本文關(guān)鍵詞：慢病毒載體介導(dǎo)CD25siRNA在大鼠高危角膜移植免疫排斥反應(yīng)中的研究　出處：《重慶醫(yī)科大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

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【摘要】：目的 通過慢病毒介導(dǎo)CD25siRNA（LV-CD25siRNA）來轉(zhuǎn)染高危角膜移植術(shù)后的大鼠角膜，探討CD25siRNA在角膜移植排斥中的作用。 本實(shí)驗(yàn)分為兩部分： 實(shí)驗(yàn)一：慢病毒介導(dǎo)增強(qiáng)型綠色熒光蛋白（LV-EGFP）在不同濃度和不同轉(zhuǎn)染途徑下對(duì)大鼠角膜進(jìn)行轉(zhuǎn)染的有效性和毒性的研究 實(shí)驗(yàn)二：通過慢病毒介導(dǎo)CD25siRNA來干預(yù)高危角膜移植術(shù)后的大鼠角膜，探討CD25siRNA在角膜移植排斥中的作用 方法 實(shí)驗(yàn)一：SD大鼠25只，隨機(jī)分為5組。分別是感染復(fù)數(shù)(multiplicity of infection，，MOI)=5滴眼組（A）、 MOI=5結(jié)膜下注射組（B）、MOI=10點(diǎn)眼組（C）、MOI=10結(jié)膜下注射組（D）和MOI=10離體轉(zhuǎn)染組（E）。各組角膜按各自條件轉(zhuǎn)染后進(jìn)行包埋、切片后置于熒光顯微鏡下觀察，并分析熒光強(qiáng)度，然后通過HE染色觀察角膜各層組織的變化。 實(shí)驗(yàn)二：用NaOH法制備SD大鼠中到重度角膜堿燒傷動(dòng)物模型，以Lewis大鼠為供體在堿燒傷模型上行穿透性角膜移植來構(gòu)建高危角膜移植模型，角膜移植術(shù)后通過4種不同的方式干預(yù)：LV-CD25siRNA滴眼（A）、環(huán)孢素A滴眼液（B）、陰性對(duì)照病毒（含陰性對(duì)照siRNA的慢病毒）（C）、生理鹽水（D）。轉(zhuǎn)染后，我們主要通過以下四個(gè)方面進(jìn)行觀測(cè)。除裂隙燈每天檢查外，其余三項(xiàng)檢查檢查在3、7、14天三個(gè)時(shí)間點(diǎn)進(jìn)行檢測(cè)： 1.裂隙燈下觀察各個(gè)實(shí)驗(yàn)組的角膜術(shù)后情況，包括：角膜水腫、渾濁情況，角膜新生血管及前房炎癥。 2.HE染色病理切片下角膜組織的變化。 3.運(yùn)用RT-qPCR檢測(cè)術(shù)后3個(gè)時(shí)間點(diǎn)角膜中CD25和VEGF-A的mRNA表達(dá)情況。 4.運(yùn)用Western blot檢測(cè)術(shù)后3個(gè)時(shí)間點(diǎn)角膜中CD25和VEGF-A的蛋白質(zhì)表達(dá)情況。 結(jié)果 實(shí)驗(yàn)一：熒光顯微鏡觀察結(jié)果顯示，同MOI組比較，點(diǎn)眼方式較結(jié)膜下注射角膜的熒光分布更加均勻。各組相對(duì)熒光強(qiáng)度值分別為：A組0.1803±0.0440，B組0.1061±0.0434，C組0.2369±0.0157，D組0.2002±0.0307，E組0.2434±0.0173，表明點(diǎn)眼方式較結(jié)膜下注射角膜的熒光表達(dá)更加強(qiáng)烈(P0.05)，MOI增加時(shí)EGFP表達(dá)增強(qiáng)(P0.05)。E組與C組表達(dá)出最強(qiáng)的的熒光強(qiáng)度，相對(duì)比之下兩組間不具有統(tǒng)計(jì)學(xué)差異(P0.05)。各組角膜細(xì)胞形態(tài)正常，未見明顯凋亡細(xì)胞。離體轉(zhuǎn)染角膜經(jīng)過7d培養(yǎng)液轉(zhuǎn)染后，角膜內(nèi)皮細(xì)胞仍然生長良好，未見內(nèi)皮細(xì)胞出現(xiàn)皺縮、變形及缺失等病理改變。 實(shí)驗(yàn)二：裂隙燈檢查結(jié)果：術(shù)后10天觀察到A組角膜植片中央輕度水腫、混濁，未見明確新生血管長入植片，C組和D組可見角膜完全混濁，明顯水腫，大量新生血管成簇彎曲長入角膜植片，B組角膜輕度水腫、混濁，6點(diǎn)到9點(diǎn)位可見大量新生血管長入植片。 HE染色檢查結(jié)果：手術(shù)后14天各組角膜HE染色可見，角膜植片均有水腫，植片增厚明顯，以角膜基質(zhì)層增厚為主，A組（CD25組）可見角膜上皮層細(xì)胞排列規(guī)則、緊密，無明顯水腫，角膜基質(zhì)基質(zhì)中炎性細(xì)胞浸潤和新生血管較少，角膜內(nèi)皮層細(xì)胞整齊，形態(tài)無明顯改變，無明顯缺失。