Effects Of Bone Marrow Mesenchymal Stem Cells Derived From Recipients On The Rejection In Rat Cardiac Transplantation Models

Partâ… Establishment of a Cervical Heterotopic Cardiac Transplantation Model of RatsObjectives:To establish a modified model of rat cervical heterotopic cardiac transplantation for high achievement ratio.Methods:A right cervical heterotopic cardiac transplantation model of rats was established.The allograft blood circulation was established with cuff technique using home-made cannulas.Through the cannulas,the donor innominate artery was anastomosed to the recipient's right common carotid artery and donor left pulmonary artery to the recipient's external jugular vein.The successful standard of transplantation was the transplanted heart could beat rhythmically over 24 hours.Results:Successful rate of heterotopic cardic transplantation is 88%(30/34).Conclusions:A modified model of rat cervical heterotopic cardiac transplantation was established with cuff technique.Partâ…¡Isolation,Culturing and Identification of Rat MSC From Bone Marrow in vitroObjectives:To establish the methods for the isolation,culturing and identification of rat MSC from bone marrow in vitro. Methods:One month old male Wistar rats were killed to get the tibias and femurs.After the long bones were dissected from rats,the marrows were flushed out with DMEM medium(supplemented with 15%fetal bovine serum) under aseptic condition and purified by Percoll gradient centrifugation to get the mononuclear cells.After the mononuclear cells were cultured,the adherent cells were chosen for serial subcultivation.The third generation cells were harvested and the cell phenotypes were identified by flow cytometry.Results:The mesenchymal stem cells were adhered to plastic surface within 24h.The cell growth was obvious on the 3～4 days.Nonadherent cells were removed by changing the culture medium.After passage,the cells gown rapidly to fill the floor of cell culture flasks and achieved confluence within 6～8 days. Purified MSC fibroblast-like cells could be harvested after 3 generations.There was no obvious influence to cell growth when reserved in frozen condition.The cells could passage for at least 10 generations.The cell phenotypes identified by flow cytometry showed CD29(+),CD34(-),CD45(-),CD11B(-),CD90 (+),CD106(+).Conclusions:The methods of obtaining rat MSC with density gradient centrifugation and adherence cultivation was convenient,and could provide immortalized rat MSC lines.Partâ…¢Effects of Bone Marrow Mesenchymal Stem Cells Derived from Recipients on the Acute Rejection in Rat Cardiac Transplantation ModelsObjectives:To investigate the effects of MSC derived from recipients' bone marrow on the acute rejection of rat cardiac transplantation and study the possible underlying mechanisms.Methods:Twenty Wistar rats beating transplanted heart from Lewis rats were divided randomly into 2 groups(10 rats each group ):(1)Control group (group A3):3mL 0.9%NaCl solution was injected through tail vein at 24h after heart transplantation.(2) Treatment group(group B₃):2×10⁶ MSC in 3mL 0.9%NaCl solution was injected through tail vein at 24h after heart transplantation. One week after transplantation,4 rats randomly from each group were anaesthetized to get blood from inferior canal vein and allografts.Grafts pathology was studied.Serum levels of IL-2 and IL-10 was detected by method of ELISA.Flow cytometry was used to detect the ratio of CD4⁺ T cells,CD8⁺ T cells, CD4⁺CD25^high T cells and CD4⁺CD25^highfoxp3⁺ T cells in lymphocytes in the blood and grafts.The rest 6 rats in each group were observed survival time of grafts.Two other cardiac transplantation models were established.At 24h after transplantation,2×10⁶ MSC^GFP were injected throug tail vein,respectively.The two grafts were harvested on 7^th day after transplantation to get frozen sections for observing the fluorescence cells in grafts.Results:(1) The survival time of allografts:group A₃ was 7.2±1.3d,group B₃ was 14.8±2.9d.P＜0.01.(2)Concentrations of serum IL-2:group A₃ was 233.99±30.19pg/mL,group B₃ was 249.11±20.34 pg/mL,P＞0.1;IL-10:group A₃ was 81.44±11.01 pg/mL,group B₃ was 158.39±18.70 pg/mL,P＜0.01. (3)Pathology grade:group A₃:1 case was Grade 3B,3 cases were Grade 4;group B₃:1 case was Grade 2,2 cases were Grade 3A,1 case was Grade 3B.(4) Ratios of CD4⁺/CD8⁺,CD4⁺CD25^high T cells/total lymphocytes and CD4⁺CD25^highFoxp3⁺ T cells/total lymphocytes in blood:group B3 was higher than group A₃,P＜0.01.(5) Ratios of CD4⁺/CD8⁺ in allografts were similar in two groups,P＞0.1;CD4⁺CD25_highT cells/total lymphocytes and CD4⁺CD25^highFoxp3⁺ T cells/total lymphocytes in allografts:group B₃ was higher than group A₃, P＜0.01.(6) Fluorescence cells could be observed to concentrate to the surface of allografts,some cells had infiltrated into cardiac muscle and their shapes and distributions were just like cardiac muscle.Conclusions:Intravenous infusion with MSC from recipients could prolong the survival time of transplanted heart and inhibit acute rejection. Partâ…£Effects of Bone Marrow Mesenchymal Stem Cells Derived from Recipients on the Chronic Rejection in Rat Cardiac Transplantation ModelsObjectives:To observe the effects of mesenchymal stem cells derived from recipients' bone marrow on the chronic rejection of rat cardiac transplantation.Methods:Twenty rats bearing transplanted hearts were divided randomly into 4 groups(5 rats each group):(1)Homologous transplantation group(group A₄):Wistar-Wistar,CsA 2mg/kg·d was injected into abdominal cavity for 14 days after transplantation;(2)Chronic rejection group(group B₄):Lewis-Wistar,CsA 2mg/kg·d was injected into abdominal cavity for 14 days after transplantation; (3)MSC treatment group(group C₄):Lewis-Wistar,CsA 2mg/kg·d was injected into abdominal cavity for 14 days after transplantation.2×10⁶ MSC was injected through tail vein at the 15^th day and 30^th day after transplantation,respectively;(4) CsA treatment group(group D₄):Lewis-Wistar,CsA 2mg/kg·d was injected into abdominal cavity from after transplantation to the end of investigation.The investigation time was 120 days after transplantation.On 120^th day after transplantation,the allografts were harvested for pathological examination to analyze the depth of artery wall and the infiltration of collagenous fiber in cardiac muscle by MASSON staining.Results:All recipients and transplanted hearts survived well to the investigating ends.The mean ratio of artery wall depth to artery diameter(%): group A₄ was 19.68±2.06,group B₄ was 29.14±1.98,group C₄ was 29.88±2.28 and group D₄ was 39.82±1.84.Group B₄ and group C₄ is similar,P＞0.05.The infiltration area of collagenous fiber to cardiac muscle(%):group A₄ was 8.76±1.56,group B₄ was 17.98±2.40,group C₄ was 19.50±2.24 and group D₄ was 17.64±2.47.Group A₄ was less infiltrated with collagenous fiber than the other 3 groups,P＜0.01.Group B₄,group C₄ and group D₄ were similar in the infiltration of collagenous fiber,P＞0.05.Conclusions:Intravenous infusion with MSC from recipients did not reduce the level of chronic rejection of rat cardiac transplantation.