【摘要】： 第一部分:應(yīng)用氪離子激光建立大鼠脈絡(luò)膜新生血管模型 目的 1.探討應(yīng)用氪離子激光建立棕色挪威大鼠(Brown Norway,BN)脈絡(luò)膜新生血管(Choroidal neovascularization,CNV)模型的有效性和安全性。 2.觀察氪離子激光光凝后CNV形成及變化規(guī)律,并確定CNV形成和高峰時(shí)間,為進(jìn)一步研究CNV的發(fā)生機(jī)制及探索新的基因治療方法提供理論依據(jù)。 方法 選取6～8周雄性BN大鼠25只(50只眼),從中隨機(jī)選取一只大鼠(2只眼)作為空白對(duì)照組,其余24只大鼠(48只眼)作為實(shí)驗(yàn)組,再根據(jù)光凝后不同觀察時(shí)間,將實(shí)驗(yàn)組隨機(jī)分為6個(gè)亞組,每個(gè)亞組4只大鼠(8只眼)�？瞻讓�(duì)照組不做任何處理,只用于與實(shí)驗(yàn)組激光光凝后視網(wǎng)膜熒光血管造影(Fundus fluoresceinangiography,FFA)、病理及透射電鏡變化作對(duì)照。手術(shù)方法為用846復(fù)合麻醉劑0.5ml/Kg腹腔注射充分麻醉動(dòng)物,激光光凝前5min用復(fù)方托品酰胺眼液滴眼一次,充分散大雙眼瞳孔。固定動(dòng)物,在-53.00D角膜接觸鏡輔助下,圍繞視盤(pán)并在距視盤(pán)2PD位置等距離行氪離子激光光凝,共計(jì)8個(gè)光凝斑,激光波長(zhǎng)為647.1nm,功率為350mW,光凝斑直徑和時(shí)間分別為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天分別各隨機(jī)抽取4只實(shí)驗(yàn)組大鼠,行FFA、組織病理及透射電鏡檢查。 結(jié)果 1.通過(guò)眼底照相及FFA檢查證實(shí),光凝后第7天光凝斑部位開(kāi)始出現(xiàn)圓盤(pán)狀熒光素滲漏(190/16×20,59%),14天光凝斑熒光素滲漏增加(172/16×16,67%),21天光凝斑熒光素滲漏達(dá)高峰(162/16×12,84%)(P＜0.01);21天后光凝斑熒光素滲漏穩(wěn)定,28天光凝斑滲漏為(106/16×8,83%),56天光凝斑滲漏為(52/16×4,81%)(P＞0.05)。 2.病理組織學(xué)檢查:光凝后3天,光鏡下顯示光凝區(qū)視網(wǎng)膜色素上皮(Retinalpigment epithelial,RPE)層和Bruch膜破裂,脈絡(luò)膜毛細(xì)血管層受到破壞;電鏡下顯示脈絡(luò)膜血管管腔內(nèi)有內(nèi)皮細(xì)胞和紅細(xì)胞。在靠近視網(wǎng)膜一側(cè)血管,見(jiàn)有內(nèi)皮細(xì)胞胞突和內(nèi)皮細(xì)胞圍成的不完全管腔樣結(jié)構(gòu)。光凝后7天,光鏡下顯示光凝區(qū)視網(wǎng)膜全層水腫、隆起;電鏡下顯示脈絡(luò)膜近視網(wǎng)膜處有新生毛細(xì)血管,管腔內(nèi)附著內(nèi)皮細(xì)胞胞突,紅細(xì)胞游離于管腔中。光凝后14天,光鏡下顯示視網(wǎng)膜水腫消退,新生血管增多;電鏡下顯示色素上皮細(xì)胞內(nèi)靠近基部有明顯的空腔,色素顆粒減少;高倍視野下,色素上皮細(xì)胞胞質(zhì)內(nèi)的細(xì)胞器結(jié)構(gòu)不清,色素顆粒減少,基部的絨毛呈溶解性改變,頂部的板層小體呈溶解性改變并形成大量的空腔。光凝后21天,光鏡下顯示CNV呈現(xiàn)顯著的纖維血管增殖(Fibrovascular proliferation,FVP),其中可見(jiàn)大量新生血管,管腔內(nèi)可見(jiàn)紅細(xì)胞;電鏡下顯示脈絡(luò)膜黑色素細(xì)胞間有毛細(xì)血管呈凝聚性改變,內(nèi)皮細(xì)胞凝聚,血管腔內(nèi)多個(gè)紅細(xì)胞形似臘腸。高倍下,基膜和血管壁之間的結(jié)構(gòu)不清,見(jiàn)有大量膠原纖維和彈力纖維。21天后,光鏡顯示CNV保持穩(wěn)定FVP狀態(tài)。