TLR2在HCMV宫内感染新生小鼠脑损害中的表达
发布时间：2018-11-24 07:30
【摘要】：目的:通过HCMV宫内感染致新生小鼠脑损害模型的建立,检测新生小鼠脑组织病理学改变、TLR2表达、My D88、IL-8及IFN-β水平,探讨TLR2在HCMV宫内感染致新生小鼠脑损害中的作用机制。方法:1.动物模型的构建与评估:随机选取8~10周龄清洁级(SPF)BALB/c小鼠,血清HCMV-Ig M和Ig G抗体检测均为阴性,随机分为A、B和C三组(雌雄比均为2:1)。A组小鼠经腹腔内注射一次HCMV AD169株悬液0.5ml(5.0log TCID50),B组小鼠经腹腔注射一次人胚肺纤维细胞悬液0.5ml,C组未予以病毒液及细胞悬液注射。在完成上述处理7天后分别对三组小鼠进行血清HCMV-Ig M和Ig G抗体检测(ELISA法)。检测后随机从完成上述处理的各组小鼠中选择:A组54只(雌雄2:1,血清HCMV-Ig M均为阳性),B和C组各15只(雌雄2:1,血清HCMV-Ig M均为阴性),配对饲养,待孕鼠自然分娩,获得仔鼠。观察三组雌性小鼠阴栓出现时间、死胎率;各组部分存活仔鼠于生后第一天(生后24小时,P1)低温麻醉,断头取脑和血液组织。半侧脑组织用于HCMV-Ig M(胶体金法)及病理学检测(HE染色),另半侧脑组织-80℃保存备用。ELISA法检测血清HCMV-Ig M并统计仔鼠宫内感染率及脑损害发生率。2.脑损害新生小鼠脑组织TLR2表达及对下游相关细胞因子的影响:分别选取上述A、B和C三组备用的仔鼠脑组织。随机选取A组血清及脑组织HCMV-Ig M均为阳性的备用标本,根据脑组织病理学结果分为A1组(病理学证实有脑组织损害)和A2组(病理学证实无脑组织损害);B和C组各随机选择16例备用脑组织标本分别为B1(空白对照)和C1组(正常对照),两组血清HCMVIg M与脑组织HCMV-Ig M检测结果为阴性且病理学证实无脑组织损害。A、B、C三组部分新生小鼠于生后第7天按第1天的方法处理及分组。分别检测各组(第1天、第7天)新生小鼠脑组织TLR2m RNA表达(RT-q PCR)及My D88、IL-8、IFN-β水平(ELISA)。结果:(1)注射(病毒液、细胞上清液、未注射)7天后三组雌鼠体重比较仍无差异(F=0.61,P=0.51),血清HCMVIg M抗体检测阳性率A组明显高于B组及C组,比较差异有统计学意义(P=0.00)。配对饲养后三组雌鼠产生阴栓的时间(天)分别为(8.7±1.50、6.5±1.1、6.4±0.80),平均每次产新生鼠数(只)分别为(6.5±1.30、8.0±1.20、7.7±1.00),新生小鼠血清HCMV-Ig M阳性率(%)分别为(40.1%、4.0%、3.0%),新生小鼠脑组织HCMV-Ig M阳性率(%)分别为(22.7%、0%、0%),A组均与B和C组有差异,且差异有统计学意义(P0.05)。A组新生小鼠血清及脑组织HCMV-Ig M阳性的脑组织病理切片可见明显的病理改变为16例,余两组新生小鼠脑组织切片均未见病理改变。(2)第1天证实有脑损害的A1新生小鼠脑组织TLR2m RNA(Ct)、My D88、IL-8、IFN-β的表达水平检测分别为(9.7±1.01、5.41±0.91 ng/ml、39.5±2.78 pg/ml、19.61±2.18 ng/l)明显高于A2、B1及C1组且差异有统计学意义(P0.05)。第7天证实有脑损害的A1新生小鼠脑组织TLR2m RNA(Ct)、My D88、IL-8、的表达水平检测分别为(13.33±1.75、3.77±0.49ng/ml、30.05±1.95pg/ml),与其他三组比较差异无统计学意义,但IFN-β与其他三组比较差异有统计学意义,其中A1高于其他3组,且A2、B1及C1组之间差异无统计学意义。第1天A1组新生小鼠脑组织的TLR2m RNA(Ct)相对表达量较第7天的高,且两组比较差异有统计学意义(t=3.43,P=0.009),其余三组两时间段表达无差异。结论:1.SPF 8~10周龄BALB/c小鼠经腹腔注射HCMV AD169病毒,可诱导宫内感染的发生,且通过胎盘垂直感染子代并造成其脑损害。2.TLR2受体主要通过My D88依赖途径,诱导炎性细胞因子释放,启动局部的炎症反应,参与HCMV宫内感染致新生小鼠脑损害的病理过程。
[Abstract]:Objective: To establish the brain damage model of neonatal mice by HCMV intrauterine infection, to detect the pathological changes, TLR2 expression, My D88, IL-8 and IFN-1 levels in neonatal mice, and to explore the mechanism of TLR2 in the brain damage induced by HCMV intrauterine infection. Method: 1. The animal model was constructed and evaluated: the BALB/ c mice were randomly selected from 8 to 10 weeks old (SPF), and the serum HCMV-IgM and Ig G antibodies were all negative, and they were randomly divided into three groups: A, B and C (both sexes were 2: 1). A group of mice were intraperitoneally injected with a single HCMV AD169 suspension of 0.5ml (5. 