the aim of KBP to the human retinal capillary blood induced by high glucose Methods 1. Human retinal endothelial cells (HRECs) of human retinal capillary endothelial cells (HRECs) was divided into two groups, and 5m M and 30m M concentration solution were added to 24h to extract cell RNA, and the effects of different sugar concentrations on the expression of KBP and vascular endothelial growth factor were detected. 4 groups: 5m M concentration glucose, 30m M concentration glucose, 30m M concentration glucose and 100N M KBP or 1000N M KBP., cells were added to 96 Kong Banzhong, about 1000 cells per pore. 50nm wavelength absorbance (OD), recording analysis, collecting cells, Western blot detection of VEGF protein expression.3. into 3 groups: 5m M concentration of glucose, 30m M concentration of glucose, 30m M concentration glucose and 1000N cells migrating 24 orifice, cell starvation overnight into the upper layer, the lower layer added After different concentrations of glucose and KBP, 24h, the number of cells recorded in the lower layer of the membrane was divided into 3 groups: 5m M concentration glucose, 30m M concentration of glucose, 30m M concentration glucose and 1000N M KBP., and different concentrations of glucose and added into the 96 pore plate containing the matrix glue, culture box culture and the number of photographs Statistical analysis.5. divided the cells into two groups: one group introduced Si RNA that interfered with the expression of KBP gene, and a group of Si RNA. was introduced into the 5m M glucose environment for CCK-8 experiment, cell migration experiment, and lumen formation experiment. The results were recorded. Results 1.RT-PCR results showed that the concentration of saccharide concentration was compared with 5m M sugar concentration. Compared with the concentration of 5m M, the concentration of 30m M sugar promoted the proliferation and migration of the cells and the formation of the lumen. After adding 100N M KBP or 1000N M KBP, these effects were inhibited by the 100N M KBP or 1000N M KBP. The expression can improve the ability of cell proliferation and migration (P0.05).5. in 5m M glucose concentration environment, KBP Si RNA interferes with cell KBP gene and VEGF protein expression increases. Conclusion 1. high sugar promotes HRECs KBP and VEGF genes to inhibit high sugar stimulation induced proliferation inhibition of high glucose stimulation The expression of VEGF in induced HRECs,.4. inhibited KBP gene expression and increased the proliferation of HRECs..5. inhibited KBP gene and increased VEGF expression in HRECs.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R774.1;R587.2

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