【摘要】：第一章Caveolin-1在鼠視網(wǎng)膜新生血管模型中的表達(dá) 目的建立氧誘導(dǎo)視網(wǎng)膜新生血管小鼠模型,觀察小窩蛋白-1 (caveolin-1)在正常鼠和氧誘導(dǎo)視網(wǎng)膜新生血管模型鼠視網(wǎng)膜中的表達(dá),探討caveolin-1在視網(wǎng)膜新生血管形成過(guò)程中的作用。 方法取鼠齡7天(P7)的健康C57BL/6J小鼠(144只)隨機(jī)分為模型組(72只)和正常對(duì)照組(72只),模型組置于75%±2%氧氣濃度的密閉氧艙中5天,鼠齡12天時(shí)(P12)返回正�？諝猸h(huán)境以建立氧誘導(dǎo)鼠視網(wǎng)膜新生血管模型；正常對(duì)照組置于正常空氣環(huán)境中飼養(yǎng)不予任何處理。模型組和正常對(duì)照組均在鼠齡17天(P17)行FITC-Dextran左心室灌注視網(wǎng)膜鋪片,熒光顯微鏡下觀察視網(wǎng)膜血管分布和形態(tài)；組織切片HE染色觀察突破視網(wǎng)膜內(nèi)界膜的血管內(nèi)皮細(xì)胞核數(shù)量。提取不同鼠齡(P13、P15、P17)模型組和正常對(duì)照組視網(wǎng)膜總RNA及蛋白質(zhì),采用RT-PCR及western blot技術(shù)檢測(cè)視網(wǎng)膜組織中caveolin-1的表達(dá)；Western blot技術(shù)檢測(cè)視網(wǎng)膜白蛋白(albumin)含量。 結(jié)果熒光造影結(jié)果顯示模型組視網(wǎng)膜有大量新生血管形成及明顯的熒光素滲漏；組織切片顯示模型組有大量突破視網(wǎng)膜內(nèi)界膜的新生血管內(nèi)皮細(xì)胞核。模型組視網(wǎng)膜caveolin-1 mRNA和蛋白質(zhì)表達(dá)水平隨缺氧時(shí)間延長(zhǎng)而上調(diào),于P17明顯上調(diào)；同時(shí)視網(wǎng)膜組織中白蛋白含量逐漸增加,與caveolin-1表達(dá)呈基本相同的上升趨勢(shì),兩者在時(shí)間和表達(dá)趨勢(shì)上是一致的。不同鼠齡正常對(duì)照組caveolin-1 mRNA和蛋白質(zhì)表達(dá)無(wú)明顯變化。 結(jié)論在氧誘導(dǎo)鼠視網(wǎng)膜新生血管形成過(guò)程中caveolin-1表達(dá)明顯上調(diào),其變化趨勢(shì)與血-視網(wǎng)膜屏障破壞及視網(wǎng)膜新生血管形成具有時(shí)間上的一致性。caveolin-1可能參與調(diào)控視網(wǎng)膜新生血管形成。 第二章Caveolin-1 shRNA抑制鼠視網(wǎng)膜新生血管的實(shí)驗(yàn)研究 目的觀察caveolin-1小發(fā)夾RNA (caveolin-1 shRNA)對(duì)血-視網(wǎng)膜屏障破壞和視網(wǎng)膜新生血管形成的抑制作用,探討caveolin-1對(duì)視網(wǎng)膜新生血管的調(diào)控作用,為視網(wǎng)膜新生血管性疾病的治療提供新的治療靶點(diǎn)。 方法取鼠齡7天(P7)的健康C57BL/6J小鼠(144只)隨機(jī)分為正常對(duì)照組(36只)、模型對(duì)照組(36只)、陰性對(duì)照組(36只)和caveolin-1 shRNA注射組(36只)。正常對(duì)照組置于正常空氣環(huán)境中飼養(yǎng)不予任何處理。模型對(duì)照組、陰性對(duì)照組和caveolin-1 shRNA注射組置于75%±2%氧氣濃度的密閉氧艙中5天,鼠齡12天時(shí)(P12)返回正常空氣環(huán)境以建立氧誘導(dǎo)鼠視網(wǎng)膜新生血管模型；其中,模型對(duì)照組不做任何治療；caveolin-1 shRNA注射組在P12時(shí)予玻璃體腔注射1ul脂質(zhì)體-caveolin-1 shRNA重組質(zhì)粒混合物；陰性對(duì)照組在P12時(shí)玻璃體腔注射1ul脂質(zhì)體-陰性對(duì)照質(zhì)�；旌衔铩Ｒ陨细鹘M在鼠齡17天(P17)分別采用RT-PCR和western blot技術(shù)檢測(cè)視網(wǎng)膜caveolin-1 mRNA和蛋白質(zhì)表達(dá),western blot技術(shù)檢測(cè)視網(wǎng)膜白蛋白表達(dá)。行FITC-Dextran造影視網(wǎng)膜鋪片方法觀察血管形態(tài)變化,組織切片HE染色觀察并計(jì)數(shù)突破視網(wǎng)膜內(nèi)界膜的血管內(nèi)皮細(xì)胞核數(shù)目, 結(jié)果Caveolin-1 shRNA注射組視網(wǎng)膜caveolin-1 mRNA和蛋白質(zhì)表達(dá)分別較模型對(duì)照組下調(diào)47.94%和54.76%,差異均有統(tǒng)計(jì)學(xué)意義(P0.05)；caveolin-1 shRNA注射組視網(wǎng)膜組織白蛋白含量較模型對(duì)照組下調(diào)56.32%,差異有統(tǒng)計(jì)學(xué)意義(P0.05)；熒光造影顯示：caveolin-1 shRNA注射組較模型對(duì)照組視網(wǎng)膜新生血管明顯減少,熒光滲漏明顯減輕；組織切片顯示caveolin-1 shRNA注射組突破視網(wǎng)膜內(nèi)界膜的血管內(nèi)皮細(xì)胞核數(shù)目較模型對(duì)照組減少51.3%,差異有統(tǒng)計(jì)學(xué)意義(P0.01)。 結(jié)論Caveolin-1 shRNA可以有效地抑制視網(wǎng)膜血管白蛋白滲漏,并抑制視網(wǎng)膜新生血管形成,進(jìn)一步證明了caveolin-1在視網(wǎng)膜新生血管發(fā)生中的調(diào)控作用,為視網(wǎng)膜新生血管性疾病的治療提供了新的靶點(diǎn)。
[Abstract]:Chapter 1 expression of Caveolin-1 in rat retinal neovascularization model
Objective to establish a mouse model of oxygen induced retinal neovascularization, and to observe the expression of fossa protein -1 (caveolin-1) in normal rat and oxygen induced retinal neovascularization model rat retina, and to explore the role of caveolin-1 in the formation of retinal neovascularization.
