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

Human Leukocyte Antigen-G Regulates Biological Behaviors of Trophoblast-derived Cell Line HTR-8/SV Neo Via MAPK Signaling Pathways

Author LiHuiJian
Tutor GuWeiRong
School Fudan University
Course Perinatal Medicine
Keywords HLA antigens Histocompatibility antigen class I Mitogen-activated protein kinase Small interfering RNA Preeclampsia
CLC R714.2
Type Master's thesis
Year 2011
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Trophoblast cells are in direct contact with the part of the embryo and the mother, including cytotrophoblast cells, syncytiotrophoblast, extravillous cytotrophoblast cells three subsets. Early pregnancy trophoblast cells with high proliferative capacity and similar unique biological features of the malignant tumor cell invasion ability. Trophoblast cells invade the maternal decidua, maternal exclusion rapid proliferation is one of the key factors to successful pregnancy, trophoblastic sparingly invasive required endometrial stromal pregnancy is normal, abnormal invasive process will result in pathological pregnancy such as spontaneous abortion, intrauterine growth retardation, hypertensive disorders in pregnancy. Mother - the interaction between the the fetal interface micro-environment of Cellular and Molecular you can adjust the trophoblast cells invade the endometrium and decidua. Therefore, the mother - fetal interface associated molecules involved in the regulation of trophoblast cell biological behavior, can affect the normal implantation of the blastocyst and growth. Human leukocyte antigen G (human leukocyte antigen-G, HLA-G) gene is a non-classical major histocompatibility complex class I molecules (major histocompatibility complex, MH). Previous studies that HLA-G maternal-fetal immune tolerance induced by a variety of mechanisms, in particular the immune cells in the maternal-fetal interface, such as NK cells, T cells, antigen-presenting cells, such as regulatory role, so that the embryos for Free by decidual NK cells and T lymphocytes attack and maintain a normal pregnancy. However, our previous work suggests that the decline in HLA-G gene expression can be directly lead to reduced ability of trophoblastic invasion, decreased proliferation and increased apoptosis, which may lead to a series of pathological pregnancy, such as preeclampsia and fetal growth restriction, the recurrence spontaneous abortion occurred. However, the specific mechanisms of the HLA-G gene and pathological pregnancy is not clear. Mitogen-activated protein kinase (mitogen-activated protein kinase, MAPK) is an important intracellular signal transduction, including extracellular signal-regulated kinase (extracellular signal-regulated kinase, ERK), c-Jun N-terminal kinase ( c-Jun N-terminal kinase, JNK) and p38MAPK members, involved in the regulation of cell proliferation, differentiation, apoptosis and cell adhesion and migration. It has been reported that the placental tissue p38MAPK widely distributed p38MAPK activation and expression of a change of state may be closely related to the incidence of pregnancy with preeclampsia and other pathological. In this study, RNA interference technique to suppress the expression of HLA-G gene HTR-8/SVneo cells, HLA-G expression decreased trophoblastic biological behavior, MAPK and MAPK phosphorylation of key protein kinase expressions, to further clarify eclampsia The early pathological pregnancy in the pathogenesis and provide new ideas for the treatment. The first part of the chemically modified siRNA in vitro down to nourish the cells of HLA-G expression purposes vitro to silence HTR-8/SVneo nourish the cells of HLA-G expression. By siRNA interference technology, transfected HTR-8/SVneo trophoblast cells, respectively, with a fluorescent oligo transfected cells, RT-PCR, Western-blot detection nourish the cells of HLA-G expression levels. Results HTR-8/SVneo trophoblastic fluorescent transfection efficiency up to 70% -90%. HLA-G mRNA expression: the experimental group to 0.26 ± 0.08, negative control group (0.71 ± 0.11) in the control group was 0.79 ± 0.07. Experimental group and negative control group, the difference was statistically significant (P lt; 0.01); negative control group and blank control group, the difference was not statistically significant (P gt; 0.05); experimental group HLA-G mRNA expression inhibition rate is (69.8 ± 6.3)%, the negative control group HLA-G mRNA expression inhibition rate (14.9 ± 2.2)%, control group. HLA-G protein expression: experimental group to 0.20 ± 0.15, negative control group, 0.75 ± 0.12 in the control group was 0.76 ± 0.21. Experimental group and negative control group, the difference was statistically significant (P lt; 0.01); negative control group and blank control group, the difference was not statistically significant (P gt; 0.05); experimental group HLA-G protein expression inhibition rate (81.1 ± 14.4)%, negative control group of HLA-G protein expression inhibition rate (18.0 ± 7.7)%, the control group, the experimental group and negative control group, the difference was statistically significant (P lt; 0.01 ). Experimental group HLA-G mRNA expression levels and protein expression levels than the negative control and blank control group was significantly reduced. Conclusion siRNA can effectively inhibit trophoblastic Department HTR-8/SVneo cells of HLA-G expression. The second part of the HLA-G expression decreased Objective trophoblastic biological behavior observed HLA-G expression decreased HTR-8/SVneo trophoblast invasion, proliferation and apoptosis. By siRNA interference to silence HTR-8/SVneo nourish the cells of HLA-G expression and Matrigel invasion assay trophoblast invasion force, BrdU proliferation assay cell proliferation, Annexin V / P double staining to detect cell apoptosis rate. Success to silence HTR-8/SVneo nourish the cells of HLA-G expression, observed under high magnification (× 200) through the membrane to reach the lower surface of the cells, the number of invasive cells of the lower the Transwell small room: the experimental group (57 ± 38), the negative control group (3644 ± 79), the control group (260 ± 84). Experimental group and negative control group, the difference was statistically significant (P lt; 0.01); negative control group and blank control group, the difference was not statistically significant (P gt; 0.05) The number of cells of the experimental group as compared to the negative control group and blank control group decreased significantly. 