小鼠巨细胞病毒宫内感染实验模型研究

发布时间：2018-05-06 06:30

本文选题：鼠巨细胞病毒 + 宫内感染　；参考：《华中科技大学》2006年博士论文

【摘要】： 第一部分MCMV宫内感染对妊娠和胎仔生长发育的影响【目的】建立小鼠MCMV宫内感染模型，，观察MCMV宫内感染对妊娠结局及胎仔生长发育的影响。【方法】(1)MCMV Smith毒株体外细胞培养法传毒增殖，Reed-Muench法计算病毒半数细胞感染剂量TCID_(50)。(2)ELISA法筛查MCMV Ig M、IgG双阴性BALB／C小鼠，雌雄小鼠交配受孕后随机选择孕龄10～11d孕鼠分组：高、中、低病毒感染剂量组各14、12、12只，经胎盘分别接种1×10~7TCID_(50)、1×16~6TCID_(50)、1×10~5TCID_(50) MCMV悬液；模型对照组12只，同法注射等量无菌3％FCS DMEM培养液，空白对照组12只，开腹只清点孕囊数，不做其他特殊处理。①观察孕鼠一般情况。②处死当日留取母鼠血液行ELISA Ig M检测。③留取胎盘及胎仔脏器组织(脑、肺、肝脏、肾脏)，各脏器部分组织行病理学检查，部分组织匀浆液上清体外培养法行病毒学分离。④部分胎盘组织RT-PCR法检测MCMV mRNA和胎仔脏器PCR法检测MCMV DNA。⑤记录各组母鼠妊娠进展及结局。⑥观察仔鼠生长发育情况，称各组胎仔出生时体重及测量身长、头围。【结果】(1)MCMV Smith毒株半数细胞感染剂量为5.62×10~7TCID_(50)／ml。(2)①高、中、低病毒滴度组孕鼠均出现非特异性病毒血症征象，所有孕鼠MCMV Ig M检测均阳性；模型对照组和空白对照组均为阴性。②感染组胎盘及胎仔脏器组织病毒学分离阳性，相应组织标本出现病理学改变。③感染组胎盘MCMV mRNA检测阳性率为58.49％(30／53)，胎仔各脏器MCMV DNA检测阳性率分别为：脑37.73％(20／53)、肺32.07％(17／53)、肝脏22.64％(12／53)、肾脏22.64％(12／53)。(3)①各组孕鼠异常妊娠发生率比较，高、中、低病毒滴度组均要高于模型对照组，差异有统计学意义(P＜0.05)。②各组活胎率比较，高、中、低病毒滴度组均要低于模型对照组，差异有极显著性意义(P＜0.001)。③各组活胎仔出生身长、头围、体重比较，高、中、低病毒滴度组均要低于模型对照组，差异有显著性意义(P＜0.01)；④各组活胎中小头畸形胎仔率比较，高、中、低病毒滴度组高于模型对照组，差异有显著性意义(P＜0.01)⑤各组活胎仔低出生体重胎仔率比较，高、中、低病毒滴度组高于模型对照组，差异有统计学意义(P＜0.01)。以上各观测指标，模型对照组与空白对照组间差异均无统计学意义(P＞0.05)。【结论】经胎盘接种MCMV可以建立小鼠宫内感染动物模型；MCMV宫内感染增加孕鼠异常妊娠发生率，严重影响宫内胎仔生长发育并导致低出生体重及小头畸形胎仔的发生率增高。第二部分MCMV感染孕鼠T淋巴细胞亚型的影响【目的】观察MCMV感染不同时相孕鼠T淋巴细胞增殖和亚型的改变，了解孕鼠感染MCMV后机体免疫功能的变化规律。【方法】ELISA法筛查MCMV Ig M、IgG双阴性BALB／C小鼠，雌雄小鼠交配受孕后随机选择孕10～11d天孕鼠分组：感染组：20只，经胎盘接种1×10~6TCID_(50) MCMV悬液；模型对照组：20只，同法注射等量无菌3％FCS DMEM培养液；空白对照组：20只，不做任何特殊处理的正常孕鼠。于孕第14d、17d、20d、自然分娩当日随机取孕鼠，①经心脏采血行ELISA检测MCMV—IgM。②无菌取脾脏，称取脾脏湿重并行病理学观察。③制备脾细胞，分离单个核细胞(PBMC)，培养过夜取悬浮细胞，CCK-8法检测T淋巴细胞体外增殖反应。④流式细胞术检测T淋巴细胞亚型CD3~+、CD4~+、CD8~+T细胞的相对数并计算CD4~+／CD8~+比值。