Methods the healthy C57BL/6J mice of 7 days of age (P7) were randomly divided into model group (72 rats) and normal control group (72 rats). The model group was placed in the closed oxygen chamber of 75% + 2% oxygen concentration for 5 days, and the rat age 12 days (P12) returned to the normal air environment to establish the oxygen induced retinal neovascularization model in the oxygen induced rat, and the normal control group was placed in the normal air environment. In the model group and the normal control group, the retinal vasculature was perfused in the left ventricle of FITC-Dextran at 17 days (P17), and the distribution and morphology of the retinal vessels were observed under the fluorescence microscope. The number of vascular endothelial cells that broke through the inner boundary membrane of the retina was observed by HE staining. The models of different age of rat (P13, P15, P17) were extracted. The total RNA and protein in the retina of the group and the normal control group were detected by RT-PCR and Western blot, and the content of retina albumin (albumin) was detected by Western blot.
The results showed that the retina of the model group had a large number of neovascularization and obvious fluorescein leakage, and the tissue section showed that the model group had a large number of neovascular endothelial nuclei breaking through the inner boundary membrane of the retina. The expression level of caveolin-1 mRNA and protein in the model group was up-regulated with the prolongation of the time of hypoxia and was obviously on the P17. At the same time, the content of albumin in retinal tissue increased gradually, and the expression of caveolin-1 showed the same upward trend, both in time and in the expression trend. The expression of caveolin-1 mRNA and protein in the normal control group had no obvious change.
Conclusion the expression of caveolin-1 is obviously up-regulated during the formation of retinal neovascularization in oxygen induced retina. The trend of the change is consistent with the disruption of the blood retinal barrier and the formation of retinal neovascularization in time..caveolin-1 may be involved in the regulation of the formation of retinal neovascularization.
Second chapter Caveolin-1 shRNA inhibits retinal neovascularization in rats
Objective To observe the inhibitory effect of caveolin-1 small hairpin RNA (caveolin-1 shRNA) on the destruction of blood retinal barrier and the formation of retinal neovascularization, and to explore the regulation of caveolin-1 on retinal neovascularization, and to provide new therapeutic targets for the treatment of retinal neovascularization.
Methods the healthy C57BL/6J mice of 7 days of age (P7) were randomly divided into normal control group (36 rats), model control group (36 rats), negative control group (36) and caveolin-1 shRNA injection group (36 rats). Normal control group was placed in normal air environment for no treatment. Model control group, negative control group and caveolin-1 shRNA injection group were set up. In the 75% + 2% oxygen concentration airtight oxygen tank 5 days, the rat age 12 days (P12) returned to the normal air environment to establish the oxygen induced rat retinal neovascularization model, and the model control group did not do any treatment; the caveolin-1 shRNA injection group injected the vitreous cavity with the 1ul liposome -caveolin-1 shRNA recombinant plasmid mixture at P12; negative pairs. The mixture of 1ul liposome negative control plasmid was injected into the vitreous cavity at P12. The expression of retina caveolin-1 mRNA and protein was detected by RT-PCR and Western blot in the 17 days of age (P17), and the expression of retinal albumin was detected by Western blot. Tissue sections were stained with HE to observe and count the number of endothelial cells that broke through the inner limiting membrane of the retina.
Results the expression of caveolin-1 mRNA and protein in the retina of the Caveolin-1 shRNA injection group was down 47.94% and 54.76%, respectively, and the difference was statistically significant (P0.05). The albumin content of retinal tissue in caveolin-1 shRNA injection group was 56.32% lower than that in the model control group, and the difference was statistically significant (P0.05); fluorescein angiography showed ca. Compared with the model control group, the retinal neovascularization in the veolin-1 shRNA injection group was significantly reduced and the fluorescence leakage was significantly reduced. The number of vascular endothelial nuclei in the caveolin-1 shRNA injection group was 51.3% less than that in the model control group, and the difference was statistically significant (P0.01).
Conclusion Caveolin-1 shRNA can effectively inhibit the leakage of retinal blood vessel albumin and inhibit the formation of retinal neovascularization, and further demonstrate the regulatory role of caveolin-1 in the development of retinal neovascularization, which provides a new target for the treatment of retinal neovascularization.
【學(xué)位授予單位】：中南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R774.1

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