人巨细胞病毒对早孕绒毛外细胞滋养细胞免疫功能影响的体外研究
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摘要
第一部分T细胞免疫对感染HCMV的绒毛外细胞滋养细胞保护作用研究
[目的]体外研究T细胞对感染HCMV的绒毛外细胞滋养细胞的免疫效应。
[方法]①选择人早孕胎盘绒毛滋养细胞株HPT-8进行研究,建立感染HCMV的HPT-8细胞模型：以100TCID50HCMV攻击HPT-8细胞后,应用免疫荧光细胞化学法检测HCMVpp65抗原表达及病毒滴定法观察HPT-8感染情况并绘制生长曲线,判定HPT-8细胞对HCMV的易感性,感染状态以及HCMV在细胞内生长情况。②收集门诊孕前咨询健康妇女外周血,提取外周血单个核细胞,尼龙毛柱法分离纯化获得T细胞。③建立T细胞与感染HCMV的HPT-8细胞共培养模型,应用实时荧光定量PCR法检测HPT-8细胞内HCMV DNA负荷量。
[结果]①加入HCMV48h后,HPT-8细胞胞质内出现大量HCMV pp65抗原信号；感染3d后的HPT-8细胞培养液(Trophoblast conditioned medium, TCM)可使HEL细胞发生细胞病变；HCMV在HPT-8细胞内的复制于第4d开始增高,6d达到高峰。②T细胞具有下调HPT-8细胞内HCMV DNA负荷量的作用(p<0.05)；T细胞与感染的HPT-8细胞共培养3d后的细胞上清,也具有下调HPT-8细胞内HCMV DNA负荷量的作用(p<0.05)；共培养3天后的T细胞则失去了下调HPT-8细胞内HCMV DNA负荷量的作用(p>0.05)。
[结论]T细胞在母-胎界面HCMV感染初期具有一定的免疫防御作用,其机制可能与T细胞的直接溶解杀伤作用及其分泌的细胞因子相关；但T细胞的免疫功能很快受到抑制,其中的机制有待进一步研究。
第二部分HCMV感染后绒毛外细胞滋养细胞分泌产物对外周血T细胞Treg/Th17平衡的影响
[目的]体外研究HCMV感染后绒毛外细胞滋养细胞分泌产物对外周血T细胞Treg/Th17平衡的影响
[方法]①选择人早孕胎盘绒毛滋养细胞株HPT-8进行研究,建立感染HCMV的HPT-8细胞模型。②尼龙毛柱法分离纯化获得T细胞。③建立TCM与T细胞共培养体系：100TCIDso HCMV攻击HPT-8细胞后,收集HCMV感染72h以及相应时间点对照组的TCM,过滤离心后紫外灯照射15mmin灭活HCMV;根据T细胞培养液组分的不同,分为感染组(HCMV感染72h的TCM)、TCM对照组(对照组的TCM)及空白对照组(不含TCM),置于C02培养箱(5%C02、37℃)中继续培养72h。④应用流式分析法检测HCMV感染后绒毛外细胞滋养细胞分泌产物对T细胞内CD4+CD25+CD12-/CD4+、CD4+IL17A+/CD4+比值的影响。⑤应用实时荧光定量PCR法检测HCMV感染后绒毛外细胞滋养细胞分泌产物对T细胞分化相关转录因子Foxp3、 IL-6、IL-17A、TGF-p及RORyt mRNA表达的影响；⑥应用ELISA法检测各组T细胞培养上清中TGF-β、IL-17A的浓度。
[结果]①与空白对照组相比,TCM对照组Treg/Th17比值增高(p<0.01)；与TCM对照组相比,感染组Treg/Th17比值降低(p<0.01)。②与空白对照组比较,TCM对照组T细胞内Foxp3, RORyt及TGF-p mRNA的表达均明显升高(p<0.01)；与TCM对照组比较,感染组T细胞内Foxp3, RORyt及TGF-β mRNA表达均明显减少(p<0.01)。③与空白对照组相比,TCM对照组T细胞内IL-17A和IL-6mRNA的表达均明显降低(p<0.01)。与TCM对照组相比,感染组T细胞内IL-17A和IL-6mRNA的表达均明显增加(p<0.01)。④与空白对照组相比,TCM对照组上清中TGF-β1浓度增加(p<0.01)；与TCM对照组比较,感染组上清中TGF-β1浓度减少(p<0.01)。⑤与空白对照组相比,TCM组上清中IL-17A浓度减少(p<0.01);和TCM对照组相比,感染组IL-17A的浓度增加(p<0.01)。
[结论]绒毛外细胞滋养细胞分泌物可促使外周血T细胞Treg/Th17型免疫平衡向Treg细胞偏倚,有利于妊娠的维持,而HCMV可能通过影响绒毛外细胞滋养细胞合成及分泌功能从而使外周血T细胞内Treg/Th17平衡向Th17细胞偏倚,导致不良妊娠结局。
第三部分HCMV感染对绒毛外细胞滋养细胞合成分泌V][P表达水平的影响
[目的]体外研究HCMV对绒毛外细胞滋养细胞合成分泌血管活性肠肽(vasoactive intestinal peptide, VIP)表达水平的影响
