Bone Marrow Mesenchymal Stem Cells Prolong The Transplant Survival By Treg Cells And IL-10 In Heterotopic Heart Transplant Rat

BACKGROUND AND OBJECTIVE:Heart transplantation is an effective method for treatment of end-stage heart failure,but immune rejection following heart transplantation seriously impact therapeutic effectiveness.Therefore,seeking novel and effective methods for immunosuppression is of great importance to clinical practice.To investigate the effect of bone marrow mesenchymal stem cells?BMSCs?on the survival time of heart and the regulatory effect on the immune function after heterotopic heart transplantation in rats.METHODS:1.Isolation and culture of the rat BMSCs:Ten wistar rats were euthanized with ether?5ml?and sterilized by ethanol solution before dissection of femurs and tibias.Bone marrow was extruded by flushing with DMEM media containing 15%fetal bovine serum repeatedly.Then flushing liquid was collected to obtain single cell suspension by suctioning and blowing with pipettes,and the cell suspension was centrifuged with 10-ml centrifuge tube.Supernatant was aspirated,and the pellet was suspended in the DMEM media with 15%fetal bovine serum,and then Ficoll lymphocytes separating solution with a density of 1.085 g/ml added.After centrifuge stratification,the milky white mononuclear cell layer was removed and suspended in the DMEM media with 15%fetal bovine serum,incubated in 25 cm²flasks,at 37°C in 5%humidified CO₂ incubator.After 48h incubation,the primary culture was washed with PBS,and non-adherent cells and medium were removed and replaced with fresh medium.The medium was changed twice per week until the adherent cells achieved 80%confluence.The MSCs were detached from the culture flask with 0.2%Trypsin-EDTA solution.Following enumeration,aliquots of the cell suspension were frozen and stored while the remaining cells were maintained for passage 10.2.Identification of MSCs:The MSCs were identified according to their self-renewal ability,morphological characteristics and surface markers.Proliferation and morphological characteristics of MSCs were observed by using inverted microscope.Single-cell suspension of MSCs were prepared and characterized for expression of cell surface antigens with the following anti-rat mAbs:CD29?CD90?CD34,and CD45.The analyses were performed on LSR II flow cytometer.3.MSCs transplantation:Twenty Wistar Rats were prepared and randomly divided into MSCs transplantation group and control group with 10 rat of each group.Rats in the MSCs transplantation group were given MSCs suspension?0.5mL,2×10⁵ cells?via the caudal vein every other day one week,before heart transplantation,on the day of heart transplantation and 3 days after transplantation.while rats in the control group were given 0.5mL sodium chloride.4.Preparation of rat heterotopic heart transplantation model:Rats cervical heart transplantation models,enrolling a total of 20 Lewis rats as donors and 20 Wistar rats as recipients,were established by using a cuff technique.Firstly,the heart of Lewis rats was taken out,and the roots of the ascending aorta and pulmonary artery were separated from the surrounding tissue.After washing the lumen with 50U/ml of?0-4centigrade?heparin saline,the heart was wrapped by the brain cotton to prepare for transplantation.Secondly,the external jugular vein and common carotid artery of Wistar rats were separated and cut at the distal end,which were Ligated and fixed with an external sheath tube of a vascular puncture.After anastomosing and fixing the heart aorta of donor to the right common carotid artery of recipient,and the pulmonary artery of donor to the right external jugular vein of the recipient,the transplanted heart was placed in the subcutaneous tissue of right neck.The heart rate and mental state of rats was monitored every day.If the transplanted heart stopped beating within 48 hours of transplantation,the operation is unsuccessful.Otherwise,it can be regarded as the success of the model if the transplanted heart beat above 48h.5.Detection of serum interleukin 2?IL-2?cytokine IL-2,interleukin 10?IL-10?,and T cell subsets:The expressing level of IL-2 and IL-10 in serum of MSCs transplantation group and control group was detected by ELISA,and the proportion of T cell subsets in blood of rats was detected by flow cytometry seven days after heart transplantation.6.Observation of the survival time of transplanted heart and pathological diagnosis of transplanted heart tissue:Five rats from each group were sacrificed,specimens were obtained for pathological observation seven days after heart transplantation,and the remaining 5 rats in each group were maintained to observe the survival time of transplanted heart by recording the mental state of rats and the amplitude,rhythm and rate of heartbeat.RESULTS:1.Detection of MSCs morphology,proliferation and cell surface markers of MSCs:After 1-3 days incubation,the bone marrow mononuclear cells attached and proliferated on plastic culture plates and subsequently presented polygonal shapes.At passage 3,the cell had typical flat,adherent,long cord or polygonal shape,and the cell proliferation was accelerated.With the increase of passage times,the cell morphology was homogeneous,and passages 4 to 6 were collected to detect the cell surface markers.The results showed that the positive rates of CD29 and CD90 were98.42%and 97.73%,respectively,while the positive rates of CD34 and CD45 were1.35%and 2.52%,which were in line with the characteristics of bone marrow MSCs.2.The effect of MSCs on the survival time of the transplanted heart:The survival time of transplanted heart in two groups was statistically analyzed.The results showed that the survival time of transplanted heart in the MSCs transplantation group?14.25±2.15 days?was remarkably longer than that in the control group?7.15±1.35days,P<0.05?.3.The effect of MSCs on IL-2,IL-10 and T cell subsets in heart transplant rats:Seven days after heart transplantation,The expressing level of IL-2 and IL-10 in serum of two groups was detected by ELISA.The result showed that no statistical difference in the expressing level of IL-2 and IL-10 in MSCs transplantation group?248.15±25.23 pg/ml,P>0 05?was found as compared to control group?230.25±20.35 pg/ml?.However,the expressing level of IL-10 in the MSCs transplantation group?160.23±18.75 pg/ml?was remarkably higher than that in control group?80.25±12.26 pg/ml,P<0.05?.The proportion of T cell subsets in blood of rats in two groups was detected by flow cytometry seven days after heart transplantation.The proportion of CD4⁺,CD8⁺,CD4⁺/CD8⁺,CD4⁺CD25^high,and CD4⁺CD25^highigh Foxp³⁺T cell in the MSCs transplantation group was significantly higher than that in the control group?P<0.05?.4.Pathological analysis of transplanted heart tissue:Seven days after heart transplantation,the histological changes showed that the hyperacute rejection occurs in control group:the transplanted heart tissue with a large number of lymphocytes and mononuclear cells infiltration,thrombus formation in the capillary,obvious edema and bleeding in cardiac interstitial,different degrees of Basophilic?mucoid?degeneration of the myocardium and degeneration and necrosis of cardiac myocytes.However,Inflammatory response of the transplanted heart tissue in MSCs transplantation group is slighter,a small number of lymphocytes infiltration,focal damage of the myocardium,mild hemorrhage and edema in the myocardial interstitium,as compared to control group.CONCLUSION:MSCs transplantation could prolong the survival time of of the transplanted heart,effectively regulate the immune function of rats,and reduce the immune rejection after heterotopic heart transplantation.