光凝后56天,光鏡下顯示光凝斑中央外層視網(wǎng)膜向內(nèi)層凹陷,CNV中存在大量的纖維細(xì)胞和新生血管。 3.CNV中央最大厚度:光凝后7天至21天CNV中央最大厚度顯著增加(P＜0.01),21天后無(wú)明顯改變(P＞0.05)。 4.并發(fā)癥:少數(shù)激光光凝斑處可見(jiàn)視網(wǎng)膜下少量出血(8/384),余未見(jiàn)玻璃體出血,視網(wǎng)膜脫離,脈絡(luò)膜脫離及視網(wǎng)膜表面膜形成等并發(fā)癥。 結(jié)論 1.應(yīng)用氪離子激光成功建立BN大鼠脈絡(luò)膜新生血管模型。 2.氪離子激光光凝后第7天CNV開(kāi)始形成,14天增加,21天達(dá)到高峰,21天后趨于穩(wěn)定。 3.本試驗(yàn)所建立BN大鼠脈絡(luò)膜新生血管模型成模時(shí)間短,持續(xù)時(shí)間長(zhǎng),成模率高,為進(jìn)一步研究CNV發(fā)生機(jī)制及探索Ad-PEDF治療提供穩(wěn)定可靠的動(dòng)物模型。 第二部分:重組腺病毒Ad-PEDF構(gòu)建及其真核表達(dá)的實(shí)驗(yàn)研究 目的 建立攜帶色素上皮衍生因子(pigment epithelium-derived factor,PEDF)基因的重組腺病毒,為進(jìn)一步基因治療脈絡(luò)膜新生血管(choroidal neovasculrization,CNV)奠定基礎(chǔ)。 方法 1.從棕色挪威大鼠(Brown Norway,BN)視網(wǎng)膜組織中提取總RNA。巢式RT-PCR擴(kuò)增PEDF基因cDNA,將PEDF克隆入T載體,行DNA序列分析。提取PEDF質(zhì)粒和腺病毒穿梭質(zhì)粒pDC316,以EcoRⅠ和HindⅢ進(jìn)行雙酶切,構(gòu)建重組腺病毒穿梭載體pDC316/PEDF,提取重組質(zhì)粒后以SacⅠ酶進(jìn)行酶切鑒定并進(jìn)行DNA序列分析。脂質(zhì)體法將pDC316/PEDF質(zhì)粒與腺病毒基因組骨架質(zhì)粒共轉(zhuǎn)染293細(xì)胞,利用Cre/loxp位點(diǎn)同源重組方法構(gòu)建重組腺病毒Ad-PEDF,行PCR鑒定后,大量擴(kuò)增并以氯化銫(Cesium chloride,Cscl)梯度法純化,檢測(cè)重組腺病毒滴度。 2.檢測(cè)PEDF在真核細(xì)胞中的表達(dá)。以Ad-PEDF感染體外培養(yǎng)的HepG2細(xì)胞,1h后更換為等量無(wú)血清培養(yǎng)液,培養(yǎng)48h后收集上清,同時(shí)以未感染Ad-PEDF的HepG2細(xì)胞培養(yǎng)上清作為對(duì)照行Western blot檢測(cè)PEDF的表達(dá)。 結(jié)果 1.基因測(cè)序結(jié)果表明,所克隆的PEDF基因包含了大鼠PEDF基因閱讀框內(nèi)的全部序列,與NCBI Sequence Viewer中公布的大鼠PEDF mRNA序列(NM-177927)完全一致。成功構(gòu)建了重組腺病毒穿梭載體pDC316/PEDF,測(cè)序驗(yàn)證了其可靠性,以其與腺病毒基因組骨架質(zhì)粒共轉(zhuǎn)染293細(xì)胞,應(yīng)用Cre/loxp位點(diǎn)細(xì)胞內(nèi)同源重組方法構(gòu)建Ad-PEDF,經(jīng)PCR鑒定證實(shí)重組腺病毒構(gòu)建成功。大量擴(kuò)增后進(jìn)行CsCl梯度離心純化,并行病毒滴度測(cè)定,滴度達(dá)3.08×10~(10)pfu/mL,滿足了體內(nèi)和體外試驗(yàn)要求。 2.Western blot結(jié)果顯示感染組細(xì)胞上清液有PEDF表達(dá),對(duì)照組沒(méi)有PEDF表達(dá)。證明Ad-PEDF能夠轉(zhuǎn)染真核細(xì)胞使其表達(dá)PEDF,并分泌到細(xì)胞外。 結(jié)論 成功構(gòu)建攜帶PEDF基因的重組腺病毒Ad-PEDF,為基因治療CNV奠定了基礎(chǔ)。 