| Objective: Unexplained recurrent spontaneous abortions(URSA) seriously threatens the physical and psychological health of the maternal, but the means of preventen and treatment need further perfection. Currently, adoptive immunity of parternal lymphocytes was applied to treat URSA through inducing effective immunotolerance between the pregnant women and the embryo. However, the shortcomings of this method limit its clinical application and dissemination, such as the lower standardization of preparation, the instability of therapeutic effect and etc. T lymphocytes can secrete and release a kind of micro-capsule structure called Exosomes during its growth and proliferation. It was indicated in previous animal experiments that it can produce better effect than lymphocyte immunotherapy in improving pregnancy outcome of mice with pregnancy loss, and overcome the limitations of lymphocyte immunotherapy.In this study, based on experimental animal models, effects of Exosomes from T lymphocytes on embryo development, immunofunction of peripheral NK cells from mice with pregnancy loss were analyzed. It is aimed at exploring the immunomechanism of non-cellular component on inducing maternal-fetal immunotolerance and establishing a new idea for immunotherapy of recurrent pregnancy loss.Methods:1 Preparation of splenic mononuclear cells and Exosomes from the unrelated: BALB/c male mice were executed, the spleen were taken out by aseptic dissection, and the suspension of splenocytes was collected. After co-innoculated with Con A for 72 h, Exosomes were extracted by sucrose density gradient ultracentrifugation and ultrafiltration.2 Establishment of animal model and experimental groups: The female and male mice were randomly divided into groups with mating 2:1, CBA/J(♀) ×BALB/c(♂) as control group of normal pregnancy(Control) and CBA/J(♀) ×DBA/2(♂) as URSA experimental animal model. The CBA/J mice from URSA group were randomly divided into URSA group(On the fourth day after pregnancy, were injected intravenously with sodium chloride), Cellular Therapy group(On the fourth day after pregnancy, were injected intravenously with splenocytes from unrelated BALB/c male mice), Non-cellular Therapy group(On the fourth day after pregnancy, were injected intravenously with Exosomes derived from splenocytes).3 Observation of embryo development: The CBA/J pregnant mice in each group were executed respectively on the fourteenth day of pregnancy, the volumes of placenta were measured, the numbers of absorbed embryo and the survival embryo were counted respectively, the rates of fetal absorption and pregnancy loss were calculated.4 Analysis of the expressions of peripheral NK cell’s immuno-phenotype, surface receptors and effector molecules: The CBA/J pregnant mices in each group were executed respectively in the fourteenth day of pregnancy, the spleen were taken out by aseptic dissection, and the suspension of splenocytes was collected, the expression percentages of characteristic differentiation antigens(CD3-CD56+, CDl6+CD56+, CDl6-CD56+), inhibitory and activative surface receptors(CD56+NKG2A+, CD56+NKG2D+), signal transduction molecule(CD3-CD247+) and killer effector molecule(CD56+Perforin+) of peripheral NK cell were measured respectively by fluorescence-labelled flow cytometry.5 Statistical analysis: SPSS13.0 software was used. After analysis of normality and equality of variances, the chi-square test was applied to analyze the rates of embryo absorption, pregnancy loss in placental tissues. One-way ANOVA of F-test was used to analyze the expressions of characteristic differentiation antigens, surface receptors, signal transduction and effector molecules of peripheral NK cells, S-N-K test was applied to make further comparasion among the averages of different samples. The significance test level α is 0.05.Results: 1 The effect on embryo development from mice with preganancy lossThe rate of embryo absorption in group of normal pregnancy and URSA were 5.78% and 21.17% respectively, and that in group of cellular therapy and non-cellular therapy were 6.93% and 3.25% respectively. Compared with untreated URSA group, the rate of embryo adsorption after both of cellular and non-cellular adoptive transferation decreased significantly(Both P<0.005) to the level of normal pregnancy(Both P>0.05). Compared with cellular therapy group, the rate of embryo adsorption in non-cellular therapy group decreased significantly(P<0.05).The rate of pregnancy loss in Control group and URSA were 20% and 64% respectively, and that in group of cellular therapy and non-cellular therapy were 24% and 16% respectively. Compared with untreated URSA group, the rate of pregnancy loss after both of cellular and non-cellular adoptive transferation decreased significantly(Both P<0.01) to the level of normal pregnancy(Both P>0.05), and there was no significant difference between the two groups of treatment(P>0.05). 