【结果】①感染组孕鼠MCMV—IgM检测均阳性，模型对照组和空白对照组均为阴性。②感染组孕鼠脾脏湿重与相应时相模型对照组较，均高于模型对照组，并随感染时间的延长逐渐增加，差异有统计学意义(P＜0.01)。③感染组孕鼠T淋巴细胞增殖反应和相应时相模型对照组比较，要低于模型对照组，差异有显著性意义(P＜0.01)。④感染组孕鼠CD3~+T细胞的相对百分数与相应时相模型对照组比较，差异无统计学意义(P＞0.05)；CD4~+T细胞的相对百分数与相应时相模型对照组比较，孕17d、孕20d、分娩当日均要低于模型对照组，并随着感染时间的延长逐渐降低，差异有显著性意义(P＜0.01)；CD8~+T细胞的相对数与相应时相模型对照组比较，孕17d、孕20d、分娩当日均要高于模型对照组，并随着感染时间的延长逐渐升高，差异有显著性意义(P＜0.01)；感染组CD4~+／CD8~+比值与相应时相模型对照组比较，各时相均要低于模型对照组，并随感染时间的延长逐渐降低，差异有显著性意义(P＜0.01)。以上各检测指标，模型对照组与空白对照组间差异无统计学意义(P＞0.05)。【结论】MCMV感染孕鼠，脾脏T淋巴细胞的变化规律是：CD4~+T细胞降低，CD8~+T细胞升高，CD4~+／CD8~+比值下降，并与感染时间呈现出一定的时间—效应关系，机体免疫功能处于抑制状态，此免疫抑制状态可能是MCMV垂直传播的免疫机制之一。第三部分MCMV感染对孕鼠NK细胞和CTL活性的影响【目的】观察MCMV感染不同时相孕鼠NK细胞和CTL活性改变，了解孕鼠感染MCMV后体内细胞免疫功能的变化规律。【方法】ELISA法筛查MCMV Ig M、IgG双阴性BALB／C小鼠，雌雄小鼠交配受孕后随机选择孕10～11d天孕鼠分组：感染组：20只，经胎盘接种1×10~6TCID_(50) MCMV悬液；模型对照组：20只，同法注射等量无菌3％FCS DMEM培养液；空白对照组：20只，只开腹，不做其它特殊处理的正常孕鼠。于孕第14d、17d、20d、自然分娩当日随机取孕鼠①经心脏采血行ELISA检测MCMV—IgM。②制备脾细胞，分离单个核细胞(PBMC)，培养过夜取贴壁细胞，CCK-8法检测NK细胞的杀伤活性。③制备脾细胞，分离单个核细胞(PBMC)，培养过夜取悬浮细胞，ELISPOT法检测CTL活化功能及CCK-8法检测CTL的杀伤活性。【结果】①感染组孕鼠MCMV—IgM检测均阳性，模型对照组和空白对照组均为阴性。②感染组孕鼠NK细胞的杀伤活性与相应时相的对照组比较，孕14d时要高于对照组，其余时相均低于模型对照组，并随感染时间的延长逐渐降低，差异有极显著性意义(P＜0.001)。④感染组孕鼠CTL产生的斑点频率与相应时相模型对照组比较，均低于模型对照组，并随感染时间的延长逐渐降低，差异有极显著性意义(P＜0.001)；CTL的杀伤功能与相应时相的模型对照组比较，孕17d至分娩各时相均低于模型对照组，并随感染时间的延长逐渐降低，差异有极显著性意义(P＜0.001)。【结论】MCMV感染孕鼠，NK细胞的杀伤活性受到抑制，CTL的活化和杀伤功能亦明显受到抑制，并与感染时间呈现一定的时间—效应关系，母体抗MCMV的细胞免疫功能处于抑制状态，此免疫抑制状态可能是MCMV宫内传播免疫机制之一。
[Abstract]:Part one the effect of MCMV intrauterine infection on pregnancy and fetal growth and development
[Objective] to establish mouse MCMV intrauterine infection model and observe the effect of MCMV intrauterine infection on pregnancy outcome and fetal growth and development.