[方法]①选择人早孕胎盘绒毛滋养细胞株HPT-8进行研究,建立感染HCMV的HPT-8细胞模型。②收集HCMV感染72h以及相应时间点对照组的细胞及TCM,TCM过滤离心后紫外灯照射15min灭活HCMV。③应用实时荧光定量PCR法检测HCMV感染对HPT-8细胞VIP mRNA表达水平的影响。④应用免疫细胞组织化学及免疫印迹法检测HCMV感染对HPT-8细胞VIP蛋白质表达水平的影响。⑤应用ELISA法检测HCMV感染对HPT-8细胞分泌VIP浓度的影响。
[结果]①与空白对照组相比,感染组HPT-8细胞VIP mRNA表达水平明显降低(p<0.01)。②空白对照组及感染组HPT-8细胞的胞质和胞浆均有VIP蛋白表达,呈棕黄色颗粒；感染组HPT-8细胞VIP蛋白表达水平明显低于空白对照组p<0.01)。③感染组HPT-8细胞分泌VIP浓度明显低于空白对照组p<0.01)。
[结论]母-胎界面HCMV感染抑制了绒毛外细胞滋养细胞合成和分泌的VIP表达水平。
第四部分黄芩素对感染HCMV的绒毛外细胞滋养细胞VIP表达水平的影响
[目的]探讨黄芩素阻断HCMV感染绒毛外细胞滋养细胞的作用及对HCMV感染的绒毛外细胞滋养细胞VIP表达水平的影响。
[方法]①选择人早孕胎盘绒毛滋养细胞株HPT-8进行研究,建立感染HCMV的HPT-8细胞模型。②收集HCMV感染72h以及相应时间点对照组、黄芩素组的HPT-8细胞及TCM, TCM过滤及离心后用紫外灯照射15min灭活HCMV。③应用实时荧光定量PCR法检测黄芩素对感染HCMV的HPT-8细胞HCMV DNA负荷量的影响。④应用实时荧光定量PCR法检测黄芩素对感染HCMV的HPT-8细胞内VIP mRNA表达的影响。⑤应用免疫细胞组织化学及免疫印迹法检测黄芩素对感染HCMV的HPT-8细胞内VIP蛋白质表达水平的影响。⑥ELISA法检测黄芩素对感染HCMV的HPT-8细胞分泌VIP浓度的影响。
[结果]①黄芩素减少感染的HPT-8细胞内HCMV DNA负荷量。②与空白对照组相比,感染组HPT-8细胞内VIP蛋白转录,合成及分泌的表达均明显下降,差异均具有统计学意义(p<0.05);黄芩素组HPT-8细胞内VIP蛋白转录,合成及分泌的表达与正常对照组相比,差异均无统计学意义(p>0.05)
[结论]黄芩素对母-胎界面的HCMV感染及其导致的VIP表达异常具有一定积极影响。
Part1T cell-mediated immunological effect in human cytomegalovirus infected extravillous cytotrophoblasts
[Objective] To explore T cell-mediated restriction of human cytomegalovirus (HCMV) in extravillous cytotrophoblasts (EVT) in vitro
[Methods]①A human trophoblast cell line (HPT-8) was chosen to conduct the research, and then to established a HCMV infected HPT-8cells model:HPT-8cells were infected with100TCID50HCMV. The following methods were used to observe infection susceptibility and status of HPT-8cells to HCMV, and growth situation of HCMV in cells: Immunofluorescence cytochemistry method was used to detect HCMV pp65, and method of viral titres was used to observe infection, drawing a growth curve of HCMV.②T cells were isolated and purified by using nylon column from the peripheral blood of normal reproductive women for preconception counseling in outpatient.③A T cells-HCMV infected HPT-8cells co-culture system was established, real-time quantitative PCR was used to detect the HCMV DNA in HPT-8cells.
