大鼠脉络膜新生血管模型建立及应用Ad-PEDF治疗的实验研究
发布时间：2018-08-07 14:17
【摘要】： 第一部分:应用氪离子激光建立大鼠脉络膜新生血管模型 目的 1.探讨应用氪离子激光建立棕色挪威大鼠(Brown Norway,BN)脉络膜新生血管(Choroidal neovascularization,CNV)模型的有效性和安全性。 2.观察氪离子激光光凝后CNV形成及变化规律,并确定CNV形成和高峰时间,为进一步研究CNV的发生机制及探索新的基因治疗方法提供理论依据。 方法 选取6～8周雄性BN大鼠25只(50只眼),从中随机选取一只大鼠(2只眼)作为空白对照组,其余24只大鼠(48只眼)作为实验组,再根据光凝后不同观察时间,将实验组随机分为6个亚组,每个亚组4只大鼠(8只眼)。空白对照组不做任何处理,只用于与实验组激光光凝后视网膜荧光血管造影(Fundus fluoresceinangiography,FFA)、病理及透射电镜变化作对照。手术方法为用846复合麻醉剂0.5ml/Kg腹腔注射充分麻醉动物,激光光凝前5min用复方托品酰胺眼液滴眼一次,充分散大双眼瞳孔。固定动物,在-53.00D角膜接触镜辅助下,围绕视盘并在距视盘2PD位置等距离行氪离子激光光凝,共计8个光凝斑,激光波长为647.1nm,功率为350mW,光凝斑直径和时间分别为50μm及0.05s。光凝后立刻行眼底照相。于光凝后3、7、14、21、28及56天分别各随机抽取4只实验组大鼠,行FFA、组织病理及透射电镜检查。 结果 1.通过眼底照相及FFA检查证实,光凝后第7天光凝斑部位开始出现圆盘状荧光素渗漏(190/16×20,59%),14天光凝斑荧光素渗漏增加(172/16×16,67%),21天光凝斑荧光素渗漏达高峰(162/16×12,84%)(P＜0.01);21天后光凝斑荧光素渗漏稳定,28天光凝斑渗漏为(106/16×8,83%),56天光凝斑渗漏为(52/16×4,81%)(P＞0.05)。 2.病理组织学检查:光凝后3天,光镜下显示光凝区视网膜色素上皮(Retinalpigment epithelial,RPE)层和Bruch膜破裂,脉络膜毛细血管层受到破坏;电镜下显示脉络膜血管管腔内有内皮细胞和红细胞。在靠近视网膜一侧血管,见有内皮细胞胞突和内皮细胞围成的不完全管腔样结构。光凝后7天,光镜下显示光凝区视网膜全层水肿、隆起;电镜下显示脉络膜近视网膜处有新生毛细血管,管腔内附着内皮细胞胞突,红细胞游离于管腔中。光凝后14天,光镜下显示视网膜水肿消退,新生血管增多;电镜下显示色素上皮细胞内靠近基部有明显的空腔,色素颗粒减少;高倍视野下,色素上皮细胞胞质内的细胞器结构不清,色素颗粒减少,基部的绒毛呈溶解性改变,顶部的板层小体呈溶解性改变并形成大量的空腔。光凝后21天,光镜下显示CNV呈现显著的纤维血管增殖(Fibrovascular proliferation,FVP),其中可见大量新生血管,管腔内可见红细胞;电镜下显示脉络膜黑色素细胞间有毛细血管呈凝聚性改变,内皮细胞凝聚,血管腔内多个红细胞形似腊肠。高倍下,基膜和血管壁之间的结构不清,见有大量胶原纤维和弹力纤维。21天后,光镜显示CNV保持稳定FVP状态。光凝后56天,光镜下显示光凝斑中央外层视网膜向内层凹陷,CNV中存在大量的纤维细胞和新生血管。 3.CNV中央最大厚度:光凝后7天至21天CNV中央最大厚度显著增加(P＜0.01),21天后无明显改变(P＞0.05)。 4.并发症:少数激光光凝斑处可见视网膜下少量出血(8/384),余未见玻璃体出血,视网膜脱离,脉络膜脱离及视网膜表面膜形成等并发症。 结论 1.应用氪离子激光成功建立BN大鼠脉络膜新生血管模型。 2.氪离子激光光凝后第7天CNV开始形成,14天增加,21天达到高峰,21天后趋于稳定。 3.本试验所建立BN大鼠脉络膜新生血管模型成模时间短,持续时间长,成模率高,为进一步研究CNV发生机制及探索Ad-PEDF治疗提供稳定可靠的动物模型。 第二部分:重组腺病毒Ad-PEDF构建及其真核表达的实验研究 目的 建立携带色素上皮衍生因子(pigment epithelium-derived factor,PEDF)基因的重组腺病毒,为进一步基因治疗脉络膜新生血管(choroidal neovasculrization,CNV)奠定基础。 方法 1.从棕色挪威大鼠(Brown Norway,BN)视网膜组织中提取总RNA。巢式RT-PCR扩增PEDF基因cDNA,将PEDF克隆入T载体,行DNA序列分析。提取PEDF质粒和腺病毒穿梭质粒pDC316,以EcoRⅠ和HindⅢ进行双酶切,构建重组腺病毒穿梭载体pDC316/PEDF,提取重组质粒后以SacⅠ酶进行酶切鉴定并进行DNA序列分析。