Inductive Effect Of Qidantongmai Tablet On Differentiation Of Rat Marrow Mesenchymal Stem Cells Into Endothelial Cells

Research background: For the past few years, Marrow Mesenchymal Stem Cells transplantation for the treatment of ischemic heart disease has been the hot spot of cardial vascular research field. However, survival of transplanted cells may be difficult in ischemic zones. Previous research has suggested that a key point in the treatment of myocardial ischemia was the re-establishment of circulation in the ischemic zone. Consequently, the use of MSC differentiation into endothelial cells has become a hot topic of research in the treatment of myocardial ischemia.Recent research has suggested that herbs used in traditional Chinese medicine (TCM) may have an induction effect on MSC differentiation. principle of yiqihuoxue is a major therapeutic principle of TCM in the treatment of myocardial ischemia , the use of Qidantongmai tablets in that treatment was developed based on the principle of yiqihuoxue. Research in our laboratory previously demonstrated that Qidantongmai tablets increased the expression of VEGF and bFGF in ventricles of myocardial ischemia rats and improved the microvessel density (MVD)in the ischemia zones . Both VEGF and bFGF have been demonstrated to induce MSC differentiation into endothelial cells. In this current study, Rat marrow mesenchymal stem cells were isolated . Eeffects of Qidantongmai containing serum and Qidantongmai tablet on MSCs and transplanted MSCs were observed respectively in vitro and in vivo to investigate the inductive effects of Qidantongmai tablets on the differentiation of MSCs into endothelial cells as well as the effects on microvessel density (MVD), myocardial cell apoptosis and heart function in acute myocardial ischemia rats. the vasculogenesis effect of yiqihuoxue principle was investigated from a new view of MSC differentiation to provide some new theoretical and experimental basisi for treatment of myocardial ischemia using yiqihuoxue principle.It might be provide an efficacious adjunct to MSC implantation therapy in the treatment of acute myocardial infarction.Part 1 Isolation and Culture of Rat MSCs in vitro Aim Using adherence separation and density gradient centrifugation to identify the effect of different culture condition on acquired rat bone marrow mesenchymal stem cells, trying to find suitable culture condition for acquiring high quality and high activity bone marrow mesenchymal stem cells.Methods 12 cases from 18 SPF male SD rats were used in adherence Separation, which divided into 4 groups based on different serum and nutritive medium : import serum + DMEM group, import serum + F12-DMEM group, domestic serum + DMEM group, domestic serum + F12-DMEM group, 3 rats each group . the other 6 rats were used in density gradient centrifugation which divided into Ficoll and Percoll separating medium group based on different separating medium , 3 rats each group. Growth information and morphologic feature were observed in different culture condition of adherence Separation and density gradient centrifugation groups under inverted microscope. Congo blue exclusion test was carried out to count the cytoactive.Drawing growth curves.Results①O bservation results of morphologic feature : Adherence separation : Adhering spindle- shaped MSCs with higher cytoactive were both obtained in adherence separation group by using the import FBS. The MSCs of primary culture in F12-DMEM had more rapid proliferation, earlier colony confluence and shorter time for passage than in DMEM. Adhering spindle- shaped MSCs was also obtained in adherence Separation group by using domestic FBS cultured in F12-DMEM, but many myeloid cell witch similar to round shape and few spindle- shaped MSCs were obtained in DMEM group. Density gradient centrifugation: Adhering spindle- shaped MSCs were both obtained in difference separating medium Ficoll and Percoll. Both cells have good cytoactive. But the time of colony confluence and passage was longer than adherence Separation group.②Detection results of cytoactive: : Adhence separation :The ratio of living cells was 98.3%,98.7%,97.1%,97.7% respectively in import serum + DMEM group, import serum + F12-DMEM group, domestic serum + DMEM group, domestic serum + F12-DMEM group. There was no obviously difference of cytoactive in each group (p>0.05). Density gradient centrifugation: The ratio of living cells in Ficoll separating medium group was similar to the ratio of living cells in Percoll separating medium group. (96.9%,97.1%,p>0.05)③Detection results of growth curves of cells from the third passage: The growth curves of cells from the third passage were similar in each groups under different culture condition. The cell population was decreased slightly after 1 day incubatio and increased quickly after 4 days incubation. It reached plateau phase on the day 8, the cell multiplication was slow down.Conclusion MSCs can obtained both with adherence separation and density gradient centrifugation as long as choosing the suitable culturing condition. Import FBS and F12-DMEM nutritive medium are better choice. PartⅡInductive Effect of Qidantongmai Containing Serum on Differentiation of Rat Marrow Mesenchymal Stem Cells into Endothelial Cells in VitroAim To observe the inductive effects of Qidantongmai