The Study On Immune Properties Of Early Placental Extravillous Cytotrophoblasts With Human Cytomegalovirus In Vitro

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.