脂质体法将pDC316/PEDF质粒与腺病毒基因组骨架质粒共转染293细胞,利用Cre/loxp位点同源重组方法构建重组腺病毒Ad-PEDF,行PCR鉴定后,大量扩增并以氯化铯(Cesium chloride,Cscl)梯度法纯化,检测重组腺病毒滴度。 2.检测PEDF在真核细胞中的表达。以Ad-PEDF感染体外培养的HepG2细胞,1h后更换为等量无血清培养液,培养48h后收集上清,同时以未感染Ad-PEDF的HepG2细胞培养上清作为对照行Western blot检测PEDF的表达。 结果 1.基因测序结果表明,所克隆的PEDF基因包含了大鼠PEDF基因阅读框内的全部序列,与NCBI Sequence Viewer中公布的大鼠PEDF mRNA序列(NM-177927)完全一致。成功构建了重组腺病毒穿梭载体pDC316/PEDF,测序验证了其可靠性,以其与腺病毒基因组骨架质粒共转染293细胞,应用Cre/loxp位点细胞内同源重组方法构建Ad-PEDF,经PCR鉴定证实重组腺病毒构建成功。大量扩增后进行CsCl梯度离心纯化,并行病毒滴度测定,滴度达3.08×10~(10)pfu/mL,满足了体内和体外试验要求。 2.Western blot结果显示感染组细胞上清液有PEDF表达,对照组没有PEDF表达。证明Ad-PEDF能够转染真核细胞使其表达PEDF,并分泌到细胞外。 结论 成功构建携带PEDF基因的重组腺病毒Ad-PEDF,为基因治疗CNV奠定了基础。 第三部分:重组腺病毒Ad-PEDF抑制大鼠脉络膜新生血管的实验研究 目的 研究重组腺病毒Ad-PEDF对棕色挪威大鼠(Brown Norway,BN)脉络膜新生血管(CNV)的抑制作用;观察CNV减少或消退的变化规律;并比较不同给药方式的治疗效果,从而明确药物疗效及最佳给药途径,为进一步临床实验奠定基础。 方法 选取6～8周雌性BN大鼠68只(136只眼),从中随机选取4只大鼠(8只眼)作为空白对照组(N组),其余64只大鼠(128只眼)作为实验组。根据给药方式不同将实验组随机分为4组:玻璃体腔注射组(A组)、玻璃体腔注射对照组(B组)、球周注射组(C组)、球周注射对照组(D组),每组16只大鼠(32只眼),再根据给药后观察时间不同,将每组随机分为4个亚组:3天组、7天组、14天组、28天组,每个亚组4只大鼠(8只眼)。所有大鼠通过氪离子激光光凝建立脉络膜新生血管模型,激光参数为波长647nm,功率350～380mW,光斑直径50μm,曝光时间0.05s。N组:光凝后14天不做任何处理,只用于与两实验组给药后眼底荧光血管造影(FFA)、病理及TUNEL法检测新生血管内皮细胞凋亡作对照。各实验组于光凝后14天先行FFA检查,然后给予不同处理。光凝后14天,A组玻璃体腔注射Ad-PEDF 1μl;B组玻璃体腔注射AdNull 1μl;C组球周注射Ad-PEDF 1μl;D组球周注射AdNull 1μl。各实验组于给药后3天、7天、14天及28天分别各随机抽取4只大鼠,FFA检查后处死,摘取眼球行组织病理学及TUNEL检查,观察CNV消退及新生血管内皮细胞凋亡情况。FFA记录光斑渗漏程度,光镜200倍视野下取连续切片的CNV中央最大厚度进行测量,应用SPSS11.5软件进行统计分析。 结果 1.A组(54.7%)C组(56.3%)给药后比给药前荧光素渗漏减轻(t=2.75;t=3.15,P＜0.01)。 2.给药后7天,A组(57.3%)、C组(57.8%)CNV数量减少;B、D组CNV呈显著纤维血管增殖。 3.给药后A组(44.51±0.53μm)、C组(44.37±0.48μm)CNV中央最大厚度比N组(46.35±0.93μm)减小(F=7.57,8.85;P＜0.01),并且随时间延长而减小(F=4.31,5.25;P＜0.05)。给药后3天A组(46.35±0.62μm)CNV中央最大厚度比C组(44.90±0.44μm)大(F=3.55,P＜0.05);给药后14及28天,A组CNV中央最大厚度比C组减少(F=6.54,P＜0.01;F=4.41,P＜0.05)。 4.给药后A、C组CNV内皮细胞出现部分TUNEL阳性细胞。 5.玻璃体腔注射除5只眼发生白内障外,无其他并发症。球周注射术后未发现明显并发症。 结论 1.Ad-PEDF对BN大鼠脉络膜新生血管具有抑制作用。 2.BN大鼠脉络膜新生血管于治疗后7天起效,14天抑制作用最强,持续28天。 3.玻璃体腔注射方式比球周注射方式起效慢,但玻璃体腔注射方式抑制作用强。 4.球周注射方法简单易行,并可重复操作;玻璃体腔注射方法较为复杂,可重复性差,并可引起白内障等并发症。
