【摘要】：目的:通過(guò)HCMV宮內(nèi)感染致新生小鼠腦損害模型的建立,檢測(cè)新生小鼠腦組織病理學(xué)改變、TLR2表達(dá)、My D88、IL-8及IFN-β水平,探討TLR2在HCMV宮內(nèi)感染致新生小鼠腦損害中的作用機(jī)制。方法:1.動(dòng)物模型的構(gòu)建與評(píng)估:隨機(jī)選取8~10周齡清潔級(jí)(SPF)BALB/c小鼠,血清HCMV-Ig M和Ig G抗體檢測(cè)均為陰性,隨機(jī)分為A、B和C三組(雌雄比均為2:1)。A組小鼠經(jīng)腹腔內(nèi)注射一次HCMV AD169株懸液0.5ml(5.0log TCID50),B組小鼠經(jīng)腹腔注射一次人胚肺纖維細(xì)胞懸液0.5ml,C組未予以病毒液及細(xì)胞懸液注射。在完成上述處理7天后分別對(duì)三組小鼠進(jìn)行血清HCMV-Ig M和Ig G抗體檢測(cè)(ELISA法)。檢測(cè)后隨機(jī)從完成上述處理的各組小鼠中選擇:A組54只(雌雄2:1,血清HCMV-Ig M均為陽(yáng)性),B和C組各15只(雌雄2:1,血清HCMV-Ig M均為陰性),配對(duì)飼養(yǎng),待孕鼠自然分娩,獲得仔鼠。觀察三組雌性小鼠陰栓出現(xiàn)時(shí)間、死胎率;各組部分存活仔鼠于生后第一天(生后24小時(shí),P1)低溫麻醉,斷頭取腦和血液組織。半側(cè)腦組織用于HCMV-Ig M(膠體金法)及病理學(xué)檢測(cè)(HE染色),另半側(cè)腦組織-80℃保存?zhèn)溆�。ELISA法檢測(cè)血清HCMV-Ig M并統(tǒng)計(jì)仔鼠宮內(nèi)感染率及腦損害發(fā)生率。2.腦損害新生小鼠腦組織TLR2表達(dá)及對(duì)下游相關(guān)細(xì)胞因子的影響:分別選取上述A、B和C三組備用的仔鼠腦組織。隨機(jī)選取A組血清及腦組織HCMV-Ig M均為陽(yáng)性的備用標(biāo)本,根據(jù)腦組織病理學(xué)結(jié)果分為A1組(病理學(xué)證實(shí)有腦組織損害)和A2組(病理學(xué)證實(shí)無(wú)腦組織損害);B和C組各隨機(jī)選擇16例備用腦組織標(biāo)本分別為B1(空白對(duì)照)和C1組(正常對(duì)照),兩組血清HCMVIg M與腦組織HCMV-Ig M檢測(cè)結(jié)果為陰性且病理學(xué)證實(shí)無(wú)腦組織損害。A、B、C三組部分新生小鼠于生后第7天按第1天的方法處理及分組。分別檢測(cè)各組(第1天、第7天)新生小鼠腦組織TLR2m RNA表達(dá)(RT-q PCR)及My D88、IL-8、IFN-β水平(ELISA)。結(jié)果:(1)注射(病毒液、細(xì)胞上清液、未注射)7天后三組雌鼠體重比較仍無(wú)差異(F=0.61,P=0.51),血清HCMVIg M抗體檢測(cè)陽(yáng)性率A組明顯高于B組及C組,比較差異有統(tǒng)計(jì)學(xué)意義(P=0.00)。配對(duì)飼養(yǎng)后三組雌鼠產(chǎn)生陰栓的時(shí)間(天)分別為(8.7±1.50、6.5±1.1、6.4±0.80),平均每次產(chǎn)新生鼠數(shù)(只)分別為(6.5±1.30、8.0±1.20、7.7±1.00),新生小鼠血清HCMV-Ig M陽(yáng)性率(%)分別為(40.1%、4.0%、3.0%),新生小鼠腦組織HCMV-Ig M陽(yáng)性率(%)分別為(22.7%、0%、0%),A組均與B和C組有差異,且差異有統(tǒng)計(jì)學(xué)意義(P0.05)。A組新生小鼠血清及腦組織HCMV-Ig M陽(yáng)性的腦組織病理切片可見(jiàn)明顯的病理改變?yōu)?6例,余兩組新生小鼠腦組織切片均未見(jiàn)病理改變。(2)第1天證實(shí)有腦損害的A1新生小鼠腦組織TLR2m RNA(Ct)、My D88、IL-8、IFN-β的表達(dá)水平檢測(cè)分別為(9.7±1.01、5.41±0.91 ng/ml、39.5±2.78 pg/ml、19.61±2.18 ng/l)明顯高于A2、B1及C1組且差異有統(tǒng)計(jì)學(xué)意義(P0.05)。第7天證實(shí)有腦損害的A1新生小鼠腦組織TLR2m RNA(Ct)、My D88、IL-8、的表達(dá)水平檢測(cè)分別為(13.33±1.75、3.77±0.49ng/ml、30.05±1.95pg/ml),與其他三組比較差異無(wú)統(tǒng)計(jì)學(xué)意義,但I(xiàn)FN-β與其他三組比較差異有統(tǒng)計(jì)學(xué)意義,其中A1高于其他3組,且A2、B1及C1組之間差異無(wú)統(tǒng)計(jì)學(xué)意義。第1天A1組新生小鼠腦組織的TLR2m RNA(Ct)相對(duì)表達(dá)量較第7天的高,且兩組比較差異有統(tǒng)計(jì)學(xué)意義(t=3.43,P=0.009),其余三組兩時(shí)間段表達(dá)無(wú)差異。結(jié)論:1.SPF 8~10周齡BALB/c小鼠經(jīng)腹腔注射HCMV AD169病毒,可誘導(dǎo)宮內(nèi)感染的發(fā)生,且通過(guò)胎盤垂直感染子代并造成其腦損害。2.TLR2受體主要通過(guò)My D88依賴途徑,誘導(dǎo)炎性細(xì)胞因子釋放,啟動(dòng)局部的炎癥反應(yīng),參與HCMV宮內(nèi)感染致新生小鼠腦損害的病理過(guò)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，

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