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Human Cytomegalovirus Induced B7-H1 Expression:an in Vitro Study in Trophocyte

Author: ZhaoJianHua
Tutor: ChenSuHua
School: Huazhong University of Science and Technology
Course: Obstetrics and Gynaecology
Keywords: human cytomegalovirus trophocyte B7-H1 MAPK-mediated signal transduction pathway miR-513 siRNA
CLC: R714.2
Type: Master's thesis
Year: 2011
Downloads: 9
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Abstract


ObjectiveIn order to provide a theoretical basis for the prevention and treatment of intrauterine infection of HCMV. We investigated if human cytomegalovirus(HCMV) induces the expression of B7-H1 in cytotrophoblast and explored the role of MAPK-mediated signal transduction pathway/ microRNA in this process. Furthermore we used B7-H1 siRNA to interfer this process.Methods1. The human fetal lung fibroblast cells(HEL) and the cytotrophoblast cell line(HPT-8) were routinely cultured with the method for adherent cell in the laboratory.2. The AD169 strain of HCMV was passaged by cell culture and the TCID50 was quantitated by the method of Reed-Muench.3. Real-Time PCR detected the expression of B7-H1 mRNA in HPT-8 which were infected by HCMV with different titers.4. Flow cytometry(FCM) detected the expression of B7-H1 protein in HPT-8 which were infected by HCMV with different titers.5. Western-blot detected the phosphorylation of MAPK during the process of HCMV infection in HPT-8 and its relation to B7-H1 expression by using different MAPK inhibitors;6. Real-Time PCR detected the expression of miR-513 in HPT-8 which were infected by HCMV with different titers; Flow Cytometry(FCM) detected the expression of B7-H1 protein in HPT-8 which were transfected with miR-513 mimics or inhibitor, respectively. 7. Designed the B7-H1-specific siRNAs plasmid and construct the expression vector for eukaryotic cell; Flow Cytometry(FCM) detected the expression of B7-H1 protein in HPT-8 after transfection with the B7-H1-specific siRNAs expression vector.Result1. The TCID50 of the AD169 strain of HCMV was 10-3.29/100ul.2. The expression of B7-H1 mRNA and protein were increased in HPT-8 which were infected by HCMV with different titers,the group of 100TCID50 increased B7-H1 mRNA most significantly(compared with the blank control group, P<0.05).3. HCMV could induce the phosphorylation of ERK signal transduction pathway in HPT-8; Low concentration of DMSO、JNK-inhibitor SB203580(20μM) and P38-inhibitor SP600125(10μM) did not affect the expression of B7-H1 protein in HPT-8 which were infected by HCMV(compared with the blank control group, P>0.05). However ,ERK1/2-inhibitor U0126(10μM) increased the expression of B7-H1 protein in HPT-8 which were infected by HCMV(compared with the blank control group, P<0.05).4. The expression of miR-513 was reduced in HPT-8 which were infected by HCMV with different titers(compared with the blank control group, P<0.05); HPT-8 which transfected with miR-513-inhibitor expressed even more B7-H1 protein(compared with the negative control group, P<0.05),this demenstrated that miR-513 could affect the expression of B7-H1 protein in HPT-8; HPT-8 which transfected with miR-513-mimics expressed lower B7-H1 protein,even with the infection of HCMV(compared with the negative control group, P<0.05),this demenstrated that miR-513 could affect the process of HCMV induce HPT-8 to expression B7-H1 protein.5. We designed the B7-H1 siRNA-1、B7-H1 siRNA-2、B7-H1 siRNA-3.All of them could restrain the expression of B7-H1 protein in HPT-8 which infected by HCMV(compared with the negative control group, P<0.05). Conclusion①HCMV can induce the expression of B7-H1 mRNA and protein in HPT-8;②In this prosess,HCMV may be influence the B7-H1 expression through activaties of the ERK 1/2signal transduction pathway,also may be inhibit the composition of miR-513 to increase the expression of B7-H1 in HPT-8;③The intervention of B7-H1 siRNA can restrain this contribution of HCMV.

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CLC: > Medicine, health > Obstetrics and Gynaecology > Obstetrics > Pathological pregnancy ( abnormal pregnancy )
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