B、C、D組發(fā)現(xiàn)角膜上皮層部分細(xì)胞脫失、排列相對(duì)疏松，基質(zhì)中觀察到大量炎癥細(xì)胞浸潤和新生血管管腔形成，內(nèi)皮細(xì)胞結(jié)構(gòu)基本完整。 RT-qPCR結(jié)果：A組CD25的mRNA表達(dá)在各個(gè)時(shí)間點(diǎn)均比其余三組有統(tǒng)計(jì)學(xué)差異，VEGF的mRNA表達(dá)與CsA組比較發(fā)現(xiàn)差異無統(tǒng)計(jì)學(xué)意義（P0.05）。 Western blot結(jié)果：A組（0.362±0.09）與C組（0.994±0.19）及D組（1.07±0.15）兩對(duì)照組相比VEGF-A蛋白表達(dá)減少。B組(0.384±0.13)與A組對(duì)比，VEGF-A蛋白表達(dá)不具有統(tǒng)計(jì)學(xué)上的差異。 結(jié)論 1. LV-EGFP能在較低MOI下有效轉(zhuǎn)染大鼠角膜，點(diǎn)眼轉(zhuǎn)染比結(jié)膜下注射轉(zhuǎn)染效率更高，提高M(jìn)OI能提高角膜轉(zhuǎn)染效率，大鼠角膜在較低MOI的持續(xù)轉(zhuǎn)染下有良好的安全性。 2. CD25siRNA能有效干擾角膜移植術(shù)后角膜植片中CD25的合成，延遲了角膜移植免疫排斥反應(yīng)的發(fā)生，并且還一定程度上抑制了角膜新生血管的發(fā)生、發(fā)展，我們發(fā)現(xiàn)這種抑制可能不是通過VEGF-A的控制來實(shí)現(xiàn)，而可能是多種細(xì)胞因子間的相互聯(lián)系來完成的。
[Abstract]:objective
The role of CD25siRNA in corneal graft rejection was investigated by using lentivirus mediated CD25siRNA (LV-CD25siRNA) to transfect the rat cornea after high risk corneal transplantation.
The experiment is divided into two parts:
Experiment 1: efficacy and toxicity of lentivirus mediated enhanced green fluorescent protein (LV-EGFP) transfection on rat corneas under different concentrations and different transfection routes.
Experiment two: the role of lentivirus mediated CD25siRNA to intervene in the cornea of rats after high risk corneal transplantation and to explore the role of CD25siRNA in corneal graft rejection
Method
Experiment one: 25 SD rats were randomly divided into 5 groups respectively. The multiplicity of infection (multiplicity of infection, MOI) =5 eyedrop group (A), MOI=5 subconjunctival injection group (B), MOI=10 treatment group (C), MOI=10 subconjunctival injection group (D) and MOI=10 in vitro transfection group (E) groups according to their respective conditions. Cornea after transfection was embedded in observation sections under fluorescence microscope and fluorescence intensity analysis, and then through the HE staining to observe changes of the corneas.