[Results]①A large number of HCMV pp65antigen signals could be seen in infected HPT-8cells at the48time point. TCM of3d after infection induced typical cell lesion in HEL cells; HCMV replication began to increase4d after infection and peaked at6d.②HCMV DNA load in infected HPT-8cells was significantly decreased in T cells co-cultured group (p<0.05). Cell-free co-culture supernatant was also diminished the HCMV DNA load (p<0.05). However, when co-cultured T cells was added to fresh HCMV infected HPT-8cells, there was no significant difference in HCMV DNA load with HCMV infected group (p>0.05).
[Conclusions] T cells had antiviral effect during the early stage of infected maternal-fetal interface, and the mechanism may be associated with both direct killing effect and secreting soluble factors. However, the immune function of T cells was rapidly inhibited, of which the underlying mechanisms remain unclear and need further study.
Part2The effect of secretory products of human cytomegalovirus infected extravillous cytotrophoblasts on Treg/Th17balance in peripheral blood T cell
[Objective]To investigated the effect of secretory products of HCMV-infected EVT on the Treg/Th17balance in peripheral blood T cell.
[Methods]①A human trophoblast cell line (HPT-8) was chosen to conduct the research, and then to establish a HCMV infected HPT-8model.②T cells were isolated and purified by using nylon column.③The co-culture system of TCM and T cells was established in vitro:HPT-8cells were infected with100TCID50HCMV, and then TCM was collected after HCMV infection within72h and that of control group at the same time point, after centrifugation HCMV in the TCM inactivated by ultraviolet light irradiating15min; the study was grouped according to various components of TCM:infection group (including TCM from infected HCMV within72h), TCM control group (including non-infected TCM) and control group (without TCM), and then put them into incubator (37℃,5%CO2) for72h.③Flow cytometric analysis was applied to detect the change of CD4+CD25+CD127-/CD4+CD4+IL17A+/CD4+in T cells after HCMV infection.⑤Real-time quantitative PCR was used to detect the effect of secretory products of HCMV infected EVT on the expression of the associated transcription factors of T cells differentiation Foxp3、IL-6、IL-17A、TGF-β and RORyt.⑥TGF-β1and IL-17A excreted by T cells were detected with ELISA method.
[Results]①Compared with control group, The ratio of Treg/Th17in TCM control group was significantly higher (p<0.01); The ratio of Treg/Th17was significantly lower in infection group than that in TCM control group (p<0.01).②Compared with control group, the mRNA expression in TCM control group of the three associated transcription factors of T cells differentiation Foxp3、TGF-β and RORyt were significantly increased (p<0.01); compared with TCM control group, the mRNA expression of the three factors was decreased in infection group (p<0.01).③Compared with control group, the mRNA expression levels in TCM control group of the two associated transcription factors of T cells differentiation IL-17A and IL-6were significantly decreased (p<0.01); the mRNA expression levels of the two transcription factors was significantly higher in infection group than that in TCM control group (p<0.01).④TGF-β1concentration in TCM control group was higher than that in control group (p<0.01); compared with TCM control group, TGF-β1concentration in infection group was significantly decreased (p<0.01).⑤The concentration of IL-17A in control group was significantly lower than that in TCM control group (p<0.01); compared with TCM control group, IL-17A concentration was significantly increased (p<0.01)
[Conclusions] EVT secretion can promote the balance of Treg/Th17to Treg cells in peripheral blood T cell, which is conductive to pregnancy. However, HCMV may boost the balance of Treg/Th17to Th17cells in peripheral blood T cell by disturbing the secretory function of EVT, which may induce adverse pregnancy outcomes.
Part3Effect of human cytomegalovirus infection on VIP synthesized and excreted by extravillous cytotrophoblasts
[Objective]To study the effect of HCMV infection on VIP synthesized and excreted by EVT.