[Abstract]:Part one: establishment of rat choroidal neovascularization model using krypton laser.
objective
1. to explore the effectiveness and safety of krypton ion laser for the establishment of Brown Norway (BN) choroidal neovascularization (Choroidal neovascularization, CNV) model in the brown Norway rat.
2. the formation and change rules of CNV after krypton laser photocoagulation were observed, and the formation and peak time of CNV were determined, which provided a theoretical basis for further study of the mechanism of CNV and the exploration of new gene therapy methods.
Method
In 6~8 weeks, 25 male BN rats (50 eyes) were selected and one rat (2 eyes) was selected randomly as the blank control group, and the other 24 rats (48 eyes) were used as the experimental group. The experimental group was randomly divided into 6 subgroups and 4 rats in each subgroup (8 eyes). The blank control group was only used for the control group. In the experimental group, the retinal fluorescence angiography (Fundus fluoresceinangiography, FFA) after laser photocoagulation was used as the control. The operation method was to intraperitoneal injection of 846 compound anesthetic agent 0.5ml/Kg intraperitoneally to anesthetized animals. Before laser photocoagulation, the eye drops of compound toppinamide eye were used once before laser photocoagulation, and the Large Binocular pupil was dispersed, fixed animal, With the aid of -53.00D corneal contact lens, krypton laser photocoagulation was performed around the disc and at the distance from the 2PD position of the disc, with 8 photocoagulation spots, the laser wavelength was 647.1nm, the power was 350mW, the diameter and time of the photocoagulation spot and the time were 50 m and 0.05s. photocoagulation respectively. After the photocoagulation, 4 real samples were randomly selected from 3,7,14,21,28 and 56 days respectively. The rats in the control group were examined by FFA, histopathology and transmission electron microscopy.