0log TCID50), and the group B mice were injected intraperitoneally with a human embryo lung fibroblast suspension of 0.5ml, and the C group was not injected with the virus liquid and the suspension liquid of the cell. Serum HCMV-Ig M and Ig G antibody detection (ELISA) were performed on three groups of mice after the above-mentioned treatment for 7 days. In group A, 54 (male and female 2: 1, serum HCMV-Ig M were positive), 15 (male and female 2: 1, serum HCMV-Ig M were negative) in group A and 15 in group B and C (male and female 2: 1, serum HCMV-Ig M were all negative). The time and stillbirth rate of female mice in three groups of female mice were observed. The half-side brain tissue was used for HCMV-Ig M (colloidal gold method) and pathological examination (HE staining), and the other half-side brain tissue was preserved for use at 80 鈩，
本文编号：2352793
[Abstract]:Objective: To establish the brain damage model of neonatal mice by HCMV intrauterine infection, to detect the pathological changes, TLR2 expression, My D88, IL-8 and IFN-1 levels in neonatal mice, and to explore the mechanism of TLR2 in the brain damage induced by HCMV intrauterine infection. Method: 1. The animal model was constructed and evaluated: the BALB/ c mice were randomly selected from 8 to 10 weeks old (SPF), and the serum HCMV-IgM and Ig G antibodies were all negative, and they were randomly divided into three groups: A, B and C (both sexes were 2: 1). A group of mice were intraperitoneally injected with a single HCMV AD169 suspension of 0.5ml (5. 0log TCID50), and the group B mice were injected intraperitoneally with a human embryo lung fibroblast suspension of 0.5ml, and the C group was not injected with the virus liquid and the suspension liquid of the cell. Serum HCMV-Ig M and Ig G antibody detection (ELISA) were performed on three groups of mice after the above-mentioned treatment for 7 days. In group A, 54 (male and female 2: 1, serum HCMV-Ig M were positive), 15 (male and female 2: 1, serum HCMV-Ig M were negative) in group A and 15 in group B and C (male and female 2: 1, serum HCMV-Ig M were all negative). The time and stillbirth rate of female mice in three groups of female mice were observed. The half-side brain tissue was used for HCMV-Ig M (colloidal gold method) and pathological examination (HE staining), and the other half-side brain tissue was preserved for use at 80 鈩，
本文编号：2352793
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