RNAi沉默卡氏肺孢子MSG-UCS基因表达的研究

发布时间：2018-01-01 22:34

本文关键词：RNAi沉默卡氏肺孢子MSG-UCS基因表达的研究　出处：《重庆医科大学》2008年硕士论文　论文类型：学位论文

【摘要】： 目的:本研究旨在应用RNA干扰(RNA interference, RNAi)技术,构建卡氏肺孢子(Pneumocystis carinii,PC)主要表面糖蛋白(Major surface glycoprotein,MSG)表达调控基因上游保守序列(Upstream conserved sequence,UCS)的小干扰RNA(short interfering RNA,siRNA)表达载体,并将其转染到PC中有效表达后,研究其对PC的抑制作用。方法:设计并人工合成针对PC MSG-UCS基因的短发夹状(Short hairpin RNA,shRNA)序列并定向克隆到siRNA表达载体pTZU6+1上,采用双酶切与测序法进行鉴定;将构建成功的主要表面糖蛋白UCS基因siRNA表达载体pPC-UCS体外转染到PC中,用RT-PCR检测转染48小时后UCS基因的表达,在扫描电镜下观察PC的超微结构变化;将表达载体pPC-UCS用水动力法注射到PCP大鼠体内,48小时后进行以下检查:大鼠肺印片记数包囊、观察肺组织病理改变、电镜检测PC的超微结构改变。结果:酶切和测序鉴定得到的产物与预期目的基因一致;体外转染pPC-UCS的PC中UCSmRNA表达水平降低,与对照组相比,结果具有显著性差异,UCSmRNA的抑制率为77.47%;转染pPC-UCS48小时后,在扫描电镜中观察PC,可见PC水肿胀大,微绒毛脱落,表膜破裂,出现破损。体内转染pPC-UCS 48h后,包囊减少率为67.88%,与对照组相比,结果具有显著性差异;大鼠肺泡炎减轻,与对照组相比,结果具有显著性差异;扫描电镜观察可见PC水肿胀大,微绒毛脱落,表膜破裂,出现破损。结论:成功构建和鉴定卡氏肺孢子主要表面糖蛋白UCS基因的siRNA表达载体;所构建的PC MSG-UCS siRNA表达质粒能在体外有效抑制PC中UCS基因的表达,导致PC细胞壁的破坏并杀灭PC;所构建的PC MSG-UCS siRNA表达质粒能在体内有效抑制PC增长并杀灭PC,减轻肺部炎症。
[Abstract]:Objective: the purpose of this study is to use RNA interference (RNA interference RNAi) technology, construction of Pneumocystis carinii (Pneumocystis carinii, PC) major surface glycoprotein (Major surface glycoprotein, MSG) expression of conserved sequences upstream regulatory genes (Upstream conserved sequence UCS RNA (siRNA) small interfering short expression vector, interfering RNA) and transfected into the effective expression of PC, to study its inhibitory effect on PC.
Methods: designed and synthesized according to PC gene MSG-UCS short hairpin (Short hairpin RNA, shRNA) sequence and cloned into siRNA pTZU6+1 expression vector and identified by enzyme digestion and sequencing; the construction of major surface glycoprotein UCS gene siRNA expression vector pPC-UCS was transfected into PC, with the expression of 48 RT-PCR hours after transfection was detected in the UCS gene, to observe the ultrastructural changes of PC in the scanning electron microscope; the expression vector of pPC-UCS water power injection to PCP rats after 48 hours, check the following: India Pianji number of rat lung cysts, observe the change of lung tissue pathology and ultrastructural changes of electron microscopy for detection of PC.
Results: the target gene product with consistent enzyme digestion and sequencing; reduce the level of UCSmRNA expression in vitro transfection of pPC-UCS PC, compared with the control group, there was significant difference between UCSmRNA, the inhibition rate was 77.47%; pPC-UCS48 hours after transfection, PC was observed in scanning electron microscopy, PC visible edema swell microvilli table, membrane rupture, breakage. In vivo transfection of pPC-UCS 48h after cyst reduction rate of 67.88%, compared with the control group, the results were significant differences; rat alveolar inflammation reduced, compared with the control group, there was significant difference between observed; scanning electron microscopy PC edema swell, microvilli membrane rupture, appear damaged.
Conclusion: siRNA and identification of Pneumocystis major surface glycoprotein UCS gene expression vector was successfully constructed; the constructed PC MSG-UCS expression plasmid siRNA can effectively inhibit the expression of PC UCS gene in vitro, cause PC cell wall damage and kill PC; the PC MSG-UCS siRNA expression plasmid could effectively inhibit the PC in vivo growth and kill PC, reduce the inflammation in the lungs.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R531.5;R346

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