Result
1. through fundus photography and FFA examination, it was confirmed that the disc like fluorescein leakage (190/16 x 20,59%) began to appear on seventh days after photocoagulation, the fluorescein leakage increased (172/16 x 16,67%) in 14 days (172/16 x 16,67%), and the fluorescein leakage reached the peak (P < 0.01) in the 21 day (P < 0.01), and the fluorescein leakage was stable and 28 days after 28 days. For (106/16 * 8,83%), the leakage of 56 day light spot was (52/16 * 4,81%) (P > 0.05).
2. histopathological examination: 3 days after photocoagulation, the retinal pigment epithelium (Retinalpigment epithelial, RPE) layer and Bruch membrane were ruptured under light microscope, and the choroidal capillary layer was damaged; under electron microscope, there were endothelial cells and red cells in the choroidal vascular cavity. The incomplete lumen like structure of the endothelial cells. 7 days after photocoagulation, the retinal edema and protruding of the retina were revealed under light microscope. Under electron microscope, the retina of the choroid myopia had newborn capillaries, the endothelium was attached to the endothelium in the cavity, and the red cells were free in the lumen. After 14 days of light coagulation, the retinal edema subsided and newborn blood was revealed under light microscope. Guan Zengduo; under the electron microscope, there was a clear cavity near the base of the pigment epithelium and the decrease of the pigment granules. In the high field of vision, the organelle structure in the cytoplasm of the pigment epithelial cells was unclear, the pigment granules decreased, the villi in the base were dissolving, the lamellar body in the top was dissolved and formed a large number of cavity. After 21 days of light coagulation, light Under the microscope, CNV showed significant fibrovascular proliferation (Fibrovascular proliferation, FVP), which showed a large number of new blood vessels and red blood cells in the lumen. Under electron microscope, the capillary vessels in the choroid melanocytes were condensed, the endothelial cells condensed, and many red cells in the blood vessels resembled the sausage. High times, the basement membrane and the blood vessels. The structure between the walls was not clear, and a large number of collagen fibers and elastic fibers were found for.21 days. The light microscopy showed that CNV remained stable in FVP state. After 56 days of photocoagulation, the retinal detachment of the central outer layer of the retina to the inner layer was revealed under light microscope, and there were a large number of fibrous cells and neovascularization in CNV.
3. Central maximum thickness of CNV: The central maximum thickness of CNV increased significantly from 7 days to 21 days after photocoagulation (P < 0.01), but did not change significantly after 21 days (P > 0.05).
4. complications: a few laser speckles showed a small amount of hemorrhage under the retina (8/384). There were no complications such as vitreous hemorrhage, retinal detachment, choroidal detachment and retina surface film formation.
conclusion
1. the BN rat choroidal neovascularization model was successfully established by krypton laser.
After 2. krypton laser photocoagulation, CNV began to form on the seventh day, increased on the 14 day, reached the peak in 21 days, and became stable after 21 days.
3. the model of BN rat choroidal neovascularization model established in this experiment was short, long duration and high mold forming rate, which provided a stable and reliable animal model for further study of the mechanism of CNV and the exploration of Ad-PEDF treatment.
The second part: Construction of recombinant adenovirus Ad-PEDF and its eukaryotic expression.
objective
A recombinant adenovirus carrying pigment epithelium-derived factor (PEDF) gene is established to lay a foundation for further gene therapy of choroidal neovascularization (choroidal neovasculrization, CNV).
Method
1. the PEDF gene cDNA was amplified from the brown Norway rat (Brown Norway, BN) retina, and the PEDF gene cDNA was amplified by the total RNA. nested RT-PCR. The PEDF was cloned into the T carrier, and the DNA sequence was analyzed. The PEDF plasmid and the Adenovirus Shuttle Plasmid pDC316 were extracted. Sac I enzyme was identified by enzyme digestion and DNA sequence analysis. The liposome method co transfected 293 cells with pDC316/PEDF plasmid and adenovirus genome skeleton plasmid, and constructed recombinant adenovirus Ad-PEDF using Cre/loxp locus homologous recombination method. After PCR identification, the recombinant plasmid was amplified and purified with cesium chloride (Cesium chloride, Cscl) gradient method to detect the recombinant gland. Virus titer.
