Objective To study the effect of allogeneic canine cord blood mesenchymal stem cells（cbMSC）transplantation on the distribution of CD4+T and CD8+T in infracted area of hearts. Methods Mononuclear cells of cord blood were isolated by density gradient centrifugation and amplified by adherent culture. 36 adult male dogs were divided into experimental group and control group. Animal models of acute myocardial infarction were established by ligating anterior descending coronary artery. The fourth generations of mesenchymal stem cells (MSC) were transplanted into infarcted area of hearts by left anterior descending coronary artery after 72h induced by 5-aza and transfected by LacZ. The survival of transplanted cells in hearts can be confirmed by βgal expression. CD4+T and CD8+T cells distributed in infarcted area were detected by immunohistochemical staining method. The ImagePlus 5.1 software was used to analyze the images. Results Cells transplanted into infarcted area could survive for a long time. 2, 4, 8 weeks after transplantation, the IOD of CD4+T in experimental group were 44.35±7.03, 19.29±4.11 and 20.27±3.51 respectively, and the CD4+T/CD8+T ratios were 0.63±0.12, 0.51±0.15 and 0.66±0.08. In control group, the IOD of CD4+T at 2, 4, 8 weeks after transplantation were 65.78±10.27, 28.02±2.59, 29.79±6.83, and the CD4+T/CD8+T ratios were 1.28±0.20, 1.34±0.09 and 1.50±0.16. The IOD of CD4+T and CD4+T/CD8+T ratio in experimental group were significantly lower than that in control group. In experimental group the IOD of CD8+T at 2, 4, 8 weeks after transplantation were 69.88±7.84 , 37.80±8.83 and 30.81±7.42, higher than that in control group which were 51.28±10.01, 20.87±4.50 and 19.91±2.87. Conclusion The preliminary results indicated that allogeneic cbMSC transplanted in infarcted area can escape from immune rejection, its mechanism may be associated withdecreasing the amount of CD4+T cells infiltrated in periphery of infarcted area and maintaining CD4+T/CD8+T ratios at a lower level.
Objective To investigate the influence of infarcted myocardial construction by umbilical cord blood mesenehymal stem cells（MSC） with induced to myo-derived stem cells and implanted into infarcted myocardium. Methods Thirty-six adult mongrel canines were randomly divided into MSC transplant group and control group （18 each group）. Transplant group： the umbilical cord blood MSC differented to myo-derived stem cells induced by 5-azacytidine（5-aza） were implanted into the acute myocardial infarct site via the ligated left anterior descending （LAD） artery. Control group： administer the same volume of IMDM culture medium containing 0. 02% 4,6- diamidino-2-phenylindone （DAPI）. At 2, 4, 8 weeks after implantation, the change of basic myocardial construction and the distribution of desmin were observed by using Nagar-Olsen staining and immunohistology respectively. Results With less fusing, the arrangement of gelatine fibers and elastic fibers were in order in transplant group,and they were partly fused in control group by Nagar-Olsen staining. The expression of desmin of infarcted myocardial cell in transplant group was much higher than those in control group （P〈0. 01）. No significant difference was detected in the expression of desmin in normal site of both groups （P〉0. 05）. Conclusion There is an protective effect on the basic myocardial construction in infarcted myocardium after the umbilical cord blood MSC was differented to myo-derived stem cell by induced with 5-aza in vitro and implanted into the acute myocardial infarction.
Abstract： Objective To investigate and improve the method of isolation, induction and culture, amplification in vitro of human endothelial progenitor cells (EPCs) in human bone marrow, thus establish a foundation for EPCs to participate in basic research and clinical application. Methods WHuman bone marrow mononuclear cells (hBMMNCs) were isolated by density gradient centrifugation and cultured in the 6plateds coating human fibronections(HFN group), coating gelatinum (coating gelatinum group) and coating nothing (coating nothing group) respectively. After culturing for 4-7d endothelial cell basal medium-2(EBM-2) cell colonyforming units (CFUs) appeared, then select EPCsliked CFUs for cultivation which was named the pick method, CD34+ KDR+ and CD133+ KDR+ double positive cells were detected in flow cytometry, and CD133, CD34, CD31, vWF and KDR expression were detected with cell immunochemical test. Results hBMMNCs were isolated from human bone marrow more effectively with OptiprepTM cell separating medium, and induced and obtained more EPCs, and cultured and amplificated in vitro. Flow cytometer showed CD133+ KDR+ double positive cells reaching up to 70.4%±5.4%, CD34+ KDR+ double positive cells reaching up to 69.1%±8.7%. EPCs grew vigorously in coating HFN group and coating gelatinum group, both HFN and gelatinum promote EPCs adherence and growth, but there were no statistically difference in two groups (Pgt;0.05).Surface mark of adherent cells cultured 7d such as CD133, CD31, vWF and KDR showed positive, and cells cultured 14d such as CD34 showed positive. Conclusion The method of picking CFUs can obtain more EPCs from human bone marrow with success and can amplificate EPCs in vitro, thus introducing another simple and effective method to purify EPCs, further widening range and increasing method to purify EPCs.
Abstract: Objective To study the integration of transplanted cells and host cells by detection of the cellcell junction after transplantation of the myocardiumlike cell derived from the canine umbilical cord blood. Methods The mesenchymal stem cell(MSCs) was transfected by Laz-Z after harvest, culture, induced by 5-azacytidine(5-aza). Thirty-six adult hybrid dogs were randomly divided into cell transplantation group and control group. The canine of myocardium infarction was established. 107 MSCs were transplanted into dogs with acute myocardium infarction by coronary artery infusion and local injection in cell transplantation group and physiologic saline was used in the control group. The specimens were harvested and detected by immunofluorescence for 2, 4 and 8 weeks respectively. Results The umbilical cord blood MSCs were fusiform or spindleshaped. They presented clonal and knittinglike growth.The MSCs could differentiate into myocardium-like cell by the induction of 5-aza and express α-actin, desmin, connexin43.The transplanted cells could survive more than 8 weeks after transplantation. Cadherin and connexin 43 were found in the position of cellcell junction of transplanted cells group and between transplanted cells and host cells. Cadherin and connexin 43 were found in the hose cells of the control group. Conclusion The umbilical cord blood MSCs is able to differentiate into myocardiumlike cell in vitro and form cellcell junction in vivo to communicate with surrounding cells.