[Methods]①A human trophoblast cell line (HPT-8) was chosen to conduct the research, and then a HCMV infected HPT-8model was established.②HPT-8cells and TCM were collected after HCMV infection within72h and that of control group at the same time point, after centrifugation HCMV in the TCM inactivated by ultraviolet light irradiating15min;③The mRNA expression of VIP was detected by real-time quantitative PCR.④The protein expression level of VIP was detected by both immunocytochemistry and Western blot.⑤VIP excreted by HPT-8cells was detected with ELISA method.
[Results]①Comparing to control group, the mRNA expression level of VIP was lower in infection group (p<0.01).②VIP protein expressed in cytoplast and cell plasma of HPT-8cells in both control group and infection group with the expression presenting yellow-brown; the protein expression of VIP in infection group was lower than that in control group (p<0.01).③VIP concentration in TCM from infection group was lower than that from control group (p<0.01)
[Conclusions] The expression of VIP synthesized and excreted by EVT was decreased after HCMV infection at maternal-fetal interface.
Part4Effect of baicalein on the expression of VIP in extravillous cytotrophoblasts infected with human cytomegalovirus in vitro
[Objective] to study the function of baicalein to interrupt HCMV infected EVT and its effect on the expression of VIP of infected EVT.
[Methods]①A human trophoblast cell line (HPT-8) was chosen to conduct the research, and a HCMV infected HPT-8model was established.②After infected72h, the cultured HPT-8cells and TCM were collected respectively. The TCM was exposed to ultraviolet rays for15min to inactivate CMV, after filtration and centrifugalization.③The effect of baicalein on HCMV DNA load in infected HPT-8cells was detected by real-time quantative PCR.④The effect of baicalein on the expression of VIP mRNA in infected HPT-8cells was also detected by real-time quantative PCR.⑤The effect of baicalein on the protein expression of VIP was detected by immunocytochemistry and Western blot.⑥The effect of baicalein on VIP concentration in supernatant was observed with ELISA method.
[Results]①Beicalein can reduce HCMV DNA load in infected HPT-8cells.②Compared to control group, the mRNA and protein expression levels of VIP synthesized and excreted by HPT-8cells in infection group was decreased (p<0.05). The differences in the mRNA, protein and excretion expression levels of VIP between control group and beicalein group were not significant statistically (p>0.05)
[Conclusions] Baicalein has a positive impact on the abnormal VIP expression by HCMV infection at maternal-fetal interface.
[目的]体外研究T细胞对感染HCMV的绒毛外细胞滋养细胞的免疫效应。
[方法]①选择人早孕胎盘绒毛滋养细胞株HPT-8进行研究,建立感染HCMV的HPT-8细胞模型：以100TCID50HCMV攻击HPT-8细胞后,应用免疫荧光细胞化学法检测HCMVpp65抗原表达及病毒滴定法观察HPT-8感染情况并绘制生长曲线,判定HPT-8细胞对HCMV的易感性,感染状态以及HCMV在细胞内生长情况。②收集门诊孕前咨询健康妇女外周血,提取外周血单个核细胞,尼龙毛柱法分离纯化获得T细胞。③建立T细胞与感染HCMV的HPT-8细胞共培养模型,应用实时荧光定量PCR法检测HPT-8细胞内HCMV DNA负荷量。