2. the expression of PEDF in the eukaryotic cells was detected. The HepG2 cells cultured in vitro by Ad-PEDF were replaced with the same amount of serum free culture medium after 1h, and the supernatant was collected after the culture of 48h, and the expression of Western blot detection PEDF was taken as the control of the HepG2 cell culture supernatant that did not infect Ad-PEDF.
Result
The 1. gene sequencing results showed that the cloned PEDF gene contained all the sequence in the PEDF gene reading frame of the rat and identical with the rat PEDF mRNA sequence (NM-177927) published in NCBI Sequence Viewer. The Recombinant Adenovirus Shuttle Carrier pDC316/PEDF was successfully constructed, and its reliability was verified by sequencing. The plasmid co transfected 293 cells and used the homologous recombination method of Cre/loxp site to construct Ad-PEDF. The recombinant adenovirus was successfully constructed by PCR identification. The recombinant adenovirus was successfully purified by CsCl gradient centrifugation, and the titer was measured in parallel with the titer of 3.08 x 10~ (10) pfu/mL, which met the requirements of the test in vivo and in vitro.
The results of 2.Western blot showed that the cell supernatant of the infection group had PEDF expression, and the control group had no PEDF expression. It was proved that Ad-PEDF could transfect eukaryotic cells to express PEDF and secrete to the extracellular.
conclusion
The recombinant adenovirus Ad-PEDF carrying PEDF gene was successfully constructed, which laid the foundation for gene therapy of CNV.
The third part: recombinant adenovirus Ad-PEDF inhibits choroidal neovascularization in rats.
objective
To study the inhibitory effect of recombinant adenovirus Ad-PEDF on the choroidal neovascularization (CNV) of brown Norway (Brown Norway, BN), observe the change of CNV reduction or regression, and compare the therapeutic effect of different ways of administration, so as to clarify the effect of the drug and the best way of administration, and lay the foundation for further clinical experiment.
Method
68 rats (136 eyes) of 6~8 weeks female BN rats were selected and 4 rats (8 eyes) were selected randomly as the blank control group (group N), and the rest 64 rats (128 eyes) were used as experimental group. According to the different way of administration, the experimental group was randomly divided into 4 groups: glass cavity injection group (group A), glass cavity injection control group (group B), week of ball injection group (group C), ball week The injection control group (group D), 16 rats in each group (32 eyes), then divided each group randomly into 4 subgroups: 3 days group, 7 day group, 14 day group, 28 day group, 4 rats in each subgroup (8 eyes). All rats were constructed by krypton laser photocoagulation to build up choroidal neovascularization model, laser parameters were wavelength 647nm, power 350~38 0mW, the diameter of the spot 50 mu m, exposure time 0.05s.N group: 14 days after photocoagulation, no treatment, only used with the two experimental group after the fundus fluorescein angiography (FFA), pathological and TUNEL method of detection of neovascular endothelial cell apoptosis as control. The experimental group in the 14 days after photocoagulation before the FFA examination, and then given different treatments, 14 days after photocoagulation, A group glass The body cavity was injected with Ad-PEDF 1 mu L, group B was injected with AdNull 1 mu L, and group C was injected with Ad-PEDF 1 u l in group C; D group was injected with AdNull 1 mu L., each group was randomly selected for 3 days, 7 days, 14 days and 28 days respectively. The extent of spot leakage was recorded by.FFA, and the maximum central thickness of CNV was measured under 200 times of light microscope, and the SPSS11.5 software was used for statistical analysis.
Result
Group 1.A (54.7%) C group (56.3%) had less leakage of fluorescein than before administration (t=2.75, t=3.15, P < 0.01).
2. on the 7 day after administration, the number of CNV in group A (57.3%) and group C (57.8%) decreased; CNV in group B and D showed significant proliferation of fibrous vessels.
