CFB-siRNA抑制大鼠脉络膜新生血管生成的实验研究

发布时间：2018-04-26 09:32

本文选题：脉络膜新生血管 + 基因疗法　；参考：《河北医科大学》2011年硕士论文

【摘要】：目的:脉络膜新生血管( choroidal neovascularization, CNV)是年龄相关性黄斑变性等眼底疾病造成患者视力严重受损的主要原因之一,其治疗较为困难,深入研究CNV的发病机制和提供新的治疗方法成为目前眼科的研究热点。目前临床上常用的治疗方法对于控制疾病的发展具有一定的作用。但是所有的治疗方法主要是针对已生成的CNV,尚不能从根本上防止CNV的发生、发展和复发。其发生机制目前尚未完全阐明,通常认为,CNV的生成与RPE-Bruch膜-脉络膜毛细血管复合体的改变有关。它的生成和发展是一个多细胞参与、多因子调控的复杂过程,炎症介质及补体系统在CNV生成中起到一定的作用。本研究采用RNA干扰(RNA interference, RNAi)技术,使用前期研究中成功构建的CFB-siRNA将CFB沉默,从而阻断旁路途径,观察CFB-siRNA对激光诱导的大鼠CNV及其相关生长因子VEGF、TGF-β2的抑制作用,为从根本上治愈CNV提供可能性。方法: 1不同浓度CFB-siRNA体内转染效果评价。 1.1 8-10周健康棕色挪威(Brown Norway,BN)大鼠60只,随机分为4组,每组15只。氪激光(波长647nm,光斑直径200μm ,功率260mW,曝光时间0.05s)围绕视盘均匀光凝9-10个点,以见到有气泡产生提示Bruch膜被击穿为准建立大鼠CNV模型。 1.2随机分为实验对照组(尾静脉生理盐水注射),低剂量组(25μgB因子siRNA),中剂量组(50μgB因子siRNA),高剂量组(75μgB因子siRNA)。实验对照组和治疗组均给予激光光凝建立大鼠CNV模型。治疗组通过尾静脉注射给药。注射时间:B因子表达高峰的前日即第2天;注射方式:尾静脉注射,隔天注射一次,共注射三次,观察时间为激光后3、7、14、21、28d。 1.3各组大鼠分别于光凝后第3、7、14、21、28d进行荧光素眼底血管造影(FFA)。 1.4造影后处死大鼠,摘除眼球取眼后段组织制作石蜡切片。选取有明确血管化的激光斑处做切片,常规HE染色。 1.5随机选取光凝后第3、7、14、21、28d切片,免疫组织化学方法观察VEGF、FactorⅧ表达情况。 1.6图像分析与统计学处理采用SPSS16.0软件对所得数据各时间点灰度值进行分析,观察其随浓度变化的抑制效应。 2观察激光后大鼠CFB与CNV生成有关的VEGF、TGF-β_2的表达关系。 2.1随机选取实验对照组与高剂量组(75μgB因子siRNA),部分组织标本来源于第一部分。 2.2分别于7、14、21、28d行B因子、VEGF、TGF-β_2免疫组化及反转录聚合酶链式反应(RT-PCR)观察CFB与VEGF、TGF-β的表达强度关系。 2.3采用SPSS16.0软件对所得数据进行统计学分析。 3观察CFB-siRNA对视网膜的毒性作用 3.1随机选取实验对照组与高剂量组(75μgB因子siRNA),组织标本来源于第一部分。并增设空白对照组。 3.2分别于7、14、21、28d处死大鼠,摘除眼球取眼后段组织制作石蜡切片。选取远离激光斑点处组织做切片,常规HE染色及透射电镜观察。结果: 1不同浓度CFB-siRNA体内转染效果评价 1.1试验对照组光凝后7d出现CNV,14d逐渐增多,21d达高峰。 1.2 FFA表现为造影早期强荧光斑,晚期出现与视网膜血管无关的荧光素渗漏。 1.3试验治疗组大鼠眼光凝后21dCNV发生率到达高峰,但较对照组明显降低,各组FFA进行比较,高剂量治疗组CNV发生率明显低于低剂量治疗组(P0.05)。 1.4光镜可见到CNV自脉络膜穿过破裂的Bruch膜突向视网膜下,以及巨噬细胞浸润、RPE细胞迁移增生和纤维母细胞增多等。 1.5免疫组织化学染色结果显示VEGF、FactorⅧ表达较对照组均减弱,且高剂量治疗组减弱程度高于低剂量治疗组。 2观察光凝后大鼠CFB与CNV生成有关的VEGF、TGF-β_2的表达关系 2.1免疫组化结果显示:实验对照组CFB提前于VEGF、TGF-β_2出现,7d后表达减少,光凝后14天仍有少量表达。VEGF、TGF-β_2表达高峰均落后于CFB表达高峰,前两者在光凝后14-21d出现明显表达增多,之后略有下降。与对照组相比,实验治疗组的CFB在7d时表达减少,其后逐渐接近对照组,VEGF与TGF-β_2表达均显著降低。 2.2 RT-PCR结果显示:在实验对照组中,CFB表达持续增加,VEGF、TGF-β_2呈曲线变化,在21 d表达高峰;在实验治疗组中,CFB、VEGF、TGF-β_2表达显著减少,且各时间点无显著变化。 3观察CFB-siRNA对视网膜的毒性作用各组HE染色后观察远离激光斑点处组织视网膜各层结构清晰,排列规则,无明显异常。透射电镜检查结果显示:视杆、视锥细胞完好,但膜盘结构轻度紊乱,间隙稍不规则。神经节细胞层细胞间轻度水肿,神经纤维层轻度水肿,线粒体结构清晰,内质网轻度扩张。结论: 1尾静脉注射CFB-SiRNA能够有效抑制激光诱导的大鼠CNV的生成,其抑制效果随着注射剂量的增多而更加明显。 2补体激活旁路途径在CNV生成中扮演重要角色,抑制CFB可减少CNV中VEGF和TGF-β_2的表达。 3尾静脉快速注射CFB-SiRNA对眼部并无明显的毒副作用。
