| Objective: During pregnancy, Fetus can be viewed as allogeneic transplant which has not been rejected by its mother because of the immune balance on the maternal-fetal interface. The changes of decidual immune micro-environment during pregnancy are critical. Particularly, the changes of NK-like large granular lymphocytes on decidua are very important for normal pregnancy. The study of the relationship and mechanism between the changes of cluster differentiation and killing activity of NK-like cells on maternal decidua need to be analyzed deeply.In this study, dynamic changes of characteristic cluster differentiation phenotypes and the expression of NK-like cell receptors (NK inhibitory receptor NKG2A and killing receptor NKG2D), the intracellular perforin of NK-like cells during the normal pregnancy were analyzed by Flow cytometry (FCM), which planned to understanted the mechanism of the phenotype shift of decidual NK-like cells during pregnancy, and its relationship with local immune endometrial micro-environment. It was expected to reveal the immune tolerance of pregnancy and the mechanism of pathological pregnancy.Methods:1 20 normal pregnant women who require artificial abortion were selected randomly as the early pregnancy group. 20 normal pregnant patients who require artificial abortion and uterine-incision delivery in 13～27 weeks were selected randomly as the metaphase group. 20 term pregnancy women who require uterine-incision delivery were selected randomly as the last pregnancy group. 20 non-pregnant women with the husband who have unusual spermatozoa were selected randomly as the control group. Each group of women had no clinical complications and pregnancy complication. 2 The endometrium and decidua tissues were collected aseptic selection and non-walked monocyte were prepared by mechanical separation. The quantity of CD3-CD56+NK-like cell, the quantity of CD16+CD56+NK-like cell, the quantity of CD16-CD56+NK-like cell, the expression of the inhibitory inhibitery receptor CD56+NKG2A+, the expression of the activity surface receptor CD56+NKG2D+ and the expression of anti-characteristic fuction CD56+perforin+ of NK-like cells on endometrium and decidua in non-pregnancy and normal groups detected by derect fluorescence-labelled flow cytometry respectivity.3 SPSS13.0 sofeware was used for statistics. After normality plots with tests, analyzed the expression of the quantity of CD3-CD56+NK-li-ke cell, the quantity of CD16+CD56+NK-like cell, the quantity of CD16-CD56+NK-like cell, the expression of the inhibitory receptor CD56+NKG2A+, the expression of the activity receptor CD56+NKG2D+ and the exp-ression of killing activity CD56+perforin+ of NK-like cells on endometri-um and decidua wether were changed between the non-pregnancy and t-he normal groups by One-Way ANOVA.Results:1 The expressing of the cluster differentiation of NK-like cell in normal pregnancy decidua1.1 The expressing of CD3-CD56+NK-like cellsCompared with non-pregnancy control group (18.08%±4.76%), the percentage of CD3-CD56+NK-like cells of the early pregnancy group and the metaphase pregnancy group were respectively (38.94%±4.82%, 75.55%±6.25%, both P <0.01), which increased remarkably compared with control group. That of late pregnancy group (4.06%±1.26%, P <0.01) decreased significantly. There were significant differences between every two groups (P <0.01).1.2 The expressing of CD16+CD56+NK-like cellsCompared with control group (5.72%±2.11%), the percentage of CD16+ CD56+NK-like cells in the early pregnancy group and the metaphase pregnancy group were respectively (5.02%±1.81%, 6.03%±1.25%, both P >0.05), there was no difference. That of late pregnancy group (1.40%±0.36%, P <0.01) decreased significantly. Compared with control group (31.31%±5.63%), the rate of CD16+CD56+/CD3-CD56+ in the early pregnancy group, the metaphase pregnancy group were respectively (12.75%±3.76%, 8.01%±1.89%, both P <0.01), which decreased remarkably compared with control group. That of late pregnancy group (33.05%±4.63%, P > 0.05) increased significantly.1.3 The expressing of CD16-CD56+NK-like cellsCompared with control group (12.36%±2.99%), the percentage of CD16-CD56+NK-like cells in the early pregnancy group and the metaphase pregnancy group were respectively (33.38%±2.80%, 69.52%±6.58%, both P <0.01), which increased remarkably compared with control group. That of late pregnancy group (2.91%±0.94%, P <0.01) decreased significantly. Compared with control group (68.69%±5.63%), the rate of CD16-CD56+/ CD3-CD56+ in the early pregnancy group, the metaphase pregnancy group were respectively (87.22%±3.73%, 91.94%±1.89%, both P <0.01), which increased remarkably compared with control group. That of late pregnancy group (66.46±4.42%, P > 0.05), there was no difference.2. The expressing of activaty and inhibitory surface receptor of NK-like cell in normal pregnancy decidua2.1 The expressing of CD56+NKG2D+NK-like cellsCompared with control group (5.53%±0.82%), the percentage of CD56+NKG2D+ NK-like cells in the early pregnancy group and the metaphase pregnancy group were respectively(12.05%±4.34%, 21.46%±0.67%, both P <0.01), which increased remarkably compared with control group. That of late pregnancy group (1.20%±0.36%, P <0.01) decreased significantly. Compared with control group (31.33%±5.82%), the rate of CD56+NKG2D+ / CD3-CD56+in the early pregnancy group, the metaphase pregnancy group and the last pregnancy group were respectively (30.78%±9.05%, 28.53%±1.75%, 27.90%±4.00%, both P > 0.05), there was no difference. 2.2 The expressing of CD56+NKG2A+NK-like cellsCompared with control group(3.76%±0.79%), the percentage of CD56+ NKG2A+NK-like cells in the early pregnancy group and the metaphase pregnancy group were respectively (8.53%±3.08%, 13.55%±0.78%, both P <0.01), which increased remarkably compared with control group. That of late pregnancy group (1.14%±0.36%, P <0.01) decreased significantly. Compared with control group (21.54%±4.83%), the rate of CD56+ NKG2A+ / CD3- CD56+ in the early pregnancy group was (21.67%±6.34%, P > 0.05), there was no difference. In the metaphase pregnancy group was (18.03%±0.82%, P <0.01), decreased significantly. That of late pregnancy group (26.38%±4.04%, P <0.01) increased significantly.2.3 The ratio of CD56+NKG2A+ / CD56+NKG2D+Compared with control group (0.67:1±0.09), the ratio of CD56+NKG2A+/CD56+NKG2D+ in the early pregnancy was (0.70:1±0.04, P > 0.05), There was no significant change. In the metaphase pregnancy group was (0.62:1±0.20, P <0.01), decreased significantly. That of late pregnancy group (0.94:1±0.23, P <0.01) increased significantly.3. The expressing of the detection of killing activity of NK-like cells in normal pregnancy deciduaCompared with control group (4.36%±0.71%), the percentage of CD56+perforin+NK-like cells in the early pregnancy group and the metaphase pregnancy group were respectively (2.89%±0.49%, 3.01%±0.72%, both P <0.01), which decreased remarkably compared with control group. That of late pregnancy group (3.54%±0.91%, P <0.01) increased significantly. Compared with control group(24.98%±4.29%), the rate of CD56+perforin+/CD3-CD56+ in the early pregnancy group, the metaphase pregnancy group were respectively(7.42%±0.54%, 3.96%±0.70%, both P <0.01), which increased remarkably compared with control group. That of late pregnancy group (85.09%±7.05%, P <0.01) increased remarkably.Conclusions:1. During the normal pregnancy, the quantity of decidual NK-like cells began to increase from early pregnancy and reached the peak at the metaphase pregnancy, then gradually decreased, along with corresponding changes of intracellular perforin.2. CD16-CD56+ NK-like cells were dominant in the early pregnancy, which shifed to CD16+CD56+ phenotype in the last pregnancy, which is helpful to protect the fetal during early and mid-trimester pregnancy ,and to terminate the pregnancy timely.3. The mechanism of maternal-fetal immune tolerance was induced by NK-like cells was not through the function of activity receptor NKG2D and inhibitory receptor NKG2A mainly. The decreasing expression of NKG2A and the increasing expression of NKG2D played a significant role to protect placenta implantation and to maintain the fetus growth. |
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