3. group A (44.51 + 0.53 mu m), group C (44.37 + 0.48 mu m) CNV central maximum thickness decreased (F=7.57,8.85; P < 0.01) in N group (F=7.57,8.85; P < 0.01), and decreased with time (F=4.31,5.25; P < 0.05). 3 days after administration (46.35 + 0.62 Mu), the largest thickness of the central group was larger than that of the group (44.90 + 0.53). The central maximum thickness of group CNV was less than that of group C (F=6.54, P < 0.01; F=4.41, P < 0.05).
4. after administration, part of TUNEL positive cells appeared in CNV endothelial cells of group A and C.
5. intravitreal injection had no complications except for cataract in 5 eyes. No significant complications were found after balloon injection.
conclusion
1.Ad-PEDF inhibits choroidal neovascularization in BN rats.
The choroidal neovascularization in 2.BN rats was effective on the 7 day after treatment, and the strongest inhibition lasted for 28 days on the 14 day.
3. intravitreal injection is slower than peritoral injection, but intravitreal injection has a strong inhibitory effect.
4. Peribulbar injection method is simple and easy to operate, and intravitreal injection method is more complex, poor repeatability, and can cause cataract and other complications.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R774.1;R-332
本文编号：2170292
[Abstract]:Part one: establishment of rat choroidal neovascularization model using krypton laser.
objective
1. to explore the effectiveness and safety of krypton ion laser for the establishment of Brown Norway (BN) choroidal neovascularization (Choroidal neovascularization, CNV) model in the brown Norway rat.
2. the formation and change rules of CNV after krypton laser photocoagulation were observed, and the formation and peak time of CNV were determined, which provided a theoretical basis for further study of the mechanism of CNV and the exploration of new gene therapy methods.
Method
In 6~8 weeks, 25 male BN rats (50 eyes) were selected and one rat (2 eyes) was selected randomly as the blank control group, and the other 24 rats (48 eyes) were used as the experimental group. The experimental group was randomly divided into 6 subgroups and 4 rats in each subgroup (8 eyes). The blank control group was only used for the control group. In the experimental group, the retinal fluorescence angiography (Fundus fluoresceinangiography, FFA) after laser photocoagulation was used as the control. The operation method was to intraperitoneal injection of 846 compound anesthetic agent 0.5ml/Kg intraperitoneally to anesthetized animals. Before laser photocoagulation, the eye drops of compound toppinamide eye were used once before laser photocoagulation, and the Large Binocular pupil was dispersed, fixed animal, With the aid of -53.00D corneal contact lens, krypton laser photocoagulation was performed around the disc and at the distance from the 2PD position of the disc, with 8 photocoagulation spots, the laser wavelength was 647.1nm, the power was 350mW, the diameter and time of the photocoagulation spot and the time were 50 m and 0.05s. photocoagulation respectively. After the photocoagulation, 4 real samples were randomly selected from 3,7,14,21,28 and 56 days respectively. The rats in the control group were examined by FFA, histopathology and transmission electron microscopy.
Result
1. through fundus photography and FFA examination, it was confirmed that the disc like fluorescein leakage (190/16 x 20,59%) began to appear on seventh days after photocoagulation, the fluorescein leakage increased (172/16 x 16,67%) in 14 days (172/16 x 16,67%), and the fluorescein leakage reached the peak (P < 0.01) in the 21 day (P < 0.01), and the fluorescein leakage was stable and 28 days after 28 days. For (106/16 * 8,83%), the leakage of 56 day light spot was (52/16 * 4,81%) (P > 0.05).
