小鼠巨細(xì)胞病毒宮內(nèi)感染實(shí)驗(yàn)?zāi)Ｐ脱芯?/H1>
發(fā)布時間：2018-05-06 06:30
本文選題：鼠巨細(xì)胞病毒 + 宮內(nèi)感染 ； 參考：《華中科技大學(xué)》2006年博士論文
【摘要】： 第一部分MCMV宮內(nèi)感染對妊娠和胎仔生長發(fā)育的影響 【目的】建立小鼠MCMV宮內(nèi)感染模型，，觀察MCMV宮內(nèi)感染對妊娠結(jié)局及胎仔生長發(fā)育的影響。 【方法】(1)MCMV Smith毒株體外細(xì)胞培養(yǎng)法傳毒增殖，Reed-Muench法計(jì)算病毒半數(shù)細(xì)胞感染劑量TCID_(50)。(2)ELISA法篩查MCMV Ig M、IgG雙陰性BALB／C小鼠，雌雄小鼠交配受孕后隨機(jī)選擇孕齡10～11d孕鼠分組：高、中、低病毒感染劑量組各14、12、12只，經(jīng)胎盤分別接種1×10~7TCID_(50)、1×16~6TCID_(50)、1×10~5TCID_(50) MCMV懸液；模型對照組12只，同法注射等量無菌3％FCS DMEM培養(yǎng)液，空白對照組12只，開腹只清點(diǎn)孕囊數(shù)，不做其他特殊處理。①觀察孕鼠一般情況。②處死當(dāng)日留取母鼠血液行ELISA Ig M檢測。③留取胎盤及胎仔臟器組織(腦、肺、肝臟、腎臟)，各臟器部分組織行病理學(xué)檢查，部分組織勻漿液上清體外培養(yǎng)法行病毒學(xué)分離。④部分胎盤組織RT-PCR法檢測MCMV mRNA和胎仔臟器PCR法檢測MCMV DNA。⑤記錄各組母鼠妊娠進(jìn)展及結(jié)局。⑥觀察仔鼠生長發(fā)育情況，稱各組胎仔出生時體重及測量身長、頭圍。 【結(jié)果】(1)MCMV Smith毒株半數(shù)細(xì)胞感染劑量為5.62×10~7TCID_(50)／ml。(2)①高、中、低病毒滴度組孕鼠均出現(xiàn)非特異性病毒血癥征象，所有孕鼠MCMV Ig M檢測均陽性；模型對照組和空白對照組均為陰性。②感染組胎盤及胎仔臟器組織病毒學(xué)分離陽性，相應(yīng)組織標(biāo)本出現(xiàn)病理學(xué)改變。③感染組胎盤MCMV mRNA檢測陽性率為58.49％(30／53)，胎仔各臟器MCMV DNA檢測陽性率分別為：腦37.73％(20／53)、肺32.07％(17／53)、肝臟22.64％(12／53)、腎臟22.64％(12／53)。(3)①各組孕鼠異常妊娠發(fā)生率比較，高、中、低病毒滴度組均要高于模型對照組，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05)。②各組活胎率比較，高、中、低病毒滴度組均要低于模型對照組，差異有極顯著性意義(P＜0.001)。③各組活胎仔出生身長、頭圍、體重比較，高、中、低病毒滴度組均要低于模型對照組，差異有顯著性意義(P＜0.01)；④各組活胎中小頭畸形胎仔率比較，高、中、低病毒滴度組高于模型對照組，差異有顯著性意義(P＜0.01)⑤各組活胎仔低出生體重胎仔率比較，高、中、低病毒滴度組高于模型對照組，差異有統(tǒng)計(jì)學(xué)意義(P＜0.01)。以上各觀測指標(biāo)，模型對照組與空白對照組間差異均無統(tǒng)計(jì)學(xué)意義(P＞0.05)。 【結(jié)論】經(jīng)胎盤接種MCMV可以建立小鼠宮內(nèi)感染動物模型；MCMV宮內(nèi)感染增加孕鼠異常妊娠發(fā)生率，嚴(yán)重影響宮內(nèi)胎仔生長發(fā)育并導(dǎo)致低出生體重及小頭畸形胎仔的發(fā)生率增高。 