HCMV经Hippo-YAP信号通路对绒毛外细胞滋养细胞功能影响的体外研究

发布时间：2018-04-22 15:35

本文选题：人巨细胞病毒 + 绒毛外滋养细胞　；参考：《泰山医学院》2014年硕士论文

【摘要】：研究背景人巨细胞病毒（human cytomegalovirus, HCMV）又称人类疱疹病毒5型，是疱疹病毒科β亚科的一种常见病毒，广泛存在于自然界中，人是其唯一宿主。HCMV是目前已知的引起孕妇活动性感染、胚胎及胎儿宫内感染的最常见病原体。孕期母体感染HCMV后，可通过胎盘垂直传播或者生殖道逆行传播给胎儿，引起胚胎或胎儿宫内感染。HCMV宫内感染可导致胚胎停育、流产、死胎、早产、胎儿宫内发育迟缓等异常妊娠结局，以及新生儿认知能力发育落后、智力低下、感音神经性耳聋等严重远期后果。目前HCMV发生宫内传播及引起胎儿发育异常的具体机制和途径尚不清楚，HCMV感染胎盘滋养层细胞被认为是启动宫内感染的第一步。绒毛外细胞滋养细胞（extravillous cytotrophoblast, EVT）增殖、侵袭能力下降，导致螺旋动脉重铸不良，胎盘物质交换功能低下，是流产、死胎、胎儿宫内发育迟缓、脑发育落后等异常妊娠的重要病理生理机制。研究目的体外研究HCMV经Hippo-YAP信号通路对早孕EVT功能的影响。研究方法 1.无菌收集早孕绒毛组织，应用改良的复合酶消化梯度离心法分离、培养原代EVT； 2.通过免疫细胞化学染色检测CK7、Vim、c-erbB-2表达情况鉴定细胞来源； 3.常规培养人胚肺成纤维细胞MRC-5细胞株，扩增HCMVAD169病毒株，并根据Reed-Muench法测定TCID50； 4.将HCMVAD169病毒液接种于EVT培养基中，通过免疫荧光细胞化学染色检测HCMVpp65抗原，以确定HCMV感染EVT情况； 5. MTT比色法检测HCMV对EVT体外增殖的影响，为后续试验筛选最佳病毒接种剂量； 6.实验分为正常组（EVT）及病毒组（EVT+HCMV）； 7.荧光定量PCR检测HCMV经Hippo-YAP信号通路对EVT表达Mst1、Mst2、SAV、Lats1、Lats2、Mob1、YAP、TAZ、TEAD1~4mRNA水平的影响； 8.免疫荧光化学染色法、Western blot技术检测正常组及病毒组YAP蛋白水平，以了解HCMV对EVT的YAP蛋白表达的影响。 9.体外细胞侵袭实验检测HCMV对EVT侵袭功能的影响。研究结果 1.倒置显微镜下见分离的原代细胞贴壁后呈三角形或不规则多边形，平铺片状生长； 2.细胞来源鉴定结果显示，几乎所有分离培养的原代细胞均可见CK7和c-erbB-2表达，，极少数可见Vim表达，提示实验中分离培养获得的原代细胞为高纯度EVT； 3.病毒扩增后测定HCMV AD169病毒株TCID50为10-4.15/0.1ml； 4. EVT培养基中接种HCMV后，免疫荧光化学染色结果显示病毒组细胞内可见大量红色HCMV pp65抗原阳性信号表达，正常组细胞内未见阳性信号表达； 5. MTT检测显示，与正常组相比较，接种病毒48h时，30μl、40μl、50μl、70μl、100μlHCMV病毒液对EVT增殖有明显抑制作用（P0.05），30μl、40μl、50μl各组间无明显差异（P0.05），故选择30μl100TCID50HCMV/120μl培养基进行后续实验； 6.荧光定量PCR显示，两组EVT中均有Mst1、Mst2、SAV、Lats1、Lats2、Mob1、YAP、TAZ、TEAD1、TEAD2、TEAD3、TEAD4基因mRNA表达，各基因mRNA相对表达量正常组为（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）、病毒组为（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），病毒组与正常EVT组相比较，各基因mRNA表达水平明显降低（P均0.05）； 7.免疫荧光细胞化学染色法检测结果显示两组细胞均有YAP蛋白表达，正常组EVT细胞胞质和细胞核内均有YAP蛋白表达，病毒组EVT表达YAP细胞核定位减少，正常组与病毒组阳性细胞的平均光密度值（average optical density, AOD）分别为0.271±0.024和0.159±0.017，与正常EVT组相比较，病毒组EVT表达YAP蛋白水平明显降低（P0.05）； 8. Western blot检测显示两组细胞均有YAP蛋白表达，正常组与病毒组YAP蛋白表达的灰度值分别为1.02±0.056和0.65±0.071，与正常EVT组相比较，病毒组EVT表达YAP蛋白水平明显降低（P0.05）； 9.体外细胞侵袭实验结果显示正常组和病毒组EVT均有细胞穿过Matrigel基质胶，两组穿膜细胞数分别为（55.7±2.95）个和（47.9±3.21）个，与正常组相比较，病毒组穿膜细胞数减少（P0.05），提示HCMV降低EVT侵袭活力。结论 1. EVT是HCMV的允许细胞，HCMV可体外感染EVT，在细胞内复制并抑制其增殖，EVT感染HCMV可作为HCMV宫内感染的模型； 2. HCMV可能影响EVT细胞Hippo-YAP信号通路的多个环节，导致Hippo-YAP信号通路中各基因mRNA及关键蛋白YAP蛋白表达降低，进一步导致EVT侵袭功能下降。
[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 卤 1.024 and 0.159 卤 0 . 017 , respectively .

8 . Western blot showed YAP protein expression in both groups . The gray values of YAP protein expression were 1.02 卤 0.056 and 0.65 卤 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 卤 2.95 ) and ( 47.9 卤 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 .

【学位授予单位】：泰山医学院
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R714

【参考文献】