慢病毒介导的Netrin-1小发夹状RNA在视网膜新生血管形成中的调控作用

发布时间：2018-06-21 14:25

本文选题：netrin-1 + 慢病毒　；参考：《中南大学》2011年博士论文

【摘要】：第一章netrin-1与VEGF在糖尿病视网膜病变患者玻璃体腔内表达水平变化及关系目的检测增殖期糖尿病视网膜病变(Proliferative diabetic retinopathyPDR)及非糖尿病视网膜病变(Non-diabetic retinopathy NDRP)患者血清及玻璃体腔内netrin-1与VEGF的水平,并对PDR患者玻璃体腔内netrin-1与玻璃体腔内的VEGF表达水平进行相关性分析,探索研究netrin-1在增殖期糖尿病视网膜病变新生血管形成过程中可能的作用。方法共18例患者18只眼纳入该研究,其中10例为PDR患者(PDR组),8例为NDRP患者(对照组)。于后部玻璃体切割术中收集未经稀释的玻璃体,并抽取适量血液标本,应用酶联免疫吸附试验方法检测玻璃体及血清中netrin-1及VEGF水平。两组间采用Mann-Whitney U检验进行统计学分析,netrin-1与VEGF水平相关性采用Spearman's轶和相关性分析。结果PDR患者玻璃体腔内netrin-1及VEGF水平均较对照组NDRP患者明显增高,分别为509.94±138.85vs.85.91±11.72pg/ml,p0.001；762.60±143.14vs.77.52±7.75pg/ml,p0.001。但升高的netrin-1与VEGF水平无明显相关性(rho=0.200；P=0.704)。结论Netrin-1与VEGF水平在PDR患者玻璃体中均明显增高,而且,netrin-1可能独立于VEGF在新生血管形成中起着重要的作用,并可能成为PDR的治疗靶点之一。第二章慢病毒介导的靶向netrin-1的小发夹状RNA的病毒构建包装及体外转染筛选目的构建PGCSIL-GFP'慢病毒介导的netrin-1小发夹状RNA(small hairpin RNA shRNA)及阴性对照组病毒颗粒。方法构建含netrin-1shRNA序列和阴性对照Scramble shRNA序列的慢病毒载体。将慢病毒载体与pHelper1.0载体、pHelper2.0载体共转染293T细胞,转染后8h更换为完全培养基,培养48h后,收集富含慢病毒颗粒的细胞上清液,并对其浓缩而得到高滴度的慢病毒浓缩液。将所获得的慢病毒浓缩液体外转染小鼠脑微血管内皮细胞(bEnd.3),确定其最佳转染条件。再以同法转染bEnd.3细胞,72h后提取细胞总蛋白,western-blot检测netrin-1蛋白水平表达变化。结果成功构建并包装靶向netrin-1shRNA和阴性对照组shRNA的慢病毒颗粒。与阴性对照组相比,慢病毒介导的netrin-1shRNA体外转染bEnd.3细胞可成功抑制细胞内netrin-1的表达。结论慢病毒介导的netrin-1shRNA体外转染bEnd.3可成功抑制细胞内netrin-1的表达。第三章慢病毒介导的靶向netrin-1的小发夹状RNA抑制视网膜新生血管形成的研究目的检测netrin-1在缺氧诱导模型小鼠视网膜中netrin-1的表达,玻璃体腔内注射慢病毒介导的netrin-1shRNA抑制netrin-1在视网膜中的表达,并观察其在病理性视网膜新生血管形成中的作用。方法48只鼠龄为7天的C57BL/6J小鼠置于浓度为(75±2)%氧舱喂养5天后返回正常氧环境中。其中36只小鼠于出氧舱后一只眼玻璃体腔内注射1μ1慢病毒介导的靶向netrin-1的shRNA (5*105TU)病毒颗粒,对侧眼注射1μ1阴性对照(5*105TU)病毒颗粒。6只不作注射的高氧诱导小鼠作为模型组。另取6只小鼠于正常氧环境下饲养作为正常对照组。采用real-time PCR、Western blot等方法检测视网膜中netrin-1mRNA及蛋白质的表达水平。FITC-Dextran左心室造影视网膜铺片了解视网膜血管形态的改变,组织学切片并行H-E染色观察突破视网膜内界膜的血管内皮细胞核数量。结果：Netrin-1在P17氧诱导视网膜病变小鼠视网膜中的mRNA及蛋白表达水平较正常同龄对照组均明显增高。玻璃体腔内注射特异性慢病毒介导的netrin-1shRNA可有效抑制视网膜中netrin-1的表达,并可减少视网膜新生血管渗漏及视网膜血管无灌注区的形成,同时,慢病毒介导的netrin-1shRNA用药组突破视网膜内界膜的血管内皮细胞核数量为9.8±1.7,较同龄scramble shRNA转染组对照组(20.3±3.4)明显减少约50%以上(P0.05)。结论netrin-1在缺氧诱导的小鼠视网膜病变中表达上调,慢病毒介导的靶向netrin-1的shRNA可有效抑制视网膜新生血管形成,为血管增殖性视网膜病变的治疗提供了新的靶点。
[Abstract]:Chapter one netrin-1 and VEGF expression levels in vitreous cavity of patients with diabetic retinopathy and their relationship
Objective to detect the levels of netrin-1 and VEGF in serum and vitreous cavities of patients with proliferative diabetic retinopathy (Proliferative diabetic retinopathyPDR) and non diabetic retinopathy (Non-diabetic retinopathy NDRP), and to analyze the correlation of VEGF expression in the cavity of netrin-1 and glass in the vitreous cavity of patients with PDR. Objective to explore the possible role of netrin-1 in neovascularization of proliferative diabetic retinopathy.
Methods a total of 18 patients with 18 eyes were included in this study, of which 10 were PDR patients (group PDR) and 8 were NDRP patients (control group). In the posterior vitrectomy, the undiluted vitreous body was collected and a proper amount of blood samples were extracted. The level of netrin-1 and VEGF in glass body and serum was detected by enzyme linked immunosorbent assay. The two groups were used to Mann-. Whitney U test was used for statistical analysis. The correlation between netrin-1 and VEGF level was analyzed by Spearman's and correlation analysis.
Results the levels of netrin-1 and VEGF in the vitreous cavity of PDR patients were significantly higher than those of the control group NDRP, respectively, 509.94 + 138.85vs.85.91 + 11.72pg/ml, p0.001, and 762.60 + 143.14vs.77.52 + 7.75pg/ml, but there was no significant correlation between netrin-1 and VEGF level.
Conclusion the level of Netrin-1 and VEGF is significantly higher in the vitreous of PDR patients. Moreover, netrin-1 may be independent of VEGF in the formation of neovascularization, and may be one of the targets for the treatment of PDR.
