慢病毒载体介导CD25siRNA在大鼠高危角膜移植免疫排斥反应中的研究

发布时间：2017-12-31 13:24

本文关键词：慢病毒载体介导CD25siRNA在大鼠高危角膜移植免疫排斥反应中的研究　出处：《重庆医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的通过慢病毒介导CD25siRNA（LV-CD25siRNA）来转染高危角膜移植术后的大鼠角膜，探讨CD25siRNA在角膜移植排斥中的作用。本实验分为两部分：实验一：慢病毒介导增强型绿色荧光蛋白（LV-EGFP）在不同浓度和不同转染途径下对大鼠角膜进行转染的有效性和毒性的研究实验二：通过慢病毒介导CD25siRNA来干预高危角膜移植术后的大鼠角膜，探讨CD25siRNA在角膜移植排斥中的作用方法实验一：SD大鼠25只，随机分为5组。分别是感染复数(multiplicity of infection，，MOI)=5滴眼组（A）、 MOI=5结膜下注射组（B）、MOI=10点眼组（C）、MOI=10结膜下注射组（D）和MOI=10离体转染组（E）。各组角膜按各自条件转染后进行包埋、切片后置于荧光显微镜下观察，并分析荧光强度，然后通过HE染色观察角膜各层组织的变化。实验二：用NaOH法制备SD大鼠中到重度角膜碱烧伤动物模型，以Lewis大鼠为供体在碱烧伤模型上行穿透性角膜移植来构建高危角膜移植模型，角膜移植术后通过4种不同的方式干预：LV-CD25siRNA滴眼（A）、环孢素A滴眼液（B）、阴性对照病毒（含阴性对照siRNA的慢病毒）（C）、生理盐水（D）。转染后，我们主要通过以下四个方面进行观测。除裂隙灯每天检查外，其余三项检查检查在3、7、14天三个时间点进行检测： 1.裂隙灯下观察各个实验组的角膜术后情况，包括：角膜水肿、浑浊情况，角膜新生血管及前房炎症。 2.HE染色病理切片下角膜组织的变化。 3.运用RT-qPCR检测术后3个时间点角膜中CD25和VEGF-A的mRNA表达情况。 4.运用Western blot检测术后3个时间点角膜中CD25和VEGF-A的蛋白质表达情况。结果实验一：荧光显微镜观察结果显示，同MOI组比较，点眼方式较结膜下注射角膜的荧光分布更加均匀。各组相对荧光强度值分别为：A组0.1803±0.0440，B组0.1061±0.0434，C组0.2369±0.0157，D组0.2002±0.0307，E组0.2434±0.0173，表明点眼方式较结膜下注射角膜的荧光表达更加强烈(P0.05)，MOI增加时EGFP表达增强(P0.05)。E组与C组表达出最强的的荧光强度，相对比之下两组间不具有统计学差异(P0.05)。各组角膜细胞形态正常，未见明显凋亡细胞。离体转染角膜经过7d培养液转染后，角膜内皮细胞仍然生长良好，未见内皮细胞出现皱缩、变形及缺失等病理改变。实验二：裂隙灯检查结果：术后10天观察到A组角膜植片中央轻度水肿、混浊，未见明确新生血管长入植片，C组和D组可见角膜完全混浊，明显水肿，大量新生血管成簇弯曲长入角膜植片，B组角膜轻度水肿、混浊，6点到9点位可见大量新生血管长入植片。 HE染色检查结果：手术后14天各组角膜HE染色可见，角膜植片均有水肿，植片增厚明显，以角膜基质层增厚为主，A组（CD25组）可见角膜上皮层细胞排列规则、紧密，无明显水肿，角膜基质基质中炎性细胞浸润和新生血管较少，角膜内皮层细胞整齐，形态无明显改变，无明显缺失。B、C、D组发现角膜上皮层部分细胞脱失、排列相对疏松，基质中观察到大量炎症细胞浸润和新生血管管腔形成，内皮细胞结构基本完整。 RT-qPCR结果：A组CD25的mRNA表达在各个时间点均比其余三组有统计学差异，VEGF的mRNA表达与CsA组比较发现差异无统计学意义（P0.05）。 Western blot结果：A组（0.362±0.09）与C组（0.994±0.19）及D组（1.07±0.15）两对照组相比VEGF-A蛋白表达减少。B组(0.384±0.13)与A组对比，VEGF-A蛋白表达不具有统计学上的差异。结论 1. LV-EGFP能在较低MOI下有效转染大鼠角膜，点眼转染比结膜下注射转染效率更高，提高MOI能提高角膜转染效率，大鼠角膜在较低MOI的持续转染下有良好的安全性。 2. CD25siRNA能有效干扰角膜移植术后角膜植片中CD25的合成，延迟了角膜移植免疫排斥反应的发生，并且还一定程度上抑制了角膜新生血管的发生、发展，我们发现这种抑制可能不是通过VEGF-A的控制来实现，而可能是多种细胞因子间的相互联系来完成的。
[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.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R779.65

【参考文献】