[Methods] (1) the proliferation of MCMV Smith strains in vitro was transmitted by cell culture, and the dose of half cell infection was TCID_ (50) by Reed-Muench. (2) ELISA method was used to screen MCMV Ig M, IgG double negative BALB / C mice, and the male and female mice were randomly selected for pregnancy after pregnancy and were divided into groups: high, middle, and low virus infection groups. The placenta was inoculated with 1 x 10~7TCID_ (50), 1 x 16~6TCID_ (50), 1 x 10~5TCID_ (50) MCMV suspension, 12 rats in the model control group, with the same amount of sterile 3%FCS DMEM culture and 12 in the blank control group. The number of gestation sac was only counted and no special treatment was done. (1) the normal condition of pregnant rats was observed. (2) ELISA Ig M test of mother mice on the day of death was performed. Take the placenta and fetal organ tissue (brain, lung, liver, kidney), the organs part of the organs of the pathological examination, part of the homogenate liquid supernatant in vitro culture method for Virology separation. Part of the placental tissue RT-PCR method to detect MCMV mRNA and fetal viscera PCR method to detect MCMV DNA. in pregnant women mice pregnancy progress and outcome. 6 observation The growth and development of the offspring were described as the body weight, body length and head circumference of each group.
[results] (1) the dose of half cell infection in MCMV Smith strain was 5.62 x 10~7TCID_ (50) / ml. (2) high. In the middle, low virus titer group, all pregnant rats were all non specific viremia signs, all pregnant mice were positive for MCMV Ig M detection; both the model control group and the blank control group were negative. The positive rate of MCMV mRNA in the placenta of infection group was 58.49% (30 / 53), and the positive rates of MCMV DNA in fetal organs were 37.73% (20 / 53), 32.07% (17 / 53) of the lung, 22.64% (12 / 53) of the liver, kidney 22.64% (12 / 53), and the incidence of abnormal pregnancy in each group of pregnant rats was compared. The high, middle, and low virus titer groups were higher than the model control group, and the difference was statistically significant (P < 0.05). 2 groups of high, middle and low virus tires were lower than the model control group, and the difference was significant (P < 0.001). (3) the length of birth, head circumference, weight comparison, high, middle, low virus titer groups were all lower than the model group. In the control group, the difference was significant (P < 0.01); (4) compared with the model control group, the rate of high, middle and low virus titres was higher than that in the model control group. The difference had significant difference (P < 0.01). The low birth weight fetus rate of the living fetus was higher than that in the model control group, and the difference was statistically significant. (P < 0.01). There was no significant difference in the above indexes between the model control group and the blank control group (P > 0.05).
[Conclusion] the animal model of intrauterine infection can be established through the placental inoculation of MCMV, and the incidence of abnormal pregnancy in pregnant mice is increased by MCMV intrauterine infection, which seriously affects the growth and development of intrauterine fetus and leads to the increase of the incidence of low birth weight and small head deformity.
The second part is the effect of MCMV infection on T lymphocyte subtypes in pregnant mice.
[Objective] to observe the changes of T lymphocyte proliferation and subtype in MCMV infected mice at different times, and to understand the changes of immune function after MCMV infection in pregnant rats.
[Methods] the ELISA method was used to screen MCMV Ig M, IgG double negative BALB / C mice, and the male and female mice were randomly selected for pregnancy 10 to 11d days after mating and pregnancy. The infected group was infected with 1 x 10~6TCID_ (50) MCMV suspension via the placenta; the model control group was 20, the same amount of sterile 3%FCS DMEM culture was equal to the same method, and the blank control group was 20, and did not be appointed. 14d, 17D, 20d, pregnant rats were randomly selected on the day of natural childbirth. (1) the spleen was obtained by ELISA detection of MCMV - IgM. by cardiac blood collection. The spleen was taken asepsis and the spleen was taken aseptic and pathological observation. Splenocyte was prepared, single nucleus cell (PBMC) was isolated, and the suspension cells were cultured overnight, and CCK-8 method detected T lymphocyte in vitro increase. (4) flow cytometry was used to detect the relative numbers of CD3~+, CD4~+ and CD8~+T cells in T lymphocyte subtypes and calculate the ratio of CD4~+ / CD8~+.
