本文選題：脈絡(luò)膜新生血管 + 基因療法�。� 參考：《河北醫(yī)科大學(xué)》2011年碩士論文

【摘要】：目的:脈絡(luò)膜新生血管( choroidal neovascularization, CNV)是年齡相關(guān)性黃斑變性等眼底疾病造成患者視力嚴(yán)重受損的主要原因之一,其治療較為困難,深入研究CNV的發(fā)病機(jī)制和提供新的治療方法成為目前眼科的研究熱點(diǎn)。目前臨床上常用的治療方法對(duì)于控制疾病的發(fā)展具有一定的作用。但是所有的治療方法主要是針對(duì)已生成的CNV,尚不能從根本上防止CNV的發(fā)生、發(fā)展和復(fù)發(fā)。其發(fā)生機(jī)制目前尚未完全闡明,通常認(rèn)為,CNV的生成與RPE-Bruch膜-脈絡(luò)膜毛細(xì)血管復(fù)合體的改變有關(guān)。它的生成和發(fā)展是一個(gè)多細(xì)胞參與、多因子調(diào)控的復(fù)雜過(guò)程,炎癥介質(zhì)及補(bǔ)體系統(tǒng)在CNV生成中起到一定的作用。本研究采用RNA干擾(RNA interference, RNAi)技術(shù),使用前期研究中成功構(gòu)建的CFB-siRNA將CFB沉默,從而阻斷旁路途徑,觀察CFB-siRNA對(duì)激光誘導(dǎo)的大鼠CNV及其相關(guān)生長(zhǎng)因子VEGF、TGF-β2的抑制作用,為從根本上治愈CNV提供可能性。 方法: 1不同濃度CFB-siRNA體內(nèi)轉(zhuǎn)染效果評(píng)價(jià)。 1.1 8-10周健康棕色挪威(Brown Norway,BN)大鼠60只,隨機(jī)分為4組,每組15只。氪激光(波長(zhǎng)647nm,光斑直徑200μm ,功率260mW,曝光時(shí)間0.05s)圍繞視盤(pán)均勻光凝9-10個(gè)點(diǎn),以見(jiàn)到有氣泡產(chǎn)生提示Bruch膜被擊穿為準(zhǔn)建立大鼠CNV模型。 1.2隨機(jī)分為實(shí)驗(yàn)對(duì)照組(尾靜脈生理鹽水注射),低劑量組(25μgB因子siRNA),中劑量組(50μgB因子siRNA),高劑量組(75μgB因子siRNA)。實(shí)驗(yàn)對(duì)照組和治療組均給予激光光凝建立大鼠CNV模型。治療組通過(guò)尾靜脈注射給藥。注射時(shí)間:B因子表達(dá)高峰的前日即第2天;注射方式:尾靜脈注射,隔天注射一次,共注射三次,觀察時(shí)間為激光后3、7、14、21、28d。 1.3各組大鼠分別于光凝后第3、7、14、21、28d進(jìn)行熒光素眼底血管造影(FFA)。 1.4造影后處死大鼠,摘除眼球取眼后段組織制作石蠟切片。選取有明確血管化的激光斑處做切片,常規(guī)HE染色。 1.5隨機(jī)選取光凝后第3、7、14、21、28d切片,免疫組織化學(xué)方法觀察VEGF、FactorⅧ表達(dá)情況。 1.6圖像分析與統(tǒng)計(jì)學(xué)處理采用SPSS16.0軟件對(duì)所得數(shù)據(jù)各時(shí)間點(diǎn)灰度值進(jìn)行分析,觀察其隨濃度變化的抑制效應(yīng)。 2觀察激光后大鼠CFB與CNV生成有關(guān)的VEGF、TGF-β_2的表達(dá)關(guān)系。 2.1隨機(jī)選取實(shí)驗(yàn)對(duì)照組與高劑量組(75μgB因子siRNA),部分組織標(biāo)本來(lái)源于第一部分。 2.2分別于7、14、21、28d行B因子、VEGF、TGF-β_2免疫組化及反轉(zhuǎn)錄聚合酶鏈?zhǔn)椒磻?yīng)(RT-PCR)觀察CFB與VEGF、TGF-β的表達(dá)強(qiáng)度關(guān)系。 2.3采用SPSS16.0軟件對(duì)所得數(shù)據(jù)進(jìn)行統(tǒng)計(jì)學(xué)分析。 