Study Of The Expression Of Chamokine And Adhesion Molecules From Mice With Pregnancy Loss Induced By Adoptively Transferring Exosomes From T Lymphocytes

Objective: Abnormal immune tolerance of the maternal-fetal interface isimportant mechanism for unexplained recurrent spontaneous abortion.Embryos with double properties of Allograft and autograft. Fetus and theirappendages are not in utero by maternal rejection and mature, needed itsmother must develop immune tolerance. Abnormal immunological tolerancecan cause abortion. Study finds that the establishing and maintaining ofMaterno-fetal immuno-tolerance required a variety of cells and Cytokines toparticipate. Therefore, study on the the exact mechanism of maternal-fetalimmunotolerance, seeking therapy to form maternal-fetal immunotolerance,is important research to develop drug URSA, to clarify its immune mechanismand to guide clinical rational use.Lymphocyte immune therapy which can induce maternal immunetolerance to allogeneic fetal Antigen formation played a positive role inclinical prevention and treatment. But poor stability of lymphocytes, not easyto save, need preparation eachtime, coupled with the operational standards andunstable effect of clinical curative, restrict the clinical promotion. A recentstudy find that T-cell can release a film called Exosomes secretedmicro-capsule structure during its growth,which can overcome the limitationsof lymphocyte immune therapy. Experimental animal models used in thisstudy is to analyze the expression of chamokine (CXCR4/SDF-1,CXCR6)and adhesion molecules(CD29, CD109) induced by Exosomes, toexplore immunology mechanism of immunotolerance induced by Exosomes.Methods:1Preparation and elementary identification of splenic mononuclear cellsand Exosomes from unrelated individuals: BALB/c male mice were sacrificed, and the suspension of splenocytes was collected. Exosomes were isolated bysucrose gradient ultracentrifngation and ultrafiltration, and they were observedthrough TEM and SEM. The concentrations of proteins in Exosomes weremeasured by ultraviolet spectrophotometry for A280.2Establishment of animal model and experimental groups: The femaleand male mice were randomly divided into groups with mating2:1, 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 fromURSA group were randomly divided into URSA group (On the fourth dayafter pregnancy, were injected intravenously with sodium chloride), CellularTherapy group (On the fourth day after pregnancy, were injectedintravenously with splenocytes from unrelated BALB/c male mice),Non-cellular Therapy group (On the fourth day after pregnancy, were injectedintravenously with Exosomes derived from splenocytes).3Observation of embryo development: The CBA/J pregnant mice in eachgroups were sacrificed on14th days, the volumes of placenta were measured,the number of absorbed placenta and the survival placenta were counted, ratesof fetal absorption and pregnancy loss were calculated.4Analysis of the expression of chamokineand adhesion molecules onmaternal-fetal interface: The CBA/J pregnant mice in each groups weresacrificed in14th days of pregnancy, and the placenta tissue were prepared forparaffin imbedding slice. After immunohistochemical staining, the gray scale(GS) were measured under imagine analysis system to show the expression ofchamokine (CXCR4/SDF-1, CXCR6)and adhesion molecules(CD29, CD109),and there is negative relationship between value of GS and the intensity ofexpression, and the percentages of positive cells were calculated under lightmicroscope with rectangular track.5Statistical analysis: SPSS13.0sofeware was used. After analysis ofnormality and variance, the chi-square test was employed to analyze theembryo absorption, pregnancy loss and the percentage of positive cells inplacental tissue. One-way ANOVA was used to analyze the transformational T lymphocytes, the expressions of chamokine (CXCR4/SDF-1, CXCR6)andadhesion molecules(CD29, CD106) in placenta tissue.Results:1The elementary identification of Exosomes from T lymphocyte1.1The observation of appearance and ultra structureExosomes were observed through TEM and SEM, the exosomes from Tlymphocyte were spheres surrounded by the lipid bilayer with round or ovalcup-shape and complete membranes. The diameter of exosomes was about30nm~100nm. Low electron density substances were shown in the middle ofexosomes. The concentrations of protein in exosomes measured by ultravioletspectrophotometry for A280was0.343±0.029.2The effect of adoptive transfer on embryo development from mice withpreganancy lossThe rate of embryo absorption in group of normal pregnancy and URSAwere5.25%and20.78%respectively, and that in group of cellular therapy andnon-cellular therapy were7.81%and3.18%respectively. Compared withuntreated URSA group, the rate of embryo adsorption after both of cellularand non-cellular adoptive transfer decreased significantly (Both P<0.01) to thelevel of normal