48h after transfection, the proliferation of cells in each group, the experimental group to 0.19 ± 0.08, negative control group to 0.26 ± 0.08, the control group was 0.27 ± 0.09. Experimental group and negative control group, the difference was statistically significant (P lt; 0.01); negative control group and blank control group, the difference was not statistically significant (P gt; 0.05) Ability and negative control group of the experimental group, cell proliferation, or as compared to the blank control group was significantly reduced. 72 hours after transfection, apoptosis rate of the cells in each group, the experimental group (19.5 ± 0.47)%, negative control group (15.8 ± 0.83)% in the control group (16.3 ± 0.92)%. Experimental group and negative control group, the difference was statistically significant (P lt; 0.01); negative control group and blank control group, the difference was not statistically significant (P gt; 0.05) Compared to the experimental group apoptosis rate and negative control group or the control group increased significantly. Conclusion HLA-G low expression of directly affect trophoblast biological behavior. The third part of the HLA-G expression was decreased by the MAPK signaling pathway impact nourish the biological behavior of cells Objective To investigate the expression of HLA-G to drop a direct impact on the trophoblast cell biological behavior of cell signaling pathways. SiRNA interference technology to silence HTR-8/SVneo nourish the cells of HLA-G expression, and then detect the expression levels of MAPK signaling pathway critical protein kinase molecules. In order to analyze the p38 MAPK signal pathway, the p38MAPK signaling pathway-specific inhibitor SB203580, Matrigel invasion assay to detect the trophoblast invasion force, BrdU proliferation assay cell proliferation rate of apoptosis was detected by Annexin V / PI double staining to resolve the p38 MAPK signal pathway in low expression of HLA-G direct impact on the role of trophoblastic biological behavior. Results silence HTR-8/SVneo nourish the cells of HLA-G expression of p-p38MAPK and p38MAPK protein ratio: experimental group to 0.74 ± 0.04, negative control group was 0.47 ± 0.09, the control group was 0.36 ± 0.21. The expression levels experimental group p-p38MAPK/p38MAPK protein ratio than the negative control group and blank control group was significantly higher; level of p-JNK/JNK protein ratio of the experimental group was 0.46 ± 0.07 to 0.79 ± 0.08 negative control group, the control group 0.78 ± 0.13; level of p-ERK/ERK protein ratio of the experimental group was 0.89 ± 0.13, negative control group was 0.88 ± 0.21, and the control group was 0.89 ± 0.26. Inhibitor is added after p-p38MAPK and p38MAPK protein ratio: experimental group were 0.89 ± 0.09,0.16 ± 0.04, negative control group were 0.76 ± 0.08,0.14 ± 0.03, experimental group joined inhibitor SB203580 before and after p- p38MAPK and p38MAPK ratio, the difference was statistically significant (P lt; 0.01), negative control group to join inhibitor SB203580, after the p-p38MAPK and p38MAPK ratio, the difference was statistically significant (P lt; 0.01); Add to inhibit agent SB203580 ago,, after p-JNK and JNK protein ratio: experimental group were 0.62 ± 0.15,0.61 ± 0.21, negative control group were 0.86 ± 0.11,0.88 ± 0.09, experimental group was added before the inhibitor SB203580, after p -JNK JNK ratio, the difference was not statistically significant (P gt; 0.05), negative control group to join inhibitor SB203580 after the p-JNK JNK ratio, the difference was not statistically significant (P GT; 0.05); Add inhibitor SB203580 before each group after p-ERK and ERK protein ratio: the experimental group were 0.89 ± 0.18,0.91 ± 0.11, negative control group was 0.87 ± 0.17, 0.89 ± 0.23, respectively, before and after experimental group joined inhibitor SB203580 The ratio of p-ERK and ERK, the difference was not statistically significant (P gt; 0.05), negative control group to join inhibitor SB203580 after p-ERK and ERK ratio comparison, the difference was not statistically significant (P GT; 0.05). Prior to joining inhibitors, after transwell small chamber lower number of invasive cells: the experimental group (51 ± 13) months (90 ± 21), before and after, the difference was statistically significant (P lt; 0.01); negative control group (290 ± 52) months (298 ± 33), before and after, no statistically significant difference (P GT; 0.05); inhibitor is added in the experimental group the number of cells increased significantly compared to the experimental group, adding inhibitor experiments group the number of stained cells than the inhibitor is added the negative control group and blank control group decreased significantly. Inhibitor is added before and after the changes in cell proliferation: the experimental group were 0.22 ± 0.09 and 0.22 ± 0.16, before and after comparison, the difference was not statistically significant (P GT; 0.05); negative control group were 0.22 ± 0.15 and 0.25 ± 0.15, before and after, the difference was not statistically significant (P GT; 0.05): adding an inhibitor of the experimental group and negative control group of the inhibitor is added, the difference was not statistically significant (P gt; 0.05) Inhibitor is added before and after the changes in the rate of apoptosis: the experimental group (19.0 ± 0.58)% and (13.4 ± 0.28)%, respectively, before and after comparison, the difference was statistically significant (P lt; 0.01); negative control group (10.0 ± 0.80)% and (9.51 ± 2.73)%, before and after comparisons, no statistically significant difference (P GT; 0.05); inhibitor is added in the experimental group and negative control group of the inhibitor is added, the difference was statistically significance (P lt; 0.01). Conclusion HLA-G gene may be through the p38 MAPK signal pathway directly regulates the biological behavior of trophoblast cells, and thus participate in the occurrence of pathological pregnancy.

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