The second chapter is the construction and packaging of lentivirus mediated small hairpin RNA targeting netrin-1 and screening in vitro.
Objective to construct PGCSIL-GFP'lentivirus mediated netrin-1 hairpin RNA (small hairpin RNA shRNA) and negative control group virus particles.
Methods the lentivirus vector containing the netrin-1shRNA sequence and the negative control Scramble shRNA sequence was constructed. The lentivirus vector was co transfected with the pHelper1.0 vector and pHelper2.0 vector, and the 293T cells were transfected with the pHelper2.0 vector. After the transfection, the 8h was replaced as a complete medium. After the culture of 48h, the cell supernatant rich in lentivirus particles was collected and the high titer was obtained. The concentration liquid was transfected into the mouse brain microvascular endothelial cells (bEnd.3), and the optimal transfection condition was determined. The bEnd.3 cells were transfected with the same method, the total protein of the cell was extracted after 72h, and the expression of netrin-1 protein was detected by Western-blot.
Results the lentivirus particles targeting netrin-1shRNA and negative control group shRNA were successfully constructed and packaged. Compared with the negative control group, the transfection of bEnd.3 cells with netrin-1shRNA in vitro could inhibit the expression of netrin-1 in the cell successfully.
Conclusion lentivirus mediated netrin-1shRNA transfection in vitro can inhibit the expression of netrin-1 in bEnd.3 cells.
The third chapter lentivirus mediated small hairpin RNA targeting netrin-1 inhibits retinal neovascularization.
Objective to detect the expression of netrin-1 in the retina of hypoxic induced model mice, and to observe the role of netrin-1 in retinal neovascularization by intravitreal injection of lentivirus mediated netrin-1shRNA to inhibit the expression of netrin-1 in the retina.
Methods 48 C57BL/6J mice aged 7 days were placed in the normal oxygen environment after feeding for 5 days with a concentration of (75 + 2)% oxygen chamber. 36 mice were injected with 1 mu 1 lentivirus mediated shRNA (5*105TU) virus particles in the posterior vitreous cavity of the oxygen chamber and injected with 1 micron negative control (5*105TU) virus particles.6 only on the lateral eye. Mice were injected with hyperoxia as model group. Another 6 mice were taken as normal control group under normal oxygen environment. The expression of netrin-1mRNA and protein in retina was detected by real-time PCR, Western blot and so on..FITC-Dextran left ventriculography retina spread was used to understand the changes of retinal vascular morphology and histology. Slice parallel H-E staining was used to observe the number of endothelial cells that broke through the inner limiting membrane of the retina.
Results: the expression level of mRNA and protein in the retina of P17 oxygen induced retinopathy of mice was significantly higher than that of the normal control group. Intravitreal injection of specific lentivirus mediated netrin-1shRNA could effectively inhibit the expression of netrin-1 in the retina, and reduce the retinal neovascularization and retinal vascular failure in the retina. At the same time, the number of vascular endothelial nuclei of the lentivirus mediated netrin-1shRNA group broke through the inner boundary membrane of the retina was 9.8 + 1.7, compared with that of the scramble shRNA transfection group (20.3 + 3.4) of the same age group (20.3 + 3.4), which was significantly reduced by more than 50% (P0.05).
Conclusion the expression of netrin-1 is up-regulated in the retinopathy of hypoxia induced mouse retinopathy. The shRNA targeting netrin-1 mediated by lentivirus can effectively inhibit the formation of retinal neovascularization, and provides a new target for the treatment of vascular proliferative retinopathy.
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R739.72

【共引文献】