發(fā)布時間：2018-04-22 15:35

  本文選題：人巨細胞病毒 + 絨毛外滋養(yǎng)細胞��； 參考：《泰山醫(yī)學院》2014年碩士論文

【摘要】：研究背景 人巨細胞病毒（human cytomegalovirus, HCMV）又稱人類皰疹病毒5型，是皰疹病毒科β亞科的一種常見病毒，廣泛存在于自然界中，人是其唯一宿主。HCMV是目前已知的引起孕婦活動性感染、胚胎及胎兒宮內(nèi)感染的最常見病原體。孕期母體感染HCMV后，可通過胎盤垂直傳播或者生殖道逆行傳播給胎兒，引起胚胎或胎兒宮內(nèi)感染。HCMV宮內(nèi)感染可導致胚胎停育、流產(chǎn)、死胎、早產(chǎn)、胎兒宮內(nèi)發(fā)育遲緩等異常妊娠結局，以及新生兒認知能力發(fā)育落后、智力低下、感音神經(jīng)性耳聾等嚴重遠期后果。目前HCMV發(fā)生宮內(nèi)傳播及引起胎兒發(fā)育異常的具體機制和途徑尚不清楚，HCMV感染胎盤滋養(yǎng)層細胞被認為是啟動宮內(nèi)感染的第一步。絨毛外細胞滋養(yǎng)細胞（extravillous cytotrophoblast, EVT）增殖、侵襲能力下降，導致螺旋動脈重鑄不良，胎盤物質(zhì)交換功能低下，是流產(chǎn)、死胎、胎兒宮內(nèi)發(fā)育遲緩、腦發(fā)育落后等異常妊娠的重要病理生理機制。 研究目的 體外研究HCMV經(jīng)Hippo-YAP信號通路對早孕EVT功能的影響。 研究方法 1.無菌收集早孕絨毛組織，應用改良的復合酶消化梯度離心法分離、培養(yǎng)原代EVT； 2.通過免疫細胞化學染色檢測CK7、Vim、c-erbB-2表達情況鑒定細胞來源； 3.常規(guī)培養(yǎng)人胚肺成纖維細胞MRC-5細胞株，擴增HCMVAD169病毒株，并根據(jù)Reed-Muench法測定TCID50； 4.將HCMVAD169病毒液接種于EVT培養(yǎng)基中，通過免疫熒光細胞化學染色檢測HCMVpp65抗原，以確定HCMV感染EVT情況； 5. MTT比色法檢測HCMV對EVT體外增殖的影響，為后續(xù)試驗篩選最佳病毒接種劑量； 6.實驗分為正常組（EVT）及病毒組（EVT+HCMV）； 7.熒光定量PCR檢測HCMV經(jīng)Hippo-YAP信號通路對EVT表達Mst1、Mst2、SAV、Lats1、Lats2、Mob1、YAP、TAZ、TEAD1~4mRNA水平的影響； 8.免疫熒光化學染色法、Western blot技術檢測正常組及病毒組YAP蛋白水平，以了解HCMV對EVT的YAP蛋白表達的影響。 9.體外細胞侵襲實驗檢測HCMV對EVT侵襲功能的影響。 研究結果 1.倒置顯微鏡下見分離的原代細胞貼壁后呈三角形或不規(guī)則多邊形，平鋪片狀生長； 2.細胞來源鑒定結果顯示，幾乎所有分離培養(yǎng)的原代細胞均可見CK7和c-erbB-2表達，，極少數(shù)可見Vim表達，提示實驗中分離培養(yǎng)獲得的原代細胞為高純度EVT； 3.病毒擴增后測定HCMV AD169病毒株TCID50為10-4.15/0.1ml； 4. EVT培養(yǎng)基中接種HCMV后，免疫熒光化學染色結果顯示病毒組細胞內(nèi)可見大量紅色HCMV pp65抗原陽性信號表達，正常組細胞內(nèi)未見陽性信號表達； 5. MTT檢測顯示，與正常組相比較，接種病毒48h時，30μl、40μl、50μl、70μl、100μlHCMV病毒液對EVT增殖有明顯抑制作用（P0.05），30μl、40μl、50μl各組間無明顯差異（P0.05），故選擇30μl100TCID50HCMV/120μl培養(yǎng)基進行后續(xù)實驗； 6.熒光定量PCR顯示，兩組EVT中均有Mst1、Mst2、SAV、Lats1、Lats2、Mob1、YAP、TAZ、TEAD1、TEAD2、TEAD3、TEAD4基因mRNA表達，各基因mRNA相對表達量正常組為（1.000±0.014,1.000±0.036,1.000±0.025,1.001±0.047,1.000±0.016,1.001±0.067,1.002±0.067,1.000±0.017,1.001±0.053,1.001±0.050,1.000±0.039,1.009±0.160）、病毒組為（0.805±0.015,0.818±0.027,0.929±0.021,0.721±0.032,0.734±0.005,0.388±0.019,0.623±0.022,0.587±0.012,0.488±0.010,0.501±0.012,0.652±0.016,0.625±0.026），病毒組與正常EVT組相比較，各基因mRNA表達水平明顯降低（P均0.05）； 7.免疫熒光細胞化學染色法檢測結果顯示兩組細胞均有YAP蛋白表達，正常組EVT細胞胞質(zhì)和細胞核內(nèi)均有YAP蛋白表達，病毒組EVT表達YAP細胞核定位減少，正常組與病毒組陽性細胞的平均光密度值（average optical density, AOD）分別為0.271±0.024和0.159±0.017，與正常EVT組相比較，病毒組EVT表達YAP蛋白水平明顯降低（P0.05）； 8. Western blot檢測顯示兩組細胞均有YAP蛋白表達，正常組與病毒組YAP蛋白表達的灰度值分別為1.02±0.056和0.65±0.071，與正常EVT組相比較，病毒組EVT表達YAP蛋白水平明顯降低（P0.05）； 9.體外細胞侵襲實驗結果顯示正常組和病毒組EVT均有細胞穿過Matrigel基質(zhì)膠，兩組穿膜細胞數(shù)分別為（55.7±2.95）個和（47.9±3.21）個，與正常組相比較，病毒組穿膜細胞數(shù)減少（P0.05），提示HCMV降低EVT侵襲活力。 結論 1. EVT是HCMV的允許細胞，HCMV可體外感染EVT，在細胞內(nèi)復制并抑制其增殖，EVT感染HCMV可作為HCMV宮內(nèi)感染的模型； 2. HCMV可能影響EVT細胞Hippo-YAP信號通路的多個環(huán)節(jié)，導致Hippo-YAP信號通路中各基因mRNA及關鍵蛋白YAP蛋白表達降低，進一步導致EVT侵襲功能下降。
[Abstract]:Background of the study

