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Objective: In the course of a normal pregnancy, the fetus as a semi-allograft is not maternal rejection, actually reflects the maternal immune tolerance of embryos. Which conception stepmother vitro weeks - maternal interface of immune cell function change is the key to maintaining the maternal-fetal immune tolerance equilibrium. At home and abroad study lymphocyte immune therapy unexplained recurrent miscarriage the (Unexplained recurrent. Spontaneous abortion, URSA), but the use of the father's surviving in unstable cell origin, the extent of regulation is not high, the differences between individuals and not the efficacy of various laboratory to wait and surviving doubts and disagreements about its immune mechanism. In this study, by analyzing the relationship between maternal T cell function changes with pregnancy loss, explore adoptive immune intervention transfusion different sources paternal peripheral lymphocytes, maternal-fetal pregnancy loss in pregnant rats induced immune tolerance, for further study URSA immunological pathogenesis and guide clinical rational standardization Next adoptive immunotherapy experimental and theoretical basis. Method: 1 establishment of pregnancy loss experimental animal models and experimental groups: male and female mice were randomly divided into 5 groups were mated 2:1; CBA / J (♀) × DBA / 2 (♂) URSA group, CBA / J (♀) × BALB / c (♂) for the control group of normal pregnancy. In 4 days pregnant (implantation period) then URSA group CBA / J female mice were given intravenous injection of the father (the same cage DBA / 2 males), non-parent parties (unusual cage DBA / 2 males) peripheral mononuclear cells of unrelated individuals (BALB / c male mice), respectively, for the treatment group Ⅰ, Ⅱ, Ⅲ group; Group 4 CBA / J female mice injected with normal saline without immune intervention URSA group. Embryonic development observation: each group CBA / J pregnant mice were sacrificed on day 14 of gestation, measuring placental volume count absorb embryos and embryos survived to calculate the of embryonic absorption rate and abortion rate. 3 pregnant mice peripheral T-cell immune function test: 14 days of pregnancy were sacrificed in each group CBA / J pregnant mice, spleen sterile anatomy, the preparation of a single cell suspension. Thiazole blue (MTT) staining ConA-induced T cell transformation A492; direct fluorescent-labeled cells rupture treated by flow cytometry (FCM) were detected Th1-type cytokines IL-2, IFN-γ and Th2-type The cytokines IL-4, IL-10 The expression. 4 maternal-fetal interface of pregnant rats immunosuppressive analysis of the situation: 14 days of pregnancy were sacrificed groups CBA / J pregnant mice sterile anatomical organization of the placenta, preparation of paraffin-embedded sections. Light microscope after HE staining of each rectangular take shape count of 200 immune cells, the count of large granular lymphocytes (LGL), small lymphocytes, macrophages (Mφ) to calculate the percentage of various types of immune cells. Immunohistochemical staining, image analysis, analysis of TGF-β1 to the gray-scale values ??(GS), IL-10, COX-2 and other immunosuppressive molecule expression intensity GS magnitude is inversely proportional to the intensity of positive expression; under the light microscope rectangle out of shape count of 200 cells, calculate the percentage of positive cells. 5 Statistical analysis: application SPSS13.0 version of the software for each set of data normality and homogeneity of variance test, the chi-square test comparison between groups differences between embryonic absorption rate and the percentage of positive cells in placental tissue, single factor analysis of variance comparing groups of pregnant mice spleen cells in Th1/Th2 cytokines, placental tissue immune suppression molecule expression differences. Results: Adoptive transfer the different sources of paternal lymphocyte embryonic development pregnancy loss in pregnant rats pregnancy loss in pregnant rats (URSA group) embryo resorption rate of 22.58%, and animal models of normal pregnancy pregnant mice embryos (Control group) absorption rate of 5.23%, Adoptive transfer of paternal lymphocyte treatment Ⅰ, Ⅱ, Ⅲ embryonic absorption rate were 5.50%, 5.17%, 5.84%; URSA group compared with the untreated treatment Ⅰ, Ⅱ, Ⅲ group embryos The absorption rate was significantly decreased (all P lt; 0.05); among the 3 groups showed no significant difference (P gt; 0.05), and decreased to normal pregnancy, the level of the control group (all P gt; 0.05). The URSA group's abortion rate of 44%, while the Control group, the abortion rate was 20%. Adoptive paternal lymphocyte transfusion treatment Ⅰ, Ⅱ, Ⅲ abortion incidence of 24%, 16%, 20%, respectively; URSA group compared with the untreated treatment I, II, group Ⅲ abortion incidence was significantly decreased ( all P lt; 0.05) 3 groups no significant differences (P gt; 0.05), and decreased to normal pregnancy, the level of the control group (all P gt; 0.05). 