TLR2在HCMV宫内感染子代新生小鼠心肌损伤中的表达
发布时间：2018-09-06 12:44
【摘要】：目的:初步探讨人巨细胞病毒(human cytomegalovirus,HCMV)宫内感染所致子代新生小鼠心肌损伤模型构建方法;通过损伤心肌组织中TLR2基因表达及下游相关信号分子MyD88、IFN-β、IL-8水平的检测,以初步阐明TLR2信号通路在HCMV宫内感染子代新生小鼠心肌损伤过程中的激活状态。方法:随机选取8~10w的SPF级BALB/c小鼠,雌雄比为2:1,血清HCMV-IgM、Ig G均为阴性,随机分为实验组、空白对照组和正常对照组。实验组和空白对照组每只小鼠分别予一次性腹腔注射HCMV AD169株悬液(5.0log TCID50)和HELF细胞悬液0.5ml,正常对照组未予任何处理。1周后胶体金法检测三组小鼠血清HCMV-IgM,实验组随机选择血清HCMV-IgM阳性小鼠,雌性60只,雄性30只;空白对照组和正常对照组均随机选择血清HCMV-IgM阴性小鼠,分别为雌性12只,雄性6只,随后分别按雌雄比2:1配对合笼饲养,待雌鼠妊娠后取出单独饲养,自然分娩获得子代新生小鼠。对于各组活产的子代新生小鼠,胶体金法检测各组血清及心肌HCMV-IgM,免疫组织化学法(HE染色)观察心肌病理学改变,RT-PCR检测心肌TLR2 mRNA,酶联免疫法(ELISA)检测心肌MyD88、IL-8、IFN-β水平,比色法检测心肌组织Caspase 8活性。结果:(1)BALB/c小鼠腹腔内接种HCMV AD169一周后实验组血清HCMV-IgM阳性率,雌鼠为82.5%(66/80),雄鼠为87.5%(35/40);(2)实验组活产子代新生小鼠血清HMCV-IgM阳性率为53.4%(118/221),血清HMCV-IgM阳性仔鼠中心肌HMCV-IgM阳性率35.6%(42/118);(3)实验组所有心肌HMCV-IgM阳性子代新生小鼠心肌病理学改变表现为心肌纤排列维紊乱、心肌细胞水肿或(和)凋亡,空白和正常对照组可见心肌纤维排列整齐,着色均匀;(4)实验组心肌HMCV-IgM阳性子代新生小鼠心肌TLR2 mRNAΔCt值(10.63±1.06)低于空白对照组(12.88±1.55)和正常对照组(12.75±1.28),差异均有统计学意义(P0.01),MyD88水平(4.48±0.74)、IL-8(29.58±1.98)和IFN-β(16.90±1.78)水平显著高于空白对照组(3.79±0.37、21.24±1.86、12.96±1.64)和正常对照组子代小鼠(3.68±0.49、22.40±1.71、12.21±1.53),差异均有统计学意义(P0.05),且MyD88水平与IFN-β和IL-8水平均呈正相关(r=0.857、0.837,P0.05);(5)实验组心肌HMCV-IgM阳性子代新生小鼠心肌Caspase 8活性(67.25±34.57)与空白对照组(72.37±36.34)、正常对照组(63.18±33.67)比较,差异均无统计学意义(P0.05)。结论:(1)通过BALB/c小鼠腹腔内接种HCMV AD169诱导小鼠感染HCMV,再经雌雄交配、繁殖,可成功构建HCMV宫内感染所致子代新生小鼠心肌损伤模型。(2)TLR2信号通路可能参与HCMV诱导的宫内感染所致子代新生小鼠心肌损伤的病理过程。主要机制可能是通过经典的TLR2/MyD88通路介导炎性细胞因子的释放参与子代新生小鼠心肌损伤的病理过程。(3)TLR2/MyD88/caspase 8介导的细胞凋亡途径在此阶段可能尚未参与子代新生小鼠心肌损伤的病理生理过程。
[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 卤1.06) was significantly lower than that in the blank control group (12.88 卤1.55) and the normal control group (12.75 卤1.28), the difference being statistically significant (P0.01). The levels of IL-8 (29.58 卤1.98) and IFN- 尾 (16.90 卤1.78) in the experimental group were significantly higher than those in the blank control group (3.79 卤0.3721.24 卤1.86 卤12.96 卤1.64) and the normal control group (P 0.01). There was significant difference between the control group and the control group (3.68 卤0.49 卤1.71 卤12.21 卤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 卤34.57) was significantly higher than that of the control group (72.37 卤36.34) and the normal control group (63.18 卤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.
【学位授予单位】：皖南医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R714.251
本文编号：2226390
[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 卤1.06) was significantly lower than that in the blank control group (12.88 卤1.55) and the normal control group (12.75 卤1.28), the difference being statistically significant (P0.01). The levels of IL-8 (29.58 卤1.98) and IFN- 尾 (16.90 卤1.78) in the experimental group were significantly higher than those in the blank control group (3.79 卤0.3721.24 卤1.86 卤12.96 卤1.64) and the normal control group (P 0.01). There was significant difference between the control group and the control group (3.68 卤0.49 卤1.71 卤12.21 卤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 卤34.57) was significantly higher than that of the control group (72.37 卤36.34) and the normal control group (63.18 卤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.
【学位授予单位】：皖南医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R714.251
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