containing serum on differentiation of rat marrow mesenchymal stem cells into endothelial cells in vitro.Methods Qidantongmai containing serum and control serum were obtained by gastric perfusion. Marrow mesenchymal stem cells were isolated and cultured by density gradient method , the third generation of continuous MSCs were preconditioning with VEGF(10μg/L) for 24h, then induced by 15% Qidantongmai containing serum and 15% control serum respectively for 7 days. 1,3,5,7days after induction, The morphology of cells were observed under invert microscope. cells induced by Qidantongmai containing serum for 1,3,5,7days ,cells induced by control serum for 7days and umbilican vein endothelial cells were collected and the protein of CD31 and Tie2 were detected by western blot. Microstructure of the 7day cells in were observed under electron microscope, The expressions of endothelial-specific marker CD31 and factorⅧwere identified with immunofluorescence.Results①morphologic feature: the morphologic feature were not changed significantly in the 1st day in both group. In the 3rd day, the cell body of Qidantongmai containing serum group condensed slightly, spacing was extended between cells and reinforced stereological in 5th day. In the 7th day, the morphology of induced MSCs was obviously changed like cobble-stone shape in Qidantongmai containing serum group, but the morphologic feature were not changed in control group.②expression of CD31 and Tie2 protein: The protein of CD31 and Tie2 were expressed in 1d, 3d, 5d and 7d of Qidantongmai group , so as the umbilican vein endothelial Cells, but it was not expressed in control group.③Microstructure of the 7th day cells: Characteristic Weible-Palade bodies expressed under transmission electronic microscope in Qidantongmai containing serum group There were no Weible-Palade bodies expressed in control group.④expression of CD31 and factorⅧ: CD31 and factorⅧpositive cells can be find under confocal microscope in Qidantongmai containing serum group, and were not find in control group. Conclusion Qidantongmai containing serum have the inductive effects on differentiation of rat marrow mesenchymal stem cells into endothelial cells in vitro.PartⅢSynergistic Effect of Qidantongmai Tablet on Inducing transplanted MSCs Differentiate into Endothelial CellsAim To investigate the synergistic effect of Qidantongmai tablet on inducing transplanted MSCs differentiate into endothelial cells and their effect on pathological changes,microvessel density (MVD), myocardial cells apoptosis and heart function in acute myocardial ischemia rats.Methods Rat marrow mesenchymal stem cells were isolated by density gradient method, and then cultured and amplified to the third passage labeled with BrdU. 20 SD rats were divided into two groups randomly: MSCs implantation with (MTQ) and without (MT) administration of Qidantongmai tablets. An isochoric suspension of Qidantongmai tablets (2 g/kg) or normal saline was administered by gastrogavage for 14 days before the establishment of animal models. The left anterior descending coronary artery of rat was ligated to establish animal models of acute myocardial infarction. The MSCs labeled with BrdU were implanted intramyocardially into the infarct area. Heart function was then investigated 3h later by an echocardiographic system (Sonos5500, Phillips, CA) equipped with a 12-MHz probe. Assessment of heart function was also made four weeks later . Rats were euthanized four weeks after cell injection. Gross changes of ligated areas were observed by naked eyes. Additionally, pathological changes was investigated by Hemotoxylin- Eosin dyeing, the expression of Tie2 and factor VIII on labeled MSCs., MVD was investigated by immunofluorescence and myocardial cell apoptosis detected by TUNEL.Results : Gross changes: the color of cardiac muscle of ligated areas was similar with the normal cardiac muscle through naked eyes in MQT group , but it was still pale and was slightly oedema in the MT group. HE deying: The ischemia changes in MQT group was improved obviously than that of MT group. Although BrdU-positive cells were found in both groups, the ratio of Tie2 and factor VIII positive cells and MVD was greater in the MQT group. However, the number of apoptotic myocardial cells was greater in the MT group. Our data suggested that 3h after implantation, fractional shortening (FS) was 31.7±1.3 and 32.8±1.5 in the MTQ and MT groups, respectively. The wall-thickening fraction of the anterior and posterior walls was 32.1±2.1 and 46.3±1.1 for MTQ and 33.4±2.3 and 46.7±1.5 for MT, respectively, while four weeks after implantation, FS was 48.1±2.3 (MTQ) and 43.3±1.6 (MT). The wall thickening fraction of anterior wall and posterior wall were 41.8±2.2,37.9±2.0 and 46.5±1.2,46.6±1.5 respectively. Heart function were improved in both groups , but was more significant in MTQ group. Conclusion Our data demonstrated Qidantongmai tablets had a synergistic effect on the induction of transplanted MSC differentiation into endothelial cells within the ischemic myocardium. Our resulted suggested that MSC implantation, in conjunction with Qidantongmai tablets improved heart function compared to MSC implantation alone, as well as enhanced the MVD within the ischemic zone, and decreased apoptosis of myocardial cells in acute myocardial ischemia rats.