Study Of Mesenchymal Stem Cells From Diabetes Mellitus Transplantation For Myocaridal Infarction

ObjectiveTransplantation of mesenchymal stem cells(MSC) is one of selectived treatment for myocardial infarction.MSC have been shown to secrete various cytokines by paracrine effects to angiogenesis, inhibition of apoptosis and left ventricular(LV) remodeling, improveing cardiac function. The beneficial effects of MSC transplantation are well documented in animal studies. However, the effects in clinical trials are ambiguity, which might be caused by most patients with complications, such as DM. Little is known about biological characteristics and difference gene expression profile of MSC from DM patients. The aims of our study were to investigate: 1. Difference of gene expression profile and transplantation effect for MI of MSC from DM and no-DM in order to find some crucial cytokines for these differences(Bcl-2, Bcl-x L); 2. Whether MSC modified with Bcl-x L increase the viability of MSC in infarted zone and impove cardiac function or not.MethodsThe present study was designed to investigate the difference of gene expression profile and transplantation effect for MI of MSC from CAD+DM and CAD patients and modify MSC with h Bcl-x L by lentiviral transfection to identify function of h Bcl-x L on viability, angiogenesis, anti-apoptosis and improving cardiac function. There were three parts in our study.1. MSC were preared from patients underwent coronary artery bypass graft(CABG), and amplificated in vitro. Phenotype assessment, growth curve, RNA extraction was measured at P3 stage. 2. MSC from CAD+DM and CAD patients were transplanted into infarcted myocardium in rats with experimentally MI to measure the expression of Bcl-2, VEGF and to assess the treatment for MI(anti-apoptosis and improving cardiac function). 3. Lentiviral vectors were used to transfected h Bcl-x L-IRES2-EGFP into MSC to over express h Bcl-x L, IRES2-EGFP gene was transfected as control. Analysis of transfection efficiency included fluorescence microscopy and Western-Blot for detecting the expression of EGFP and h Bcl-x L in MSC; anti-apoptosis were measured by annexin V /PI, cytokines secretion under hypoxic condition were assessed by ELISA. Furthermore, in vivo, viability in infarcted myocardium, inhibition of apoptosis, capillary density, scar size assessment and cardiac funciton were measured by DAPI+???, TUNEL, immunohistochemistry, Trichrome-Masson method and Echocardiography respectively.Results1. MSC from DM patients exhibited the properties of MSC. They strongly expressed CD29 and CD44 but were negative for CD34 and CD45. In vitro, the proliferative potential of MSC from DM patients decreased compared with MSC from no-DM patients. 2. Base on Affymetrix gene arrays, 27 functional protein genes expression in DM patients compared with no-DM relating to cell apoptosis, cytokine, and signal transduction. Among them, the expression of 13 functional genes, including TNFRSF10 B, TNFRSF21,NGF, CAV2, ITGA8, TNS1, ITGA2, AKT3, MBP, MAP2, INHBA, FST, PLA2G5, increased significantly. However, the expression level of 14 genes including EPR1、BIRC5HELLS、BCL2、HGF、CASP1、SEPP1、ITGA9、MAP2K6RUNX3、TGFBR2、RUNX2、CTNNB1、CDC42, decreased significantly. 3. MSC from CAD+DM and CAD group transplanted into rat infarcted myocardium inhibited myocardial cell apoptosis and improve cardiac function. However, the effects above mentioned were impaired in CAD+DM group compared with CAD group, which might be related to the low level of Bcl-2 in CAD+DM group. 4. Analysis of transfection efficiency showed that h Bcl-x L-IRES2-EGFP fused gene, IRES2-EGFP gene could be transfected with lentivirus transfection system. Analysis of EGFP transfected was to confirm the transfection efficiency and assess the viability in vivo. Our study demonstrated that MSC transfection did not affect cell characteristic. Effects of h Bcl-x L over expression on effects of anti-apoptosis and cytokines secretion were improved. Western blot, annexin V /PI, ELISA showed that MSC modified with h Bcl-x L increased the expression of h Bcl-x L, improved the inhibition of apotosis, and increased the secretion of VEGF, IGF-1 and PDGF(P<0.05). 5. MSC modified with h Bcl-x L transplantion for MI in vivo demonstrated that over expression h Bcl-x L in MSC increased the viability, inhibit myocardial cell apoptosis, promot angiogenesis, reduce the scar size and improve cardiac function(P<0.05).Conclusion1. The proliferative potential of MSC from DM patients decreased compared with MSC from no-DM patients, with different gene expression, such as decreased Bcl-2 expression. 2. The effect of MSC from DM transplantion for MI decreased significantly compared with no-DM. 3. The method of lenticiral transfection is a safe and effective way to modify MSC and increase expression of h Bcl-x L, which improve the anti-apoptosis ability of MSC and secretion of VEGF, IGF-1, PDGF in vitro under hypoxia condition.4. MSC with high expression of h Bcl-x L transplanted into infarcted myocardium increased the viability, inhibit myocardial cell apoptosis, promot angiogenesis, reduce the scar size and improve cardiac function...