维拉帕米对视网膜Müller细胞分泌VEGF的影响
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摘要
糖尿病性视网膜病变(Diabetic Retinopathy,DR)是糖尿病常见的微血管并发症,是发生双眼盲的最主要疾病。如何有效的防治DR,是现今世界研究的热点。DR的主要病理表现为视网膜病理性新生血管形成,功能不全的新生血管伴随视网膜内皮细胞向玻璃体腔增殖,而后形成瘢痕,最终瘢痕牵拉视网膜而导致失明。因此,有效的预防新生血管的形成,可减缓糖尿病性视网膜病变的进展,对改善患者的生存质量具有重要意义。DR发生时出现的病理性血管新生,与VEGF的关系极为密切。VEGF可增加血管的通透性,促进内皮细胞迁移和增殖,从而促进新生血管的形成。视网膜Müller细胞是特化的神经胶质细胞,是人类视网膜最主要的神经胶质细胞,它贯穿整个内界膜至外界膜的视网膜,对维持视网膜内环境的稳定起重要作用。由Müller细胞衍生的VEGF,是血管渗漏、视网膜前及视网膜内病理性新生血管形成的主要因素。我们前期的实验已经证实,Müller细胞存在L-型钙离子通道,且应用L-型钙离子通道阻滞剂维拉帕米,可抑制Müller细胞外钙离子内流。本实验的目的在于研究钙通道阻滞剂维拉帕米,是否能够通过抑制Müller细胞钙离子内流,从而抑制Müller细胞VEGF的表达,探寻钙通道及VEGF表达的内在联系,为DR的防治提供新的思路。
本实验采用组织块悬浮培养的方法建立了体外兔视网膜Müller细胞的原代培养体系,并分为正常对照组、高浓度葡萄糖组、高浓度胰岛素组、高糖高胰岛素组、维拉帕米对照组、维拉帕米高糖组、维拉帕米高胰岛素组、及维拉帕米高糖高胰岛素组,应用免疫细胞化学染色法测定维拉帕米对高糖、高胰岛素环境下体外培养的视网膜Müller细胞分泌VEGF的影响。结果显示:高糖组与正常对照组相比VEGF表达在第1天有显著差异(P<0.01),第3天及第5天有差异(P<0.05);维拉帕米对照组与正常对照组相比VEGF的表达在第1天、第3天无明显差异(P>0.05),第5天有差异(P<0.05);维拉帕米高糖组与高糖组相比VEGF的表达,第1天有显著差异(P<0.01)第3天及第5天有差异(P<0.05)。结论:高糖可使体外培养的兔视网膜Müller细胞VEGF表达增加;维拉帕米可以使高糖条件下体外培养的兔视网膜Müller细胞VEGF表达下降;维拉帕米对正常对照组VEGF的表达无显著影响。本实验为钙离子及钙通道在DR形成中的作用机制及寻找新的治疗方法提供了理论和实验依据。
Diabetic retinopathy (DR) is a common microvascular complications of diabetes.DR is also one of the most important leading cause of double eye blindness. Retinal neovascularization is the main pathological manifestation of DR. Pathological neovessels accompany with retinal endothelial cells proliferate into vitreous cavity, then form scars. The scars traction result in retinal detachment, which cause blindness in the end. It is considered that VEGF is highly associated with the pathological neovascularization occurs in DR. VEGF can increase vascular permeability, promote endothelial cell migration and proliferation, thereby promoting angiogenesis. Müller cell-derived VEGF is a major factor contributes to vascular leakage and pathological neovascularization of retina. Our previous experiments have confirmed that Müller cells exist L-type calcium channel, and verapamil,the L-type calcium channel blocker,can inhibit the extracellular calcium influx of Müller cell. The purpose of this experiment is to study whether verapamil is able to inhibit the expression of VEGF in Müller cells by inhibiting the calcium influx of Müller cells, and to explore the relationship between VEGF expression and calcium channel, and to provide new ideas for the prevention and the treatment of DR .
Objective: To observe the effects of verapamil on VEGF expression in cultured retinal Müller cells.
Method:Establish primary culture system of rabbit retina Müller cell by using tissue piece suspension culture, and divided into normal control group, high concentrations of glucose group, high concentrations of insulin group, high glucose and high insulin group, verapamil control group, verapamil high glucose group, verapamil high insulin group, verapamil high glucose and high insulin group. Use immunohistochemical staining to determinate the effects of verapamil on the expression of VEGF in cultuered retinal Müller cells under normal control, high concentrations of glucose, high concentrations of insulin, high glucose and high insulin conditions.