[结果]①加入HCMV48h后,HPT-8细胞胞质内出现大量HCMV pp65抗原信号；感染3d后的HPT-8细胞培养液(Trophoblast conditioned medium, TCM)可使HEL细胞发生细胞病变；HCMV在HPT-8细胞内的复制于第4d开始增高,6d达到高峰。②T细胞具有下调HPT-8细胞内HCMV DNA负荷量的作用(p<0.05)；T细胞与感染的HPT-8细胞共培养3d后的细胞上清,也具有下调HPT-8细胞内HCMV DNA负荷量的作用(p<0.05)；共培养3天后的T细胞则失去了下调HPT-8细胞内HCMV DNA负荷量的作用(p>0.05)。
[结论]T细胞在母-胎界面HCMV感染初期具有一定的免疫防御作用,其机制可能与T细胞的直接溶解杀伤作用及其分泌的细胞因子相关；但T细胞的免疫功能很快受到抑制,其中的机制有待进一步研究。
第二部分HCMV感染后绒毛外细胞滋养细胞分泌产物对外周血T细胞Treg/Th17平衡的影响
[目的]体外研究HCMV感染后绒毛外细胞滋养细胞分泌产物对外周血T细胞Treg/Th17平衡的影响
[方法]①选择人早孕胎盘绒毛滋养细胞株HPT-8进行研究,建立感染HCMV的HPT-8细胞模型。②尼龙毛柱法分离纯化获得T细胞。③建立TCM与T细胞共培养体系：100TCIDso HCMV攻击HPT-8细胞后,收集HCMV感染72h以及相应时间点对照组的TCM,过滤离心后紫外灯照射15mmin灭活HCMV;根据T细胞培养液组分的不同,分为感染组(HCMV感染72h的TCM)、TCM对照组(对照组的TCM)及空白对照组(不含TCM),置于C02培养箱(5%C02、37℃)中继续培养72h。④应用流式分析法检测HCMV感染后绒毛外细胞滋养细胞分泌产物对T细胞内CD4+CD25+CD12-/CD4+、CD4+IL17A+/CD4+比值的影响。⑤应用实时荧光定量PCR法检测HCMV感染后绒毛外细胞滋养细胞分泌产物对T细胞分化相关转录因子Foxp3、 IL-6、IL-17A、TGF-p及RORyt mRNA表达的影响；⑥应用ELISA法检测各组T细胞培养上清中TGF-β、IL-17A的浓度。
[结果]①与空白对照组相比,TCM对照组Treg/Th17比值增高(p<0.01)；与TCM对照组相比,感染组Treg/Th17比值降低(p<0.01)。②与空白对照组比较,TCM对照组T细胞内Foxp3, RORyt及TGF-p mRNA的表达均明显升高(p<0.01)；与TCM对照组比较,感染组T细胞内Foxp3, RORyt及TGF-β mRNA表达均明显减少(p<0.01)。③与空白对照组相比,TCM对照组T细胞内IL-17A和IL-6mRNA的表达均明显降低(p<0.01)。与TCM对照组相比,感染组T细胞内IL-17A和IL-6mRNA的表达均明显增加(p<0.01)。④与空白对照组相比,TCM对照组上清中TGF-β1浓度增加(p<0.01)；与TCM对照组比较,感染组上清中TGF-β1浓度减少(p<0.01)。⑤与空白对照组相比,TCM组上清中IL-17A浓度减少(p<0.01);和TCM对照组相比,感染组IL-17A的浓度增加(p<0.01)。
[结论]绒毛外细胞滋养细胞分泌物可促使外周血T细胞Treg/Th17型免疫平衡向Treg细胞偏倚,有利于妊娠的维持,而HCMV可能通过影响绒毛外细胞滋养细胞合成及分泌功能从而使外周血T细胞内Treg/Th17平衡向Th17细胞偏倚,导致不良妊娠结局。
第三部分HCMV感染对绒毛外细胞滋养细胞合成分泌V][P表达水平的影响
[目的]体外研究HCMV对绒毛外细胞滋养细胞合成分泌血管活性肠肽(vasoactive intestinal peptide, VIP)表达水平的影响
[方法]①选择人早孕胎盘绒毛滋养细胞株HPT-8进行研究,建立感染HCMV的HPT-8细胞模型。②收集HCMV感染72h以及相应时间点对照组的细胞及TCM,TCM过滤离心后紫外灯照射15min灭活HCMV。③应用实时荧光定量PCR法检测HCMV感染对HPT-8细胞VIP mRNA表达水平的影响。④应用免疫细胞组织化学及免疫印迹法检测HCMV感染对HPT-8细胞VIP蛋白质表达水平的影响。⑤应用ELISA法检测HCMV感染对HPT-8细胞分泌VIP浓度的影响。
[结果]①与空白对照组相比,感染组HPT-8细胞VIP mRNA表达水平明显降低(p<0.01)。②空白对照组及感染组HPT-8细胞的胞质和胞浆均有VIP蛋白表达,呈棕黄色颗粒；感染组HPT-8细胞VIP蛋白表达水平明显低于空白对照组p<0.01)。③感染组HPT-8细胞分泌VIP浓度明显低于空白对照组p<0.01)。
[结论]母-胎界面HCMV感染抑制了绒毛外细胞滋养细胞合成和分泌的VIP表达水平。
第四部分黄芩素对感染HCMV的绒毛外细胞滋养细胞VIP表达水平的影响
[目的]探讨黄芩素阻断HCMV感染绒毛外细胞滋养细胞的作用及对HCMV感染的绒毛外细胞滋养细胞VIP表达水平的影响。
[方法]①选择人早孕胎盘绒毛滋养细胞株HPT-8进行研究,建立感染HCMV的HPT-8细胞模型。②收集HCMV感染72h以及相应时间点对照组、黄芩素组的HPT-8细胞及TCM, TCM过滤及离心后用紫外灯照射15min灭活HCMV。③应用实时荧光定量PCR法检测黄芩素对感染HCMV的HPT-8细胞HCMV DNA负荷量的影响。④应用实时荧光定量PCR法检测黄芩素对感染HCMV的HPT-8细胞内VIP mRNA表达的影响。⑤应用免疫细胞组织化学及免疫印迹法检测黄芩素对感染HCMV的HPT-8细胞内VIP蛋白质表达水平的影响。⑥ELISA法检测黄芩素对感染HCMV的HPT-8细胞分泌VIP浓度的影响。
[结果]①黄芩素减少感染的HPT-8细胞内HCMV DNA负荷量。②与空白对照组相比,感染组HPT-8细胞内VIP蛋白转录,合成及分泌的表达均明显下降,差异均具有统计学意义(p<0.05);黄芩素组HPT-8细胞内VIP蛋白转录,合成及分泌的表达与正常对照组相比,差异均无统计学意义(p>0.05)
[结论]黄芩素对母-胎界面的HCMV感染及其导致的VIP表达异常具有一定积极影响。
Part1T cell-mediated immunological effect in human cytomegalovirus infected extravillous cytotrophoblasts
[Objective] To explore T cell-mediated restriction of human cytomegalovirus (HCMV) in extravillous cytotrophoblasts (EVT) in vitro
