The Role Of MHLA-G In Human Embryo Implantation And Maternal-fetal Immune Tolerance

BACKGROUND: Successful embryo implantation plays a key role in a successful pregnancy, and a significant feature of embryo implantation is trophoblastic invasion into endometrium. Many factors are involved in this process. As it is expressed on extravillous trophoblast selectively, human leucocyte antigen G could play a key role in embryo implantation.OBJECTIVE: 1. To establish human trophoblast cell line with stable HLA-G expression; 2. To detect the effect of human leukocyte antigen-G(HLA-G) on maternal-fetal immunotolerance and proliferation, adhesion, invasion of trophoblast cell line in order to investigate the role of HLA-G in embryo implantation and uterine spiral arteries remodeling.METHODS: 1. To package Lentivirus containing HLA-G1 in 293 T cells and transfect the HLA-G(-) human trophoblast cell lines stably; 2. To detect the effect of human leukocyte antigen-G(HLA-G) on NK-92MI-mediated cytotoxicity to JAR cell line; 3. Cell proliferation was assessed by CCK-8 and Brd U ELISA in test group(groups of transfection) and negative control respectively, cell cycle and apoptosis was detected by flow cytometry assay; 4. Cell adhesion was assessed by ECM Array, fibronectin coating colorimetric Format and Endometrial cells coating colorimetric Format respectively; 5. Transwell invasion chamber assay was used to detect cell invasion, the effect of hypoxia and hepatocyte growth factor(HGF) on trophoblast cell line invasiveness was detected as well.RESULTS: 1. Two trophoblast cell lines with stable HLA-G expression were established and identified; 2. The more significant inhibition of HLA-G to NK cells-mediated cytotoxicity could be detected from E:T ratios of 1.25:1 to 10:1(P<0.05); 3. No difference about proliferation was found between the HLA-G1 transfection group and negative control group by CCK-8 assay and Brd U ELISA.(P>0.05); Compared with the negative control group(46.29±0.77)%, the ratio of S phase cells in transfection group was(4 7.07± 3.15)%, there was no different between them too(P> 0.05); No apoptosis was detected in both of two groups; 4. No difference of adhesiveness was detected between the HLA-G1 transfection group and negative control group by ECM Array, fibronectin coating colorimetric format and endometrial cells coating colorimetric format(P> 0.05);5. The invasion cells number in HLA-G1 transfection group and negative control group were 66± 19 and 67± 17 respectively, There was no difference between two groups without stimulation(P> 0.05). HGF-induced invasion could be significantly inhibited in the HLA-G1 transfection group compared with negative control group(40± 20 VS 66± 19)(P<0.05); The invasion was decreased in both groups and the inhibition of HGF could be prevente d under 3% O2.CONCLUSION: 1. HLA-G is a critical immunosuppressive molecule mediating maternal-fetal immune tolerance; 2. HLA-G could not be an independent factor contributing to proliferation, adhesion, invasion of trophoblast cell; 3. Hepatocyte growth factor-induced trophoblast invasion could be regulated by HLA-G, and the regulation will be prevented under hypoxia. It suggested HLA-G is not an independent factor for regulating the trophocytes, but HLA-G could be involved in regulating proliferation of trophoblast with other factors together, such as various growth factors, and play a indirect role in embryo implantation and uterine spiral arteries remodeling.