Result: VEGF immunohistochemical staining results:the VEGF expression in Müller cells of high glucose group were significantly higher(P<0.01) on the first day, and higher(P<0.05)on the third and the fifth day compared with the normal control group; the VEGF expression in Müller cells of verapamil high glucose group were significantly lower(P<0.01)on the first day, and lower(P<0.05)on the third and the fifth day compared with the high glucose group; the VEGF expression in Müller cells of verapamil control group compared with the control group had no significant difference (P>0.05) on the first and third day, and were lower(P <0.05)on the fifth day.
Conclusion: 1. High glucose can increase the expression of VEGF in cultured retinal Müller cells. 2. Verapamil has no significant effect on the expression of VEGF in cultured retinal Müller of normal control group. 3.Verapamil can decrease the expression of VEGF in cultured retinal Müller cells under high glucose condition.
本实验采用组织块悬浮培养的方法建立了体外兔视网膜Müller细胞的原代培养体系,并分为正常对照组、高浓度葡萄糖组、高浓度胰岛素组、高糖高胰岛素组、维拉帕米对照组、维拉帕米高糖组、维拉帕米高胰岛素组、及维拉帕米高糖高胰岛素组,应用免疫细胞化学染色法测定维拉帕米对高糖、高胰岛素环境下体外培养的视网膜Müller细胞分泌VEGF的影响。结果显示:高糖组与正常对照组相比VEGF表达在第1天有显著差异(P<0.01),第3天及第5天有差异(P<0.05);维拉帕米对照组与正常对照组相比VEGF的表达在第1天、第3天无明显差异(P>0.05),第5天有差异(P<0.05);维拉帕米高糖组与高糖组相比VEGF的表达,第1天有显著差异(P<0.01)第3天及第5天有差异(P<0.05)。结论:高糖可使体外培养的兔视网膜Müller细胞VEGF表达增加;维拉帕米可以使高糖条件下体外培养的兔视网膜Müller细胞VEGF表达下降;维拉帕米对正常对照组VEGF的表达无显著影响。本实验为钙离子及钙通道在DR形成中的作用机制及寻找新的治疗方法提供了理论和实验依据。
Diabetic retinopathy (DR) is a common microvascular complications of diabetes.DR is also one of the most important leading cause of double eye blindness. Retinal neovascularization is the main pathological manifestation of DR. Pathological neovessels accompany with retinal endothelial cells proliferate into vitreous cavity, then form scars. The scars traction result in retinal detachment, which cause blindness in the end. It is considered that VEGF is highly associated with the pathological neovascularization occurs in DR. VEGF can increase vascular permeability, promote endothelial cell migration and proliferation, thereby promoting angiogenesis. Müller cell-derived VEGF is a major factor contributes to vascular leakage and pathological neovascularization of retina. Our previous experiments have confirmed that Müller cells exist L-type calcium channel, and verapamil,the L-type calcium channel blocker,can inhibit the extracellular calcium influx of Müller cell. The purpose of this experiment is to study whether verapamil is able to inhibit the expression of VEGF in Müller cells by inhibiting the calcium influx of Müller cells, and to explore the relationship between VEGF expression and calcium channel, and to provide new ideas for the prevention and the treatment of DR .
Objective: To observe the effects of verapamil on VEGF expression in cultured retinal Müller cells.
Method:Establish primary culture system of rabbit retina Müller cell by using tissue piece suspension culture, and divided into normal control group, high concentrations of glucose group, high concentrations of insulin group, high glucose and high insulin group, verapamil control group, verapamil high glucose group, verapamil high insulin group, verapamil high glucose and high insulin group. Use immunohistochemical staining to determinate the effects of verapamil on the expression of VEGF in cultuered retinal Müller cells under normal control, high concentrations of glucose, high concentrations of insulin, high glucose and high insulin conditions.
Result: VEGF immunohistochemical staining results:the VEGF expression in Müller cells of high glucose group were significantly higher(P<0.01) on the first day, and higher(P<0.05)on the third and the fifth day compared with the normal control group; the VEGF expression in Müller cells of verapamil high glucose group were significantly lower(P<0.01)on the first day, and lower(P<0.05)on the third and the fifth day compared with the high glucose group; the VEGF expression in Müller cells of verapamil control group compared with the control group had no significant difference (P>0.05) on the first and third day, and were lower(P <0.05)on the fifth day.
Conclusion: 1. High glucose can increase the expression of VEGF in cultured retinal Müller cells. 2. Verapamil has no significant effect on the expression of VEGF in cultured retinal Müller of normal control group. 3.Verapamil can decrease the expression of VEGF in cultured retinal Müller cells under high glucose condition.
引文
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