[Abstract]:Objective: choroidal neovascularization (CNV) is one of the major causes of severe impairment of visual acuity in patients with age-related macular degeneration, which is difficult to treat. The study of the pathogenesis of CNV and the provision of new treatment methods have become a hot spot in the current ophthalmology. The treatment method plays a certain role in controlling the development of the disease. However, all the methods of treatment are mainly aimed at the generated CNV, which can not fundamentally prevent the occurrence, development and recurrence of CNV. The mechanism of its occurrence has not yet been fully elucidated. It is generally believed that the formation of CNV and the changes of the RPE-Bruch choroidal capillary complex have been changed. Its formation and development is a complex process of multi cell participation and multi factor regulation. Inflammatory mediators and complement systems play a role in the formation of CNV. This study uses the RNA interference (RNA interference, RNAi) technology to silence the CFB by the successful construction of CFB-siRNA in the previous study, thus blocking the bypass route and observing CFB-siRNA. The inhibition of laser induced CNV and its related growth factor VEGF and TGF- beta 2 in rats can provide a fundamental cure for CNV.
Method:
The effect of transfection of 1 different concentrations of CFB-siRNA in vivo was evaluated.
1.1 Brown Norway (Brown Norway, BN) rats were randomly divided into 4 groups, each group was randomly divided into 15 mice. Krypton laser (wavelength 647nm, light spot diameter 200 m, power 260mW, exposure time 0.05s) around the optic disk uniformly photocoagulation 9-10 points, to see bubbles generated to suggest that Bruch membrane was penetrated to establish rat CNV model.
1.2 randomly divided into experimental control group (caudal vein physiological saline injection), low dose group (25 gB factor siRNA), medium dose group (50 mu gB factor siRNA), high dose group (75 mu gB factor siRNA). The experimental control group and the treatment group were given laser photocoagulation to establish the rat CNV model. The treatment group was injected through the tail vein. The injection time: the peak of B factor expression. The day before yesterday is second days. The injection way is: tail vein injection, every other day, injections three times, the observation time is 3,7,14,21,28d. after laser.
1.3 rats in each group were treated with fluorescein fundus angiography (FFA) at 3,7,14,21,28d after photocoagulation.