[Methods]①A human trophoblast cell line (HPT-8) was chosen to conduct the research, and then to established a HCMV infected HPT-8cells model:HPT-8cells were infected with100TCID50HCMV. The following methods were used to observe infection susceptibility and status of HPT-8cells to HCMV, and growth situation of HCMV in cells: Immunofluorescence cytochemistry method was used to detect HCMV pp65, and method of viral titres was used to observe infection, drawing a growth curve of HCMV.②T cells were isolated and purified by using nylon column from the peripheral blood of normal reproductive women for preconception counseling in outpatient.③A T cells-HCMV infected HPT-8cells co-culture system was established, real-time quantitative PCR was used to detect the HCMV DNA in HPT-8cells.
[Results]①A large number of HCMV pp65antigen signals could be seen in infected HPT-8cells at the48time point. TCM of3d after infection induced typical cell lesion in HEL cells; HCMV replication began to increase4d after infection and peaked at6d.②HCMV DNA load in infected HPT-8cells was significantly decreased in T cells co-cultured group (p<0.05). Cell-free co-culture supernatant was also diminished the HCMV DNA load (p<0.05). However, when co-cultured T cells was added to fresh HCMV infected HPT-8cells, there was no significant difference in HCMV DNA load with HCMV infected group (p>0.05).
[Conclusions] T cells had antiviral effect during the early stage of infected maternal-fetal interface, and the mechanism may be associated with both direct killing effect and secreting soluble factors. However, the immune function of T cells was rapidly inhibited, of which the underlying mechanisms remain unclear and need further study.
Part2The effect of secretory products of human cytomegalovirus infected extravillous cytotrophoblasts on Treg/Th17balance in peripheral blood T cell
[Objective]To investigated the effect of secretory products of HCMV-infected EVT on the Treg/Th17balance in peripheral blood T cell.
[Methods]①A human trophoblast cell line (HPT-8) was chosen to conduct the research, and then to establish a HCMV infected HPT-8model.②T cells were isolated and purified by using nylon column.③The co-culture system of TCM and T cells was established in vitro:HPT-8cells were infected with100TCID50HCMV, and then TCM was collected after HCMV infection within72h and that of control group at the same time point, after centrifugation HCMV in the TCM inactivated by ultraviolet light irradiating15min; the study was grouped according to various components of TCM:infection group (including TCM from infected HCMV within72h), TCM control group (including non-infected TCM) and control group (without TCM), and then put them into incubator (37℃,5%CO2) for72h.③Flow cytometric analysis was applied to detect the change of CD4+CD25+CD127-/CD4+CD4+IL17A+/CD4+in T cells after HCMV infection.⑤Real-time quantitative PCR was used to detect the effect of secretory products of HCMV infected EVT on the expression of the associated transcription factors of T cells differentiation Foxp3、IL-6、IL-17A、TGF-β and RORyt.⑥TGF-β1and IL-17A excreted by T cells were detected with ELISA method.