2 Adoptive transfer of the impact of the different sources paternal lymphocytes pregnancy loss pregnant mice peripheral T-cell immune function 2.1 ConA induced T lymphocyte transformation function tests without ConA induced transformation and nonpregnant CBA / J female mice spleen cells for 72h A492 value (0.198 ± 0.074) compared to normal pregnancy group (Control group), the uninduced URSA group and three groups of pregnant mice spleen cells via the paternal cell immune intervention treatment group of the A492 values ??were significantly higher (P lt ; 0.05). Which was no significant difference in the normal pregnancy group with untreated URSA group (respectively 0.798 ± 0.114,0.812 ± 0.138, P gt; 0.05), treatment Ⅰ, Ⅱ, Ⅲ group was no significant difference between the (respectively 0.699 ± 0.315,0.659 ± 0.134,0.632 ± 0.132, P gt; 0.05), but lower than in normal pregnancy, untreated group were significantly lowered (all P lt; 0.05). ConA-induced transformation, not pregnant the CBA / J female mice spleen cells into the A492 value (1.221 ± 0.034) compared to normal pregnancy group (Control group) the uninduced of URSA group and three groups of paternal cellular immune intervention pregnant mouse spleen cells transformed the treatment group were significantly decreased (all P lt; 0.05 which compared with normal pregnancy group (1.011 ± 0.134), the the URSA group of untreated despite decreased, but there was no significant difference (1.001 ± 0.178, P gt; 0.05); treatment Ⅰ, Ⅱ, Ⅲ group was no significant difference (for 0.829 ± 0.211,0.859 ± 0.195,0.812 ± 0.168, P gt; 0.05), compared with normal pregnancy, untreated group were significantly lowered (all P lt; 0.05) 2.2 peripheral lymphocyte Th1/Th2 cell function testing 2.2.1 Th1-type cytokine expression and normal pregnancy control spleen cells of IL-2 expression percentage (6.40% ± 0.92% ) compared to URSA group expression percentage significantly with higher (19.73% ± 2.05%, P lt; 0.01); After following the turn lose intervention treatment group Ⅰ, group Ⅱ, group Ⅲ expression percentage significantly reduce to the level of normal pregnancy control group ( were 6.39% ± 1.21%, 6.68% ± 1.07%, 6.42% ± 1.21%, all P lt; 0.01). the percentage group Ⅰ and group Ⅱ group group III expressed no significant difference (P gt; 0.05) percentage (7.41% ± 0.73%) with normal pregnancy control group spleen cell IFN-γ expression compared to the expression of the percentage of the URSA group was significantly higher (11.48% ± 1.10%, P lt; 0.01); Following transfusion after the intervention treatment group Ⅰ, group Ⅱ, group Ⅲ expression Percentage reduce (respectively, to 7.91% ± 0.76%, 7.78% ± 0.82%, 7.43% ± 1.11%, both P lt; 0.01) to a normal pregnancy, the level of the control group. Ⅰ group group Ⅱ, Ⅲ group expression percentage between the two groups showed no significant difference (P gt; 0.05) 2.2.2 Th2 cytokine expression percentage (8.16% ± 0.92%) relative to the control group of normal pregnancy spleen cell IL-4 expression than, URSA group expression percentage significantly with reducing (5.85% ± 0.57%, P lt; 0.05); After following the turn lose intervention treatment group Ⅰ, group Ⅱ, group Ⅲ expression percentage was significantly increased (respectively, to 9.87% ± 1.04%, 9.03% ± 0.91%, 8.96% ± 1.11%, all P lt; 0.05) to the level of the control group of normal pregnancy Ⅰ and group Ⅱ group express the percentage of group III group showed no significant difference (P gt; 0.05). IL-10 expression, spleen cells of normal pregnancy control group compared to the percentage (5.06% ± 0.78%), URSA group expression percentage significantly lower (3.94% ± 0.52%, P lt; 0.05); After following the transfusion intervention treatment group Ⅰ group Ⅱ, group Ⅲ expression percentage was significantly higher (5.93% ± 0.56%, 5.94% ± 0.85%, 6.12 ± 0.96%, both P lt; 0.05) to normal pregnancy control group levels. Ⅰ group, Ⅱ group, Ⅲ group expressed in percentage group showed no significant differences (P gt; 0.05) Adoptive transfer the different sources of paternal lymphocytes 3.1 the number of immune cells in the placental tissue groups placenta impact on pregnancy loss of pregnant rats placenta local immunosuppressive state are scattered visible macrophage (Mφ), large granular lymphocyte (Large granulan lymphocyte LGL), small lymphocytes and other immune cells. various types of cells in the percentage of normal pregnancy control group was 17.85% ± 6.23%, respectively, 65.25% ± 6.68 %, 15.10% ± 4.42%, URSA group were 19.85% ± 18.86%, 64.85%, 12.60% ± 4.28% ± 4.32%, the treatment group Ⅰ were 18.85% ± 4.25%, 65.20%, 15.95% ± 6.48% ± 6.48% in the treatment group Ⅱ were 18.73% ± 5.03%, 68.11%, 13.16% ± 5.32% ± 4.65%, the treatment group Ⅲ were 17.87% ± 4.32%, 67.41% ± 4.57,14.72% ± 3.82%, in each group between no significant statistical significance (all P gt; 0.05), total lymphocyte percentage was 82.25% ± 6.28%, respectively, 80.15% ± 5.63%, 81.14% ± 4.27%, 81.28% ± 5.01%, 82.30% ± 4.63%, and no significant differences between each other (all P gt; 0.05). the 3.2 maternal-fetal interface immunosuppressive factor expression analysis groups placental trophoblast are seen scattered in the cytoplasm of cells, endothelial cells and immune cells or flake TGF -β1, IL-10, COX-2 positive expression. 