富含半胱氨酸蛋白61（CYR61）参与糖尿病视网膜病变发病的实验研究

发布时间：2019-07-02 10:08

【摘要】： 目的 1.研究CYR61 (cysteine-rich 61; CCN1)对脉络膜视网膜内皮细胞(RF／6A细胞系)增殖、迁移及形成管状结构的影响。 2.研究CYR61在糖尿病小鼠和正常小鼠视网膜的表达和分布情况及其差异。 3.研究增殖性糖尿病视网膜病变(PDR)患者玻璃体CYR61的表达水平,以及CYR61在视网膜增殖膜内的表达和分布。方法 1.通过MTT比色实验观察CYR61对RF／6A细胞增殖能力的影响：应用Transwell小室观察CYR61作用下,RF／6A细胞迁移能力的变化；采用Matrigel基质胶,观察CYR61对RF／6A细胞形成管状结构的影响。 2.腹腔注射链脲佐菌素(STZ)制作糖尿病小鼠模型,通过免疫组织化学方法观察CYR61在糖尿病小鼠视网膜的表达和分布,利用逆转录-聚合酶链反应(RT-PCR)方法分析视网膜Cyr61 mRNA的表达情况。 3.收集玻璃体切除手术患者的玻璃体液和视网膜前膜,利用酶联免疫吸附实验(ELISA)方法测定PDR患者和非糖尿病患者玻璃体CYR61的浓度,利用免疫组织化学方法观察CYR61在PDR、增殖性玻璃体视网膜病变(PVR)和特发性黄斑前膜患者的视网膜前膜中的表达和分布情况。结果 1.用不同浓度的CYR61(0,5ng/mL, 10ng/mL,50ng/mL, 100ng/mL,500ng/mL)干预RF／6A细胞72h,随着CYR61浓度增加,MTT比色实验的吸光度A490nm值升高,其差异有统计学意义(p=0.000)；400ng/mL Cry61干预RF／6A细胞不同时间(0,12h,24h,48h,72h),随着时间延长,A490nm值升高,其差异有统计学意义(Jp=0.000)。Transwell小室实验中,随着CYR61浓度增加,迁移的RF／6A细胞增加(p=0.000),400ng/ml CYR61促进细胞迁移的能力与80ng/ml VEGF相当,而加入抗CYR61抗体则可以起到显著的抑制作用。Matrigel基质胶实验中,随着CYR61浓度增加,RF／6A细胞形成的管状结构增多(p=0.000),400ng/ml CYR61与80ng/ml VEGF的作用相当,而加入抗CYR61抗体则可以起到显著的抑制作用。 2.在正常小鼠的视网膜,CYR61在神经节细胞层、内丛状层、外从状层和光感受器层均有表达,其中外从状层表达较强,其余各层弱阳性染色；在糖尿病小鼠的视网膜,CYR61同样在以上各层表达,但神经节细胞层和内丛状层的表达较正常小鼠明显增强,神经节细胞胞浆内出现团块状的阳性染色。糖尿病小鼠视网膜Cyr61 mRNA的表达较正常对照组增加,差异具有统计学意义(p=0.009)。 3.PDR组玻璃体CYR61浓度为97.53±34.13 (41.21～154.74) ng/mL,对照组玻璃体CYR61浓度为33.92±18.68 (6.24～70.04) ng/mL,两组有显著性差异(p=0.000)；视网膜和增殖膜新生血管丰富者玻璃体CYR61明显高于无明显新生血管者(p＝0.001)；未发现玻璃体CYR61与患者年龄、糖尿病病程及术前空腹血糖有关(p0.05)。CYR61分布于视网膜前膜的细胞胞浆中,在PDR视网膜前增殖膜中管腔样结构的管壁细胞高度表达,在其它细胞也可见散在的阳性染色；PVR视网膜前增殖膜及特发性黄斑前膜中,有散在的阳性染色细胞。结论 CYR61在体外可以促进脉络膜视网膜内皮细胞增殖、迁移及形成管状结构；糖尿病小鼠视网膜Cyr61 mRNA和蛋白的表达均较正常小鼠升高：PDR患者玻璃体CYR61含量明显高于非糖尿病患者,CYR61表达于PDR视网膜前增殖膜,尤其在管腔样结构的管壁细胞高度表达。CYR61很可能参与糖尿病视网膜病变的发病,在PDR新生血管的形成过程中起一定的作用。
[Abstract]:Purpose 1. Study CYR61 (cysteine-rich 61; CCN1) proliferation, migration and formation of tubular structures on choroidal retinal endothelial cells (RF/ 6A cell lines) Effect of CYR61 on the expression and distribution of retinal in diabetic mice and normal mice cloth and its differences.3. To study the level of the expression of CYR61 in the proliferative diabetic retinopathy (PDR) and CYR61 in the visual network membrane increase Methods 1. The effect of CYR61 on the proliferation of RF/ 6A cells was observed by MTT colorimetric assay. The effect of CYR61 on the proliferation of RF/ 6A cells was observed by MTT colorimetric assay. The effect of 61 on the formation of tubular structure of RF/ 6A cells was investigated.2. The model of diabetic mice was made by intraperitoneal injection of streptococcin (STZ). The expression and distribution of CYR61 in the retina of diabetic mice were observed by immunohistochemistry. RT-PCR was used to study the expression and distribution of CYR61 in the retina of diabetic mice. R) Method for the analysis of the expression of Cyr61 mRNA in the retina.3. Collect the vitreous humor and preretinal membrane of the