Study On The Expression Of CD44 In The Uterine Natural Killer Cells And Its Ligand Osteopontin At The Maternal-fetal Interface During Early Pregnancy

Pregnancy is a complex physical process.For the maternal immune system,a conceptus is a semiallograft.There is a special immulogical balance between the conceptus,tolerating the presence of a conceptus expressing paternal antigens.The materal-fetal immune equipoise is the foundation of successful human pregnany.Uterine nature killer cell(uNK cells)are the highlight of implantation immunology. There is a great number of uNK cells contacting with the trophoblast cell.The interesting phenomenum indicate that uNK cells play an important role in control maternal resistance to blastocyst.uNK cells is a special subset of NK cells.CD44 is a member of adhesion molecule family.It plays an important role in the activation of the lymphocytes,especially T and NK cell,intracellular signaling,and death of the induced cell.Many factors,for example, hormones,cytokines and chemokines take part in the growth,development and function of the uNK cells.OPN is a secretion of sugar base of phosphorus protein is important cell structure,chemical composition,sub-type of placenta and the placenta appearance,the type of placenta cells to strengthen their own movements,the placenta could be a better appearance of factors induce cell migration,cell OPN in the material,relocation and other acts regulating the cell,body tissue or organ is an important developmental process regulating factors.Osteopontin is an important ligand for CD44,and may take part in regulating the movement of the NK cells.The reason why the CD56^brightCD16^- uNK are abundant during early pregnancy is a focus.To research the mechanism of the recruitment of the uNK cell in the deciduas do well to understand the maternal-fetal immune tolerance.Meanwhile,it could provide a new theoretical basis and treatment for clinical pathology pregnancy.PartⅠThe study of CD44 on the surface of uNK cells and the OPN expression at maternal-fetal interface in early pregnancyObjective:1.To investigate the expression of CD44 on pregnant women's uterine and peripheral blood CD56^brightCD16^- natural killer cells in early pregnancy and discuss the relation between expression of CD44 on CD56^brightCD16^- uterine natural killer cells and immunotolerance at the fetal-maternal boundary.2.To investigate the expression of OPN in the decidual tissue and trophoblast,and the siganificance.Method:1.Decidual lymphocytes and peripheral blood were obtained from 20 women during 5～9 weeks of normal pregnancy who were undergoing selective termination.Peripheral blood were obtained from 20 healthy nonpregnant women as control.FACS technology was used to detect CD56^brightCD16^-NK cells number and CD44 expression.2.Fresh decidual and villus tissue were obtained from normal five cases of 5～9 weeks pregnancy women.Trizol method get the total RNA.RT-PCR technology tests OPN mRNA.Immunohistochemical technique analysis the expression of OPN protein in normal pregnancy person's specimens.Result:1.CD56^brightCD16^-natural killer cells predominate,accounting for 70%of decidual lymphocytes.FACS results indicated that CD44 was significantly decreased in uterine CD56^brightCD16^- NK cells as compared with that in peripheral blood CD56^brightCD16^-NK cells,accounting for34.65%±9.96%and 99.55%±0.35%.The diference between them in CD44 expression was significant(P＜0.05).Although the levels of CD44 in peripheral blood CD56^brightCD16^-NK cells of pregnant women were similar to that in peripheral blood CD56^brightCD16^- NK cells of non-pregnant women,accounting for 99.55%±0.35%and 99.66%±0.29%respectively,the difference between them in CD44 expression was also significant(P＜0.05)2.The OPN expression at normal early pregnancy maternal-fetal interface:In five cases of early pregnancy abortion normal specimens,RT-PCR and immunohisto -chemical technique display OPN positive expression in decidual epithelium,decidual stromal cells, cytotrophoblasts and syncytiotrophoblast.Conclusion:1.CD56^brightCD16^-natural killer cells are the predominant lymphocytes during the first trimester of decidual tissue.These cells have a great different antigenic phenotype from peripheral blood CD56^brightCD16^- natural killer cells.Uterine CD56^brightCD16^- natural killer cells have low expression of CD44.This phenotype may be responsible for the low cytoxity of uterine CD56^brightCD16^-natural killer cells and may be an important factor contributing to the immunotolerance at the fetal-maternal boundary during pregnancy.2.The positive expression of OPN at the normal early pregnancy maternal-fetal interface indicate that OPN may play an important role in the recruitment of uNK cells.PartⅡThe in vitro study of OPN regulation by progesterone in human decidual stromal cellsObjective:To investigate the effect of progesterone in the regulation of OPN in the humane decidual stromal cells and to understand the possible role of OPN in the recruitment of uNK cells.Method:1.Decidual stromal cells for the original generation to generation and transmission were obtained by mixed enzyme digestion.2.Different concentrations of Progesterone (0,10^-7,5X10^-7,10^-6mol/l)stimulatethe decidual stromal cells 72h.3.RT-PCRanalyzed OPN expression changes in decidual stromal cells.4.ELISA analyse the concentration of OPN in the superant of the culture fluid.Result: Results1.The value of semi-quantitative analysis of optical density(OPN/ beta -actin)show: contrast group 0.10±0.03,the low-dose group 0.46±0.13,the middle-dose group 0.91±0.16 dose,high-dose group 1.36±0.21.(P＜0.05).2.ELISA test in groups,low,medium and high-dose group,OPN protein content, respectively 4.56±2.16,14.16±3.26,29.62±2.78,52.86±5.68ng/ml(P＜0.05).Conclusion:Progesterone could stimulate OPN expression of the decidual stromal cells.OPN expression may be having a close relation to the recruitment of the Unk cells during early pregnancy.