第二部分MCMV感染孕鼠T淋巴細(xì)胞亞型的影響 【目的】觀察MCMV感染不同時相孕鼠T淋巴細(xì)胞增殖和亞型的改變，了解孕鼠感染MCMV后機(jī)體免疫功能的變化規(guī)律。 【方法】ELISA法篩查MCMV Ig M、IgG雙陰性BALB／C小鼠，雌雄小鼠交配受孕后隨機(jī)選擇孕10～11d天孕鼠分組：感染組：20只，經(jīng)胎盤接種1×10~6TCID_(50) MCMV懸液；模型對照組：20只，同法注射等量無菌3％FCS DMEM培養(yǎng)液；空白對照組：20只，不做任何特殊處理的正常孕鼠。于孕第14d、17d、20d、自然分娩當(dāng)日隨機(jī)取孕鼠，①經(jīng)心臟采血行ELISA檢測MCMV—IgM。②無菌取脾臟，稱取脾臟濕重并行病理學(xué)觀察。③制備脾細(xì)胞，分離單個核細(xì)胞(PBMC)，培養(yǎng)過夜取懸浮細(xì)胞，CCK-8法檢測T淋巴細(xì)胞體外增殖反應(yīng)。④流式細(xì)胞術(shù)檢測T淋巴細(xì)胞亞型CD3~+、CD4~+、CD8~+T細(xì)胞的相對數(shù)并計(jì)算CD4~+／CD8~+比值。 【結(jié)果】①感染組孕鼠MCMV—IgM檢測均陽性，模型對照組和空白對照組均為陰性。②感染組孕鼠脾臟濕重與相應(yīng)時相模型對照組較，均高于模型對照組，并隨感染時間的延長逐漸增加，差異有統(tǒng)計(jì)學(xué)意義(P＜0.01)。③感染組孕鼠T淋巴細(xì)胞增殖反應(yīng)和相應(yīng)時相模型對照組比較，要低于模型對照組，差異有顯著性意義(P＜0.01)。④感染組孕鼠CD3~+T細(xì)胞的相對百分?jǐn)?shù)與相應(yīng)時相模型對照組比較，差異無統(tǒng)計(jì)學(xué)意義(P＞0.05)；CD4~+T細(xì)胞的相對百分?jǐn)?shù)與相應(yīng)時相模型對照組比較，孕17d、孕20d、分娩當(dāng)日均要低于模型對照組，并隨著感染時間的延長逐漸降低，差異有顯著性意義(P＜0.01)；CD8~+T細(xì)胞的相對數(shù)與相應(yīng)時相模型對照組比較，孕17d、孕20d、分娩當(dāng)日均要高于模型對照組，并隨著感染時間的延長逐漸升高，差異有顯著性意義(P＜0.01)；感染組CD4~+／CD8~+比值與相應(yīng)時相模型對照組比較，各時相均要低于模型對照組，并隨感染時間的延長逐漸降低，差異有顯著性意義(P＜0.01)。以上各檢測指標(biāo)，模型對照組與空白對照組間差異無統(tǒng)計(jì)學(xué)意義(P＞0.05)。 【結(jié)論】MCMV感染孕鼠，脾臟T淋巴細(xì)胞的變化規(guī)律是：CD4~+T細(xì)胞降低，CD8~+T細(xì)胞升高，CD4~+／CD8~+比值下降，并與感染時間呈現(xiàn)出一定的時間—效應(yīng)關(guān)系，機(jī)體免疫功能處于抑制狀態(tài)，此免疫抑制狀態(tài)可能是MCMV垂直傳播的免疫機(jī)制之一。 第三部分MCMV感染對孕鼠NK細(xì)胞和CTL活性的影響 【目的】觀察MCMV感染不同時相孕鼠NK細(xì)胞和CTL活性改變，了解孕鼠感染MCMV后體內(nèi)細(xì)胞免疫功能的變化規(guī)律。 【方法】ELISA法篩查MCMV Ig M、IgG雙陰性BALB／C小鼠，雌雄小鼠交配受孕后隨機(jī)選擇孕10～11d天孕鼠分組：感染組：20只，經(jīng)胎盤接種1×10~6TCID_(50) MCMV懸液；模型對照組：20只，同法注射等量無菌3％FCS DMEM培養(yǎng)液；空白對照組：20只，只開腹，不做其它特殊處理的正常孕鼠。