patients with vitreoretinal surgery, and use the enzyme-linked immunosorbent assay (ELISA) to determine the vitreous CYR of patients with PDR and non-diabetic patients. The concentration of CYR61 in PDR, proliferative vitreoretinopathy (PV, R) And the expression and distribution of the preretinal membrane of the patients with idiopathic macular membrane. At different time (0,12 h,24 h,48 h,72 h) of the/ 6A cells, the value of A490nm increased with time, and the difference was statistically significant (Jp = 0.000). In the Transwell chamber experiment, as the concentration of CYR61 increased, the migration RF/ 6A cells increased (p = 0.000), and the ability of 400 ng/ ml CYR61 to promote cell migration was similar to that of 80. When the concentration of CYR61 increased, the tubular structure of RF/ 6A cells increased (p = 0.000),400 ng/ ml CYR61 and 80 ng/ ml as the concentration of CYR61 increased. 2. In the retina of normal mice, CYR61 was expressed in the ganglion cell layer, the inner-cluster layer, the outer-form layer and the photoreceptor layer, and the outer-form layer table In diabetic mice, CYR61 was also expressed in the above layers, but the ganglion The expression of the cell layer and the inner plexiform layer is stronger than that of the normal mouse, and the positive staining of the mass in the cytoplasm of the ganglion cell is obvious. The retina of the diabetic mouse Cyr61 mRNA expression was significantly higher than that in the control group (p = 0.009).3. The concentration of CYR61 in the PDR group was 97.53-34.13 (41.21-154.74) ng/ mL, and the concentration of CYR61 in the control group was 33.92-18.68 (6.24-70.04) ng/ mL, and there was a significant difference between the two groups (p = 0.000). Vitreous CYR61 of the retinal and proliferative membrane was significantly higher than that of the non-significant neovascular (p = 0.001); it was not found that the CYR61 was related to the age of the patient, the course of diabetes, and the pre-operative fasting blood glucose (p0.05). CYR61 was distributed in the cytoplasm of the preretinal membrane. and the cell height of the tube wall cells of the tube cavity-like structure in the preproliferative membrane of the PDR retina is highly expressed, cell Conclusion CYR61 can promote the proliferation, migration and formation of the tubular structure of the chorioretinal endothelial cells in vitro. The expression of Cyr61 mRNA and protein in the retina of the retina was higher than that in the normal mice: the vitreous CYR in the PDR The content of CYR61 was significantly higher than that of non-diabetic patients, and CYR61 was expressed in the preproliferative membrane of PDR, especially in the tube-like structure.
【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R587.1;R774.1

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