RNA干扰UL27基因对Ⅱ型单纯疱疹病毒在小鼠体内复制的影响

发布时间：2018-03-28 13:50

本文选题：RNA干扰　切入点：Ⅱ型单纯疱疹病毒　出处：《遵义医学院》2017年硕士论文

【摘要】：目的:将构建的UL27 sh RNA质粒表达载体转入BALB/c雌性小鼠生殖器疱疹(GH)动物模型体内,评价UL27sh RNA质粒对GH小鼠模型的疗效。方法:阴道栓塞注射法制备BALB/c雌性小鼠GH动物模型。选取GH模型小鼠30只,分为三组,每组10只。UL27sh RNA作为治疗组,sh RNA空载体作阴性对照组,采用脂质体lipofectamine 2000作为转染试剂,转染500 pmol UL27sh RNA或sh RNA空载体至小鼠阴道上皮。脂质体悬液作空白对照组,间隔4小时给药,每天3次,连续给药7天,第3、5、7天记录小鼠外阴病变程度的评分,给完药后解剖小鼠进行疗效观察。解剖前棉签蘸取阴道分泌物,终点滴定法测定病毒滴度,HE染色技术观察小鼠阴道组织形态学上的病理变化,实时荧光定量PCR检测UL27基因m RNA的表达水平,Western blot分析UL27基因表达的g B蛋白水平。结果:(1)阴道栓塞注射法转染0.1ml TCID5010-5的HSV-2病毒株至小鼠阴道上皮后,外阴出现红肿且分泌物增多,病理学检测有表皮内水疱,细胞内水肿。(2)给药第3天开始,UL27sh RNA治疗组外阴病变程度评分明显低于sh RNA空载体组和空白对照组(P0.05),而sh RNA空载体组评分与空白对照组相比无明显降低(P0.05)。(3)UL27sh RNA治疗组病毒滴度与sh RNA空载体组和空白对照组相比有显著下降(P0.05),而sh RNA空载体组和空白对照组相比无明显差异(P0.05)。(4)病理学观察显示经UL27sh RNA治疗的GH小鼠与sh RNA空载体组和空白对照组相比能有效抑制炎性细胞的侵润,sh RNA空载体组和空白对照组相比抑制炎性细胞侵润效果不明显。(5)实时荧光定量PCR检测结果显示UL27sh RNA治疗组的m RNA表达抑制率为37.28%,比sh RNA空载体组的6.26%和空白对照组的抑制效果明显(P0.05),sh RNA组与空白对照组相比抑制效果不明显(P0.05)。(6)Western blot检测结果显示UL27sh RNA治疗组与sh RNA空载体组和空白对照相比抑制g B蛋白表达明显(P0.05),而sh RNA空载体组与空白对照组相比抑制效果不明显(P0.05)。结论:构建的UL27sh RNA质粒表达载体能够在一定程度上抑制小鼠阴道上皮组织中炎症细胞的浸润,抑制了UL27基因在感染HSV-2的BALB/c雌性小鼠体内基因表达,对GH小鼠有一定的疗效。
[Abstract]:Objective: to transfer the constructed UL27 sh RNA expression vector into BALB/c female genital herpes herpes genitalis (BALB/c) animal model. To evaluate the therapeutic effect of UL27sh RNA plasmid on GH mouse model. Methods: the GH model of female BALB/c mice was established by vagina embolization injection. Thirty GH mice were divided into three groups, 10 rats in each group were used as empty vector of RNA in each group as negative control group. Liposome lipofectamine 2000 was used as transfection reagent to transfect 500 pmol UL27sh RNA or sh RNA empty vector into vaginal epithelium of mice. Liposome suspension was used as blank control group. The score of vulvar lesion in mice was recorded on the 7th day of 3rd 5th day, and the curative effect was observed after dissection. The vaginal secretions were dipping with cotton swabs before dissection. The histopathological changes of vagina of mice were observed by end-point titration and HE staining. Real-time fluorescence quantitative PCR was used to detect the expression level of m RNA in UL27 gene. Western blot was used to analyze the level of G B protein expressed in UL27 gene. Results after transfection of HSV-2 virus strain of 0.1ml TCID5010-5 into the vaginal epithelium of mice by vagina embolization injection, vulva appeared redness and secretion increased. Histopathological examination showed intraepidermal blister. The grade of vulvar lesion in the treatment group of UL27sh RNA was significantly lower than that in the empty carrier group of sh RNA and the blank control group (P0.05), but the score of the empty carrier group of sh RNA was not significantly lower than that of the blank control group. The titer of the virus was significantly decreased compared with the sh RNA empty vector group and the blank control group, while the sh RNA empty vector group had no significant difference compared with the blank control group. The pathological observation showed that the GH mice treated with UL27sh RNA and the sh RNA empty vector group and the sh RNA empty vector group had no significant difference. The expression of m RNA in the UL27sh RNA treatment group was detected by real-time fluorescence quantitative PCR. The results of real-time fluorescence quantitative PCR showed that the inhibition of inflammatory cell invasion was not obvious in the blank control group and the blank control group compared with the blank control group. The results of real-time fluorescence quantitative PCR analysis showed that the expression of m RNA in the UL27sh RNA treatment group was not obvious. The inhibitory rate was 37.28%. The inhibitory effect of UL27sh RNA treatment group, sh RNA empty carrier group and blank control group was lower than that of sh RNA empty carrier group and the blank control group. The results of Western blot detection showed that the effect of UL27sh RNA treatment group, sh RNA empty carrier group and blank control group was not obvious compared with the blank control group. Compared with the control group, the expression of g-B protein was significantly inhibited by p0.05, but the inhibitory effect was not obvious in the sh RNA empty vector group compared with the blank control group. Conclusion: the constructed UL27sh RNA plasmid expression vector can inhibit the infiltration of inflammatory cells in mouse vaginal epithelium to a certain extent. The gene expression of UL27 gene was inhibited in BALB/c female mice infected with HSV-2.
【学位授予单位】：遵义医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R373.11

【相似文献】