Human cytomegalovirus ( HCMV ) is also known as human Herpesviridae type 5 . It is a common virus in the family of Herpesviridae 尾 subfamily . Human cytomegalovirus ( HCMV ) is the only host in nature . HCMV is one of the most common pathogens that cause intrauterine infection , embryo and intrauterine infection . HCMV intrauterine infection can lead to fetal arrest , abortion , stillbirth , premature birth , fetal intrauterine growth retardation and other serious long - term consequences . HCMV intrauterine infection can lead to abnormal pregnancy outcomes such as fetal arrest , abortion , stillbirth , premature birth , intrauterine growth retardation , etc . HCMV intrauterine infection is considered to be an important pathological physiological mechanism of abnormal pregnancy such as abortion , fetal death , intrauterine growth retardation , and brain development .

Purpose of study

The effect of HCMV via Hippo - YAP signaling pathway on the function of early pregnancy was studied in vitro .

Research Methods

1 . sterile collection of the early pregnant chorionic villi tissue , the improved compound enzyme digestion gradient centrifugation method is applied to separate and culture the primary evt ;


2 . The expression of CK7 , Vim and c - erbB - 2 was detected by immunohistochemical staining .


3 . The human embryonic lung fibroblast MRC - 5 cell line was cultured and the HCMVAD169 strain was amplified , and was determined by the Reed - Muench method .


4 . The HCMVAD169 virus solution was inoculated in an evt medium , and HCMVpp65 antigen was detected by immunofluorescence cytochemical staining to determine the condition of HCMV infection .


5 . The effect of HCMV on the proliferation of human cytomegalovirus ( HCMV ) in vitro was detected by MTT colorimetric assay .


6 . The experiments were divided into normal group and virus group .


7 . The effect of HCMV via Hippo - YAP signal pathway on the expression of Mst1 , Mst2 , SAV , Lats1 , Lats2 , Mob1 , YAP , TAZ and TEAD1 ~ 4 mRNA was detected by fluorescence quantitative PCR .


8 . Western blot was used to detect the level of YAP protein in normal group and virus group , and to understand the effect of HCMV on the expression of YAP protein .

9 . The effect of HCMV on the invasion function in vitro was detected by in vitro cell invasion assay .

Results of the study

1 . the separated primary cell wall is inverted under an inverted microscope to form a triangular or irregular polygon , and the plate - like growth is tiled ;


2 . The results of cell culture showed that CK7 and c - erbB - 2 expression were found in almost all primary cells isolated and cultured . Vim expression was very rare , suggesting that the primary cells isolated and cultured in the experiment were high - purity EVTs ;


3 . After amplification of the virus , the HCMV AD169 strain was determined to be 10 - 4.15 / 0.1ml ;


4 . After inoculation of HCMV , the positive signal expression of HCMV pp65 antigen was found in the virus group cells , and no positive signal expression was found in the normal group cells .


5 . MTT assay showed that 30 渭l , 40 渭l , 50 渭l , 70 渭l and 100 渭L of HCMV solution had no significant difference ( P0.05 ) , 30 渭l , 40 渭l and 50 渭l ( P0.05 ) .


6 . Fluorescent quantitative PCR showed that Mst1 , Mst2 , SAV , Lats1 , Lats2 , Mob1 , YAP , TAZ , TEAD1 , TEAD2 , TEAD3 , TEAD4 mRNA expression were found in both groups .


7 . The expression of YAP was detected in both groups . The expression of YAP protein was observed in both cytoplasm and nucleus of normal group . The average optical density of YAP was 0.271 鹵 1.024 and 0.159 鹵 0 . 017 , respectively .


8 . Western blot showed YAP protein expression in both groups . The gray values of YAP protein expression were 1.02 鹵 0.056 and 0.65 鹵 0.071 , respectively , and the level of YAP protein was significantly decreased ( P0.05 ) .


9 . The results of the in vitro invasion experiment showed that both normal and viral groups had the cell cross - Matrigel matrix glue , and the number of membrane - penetrating cells in both groups were ( 55.7 鹵 2.95 ) and ( 47.9 鹵 3.21 ) , respectively . Compared with the normal group , the number of membrane - penetrating cells decreased ( P0.05 ) , suggesting that HCMV could reduce the invasion and activity .

Conclusion

1 . The evt is the permissive cell of HCMV , HCMV can infect the evt in vitro , replicate in the cell and inhibit its proliferation , and HCMV can be used as a model of HCMV intrauterine infection .


2 . HCMV could affect the multiple links of the signal pathway of the Hippo - YAP signal , which resulted in decreased expression of mRNA and protein YAP in the signal pathway of Hippo - YAP , which further led to a decrease in the invasion function .

【學位授予單位】：泰山醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R714

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相關期刊論文 前2條

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