[results] (1) the MCMV - IgM detection of pregnant rats in the infection group was positive, and both the model control group and the blank control group were negative. The spleen wet weight of the pregnant rats and the corresponding model control group were higher than those in the model control group, and the difference was statistically significant (P < 0.01). (3) the T lymphocyte of pregnant rats in the infection group. The comparison of the proliferation reaction and the corresponding phase model control group was lower than the model control group. The difference was significant (P < 0.01). (4) the relative percentage of CD3~+T cells in the infected rats was not statistically significant (P > 0.05) compared with the corresponding model control group (P > 0.05), and the relative percentage of the cell cell was compared with the corresponding model control group. Pregnancy 17D, pregnancy 20d, delivery day is lower than the model control group, and gradually decrease with the time of infection, the difference has significant significance (P < 0.01), the relative number of CD8~+T cells compared with the corresponding phase model control group, pregnant 17D, pregnant 20d, the day of childbirth should be higher than the model control group, and with the prolongation of infection time, the difference gradually increased, the difference There was significant significance (P < 0.01), and the ratio of CD4~+ / CD8~+ in the infection group was lower than that of the model control group, and the difference was significantly lower with the prolongation of the infection time (P < 0.01). The difference between the model and the blank control group was not statistically significant (P > 0.05). ).
[Conclusion] the changes of T lymphocyte in MCMV infected rats were: the decrease of CD4~+T cells, the increase of CD8~+T cells, the decrease of CD4~+ / CD8~+ ratio, and a certain time effect relationship with the time of infection, the immune function of the body was in the state of inhibition, and this immunosuppressive state may be one of the immune mechanisms of vertical transmission of MCMV.
The third part is the effect of MCMV infection on NK cell and CTL activity in pregnant rats.
[Objective] to observe the changes of NK cells and CTL activity in different pregnant mice infected with MCMV, and to understand the changes of cellular immune function in pregnant rats after MCMV infection.
[Methods] ELISA method was used to screen MCMV Ig M, IgG double negative BALB / C mice, and the male and female mice were randomly selected from 10 to 11d days after mating and pregnancy: infected group: 20 rats were inoculated with 1 x 10~6TCID_ (50) MCMV suspension via the placenta; the model control group was 20, with the same amount of aseptic 3%FCS DMEM culture, and the blank control group: 20, only open abdomen No other special treatment of normal pregnant mice. In pregnancy 14d, 17D, 20d, pregnant rats were randomly selected on the day of natural childbirth to prepare spleen cells by ELISA detection of MCMV - IgM. by cardiac blood collection, isolated mononuclear cells (PBMC), cultured for overnight adherent cells, and CCK-8 method to detect the killing activity of NK cells. (3) preparation of spleen cells and separate mononuclear cells (PBMC). The suspension cells were cultured overnight, the activation function of CTL was detected by ELISPOT, and the killing activity of CTL was detected by CCK-8.
[results] (1) the MCMV - IgM detection of pregnant rats in the infection group were all positive, and both the model control group and the blank control group were negative. The killing activity of NK cells in the pregnant rats was higher than that in the control group. The other phase was lower than the control group, and the other phase was lower than that in the model control group, and the difference was extremely obvious with the prolongation of the infection time. The difference was extremely significant. (P < 0.001). (4) the spot frequency of CTL produced by pregnant rats in the infection group was lower than that in the model control group, which was lower than that in the model control group, and gradually decreased with the prolongation of the time of infection (P < 0.001). The killing function of CTL and the model control group of the corresponding phase were lower than those of the pregnant 17D to the time of delivery. In the model control group, it gradually decreased with the prolongation of the infection time, and the difference was very significant (P < 0.001).
[Conclusion] the killing activity of NK cells in MCMV infected pregnant rats was inhibited, and the activation and killing function of CTL were also inhibited, and the time and effect relationship was presented with the time of infection. The immune function of the mother body against MCMV was in the state of inhibition. This immunosuppressive state may be one of the mechanism of the intrauterine immune immune mechanism of MCMV.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R-332

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