1.4 after the angiography, the rats were sacrificed. The eyeball was removed and the paraffin sections were made. The sections of the laser spots with clear vascularization were stained and stained with conventional HE.
1.5 the slices of 3,7,14,21,28d after photocoagulation were randomly selected to observe the expression of VEGF and Factor VIII by immunohistochemistry.
1.6 image analysis and statistical processing, SPSS16.0 software was used to analyze the gray value of the data at each time point, and observe its inhibitory effect with the concentration change.
2 to observe the expression of VEGF and TGF- beta _2 related to the generation of CFB and CNV in rats.
2.1 randomly selected the experimental control group and the high-dose group (75 gB factor siRNA), and some tissue samples came from the first part.
2.2 B factor, VEGF, TGF- beta _2 immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR) were used to observe the relationship between CFB and VEGF, TGF- beta expression in 7,14,21,28d.
2.3 SPSS16.0 software was used to analyze the data.
3 observe the toxic effect of CFB-siRNA on the retina
3.1 randomly selected the experimental control group and the high-dose group (75 gB factor siRNA). The tissue samples were obtained from the first part and a blank control group was added.
3.2 the rats were killed in 7,14,21,28d, and the paraffin sections were made from the posterior segment of the eye. The tissues were sectioned far away from the laser spots, and the routine HE staining and transmission electron microscopy were observed.
Result:
Evaluation of the transfection effect of 1 different concentrations of CFB-siRNA in vivo
1.1 in the experimental control group, 7d appeared CNV, 14d increased gradually and 21d reached the peak after photocoagulation.
1.2 FFA showed a strong fluorescein in the early stage of the angiography, and there was no leakage of fluorescein in the late stage.
In 1.3 experimental group, the incidence of 21dCNV after eye coagulation reached the peak, but compared with the control group, the rate of FFA was compared. The incidence of CNV in the high dose treatment group was significantly lower than that of the low dose treatment group (P0.05).
1.4 under light microscope, CNV can be seen from the choroid through the ruptured Bruch membrane to the subretinal layer, as well as macrophage infiltration, RPE cell migration and proliferation, and fibroblast proliferation.
1.5 immunohistochemical staining showed that the expression of VEGF and Factor VIII decreased in the control group, and the degree of attenuation in the high-dose treatment group was higher than that in the low dose treatment group.
2 to observe the expression of VEGF and TGF- beta _2 related to the generation of CFB and CNV in rats after photocoagulation.
2.1 the results of immunohistochemistry showed that CFB in the experimental control group was ahead of VEGF, TGF- beta _2 appeared, the expression of 7D decreased after 7d, and there was still a small amount of.VEGF in the 14 day after photocoagulation, and the peak of TGF- beta _2 was lagging behind CFB expression peak. The first two were obviously increased in 14-21d after photocoagulation, and then decreased slightly. Compared with the control group, CFB in 7d was in 7d. The expression decreased, and then gradually approached the control group. The expression of VEGF and TGF- beta _2 decreased significantly.
2.2 RT-PCR results showed that in the experimental control group, the expression of CFB continued to increase, VEGF, TGF- beta _2 showed a curve change and the peak of expression at 21 d. In the experimental group, the expression of CFB, VEGF, TGF- beta _2 decreased significantly, and there was no significant change at each time point.
3 observe the toxic effect of CFB-siRNA on the retina
After HE staining, each layer of the retina of the retina of each group was observed to be clear and arranged without obvious abnormality. The results of transmission electron microscopy showed that the optic rod and cone cells were intact, but the structure of the membrane was slightly irregular and the space was slightly irregular. The light edema, the mild edema of the nerve fiber layer and the mitochondria structure were clear in the ganglion cell layer. Clear, endoplasmic reticulum mild expansion.
Conclusion:
1 tail vein injection of CFB-SiRNA can effectively inhibit the formation of CNV induced by laser in rats, and its inhibitory effect is more obvious with the increase of injection dose.
2 complement activation pathway plays an important role in the generation of CNV. Inhibition of CFB can reduce the expression of VEGF and TGF- beta _2 in CNV.
Rapid injection of CFB-SiRNA into 3 caudal veins has no obvious side effects on the eye.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R774.5

【参考文献】