發(fā)布時(shí)間：2018-09-06 12:44

【摘要】：目的:初步探討人巨細(xì)胞病毒(human cytomegalovirus,HCMV)宮內(nèi)感染所致子代新生小鼠心肌損傷模型構(gòu)建方法;通過(guò)損傷心肌組織中TLR2基因表達(dá)及下游相關(guān)信號(hào)分子MyD88、IFN-β、IL-8水平的檢測(cè),以初步闡明TLR2信號(hào)通路在HCMV宮內(nèi)感染子代新生小鼠心肌損傷過(guò)程中的激活狀態(tài)。方法:隨機(jī)選取8~10w的SPF級(jí)BALB/c小鼠,雌雄比為2:1,血清HCMV-IgM、Ig G均為陰性,隨機(jī)分為實(shí)驗(yàn)組、空白對(duì)照組和正常對(duì)照組。實(shí)驗(yàn)組和空白對(duì)照組每只小鼠分別予一次性腹腔注射HCMV AD169株懸液(5.0log TCID50)和HELF細(xì)胞懸液0.5ml,正常對(duì)照組未予任何處理。1周后膠體金法檢測(cè)三組小鼠血清HCMV-IgM,實(shí)驗(yàn)組隨機(jī)選擇血清HCMV-IgM陽(yáng)性小鼠,雌性60只,雄性30只;空白對(duì)照組和正常對(duì)照組均隨機(jī)選擇血清HCMV-IgM陰性小鼠,分別為雌性12只,雄性6只,隨后分別按雌雄比2:1配對(duì)合籠飼養(yǎng),待雌鼠妊娠后取出單獨(dú)飼養(yǎng),自然分娩獲得子代新生小鼠。對(duì)于各組活產(chǎn)的子代新生小鼠,膠體金法檢測(cè)各組血清及心肌HCMV-IgM,免疫組織化學(xué)法(HE染色)觀察心肌病理學(xué)改變,RT-PCR檢測(cè)心肌TLR2 mRNA,酶聯(lián)免疫法(ELISA)檢測(cè)心肌MyD88、IL-8、IFN-β水平,比色法檢測(cè)心肌組織Caspase 8活性。結(jié)果:(1)BALB/c小鼠腹腔內(nèi)接種HCMV AD169一周后實(shí)驗(yàn)組血清HCMV-IgM陽(yáng)性率,雌鼠為82.5%(66/80),雄鼠為87.5%(35/40);(2)實(shí)驗(yàn)組活產(chǎn)子代新生小鼠血清HMCV-IgM陽(yáng)性率為53.4%(118/221),血清HMCV-IgM陽(yáng)性仔鼠中心肌HMCV-IgM陽(yáng)性率35.6%(42/118);(3)實(shí)驗(yàn)組所有心肌HMCV-IgM陽(yáng)性子代新生小鼠心肌病理學(xué)改變表現(xiàn)為心肌纖排列維紊亂、心肌細(xì)胞水腫或(和)凋亡,空白和正常對(duì)照組可見(jiàn)心肌纖維排列整齊,著色均勻;(4)實(shí)驗(yàn)組心肌HMCV-IgM陽(yáng)性子代新生小鼠心肌TLR2 mRNAΔCt值(10.63±1.06)低于空白對(duì)照組(12.88±1.55)和正常對(duì)照組(12.75±1.28),差異均有統(tǒng)計(jì)學(xué)意義(P0.01),MyD88水平(4.48±0.74)、IL-8(29.58±1.98)和IFN-β(16.90±1.78)水平顯著高于空白對(duì)照組(3.79±0.37、21.24±1.86、12.96±1.64)和正常對(duì)照組子代小鼠(3.68±0.49、22.40±1.71、12.21±1.53),差異均有統(tǒng)計(jì)學(xué)意義(P0.05),且MyD88水平與IFN-β和IL-8水平均呈正相關(guān)(r=0.857、0.837,P0.05);(5)實(shí)驗(yàn)組心肌HMCV-IgM陽(yáng)性子代新生小鼠心肌Caspase 8活性(67.25±34.57)與空白對(duì)照組(72.37±36.34)、正常對(duì)照組(63.18±33.67)比較,差異均無(wú)統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論:(1)通過(guò)BALB/c小鼠腹腔內(nèi)接種HCMV AD169誘導(dǎo)小鼠感染HCMV,再經(jīng)雌雄交配、繁殖,可成功構(gòu)建HCMV宮內(nèi)感染所致子代新生小鼠心肌損傷模型。(2)TLR2信號(hào)通路可能參與HCMV誘導(dǎo)的宮內(nèi)感染所致子代新生小鼠心肌損傷的病理過(guò)程。主要機(jī)制可能是通過(guò)經(jīng)典的TLR2/MyD88通路介導(dǎo)炎性細(xì)胞因子的釋放參與子代新生小鼠心肌損傷的病理過(guò)程。(3)TLR2/MyD88/caspase 8介導(dǎo)的細(xì)胞凋亡途徑在此階段可能尚未參與子代新生小鼠心肌損傷的病理生理過(guò)程。
[Abstract]:Objective: to study the method of constructing myocardial injury model of newborn mice induced by human cytomegalovirus (human cytomegalovirus,HCMV) intrauterine infection, and to detect the expression of TLR2 gene and the level of MyD88,IFN- 尾 -IL-8 in injured myocardium. To elucidate the activation of TLR2 signaling pathway during myocardial injury in newborn mice with HCMV intrauterine infection. Methods: BALB/c mice of SPF grade were randomly selected for 8 weeks. The ratio of female to male was 2: 1, and the serum HCMV-IgM,Ig G was negative. The mice were randomly divided into three groups: experimental group, blank control group and normal control group. Each mouse in the experimental group and the blank control group were given a single intraperitoneal injection of 0.5 ml of HCMV AD169 strain suspension (5.0log TCID50) and HELF cell suspension, while the normal control group was not given any treatment for the detection of serum HCMV-IgM, by colloidal gold method after 1 weeks of