Experiment two: preparation of NaOH of SD rats with moderate to severe corneal alkali burn animal model with Lewis rats as donor in alkali burn model upstream penetrating keratoplasty to construct high-risk corneal transplantation model, through the intervention of 4 different ways of corneal transplantation: LV-CD25siRNA eye drops (A), cyclosporine A eye drops liquid (B), negative control group (negative control virus containing siRNA lentivirus) (C), normal saline (D). After transfection, we mainly through the following four aspects were observed. In addition to the slit lamp every day to check, check the remaining three were detected in 3,7,14 days three time points:
After 1. slit lights, the postoperative conditions of the cornea were observed in the various experimental groups, including corneal edema, turbidity, corneal neovascularization and anterior chamber inflammation.
Changes of corneal tissue in pathological sections of 2.HE staining.
3. RT-qPCR was used to detect the mRNA expression of CD25 and VEGF-A in the cornea at 3 time points after operation.
4. Western blot was used to detect the protein expression of CD25 and VEGF-A in the cornea at 3 time points after operation.
Result
Experiment one: fluorescence microscopy showed that compared with the MOI group, a bit of subconjunctival injection of the cornea fluorescence distribution more uniform. The relative fluorescence intensity values were: A group 0.1803 + 0.0440, 0.1061 + 0.0434 B group, C group of 0.2369 + 0.0157, 0.2002 + 0.0307 D group, E group of 0.2434 + 0.0173 that is, the fluorescent bit of subconjunctival injection of the cornea more strongly expressed (P0.05), MOI increased expression of EGFP (P0.05).E group and C group expressed the strongest fluorescence intensity of the contrast between the two groups had no statistical difference (P0.05). Each group of corneal cells were normal, no obvious apoptosis in vitro cultured corneal cells. After cultured in 7d after transfection, corneal endothelial cells still grew well without endothelial cells appeared shrunken, change the deformation and lack of pathology.
Experiment two: slit lamp examination results: 10 days after operation were observed in the A group of central corneal opacity, mild edema, no neovascularization graft, C group and D group showed corneal edema, opacity, a large number of new blood vessels grow into clusters of bending cornea, corneal edema in B group. 6 to 9 opacity, a large number of visible neovascularization of the graft.
HE staining results: 14 days after surgery were corneal HE staining, corneal grafts were edema, thickening of the graft, with corneal stromal thickening, A group (group CD25) visible corneal epithelial cells arranged regularly, close, no obvious edema, corneal stromal matrix in inflammatory cell infiltration and neovascularization less, corneal endothelial cell layer in order, no significant morphological changes. No obvious loss of.B, C, D group found that corneal epithelial layer cell loss, arranged in a relatively loose, infiltration and blood vessels to form a large number of inflammatory cells were observed in the matrix, the basic structure of endothelial cell integrity.
RT-qPCR results: the mRNA expression of CD25 in A group was statistically different at all time points than those in the other three groups. There was no significant difference in mRNA expression between VEGF and CsA group (P0.05).
Western blot results: A group (0.362 + 0.09) compared with C group (0.994 + 0.19) and D group (1.07 + 0.15) two control group VEGF-A protein expression decreased,.B group (0.384 + 0.13) compared with A group, VEGF-A protein expression had no statistical difference.
conclusion
1. LV-EGFP in low MOI can effectively transfect rat cornea, were transfected with subconjunctival injection than higher transfection efficiency, improve corneal MOI can improve the transfection efficiency and has good safety in rat cornea at low MOI for transfection.
The synthesis of 2. CD25siRNA can effectively interfere after corneal transplantation of corneal CD25, delayed corneal allograft rejection, and also inhibit corneal neovascularization, development, we found that this inhibition may not be achieved by the VEGF-A control, and a variety of cytokines may be linked the complete.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R779.65

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