pregnancy (Both P>0.05). Compared with cellular therapygroup, the rate of embryo adsorption in non-cellular therapy group decreasedsignificantly (P<0.05).The rate of pregnancy loss in Control group and URSA were20%and62%respectively, and that in group of cellular therapy and non-cellulartherapy were22%and16%respectively. Compared with untreated URSAgroup, the rate of pregnancy loss after both of cellular and non-cellularadoptive transfer decreased significantly (Both P<0.01) to the level of normalpregnancy (Both P>0.05), and there was no significant difference between twogroups of treatment (P>0.05).3The expressions of chamokine and adhesion molecules on maternal-fetalinterface3.1Expressions of chamokine (CXCR4/SDF-1)and CXCR6on maternal-fetal interface3.1.1The expression of CXCR4/SDF-1Compared with the percentage of positive cells of CXCR4/SDF-1inplacenta tissue from normal pregnancy (47.4%±3.9%,43.6%±4.0%), thegroup of untreated URSA decreased significantly (16.4%±1.2%and18.0%±1.1%, P<0.01); the group of cellular therapy and non-cellular therapywere34.2%±4.5%,35.5%±4.9%and49.5%±7.1%,45.6%±3.2respectively,both of them increased significantly (Both P<0.01) to the level of normalpregnancy (Both P>0.05), and there was no significant difference between twogroups of treatment (P<0.05).Compared with the GS of CXCR4/SDF-1in placenta tissue from normalpregnancy (118.5±6.71,120.56±5.67), the group of untreated URSA decreasedsignificantly (142.7±3.23,140.76±4.54,P<0.01), the treatment group bypatriarchy immunologic intervention increased significantly to the level ofnormal pregnancy (129.6±4.64,127.48±5.14and119.14±9.47,111.77±5.79bothP<0.05), and there was no significant difference among three groups oftreatment (P<0.05).3.1.2The expression of CXCR6Compared with the percentage of positive cells of CXCR6in placentatissue from normal pregnancy (83.60%±3.85%), the group of untreated URSAdecreased significantly (50.40%±3.51%, P<0.01); the group of cellulartherapy and non-cellular therapy were82.00%±4.00%and82.40%±2.61%respectively, both of them increased significantly (Both P<0.01) to the level ofnormal pregnancy (Both P>0.05), and there was no significant differencebetween two groups of treatment (P>0.05).Compared with the GS of CXCR6in placenta tissue from normalpregnancy (82.68±3.91), the group of untreated URSA decreased significantly(112.68±4.20, P <0.05); the treatment group by patriarchy immunologicintervention increased significantly to the level of normal pregnancy(85.36±2.88,84.44±3.74, both P <0.05), and there was no significantdifference among three groups of treatment (P>0.05). 3.2Expressions of adhesion molecules(CD29, CD109) on maternal-fetalinterface3.2.1The expression of CD29Compared with the percentage of positive cells of CD29in placentatissue from normal pregnancy (43.20%±2.86%), the group of untreated URSAincreased significantly (83.0%±4.58%%, P<0.01); the group of cellulartherapy and non-cellular therapy were42.20%±1.92%and42.00%±2.73%respectively, both of them decreased significantly (Both P<0.01) to the levelof normal pregnancy (Both P>0.05), and there was no significant differencebetween two groups of treatment (P>0.05).Compared with the GS of CD29in placenta tissue from normalpregnancy (116.54±3.33), the group of untreated URSA decreasedsignificantly (82.9±3.97, P <0.05); the treatment group by patriarchyimmunologic intervention increased significantly to the level of normalpregnancy (120.48±2.77,121.6±2.61, both P <0.05), and there was nosignificant difference among three groups of treatment (P>0.05).3.2.2The expression of CD106Compared with the percentage of positive cells of CD106in placentatissue from normal pregnancy (30.80%±2.33%), the group of untreated URSAincreased significantly (73.30%±1.75%, P<0.01); the group of cellular therapyand non-cellular therapy were30.30%±1.78%and30.10%±1.67%respectively, both of them decreased significantly (Both P<0.01) to the levelof normal pregnancy (Both P>0.05), and there was no significant differencebetween two groups of treatment (P>0.05).Compared with the GS of CD106in placenta tissue from normalpregnancy (128.48±3.38), the group of untreated URSA decreasedsignificantly (94±4.78, P<0.05); the treatment group by patriarchyimmunologic intervention increased significantly to the level of normalpregnancy (129.58±3.10,130.7±4.66, both P<0.05), and there was nosignificant difference among three groups of treatment (P>0.05). Conclusions:1Adoptive transfer of periphery lymphocytes or their non-cellular comp-onents can induce maternal-fetal immunotolerance, which should be helpfulfor normal pregnancy.And Exosomes can induce stronger maternal-fetalimmunotolerance than T lymphocytes.2Peripheral lymphocytes from paternal individuals or non-cellular comp-onents should initiate the change of the expressions of chamokine andadhesion molecules in placenta, in order to induce efficient maternal-fetalimmunotolerance.