2. histopathological examination: 3 days after photocoagulation, the retinal pigment epithelium (Retinalpigment epithelial, RPE) layer and Bruch membrane were ruptured under light microscope, and the choroidal capillary layer was damaged; under electron microscope, there were endothelial cells and red cells in the choroidal vascular cavity. The incomplete lumen like structure of the endothelial cells. 7 days after photocoagulation, the retinal edema and protruding of the retina were revealed under light microscope. Under electron microscope, the retina of the choroid myopia had newborn capillaries, the endothelium was attached to the endothelium in the cavity, and the red cells were free in the lumen. After 14 days of light coagulation, the retinal edema subsided and newborn blood was revealed under light microscope. Guan Zengduo; under the electron microscope, there was a clear cavity near the base of the pigment epithelium and the decrease of the pigment granules. In the high field of vision, the organelle structure in the cytoplasm of the pigment epithelial cells was unclear, the pigment granules decreased, the villi in the base were dissolving, the lamellar body in the top was dissolved and formed a large number of cavity. After 21 days of light coagulation, light Under the microscope, CNV showed significant fibrovascular proliferation (Fibrovascular proliferation, FVP), which showed a large number of new blood vessels and red blood cells in the lumen. Under electron microscope, the capillary vessels in the choroid melanocytes were condensed, the endothelial cells condensed, and many red cells in the blood vessels resembled the sausage. High times, the basement membrane and the blood vessels. The structure between the walls was not clear, and a large number of collagen fibers and elastic fibers were found for.21 days. The light microscopy showed that CNV remained stable in FVP state. After 56 days of photocoagulation, the retinal detachment of the central outer layer of the retina to the inner layer was revealed under light microscope, and there were a large number of fibrous cells and neovascularization in CNV.
3. Central maximum thickness of CNV: The central maximum thickness of CNV increased significantly from 7 days to 21 days after photocoagulation (P < 0.01), but did not change significantly after 21 days (P > 0.05).
4. complications: a few laser speckles showed a small amount of hemorrhage under the retina (8/384). There were no complications such as vitreous hemorrhage, retinal detachment, choroidal detachment and retina surface film formation.
conclusion
1. the BN rat choroidal neovascularization model was successfully established by krypton laser.
After 2. krypton laser photocoagulation, CNV began to form on the seventh day, increased on the 14 day, reached the peak in 21 days, and became stable after 21 days.
3. the model of BN rat choroidal neovascularization model established in this experiment was short, long duration and high mold forming rate, which provided a stable and reliable animal model for further study of the mechanism of CNV and the exploration of Ad-PEDF treatment.
The second part: Construction of recombinant adenovirus Ad-PEDF and its eukaryotic expression.
objective
A recombinant adenovirus carrying pigment epithelium-derived factor (PEDF) gene is established to lay a foundation for further gene therapy of choroidal neovascularization (choroidal neovasculrization, CNV).
Method
1. the PEDF gene cDNA was amplified from the brown Norway rat (Brown Norway, BN) retina, and the PEDF gene cDNA was amplified by the total RNA. nested RT-PCR. The PEDF was cloned into the T carrier, and the DNA sequence was analyzed. The PEDF plasmid and the Adenovirus Shuttle Plasmid pDC316 were extracted. Sac I enzyme was identified by enzyme digestion and DNA sequence analysis. The liposome method co transfected 293 cells with pDC316/PEDF plasmid and adenovirus genome skeleton plasmid, and constructed recombinant adenovirus Ad-PEDF using Cre/loxp locus homologous recombination method. After PCR identification, the recombinant plasmid was amplified and purified with cesium chloride (Cesium chloride, Cscl) gradient method to detect the recombinant gland. Virus titer.
2. the expression of PEDF in the eukaryotic cells was detected. The HepG2 cells cultured in vitro by Ad-PEDF were replaced with the same amount of serum free culture medium after 1h, and the supernatant was collected after the culture of 48h, and the expression of Western blot detection PEDF was taken as the control of the HepG2 cell culture supernatant that did not infect Ad-PEDF.
Result
The 1. gene sequencing results showed that the cloned PEDF gene contained all the sequence in the PEDF gene reading frame of the rat and identical with the rat PEDF mRNA sequence (NM-177927) published in NCBI Sequence Viewer. The Recombinant Adenovirus Shuttle Carrier pDC316/PEDF was successfully constructed, and its reliability was verified by sequencing. The plasmid co transfected 293 cells and used the homologous recombination method of Cre/loxp site to construct Ad-PEDF. The recombinant adenovirus was successfully constructed by PCR identification. The recombinant adenovirus was successfully purified by CsCl gradient centrifugation, and the titer was measured in parallel with the titer of 3.08 x 10~ (10) pfu/mL, which met the requirements of the test in vivo and in vitro.