[Results]①Compared with control group, The ratio of Treg/Th17in TCM control group was significantly higher (p<0.01); The ratio of Treg/Th17was significantly lower in infection group than that in TCM control group (p<0.01).②Compared with control group, the mRNA expression in TCM control group of the three associated transcription factors of T cells differentiation Foxp3、TGF-β and RORyt were significantly increased (p<0.01); compared with TCM control group, the mRNA expression of the three factors was decreased in infection group (p<0.01).③Compared with control group, the mRNA expression levels in TCM control group of the two associated transcription factors of T cells differentiation IL-17A and IL-6were significantly decreased (p<0.01); the mRNA expression levels of the two transcription factors was significantly higher in infection group than that in TCM control group (p<0.01).④TGF-β1concentration in TCM control group was higher than that in control group (p<0.01); compared with TCM control group, TGF-β1concentration in infection group was significantly decreased (p<0.01).⑤The concentration of IL-17A in control group was significantly lower than that in TCM control group (p<0.01); compared with TCM control group, IL-17A concentration was significantly increased (p<0.01)
[Conclusions] EVT secretion can promote the balance of Treg/Th17to Treg cells in peripheral blood T cell, which is conductive to pregnancy. However, HCMV may boost the balance of Treg/Th17to Th17cells in peripheral blood T cell by disturbing the secretory function of EVT, which may induce adverse pregnancy outcomes.
Part3Effect of human cytomegalovirus infection on VIP synthesized and excreted by extravillous cytotrophoblasts
[Objective]To study the effect of HCMV infection on VIP synthesized and excreted by EVT.
[Methods]①A human trophoblast cell line (HPT-8) was chosen to conduct the research, and then a HCMV infected HPT-8model was established.②HPT-8cells and TCM were collected after HCMV infection within72h and that of control group at the same time point, after centrifugation HCMV in the TCM inactivated by ultraviolet light irradiating15min;③The mRNA expression of VIP was detected by real-time quantitative PCR.④The protein expression level of VIP was detected by both immunocytochemistry and Western blot.⑤VIP excreted by HPT-8cells was detected with ELISA method.
[Results]①Comparing to control group, the mRNA expression level of VIP was lower in infection group (p<0.01).②VIP protein expressed in cytoplast and cell plasma of HPT-8cells in both control group and infection group with the expression presenting yellow-brown; the protein expression of VIP in infection group was lower than that in control group (p<0.01).③VIP concentration in TCM from infection group was lower than that from control group (p<0.01)
[Conclusions] The expression of VIP synthesized and excreted by EVT was decreased after HCMV infection at maternal-fetal interface.
Part4Effect of baicalein on the expression of VIP in extravillous cytotrophoblasts infected with human cytomegalovirus in vitro
[Objective] to study the function of baicalein to interrupt HCMV infected EVT and its effect on the expression of VIP of infected EVT.
[Methods]①A human trophoblast cell line (HPT-8) was chosen to conduct the research, and a HCMV infected HPT-8model was established.②After infected72h, the cultured HPT-8cells and TCM were collected respectively. The TCM was exposed to ultraviolet rays for15min to inactivate CMV, after filtration and centrifugalization.③The effect of baicalein on HCMV DNA load in infected HPT-8cells was detected by real-time quantative PCR.④The effect of baicalein on the expression of VIP mRNA in infected HPT-8cells was also detected by real-time quantative PCR.⑤The effect of baicalein on the protein expression of VIP was detected by immunocytochemistry and Western blot.⑥The effect of baicalein on VIP concentration in supernatant was observed with ELISA method.
[Results]①Beicalein can reduce HCMV DNA load in infected HPT-8cells.②Compared to control group, the mRNA and protein expression levels of VIP synthesized and excreted by HPT-8cells in infection group was decreased (p<0.05). The differences in the mRNA, protein and excretion expression levels of VIP between control group and beicalein group were not significant statistically (p>0.05)
[Conclusions] Baicalein has a positive impact on the abnormal VIP expression by HCMV infection at maternal-fetal interface.
引文
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