于孕第14d、17d、20d、自然分娩當(dāng)日隨機(jī)取孕鼠①經(jīng)心臟采血行ELISA檢測MCMV—IgM。②制備脾細(xì)胞，分離單個核細(xì)胞(PBMC)，培養(yǎng)過夜取貼壁細(xì)胞，CCK-8法檢測NK細(xì)胞的殺傷活性。③制備脾細(xì)胞，分離單個核細(xì)胞(PBMC)，培養(yǎng)過夜取懸浮細(xì)胞，ELISPOT法檢測CTL活化功能及CCK-8法檢測CTL的殺傷活性。 【結(jié)果】①感染組孕鼠MCMV—IgM檢測均陽性，模型對照組和空白對照組均為陰性。②感染組孕鼠NK細(xì)胞的殺傷活性與相應(yīng)時相的對照組比較，孕14d時要高于對照組，其余時相均低于模型對照組，并隨感染時間的延長逐漸降低，差異有極顯著性意義(P＜0.001)。④感染組孕鼠CTL產(chǎn)生的斑點(diǎn)頻率與相應(yīng)時相模型對照組比較，均低于模型對照組，并隨感染時間的延長逐漸降低，差異有極顯著性意義(P＜0.001)；CTL的殺傷功能與相應(yīng)時相的模型對照組比較，孕17d至分娩各時相均低于模型對照組，并隨感染時間的延長逐漸降低，差異有極顯著性意義(P＜0.001)。 【結(jié)論】MCMV感染孕鼠，NK細(xì)胞的殺傷活性受到抑制，CTL的活化和殺傷功能亦明顯受到抑制，并與感染時間呈現(xiàn)一定的時間—效應(yīng)關(guān)系，母體抗MCMV的細(xì)胞免疫功能處于抑制狀態(tài)，此免疫抑制狀態(tài)可能是MCMV宮內(nèi)傳播免疫機(jī)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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2006
【分類號】：R-332
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本文選題：鼠巨細(xì)胞病毒 + 宮內(nèi)感染 ； 參考：《華中科技大學(xué)》2006年博士論文
【摘要】： 第一部分MCMV宮內(nèi)感染對妊娠和胎仔生長發(fā)育的影響 【目的】建立小鼠MCMV宮內(nèi)感染模型，，觀察MCMV宮內(nèi)感染對妊娠結(jié)局及胎仔生長發(fā)育的影響。 【方法】(1)MCMV Smith毒株體外細(xì)胞培養(yǎng)法傳毒增殖，Reed-Muench法計(jì)算病毒半數(shù)細(xì)胞感染劑量TCID_(50)。(2)ELISA法篩查MCMV Ig M、IgG雙陰性BALB／C小鼠，雌雄小鼠交配受孕后隨機(jī)選擇孕齡10～11d孕鼠分組：高、中、低病毒感染劑量組各14、12、12只，經(jīng)胎盤分別接種1×10~7TCID_(50)、1×16~6TCID_(50)、1×10~5TCID_(50) MCMV懸液；模型對照組12只，同法注射等量無菌3％FCS DMEM培養(yǎng)液，空白對照組12只，開腹只清點(diǎn)孕囊數(shù)，不做其他特殊處理。①觀察孕鼠一般情況。②處死當(dāng)日留取母鼠血液行ELISA Ig M檢測。③留取胎盤及胎仔臟器組織(腦、肺、肝臟、腎臟)，各臟器部分組織行病理學(xué)檢查，部分組織勻漿液上清體外培養(yǎng)法行病毒學(xué)分離。④部分胎盤組織RT-PCR法檢測MCMV mRNA和胎仔臟器PCR法檢測MCMV DNA。⑤記錄各組母鼠妊娠進(jìn)展及結(jié)局。⑥觀察仔鼠生長發(fā)育情況，稱各組胎仔出生時體重及測量身長、頭圍。 【結(jié)果】(1)MCMV Smith毒株半數(shù)細(xì)胞感染劑量為5.62×10~7TCID_(50)／ml。