2 The effect on the immunofunction of peripheral NK cell from mice with preganancy loss 2.1 The effect on NK cell’s characteristic differentiation antigenThe percentage of CD3-CD56+NK cells in Control group was(61.92%±4.83%), the group of untreated URSA was(57.26%±5.44%); the group of cellular therapy and non-cellular therapy were(66.86%±6.76%, 60.84%±6.88%) respectively, and there was no significant difference between every two groups(P>0.05).Compared with the percentage of CD16-CD56+NK cells in Control group(56.33%±4.10%), the group of untreated URSA decreased significantly(43.98%±4.63%, P<0.01); the group of cellular therapy and non-cellular therapy were(60.14%±4.98%, 54.77%±6.74%) respectively, both of them increased significantly(Both P<0.01) to the level of normal pregnancy(Both P>0.05), and there was no significant difference between the two groups of treatment(P>0.05).Compared with the percentage of CD16+CD56+NK cells in Control group(6.39%±1.22%), the group of untreated URSA increased significantly(13.28%±1.40%, P<0.01); the group of cellular therapy and non-cellular therapy were(4.73%±1.32%, 6.07%±0.84%) respectively, both of them decreased significantly(Both P<0.01) to the level of normal pregnancy(Both P>0.05), and there was no significant difference between the two groups of treatment(P>0.05). 2.2 The effect on inhibitory and activative surface receptorsCompared with the percentages of CD56+NKG2A+ NK cell in Control group(1.42%±0.44%), the group of untreated URSA decreased significantly(0.35%±0.09%, P<0.01); the group of cellular therapy and non-cellular therapy were(1.36%±0.30% and 4.91%±0.99%) respectively, both of them increased significantly(Both P<0.01), and there was significant difference between the two groups of treatment(P<0.05).The percentage of CD56+NKG2D+NK cells in Control group(1.29%± 0.38%), the group of untreated URSA was(1.44%±0.22%); the group of cellular therapy and non-cellular therapy were(1.16%±0.42%, 1.37%±0.26%) respectively, and there was no significant difference between every two groups(P>0.05). 2.3 The effect on signal transduction moleculeCompared with the percentage of CD3-CD247 +NK cell in Control group(3.93%±1.32%), the group of untreated URSA decreased significantly(0.14%±0.06%, P<0.01); the group of cellular therapy(3.55%±0.87%) increased significantly(P<0.01) to the level of normal pregnancy(P>0.05), and there was no significant difference between the non-cellular therapy(0.42%±0.18%) and URSA group(P>0.05). 2.4 The effect on killer effector moleculeCompared with the percentage of CD56+Perforin+NK cells in Control group(7.99%±2.51%), the group of untreated URSA increased significantly(11.44%±2.78%, P<0.01); the group of cellular therapy and non-cellular therapy were(7.51%±1.27%, 8.12%±1.32%) respectively, both of them decreased significantly(Both P<0.01) to the level of normal pregnancy(Both P>0.05), and there was no significant difference between the two groups of treatment(P>0.05).Conclusions:1 Adoptive transfer of peripheral lymphocytes or Exosomes from T lymphocytes from unrelated male individuals can effectively induce maternal-fetal immunotolerance, which should be helpful for normal pregnancy. And Exosomes exerted more stronger effect than periphery lymphocytes in improving pregnancy outcome of mice with pregnancy loss.2 Adoptive transfer of peripheral lymphocytes or Exosomes from T lymphocytes from unrelated male individuals should initiate the change of the expressions of NK cells’ characteristic differentiation antigens, surface receptors and effector molecule, to induce efficient maternal-fetal immunotolerance.3 The level of peripheral lymphocytes and Exosomes from T lymphocytes regulate immunofunction of peripheral NK cells from mice with pregnancy loss to induce maternal-fetal immunotolerance was different. The expression of activative signal transduction molecule increased significantly after cellular adoptive transferation, but it remained unchanged after non-cellular adoptive transferation. The expression of inhibitory surface receptor after both of cellular and non-cellular adoptive transferation increased, but it increased significantly after non-cellular adoptive transferation. |
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