The results of 2.Western blot showed that the cell supernatant of the infection group had PEDF expression, and the control group had no PEDF expression. It was proved that Ad-PEDF could transfect eukaryotic cells to express PEDF and secrete to the extracellular.
conclusion
The recombinant adenovirus Ad-PEDF carrying PEDF gene was successfully constructed, which laid the foundation for gene therapy of CNV.
The third part: recombinant adenovirus Ad-PEDF inhibits choroidal neovascularization in rats.
objective
To study the inhibitory effect of recombinant adenovirus Ad-PEDF on the choroidal neovascularization (CNV) of brown Norway (Brown Norway, BN), observe the change of CNV reduction or regression, and compare the therapeutic effect of different ways of administration, so as to clarify the effect of the drug and the best way of administration, and lay the foundation for further clinical experiment.
Method
68 rats (136 eyes) of 6~8 weeks female BN rats were selected and 4 rats (8 eyes) were selected randomly as the blank control group (group N), and the rest 64 rats (128 eyes) were used as experimental group. According to the different way of administration, the experimental group was randomly divided into 4 groups: glass cavity injection group (group A), glass cavity injection control group (group B), week of ball injection group (group C), ball week The injection control group (group D), 16 rats in each group (32 eyes), then divided each group randomly into 4 subgroups: 3 days group, 7 day group, 14 day group, 28 day group, 4 rats in each subgroup (8 eyes). All rats were constructed by krypton laser photocoagulation to build up choroidal neovascularization model, laser parameters were wavelength 647nm, power 350~38 0mW, the diameter of the spot 50 mu m, exposure time 0.05s.N group: 14 days after photocoagulation, no treatment, only used with the two experimental group after the fundus fluorescein angiography (FFA), pathological and TUNEL method of detection of neovascular endothelial cell apoptosis as control. The experimental group in the 14 days after photocoagulation before the FFA examination, and then given different treatments, 14 days after photocoagulation, A group glass The body cavity was injected with Ad-PEDF 1 mu L, group B was injected with AdNull 1 mu L, and group C was injected with Ad-PEDF 1 u l in group C; D group was injected with AdNull 1 mu L., each group was randomly selected for 3 days, 7 days, 14 days and 28 days respectively. The extent of spot leakage was recorded by.FFA, and the maximum central thickness of CNV was measured under 200 times of light microscope, and the SPSS11.5 software was used for statistical analysis.
Result
Group 1.A (54.7%) C group (56.3%) had less leakage of fluorescein than before administration (t=2.75, t=3.15, P < 0.01).
2. on the 7 day after administration, the number of CNV in group A (57.3%) and group C (57.8%) decreased; CNV in group B and D showed significant proliferation of fibrous vessels.
3. group A (44.51 + 0.53 mu m), group C (44.37 + 0.48 mu m) CNV central maximum thickness decreased (F=7.57,8.85; P < 0.01) in N group (F=7.57,8.85; P < 0.01), and decreased with time (F=4.31,5.25; P < 0.05). 3 days after administration (46.35 + 0.62 Mu), the largest thickness of the central group was larger than that of the group (44.90 + 0.53). The central maximum thickness of group CNV was less than that of group C (F=6.54, P < 0.01; F=4.41, P < 0.05).
4. after administration, part of TUNEL positive cells appeared in CNV endothelial cells of group A and C.
5. intravitreal injection had no complications except for cataract in 5 eyes. No significant complications were found after balloon injection.
conclusion
1.Ad-PEDF inhibits choroidal neovascularization in BN rats.
The choroidal neovascularization in 2.BN rats was effective on the 7 day after treatment, and the strongest inhibition lasted for 28 days on the 14 day.
3. intravitreal injection is slower than peritoral injection, but intravitreal injection has a strong inhibitory effect.
4. Peribulbar injection method is simple and easy to operate, and intravitreal injection method is more complex, poor repeatability, and can cause cataract and other complications.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R774.1;R-332
【参考文献】
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2 赵世红,何守志;氪激光诱导的大鼠脉络膜新生血管模型研究[J];中华眼科杂志;2003年05期
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