(2)①高、中、低病毒滴度組孕鼠均出現(xiàn)非特異性病毒血癥征象，所有孕鼠MCMV Ig M檢測均陽性；模型對照組和空白對照組均為陰性。②感染組胎盤及胎仔臟器組織病毒學(xué)分離陽性，相應(yīng)組織標(biāo)本出現(xiàn)病理學(xué)改變。③感染組胎盤MCMV mRNA檢測陽性率為58.49％(30／53)，胎仔各臟器MCMV DNA檢測陽性率分別為：腦37.73％(20／53)、肺32.07％(17／53)、肝臟22.64％(12／53)、腎臟22.64％(12／53)。(3)①各組孕鼠異常妊娠發(fā)生率比較，高、中、低病毒滴度組均要高于模型對照組，差異有統(tǒng)計(jì)學(xué)意義(P＜0.05)。②各組活胎率比較，高、中、低病毒滴度組均要低于模型對照組，差異有極顯著性意義(P＜0.001)。③各組活胎仔出生身長、頭圍、體重比較，高、中、低病毒滴度組均要低于模型對照組，差異有顯著性意義(P＜0.01)；④各組活胎中小頭畸形胎仔率比較，高、中、低病毒滴度組高于模型對照組，差異有顯著性意義(P＜0.01)⑤各組活胎仔低出生體重胎仔率比較，高、中、低病毒滴度組高于模型對照組，差異有統(tǒng)計(jì)學(xué)意義(P＜0.01)。以上各觀測指標(biāo)，模型對照組與空白對照組間差異均無統(tǒng)計(jì)學(xué)意義(P＞0.05)。 【結(jié)論】經(jīng)胎盤接種MCMV可以建立小鼠宮內(nèi)感染動物模型；MCMV宮內(nèi)感染增加孕鼠異常妊娠發(fā)生率，嚴(yán)重影響宮內(nèi)胎仔生長發(fā)育并導(dǎo)致低出生體重及小頭畸形胎仔的發(fā)生率增高。 第二部分MCMV感染孕鼠T淋巴細(xì)胞亞型的影響 【目的】觀察MCMV感染不同時相孕鼠T淋巴細(xì)胞增殖和亞型的改變，了解孕鼠感染MCMV后機(jī)體免疫功能的變化規(guī)律。 【方法】ELISA法篩查MCMV Ig M、IgG雙陰性BALB／C小鼠，雌雄小鼠交配受孕后隨機(jī)選擇孕10～11d天孕鼠分組：感染組：20只，經(jīng)胎盤接種1×10~6TCID_(50) MCMV懸液；模型對照組：20只，同法注射等量無菌3％FCS DMEM培養(yǎng)液；空白對照組：20只，不做任何特殊處理的正常孕鼠。于孕第14d、17d、20d、自然分娩當(dāng)日隨機(jī)取孕鼠，①經(jīng)心臟采血行ELISA檢測MCMV—IgM。②無菌取脾臟，稱取脾臟濕重并行病理學(xué)觀察。③制備脾細(xì)胞，分離單個核細(xì)胞(PBMC)，培養(yǎng)過夜取懸浮細(xì)胞，CCK-8法檢測T淋巴細(xì)胞體外增殖反應(yīng)。④流式細(xì)胞術(shù)檢測T淋巴細(xì)胞亞型CD3~+、CD4~+、CD8~+T細(xì)胞的相對數(shù)并計(jì)算CD4~+／CD8~+比值。 【結(jié)果】①感染組孕鼠MCMV—IgM檢測均陽性，模型對照組和空白對照組均為陰性。②感染組孕鼠脾臟濕重與相應(yīng)時相模型對照組較，均高于模型對照組，并隨感染時間的延長逐漸增加，差異有統(tǒng)計(jì)學(xué)意義(P＜0.01)。③感染組孕鼠T淋巴細(xì)胞增殖反應(yīng)和相應(yīng)時相模型對照組比較，要低于模型對照組，差異有顯著性意義(P＜0.01)。