3.2.1 immunosuppressive molecule analysis of positive expression rate compared to normal pregnancy control TGF-β1-positive cells in the placental tissue expression rate (72.35% ± 5.47%), URSA The positive cell expression rate was significantly reduced (50.45% ± 5.39%); After following the transfusion intervention treatment group Ⅰ, group Ⅱ, group Ⅲ expression percentage are significant with increased (respectively, 63.73% ± 5.12%, 70.27% ± 3.94% control group with normal pregnancy, the placenta, 73.34% ± 5.44%, P lt; 0.05) to the level of the control group of normal pregnancy Ⅰ and group Ⅱ group express the percentage of group III group showed no significant difference (P gt; 0.05). tissue of IL-10 positive cells (69.25% ± 6.37%) compared to the the URSA group of positive expression rate was significantly reduced (48.75% ± 4.32%, P lt; 0.05); After following the transfusion intervention treatment group Ⅰ group Ⅱ, group Ⅲ expression percentage significantly higher (71.25% ± 5.17%, respectively, 76.57% ± 2.98%, 65.98% ± 5.45%, both P lt; 0.05) to normal pregnancy control group levels. Ⅰ group, Ⅱ group no significant difference between the percentage of group Ⅲ group expression (P gt; 0.05). control placental tissue, COX-2 positive cells expression rate (81.05% ± 4.43%) compared with normal pregnancy, URSA group positive expression rate significantly reduce (52.24% ± 4.04% P lt; 0.05); After following the transfusion intervention treatment group Ⅰ, group Ⅱ, group Ⅲ expression percentage were significantly increased (respectively, to 71.08% ± 3.78%, 69.87% ± 4.34%, 70.69% ± 3.74%, all P lt; 0.05) to the level of the control group of normal pregnancy Ⅰ and group Ⅱ group express the percentage of group III group showed no significant difference (P gt; 0.05). 3.2.2 immunosuppressive molecules positive expression intensity analysis and normal pregnancy control placental tissue TGF-β1 expression strength (71.60 ± 7.04) compared URSA group expression strength significantly with reducing (95.84 ± 5.65, P lt; 0.05); After following the turn lose intervention treatment Ⅰ group , Ⅱ group and Ⅲ group expression intensity was significantly higher (respectively 74.36 ± 5.74,75.20 ± 8.76,72.60 ± 7.2, all P lt; 0.05) to a normal pregnancy, the level of control group. Ⅰ and group Ⅱ, Ⅲ group expression percentage group comparisons no significant difference (P gt; 0.05). control group with normal pregnancy placental tissue IL-10 expression strength (92.50 ± 6.47), URSA group compared expression intensity significantly lower (118.50 ± 8.61, P lt; 0.05); After following the transfusion intervention treatment group Ⅰ, group Ⅱ, Ⅲ group expression intensity was significantly higher (94.00 ± 6.54,100.20 ± 6.53,100.80 ± 11.49, P lt; 0.05) to normal pregnancy, the level of the control group. Ⅰ group group Ⅱ, Ⅲ group expression between the percentage of group comparison was no significant difference (P gt; 0.05). control placental tissue COX-2 expression intensity (82.60 ± 5.37%) compared with normal pregnancy, URSA group expression intensity was significantly lower ( 96.92 ± 5.42, P lt; 0.05); After following the transfusion intervention treatment group Ⅰ, group Ⅱ, Ⅲ group expression intensity was significantly higher (respectively 84.24 ± 5.08,82.70 ± 8.1,81.40 ± 6.8, all P lt; 0.05) to the level of the control group of normal pregnancy. percentage of group Ⅰ and group Ⅱ group group III expressed no significant difference (P gt; 0.05) Conclusion: Gestational T cell immune tolerance is maintained in the course of a normal pregnancy played a key role tolerated missing or inadequate is one of the important mechanisms of pregnancy loss. TGF-β1, IL-10, COX-2 and other immunosuppressive molecule expressed at low levels in the placenta and may be associated with recurrent abortion is closely related. Guo Following the outer circumference of the transfusion paternal lymphocytes induced maternal-fetal immune tolerance, conducive to normal pregnancy. outer circumference of the paternal side, non-parent parties such as different males source of lymphocytes may play a similar induction effect. 4 different sources paternal weeks lymphatic cells through a common pathway triggered maternal Th2 shift placenta moderate local immunosuppressive effective induction of maternal-fetal immune tolerance.
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