treatment. The experimental group was randomly selected. Serum HCMV-IgM positive mice, Serum HCMV-IgM negative mice were randomly selected as 12 females and 6 males in the blank control group and the normal control group. The newborn mice of offspring were obtained by natural delivery. The serum and myocardial HCMV-IgM, immunohistochemical method (HE staining) was used to detect the myocardial pathological changes of newborn mice born in each group. The level of MyD88,IL-8,IFN- 尾 in myocardium was detected by TLR2 mRNA, enzyme-linked immunosorbent assay (ELISA) by RT-PCR. Myocardial Caspase 8 activity was detected by colorimetric method. Results: (1) the positive rate of serum HCMV-IgM in the experimental group was one week after intraperitoneal inoculation of HCMV AD169 in BALB/c mice. The positive rate of serum HMCV-IgM was 82.5% (66 / 80) in female mice and 87.5% (35 / 40); (/ 2) in male mice. The positive rate of serum HMCV-IgM was 53.4% (118 / 221) in newborn mice of live birth generation in experimental group. The positive rate of HMCV-IgM in central muscle of neonatal mice with positive serum HMCV-IgM was 35.6% (42118); (3). Myocardial edema or / and apoptosis were observed in cardiomyocytes. Myocardial fibers were neatly arranged in blank and normal control groups. (4) the myocardial TLR2 mRNA 螖 Ct value of newborn mice with positive HMCV-IgM in the experimental group (10.63 鹵1.06) was significantly lower than that in the blank control group (12.88 鹵1.55) and the normal control group (12.75 鹵1.28), the difference being statistically significant (P0.01). The levels of IL-8 (29.58 鹵1.98) and IFN- 尾 (16.90 鹵1.78) in the experimental group were significantly higher than those in the blank control group (3.79 鹵0.3721.24 鹵1.86 鹵12.96 鹵1.64) and the normal control group (P 0.01). There was significant difference between the control group and the control group (3.68 鹵0.49 鹵1.71 鹵12.21 鹵1.53) (P0.05), and the level of MyD88 was positively correlated with the levels of IFN- 尾 and IL-8 (r 0.8577.37% P0.05); (5). The myocardial Caspase 8 activity of the experimental group (67.25 鹵34.57) was significantly higher than that of the control group (72.37 鹵36.34) and the normal control group (63.18 鹵33.67). The difference was not statistically significant (P0.05). Conclusion: (1) Intraperitoneal inoculation of BALB/c mice with HCMV AD169 induces HCMV, infection in mice before mating and reproduction by male and female. (2) TLR2 signaling pathway may be involved in the pathological process of myocardial injury induced by HCMV induced intrauterine infection in newborn mice. The main mechanism may be that the release of inflammatory cytokines mediated by classical TLR2/MyD88 pathway is involved in the pathological process of myocardial injury in newborn mice. (3) the apoptosis pathway mediated by TLR2/MyD88/caspase 8 may not be involved in the generation of newborn mice at this stage. The pathophysiological process of myocardial injury.
【學(xué)位授予單位】：皖南醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R714.251

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