3觀察CFB-siRNA對(duì)視網(wǎng)膜的毒性作用 3.1隨機(jī)選取實(shí)驗(yàn)對(duì)照組與高劑量組(75μgB因子siRNA),組織標(biāo)本來(lái)源于第一部分。并增設(shè)空白對(duì)照組。 3.2分別于7、14、21、28d處死大鼠,摘除眼球取眼后段組織制作石蠟切片。選取遠(yuǎn)離激光斑點(diǎn)處組織做切片,常規(guī)HE染色及透射電鏡觀察。 結(jié)果: 1不同濃度CFB-siRNA體內(nèi)轉(zhuǎn)染效果評(píng)價(jià) 1.1試驗(yàn)對(duì)照組光凝后7d出現(xiàn)CNV,14d逐漸增多,21d達(dá)高峰。 1.2 FFA表現(xiàn)為造影早期強(qiáng)熒光斑,晚期出現(xiàn)與視網(wǎng)膜血管無(wú)關(guān)的熒光素滲漏。 1.3試驗(yàn)治療組大鼠眼光凝后21dCNV發(fā)生率到達(dá)高峰,但較對(duì)照組明顯降低,各組FFA進(jìn)行比較,高劑量治療組CNV發(fā)生率明顯低于低劑量治療組(P0.05)。 1.4光鏡可見(jiàn)到CNV自脈絡(luò)膜穿過(guò)破裂的Bruch膜突向視網(wǎng)膜下,以及巨噬細(xì)胞浸潤(rùn)、RPE細(xì)胞遷移增生和纖維母細(xì)胞增多等。 1.5免疫組織化學(xué)染色結(jié)果顯示VEGF、FactorⅧ表達(dá)較對(duì)照組均減弱,且高劑量治療組減弱程度高于低劑量治療組。 2觀察光凝后大鼠CFB與CNV生成有關(guān)的VEGF、TGF-β_2的表達(dá)關(guān)系 2.1免疫組化結(jié)果顯示:實(shí)驗(yàn)對(duì)照組CFB提前于VEGF、TGF-β_2出現(xiàn),7d后表達(dá)減少,光凝后14天仍有少量表達(dá)。VEGF、TGF-β_2表達(dá)高峰均落后于CFB表達(dá)高峰,前兩者在光凝后14-21d出現(xiàn)明顯表達(dá)增多,之后略有下降。與對(duì)照組相比,實(shí)驗(yàn)治療組的CFB在7d時(shí)表達(dá)減少,其后逐漸接近對(duì)照組,VEGF與TGF-β_2表達(dá)均顯著降低。 2.2 RT-PCR結(jié)果顯示:在實(shí)驗(yàn)對(duì)照組中,CFB表達(dá)持續(xù)增加,VEGF、TGF-β_2呈曲線變化,在21 d表達(dá)高峰;在實(shí)驗(yàn)治療組中,CFB、VEGF、TGF-β_2表達(dá)顯著減少,且各時(shí)間點(diǎn)無(wú)顯著變化。 3觀察CFB-siRNA對(duì)視網(wǎng)膜的毒性作用 各組HE染色后觀察遠(yuǎn)離激光斑點(diǎn)處組織視網(wǎng)膜各層結(jié)構(gòu)清晰,排列規(guī)則,無(wú)明顯異常。透射電鏡檢查結(jié)果顯示:視桿、視錐細(xì)胞完好,但膜盤(pán)結(jié)構(gòu)輕度紊亂,間隙稍不規(guī)則。神經(jīng)節(jié)細(xì)胞層細(xì)胞間輕度水腫,神經(jīng)纖維層輕度水腫,線粒體結(jié)構(gòu)清晰,內(nèi)質(zhì)網(wǎng)輕度擴(kuò)張。 結(jié)論: 1尾靜脈注射CFB-SiRNA能夠有效抑制激光誘導(dǎo)的大鼠CNV的生成,其抑制效果隨著注射劑量的增多而更加明顯。 2補(bǔ)體激活旁路途徑在CNV生成中扮演重要角色,抑制CFB可減少CNV中VEGF和TGF-β_2的表達(dá)。 3尾靜脈快速注射CFB-SiRNA對(duì)眼部并無(wú)明顯的毒副作用。
[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.

【學(xué)位授予單位】：河北醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R774.5

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