第三部分:重組腺病毒Ad-PEDF抑制大鼠脈絡(luò)膜新生血管的實(shí)驗(yàn)研究 目的 研究重組腺病毒Ad-PEDF對(duì)棕色挪威大鼠(Brown Norway,BN)脈絡(luò)膜新生血管(CNV)的抑制作用;觀察CNV減少或消退的變化規(guī)律;并比較不同給藥方式的治療效果,從而明確藥物療效及最佳給藥途徑,為進(jìn)一步臨床實(shí)驗(yàn)奠定基礎(chǔ)。 方法 選取6～8周雌性BN大鼠68只(136只眼),從中隨機(jī)選取4只大鼠(8只眼)作為空白對(duì)照組(N組),其余64只大鼠(128只眼)作為實(shí)驗(yàn)組。根據(jù)給藥方式不同將實(shí)驗(yàn)組隨機(jī)分為4組:玻璃體腔注射組(A組)、玻璃體腔注射對(duì)照組(B組)、球周注射組(C組)、球周注射對(duì)照組(D組),每組16只大鼠(32只眼),再根據(jù)給藥后觀察時(shí)間不同,將每組隨機(jī)分為4個(gè)亞組:3天組、7天組、14天組、28天組,每個(gè)亞組4只大鼠(8只眼)。所有大鼠通過(guò)氪離子激光光凝建立脈絡(luò)膜新生血管模型,激光參數(shù)為波長(zhǎng)647nm,功率350～380mW,光斑直徑50μm,曝光時(shí)間0.05s。N組:光凝后14天不做任何處理,只用于與兩實(shí)驗(yàn)組給藥后眼底熒光血管造影(FFA)、病理及TUNEL法檢測(cè)新生血管內(nèi)皮細(xì)胞凋亡作對(duì)照。各實(shí)驗(yàn)組于光凝后14天先行FFA檢查,然后給予不同處理。光凝后14天,A組玻璃體腔注射Ad-PEDF 1μl;B組玻璃體腔注射AdNull 1μl;C組球周注射Ad-PEDF 1μl;D組球周注射AdNull 1μl。各實(shí)驗(yàn)組于給藥后3天、7天、14天及28天分別各隨機(jī)抽取4只大鼠,FFA檢查后處死,摘取眼球行組織病理學(xué)及TUNEL檢查,觀察CNV消退及新生血管內(nèi)皮細(xì)胞凋亡情況。FFA記錄光斑滲漏程度,光鏡200倍視野下取連續(xù)切片的CNV中央最大厚度進(jìn)行測(cè)量,應(yīng)用SPSS11.5軟件進(jìn)行統(tǒng)計(jì)分析。 結(jié)果 1.A組(54.7%)C組(56.3%)給藥后比給藥前熒光素滲漏減輕(t=2.75;t=3.15,P＜0.01)。 2.給藥后7天,A組(57.3%)、C組(57.8%)CNV數(shù)量減少;B、D組CNV呈顯著纖維血管增殖。 3.給藥后A組(44.51±0.53μm)、C組(44.37±0.48μm)CNV中央最大厚度比N組(46.35±0.93μm)減小(F=7.57,8.85;P＜0.01),并且隨時(shí)間延長(zhǎng)而減小(F=4.31,5.25;P＜0.05)。給藥后3天A組(46.35±0.62μm)CNV中央最大厚度比C組(44.90±0.44μm)大(F=3.55,P＜0.05);給藥后14及28天,A組CNV中央最大厚度比C組減少(F=6.54,P＜0.01;F=4.41,P＜0.05)。 4.給藥后A、C組CNV內(nèi)皮細(xì)胞出現(xiàn)部分TUNEL陽(yáng)性細(xì)胞。 5.玻璃體腔注射除5只眼發(fā)生白內(nèi)障外,無(wú)其他并發(fā)癥。球周注射術(shù)后未發(fā)現(xiàn)明顯并發(fā)癥。 結(jié)論 1.Ad-PEDF對(duì)BN大鼠脈絡(luò)膜新生血管具有抑制作用。 2.BN大鼠脈絡(luò)膜新生血管于治療后7天起效,14天抑制作用最強(qiáng),持續(xù)28天。 3.玻璃體腔注射方式比球周注射方式起效慢,但玻璃體腔注射方式抑制作用強(qiáng)。 4.球周注射方法簡(jiǎn)單易行,并可重復(fù)操作;玻璃體腔注射方法較為復(fù)雜,可重復(fù)性差,并可引起白內(nèi)障等并發(fā)癥。
[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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2008
【分類(lèi)號(hào)】：R774.1;R-332

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