④感染組孕鼠CD3~+T細(xì)胞的相對百分?jǐn)?shù)與相應(yīng)時相模型對照組比較，差異無統(tǒng)計(jì)學(xué)意義(P＞0.05)；CD4~+T細(xì)胞的相對百分?jǐn)?shù)與相應(yīng)時相模型對照組比較，孕17d、孕20d、分娩當(dāng)日均要低于模型對照組，并隨著感染時間的延長逐漸降低，差異有顯著性意義(P＜0.01)；CD8~+T細(xì)胞的相對數(shù)與相應(yīng)時相模型對照組比較，孕17d、孕20d、分娩當(dāng)日均要高于模型對照組，并隨著感染時間的延長逐漸升高，差異有顯著性意義(P＜0.01)；感染組CD4~+／CD8~+比值與相應(yīng)時相模型對照組比較，各時相均要低于模型對照組，并隨感染時間的延長逐漸降低，差異有顯著性意義(P＜0.01)。以上各檢測指標(biāo)，模型對照組與空白對照組間差異無統(tǒng)計(jì)學(xué)意義(P＞0.05)。 【結(jié)論】MCMV感染孕鼠，脾臟T淋巴細(xì)胞的變化規(guī)律是：CD4~+T細(xì)胞降低，CD8~+T細(xì)胞升高，CD4~+／CD8~+比值下降，并與感染時間呈現(xiàn)出一定的時間—效應(yīng)關(guān)系，機(jī)體免疫功能處于抑制狀態(tài)，此免疫抑制狀態(tài)可能是MCMV垂直傳播的免疫機(jī)制之一。 第三部分MCMV感染對孕鼠NK細(xì)胞和CTL活性的影響 【目的】觀察MCMV感染不同時相孕鼠NK細(xì)胞和CTL活性改變，了解孕鼠感染MCMV后體內(nèi)細(xì)胞免疫功能的變化規(guī)律。 【方法】ELISA法篩查MCMV Ig M、IgG雙陰性BALB／C小鼠，雌雄小鼠交配受孕后隨機(jī)選擇孕10～11d天孕鼠分組：感染組：20只，經(jīng)胎盤接種1×10~6TCID_(50) MCMV懸液；模型對照組：20只，同法注射等量無菌3％FCS DMEM培養(yǎng)液；空白對照組：20只，只開腹，不做其它特殊處理的正常孕鼠。于孕第14d、17d、20d、自然分娩當(dāng)日隨機(jī)取孕鼠①經(jīng)心臟采血行ELISA檢測MCMV—IgM。②制備脾細(xì)胞，分離單個核細(xì)胞(PBMC)，培養(yǎng)過夜取貼壁細(xì)胞，CCK-8法檢測NK細(xì)胞的殺傷活性。③制備脾細(xì)胞，分離單個核細(xì)胞(PBMC)，培養(yǎng)過夜取懸浮細(xì)胞，ELISPOT法檢測CTL活化功能及CCK-8法檢測CTL的殺傷活性。 【結(jié)果】①感染組孕鼠MCMV—IgM檢測均陽性，模型對照組和空白對照組均為陰性。②感染組孕鼠NK細(xì)胞的殺傷活性與相應(yīng)時相的對照組比較，孕14d時要高于對照組，其余時相均低于模型對照組，并隨感染時間的延長逐漸降低，差異有極顯著性意義(P＜0.001)。④感染組孕鼠CTL產(chǎn)生的斑點(diǎn)頻率與相應(yīng)時相模型對照組比較，均低于模型對照組，并隨感染時間的延長逐漸降低，差異有極顯著性意義(P＜0.001)；CTL的殺傷功能與相應(yīng)時相的模型對照組比較，孕17d至分娩各時相均低于模型對照組，并隨感染時間的延長逐漸降低，差異有極顯著性意義(P＜0.001)。 【結(jié)論】MCMV感染孕鼠，NK細(xì)胞的殺傷活性受到抑制，CTL的活化和殺傷功能亦明顯受到抑制，并與感染時間呈現(xiàn)一定的時間—效應(yīng)關(guān)系，母體抗MCMV的細(xì)胞免疫功能處于抑制狀態(tài)，此免疫抑制狀態(tài)可能是MCMV宮內(nèi)傳播免疫機(jī)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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2006
【分類號】：R-332
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