The Effect And Mechanism Of Human Umbilical Cord Blood Derived Mesenchymal Stem Cells For The Treatment Of CTnT^R141W Transgenic Mouse Of Dilated Cardiomyopathy

Mesenchymal stem cells (MSCs) transplantation has been widely studied in the treatment of acute myocardial infarction, the results showed that MSCs could improve cardiac function by myocardial cell regeneration, angiogenesis and paracrine effects. Stem cell of previous experiments were mainly bone marrow derived MSCs (BM-MSCs), which were low proliferating ability, easy to aging, invasive and other shortcomings. However, umbilical cord blood derived MSCs (UCB-MSCs), which could overcome these shortcomings, become a new source of MSCs. Now, there are many researches on the UCB-MSCs in treatment of ischemic cardiomyopathy, little is known about the the mechanism and effect of UCB-MSCs in treatment of dilated cardiomyopathy. In the final analysis, its clinical application is limited mainly due to the lack of basic research.To study the effect and mechanism of UCB-MSCs in the treatment of dilated cardiomyopathy,explores the role of a paracrine mechanism and drug intervention in anti-hypoxia induced apoptosis of MSCs. We optimize the methods of isolation and culture of umbilical cord blood MSCs in vitro; imitate the ischemic myocardium microenvironment of cell transplantation by hypoxia and serum deprivation (H/SD) induced MSCs apoptosis, observe the effect and mechanism of trimetazidine on the apoptosis; further simulation of myocardial infarction microenvironment by hypoxia and serum deprivation induced h9c2 cell apoptosis, observe the effect and mechanism of MSCs-conditioned medium on the apoptosis; finally, UCB-MSCs were transplanted to myocardium of cTnTR141w transgenic mice in dilated cardiomyopathy, analysis of the effect on the heart function and anti-apoptosis, angiogenesis and paracrine mechanism.Our main work includes these four aspects as follows:Part I Identification and Cultivation of umbilical cord blood mesenchymal stem cells1、Isolation and culture of umbilical cord blood MSCs. UCB was collected to isolate the mononuclear cells by density gradient centrifugation.Comparison of different fetal age, serum concentration, inoculum density, the first medium change time on the extraction effect of success rate. The results showed that the influencing factors of MSCs extraction include serum concentration, the first medium change time, gestational age, Cells in DMEM/F12 medium, containing 10%　fetal bovine serum, the first medium change time (5days), low gestational age (≤37 weeks) can elevate the culture efficiency of　UCB-MSCs.2、Identification of UCB-MSCs. Cell morphology, surface marker expression and in vitro differentiation potential were used to identify the cell. MSCs were cultured in osteogenic and adipogenic media for 3 weeks, the third passage MSCs were used for surface marker identification. The results showed (1) Morphology:Spindle shaped confluent fibroblast like cells noted 3-4 weeks after initial seeding; (2) Differentiation potential:After 3 weeks osteogenic and adipogenic inductive culture, MSCs stain positive for Alizarin Red, Adipocytic differentiation was evidenced by the formation of lipid vacuoles; (3) Surface marker identification:MSCs were positively for mesenchymal markers CD44 and CD105, but negatively for haematopoietic markers CD34 and CD45.Part I results shown that the optimized separation scheme improved the success rate of cord blood MSCs culture and shortened the culture time. MSCs were Identification by Cell morphology, surface marker expression and in vitro differentiation, which will lay the foundation for the next step of the cell and animal experiment.Part II Trimetazidine suppressed the apoptosis induced by hypoxia and serum deprivation in umbilical cord mesenchymal stem cells1、Identification and Separation of umbilical cord mesenchymal stem cells. Umbilical cord were collected by informed consent, fully eliminate of artery and vein, MSCs were isolated by tissue adherent method. The results showed that the number of adherent cells from tissue fragments increased with time, and the cells became spindle shaped within 7 days. After 2 weeks, a number of classical MSC colonies appeared, which could digestion and passage. We further identified the MSCs by cell morphology, surface marker expression and in vitro differentiation potential, which showed the successful separation of umbilical cord-MSCs.2、Hypoxia/Serum deprivation (H/SD) induced apoptosis of MSCs. MSCs were exposed to 12 hours H/SD for simulating the stem cell transplantation into myocardial microenvironment. Apoptosis of MSCs were measured by the Hoechst33342 and Annexin V/PI staining, we found that H/SD induced the MSCs apoptosis.3、Trimetazidine have protective effect on H/SD induced MSCs apoptosis. MSCs were exposed to 12 hours H/SD with Trimetazidine intervention; Apoptosis of MSCs were measured by using FACS analysis after staining with Annexin V/PI. We found that Trimetazidine could suppressed the H/SD induced MSCs apoptosis, which also promoted the proliferation and survival of MSCs, suggested that TMZ had a significant protective effect against H/SD-induced apoptosis.4、Trimetazidine could suppressed the H/SD induced Caspase-3 activity. Caspase-3 activity was measured by Western-blot after treated with H/SD. We found that MSCs apoptosis could be inhibited by Trimetazidine. It indicated that Trimetazidine might enhance MSCs anti-apoptosis by suppressing Caspase-3 activity.5、Trimetazidine up-regulated the expression of p-Akt under H/SD environmet. The expresion of p-Akt was assessed in protein level by Western-blot. We found that H/SD decreased p-Akt expression, Trimetazidine increased p-Akt expression, and Akt inhibitor, LY294002, decreased p-Akt exprseeion and increased the MSCs apoptosis. It suggested that Trimetazidine exerts protective effects on the apoptosis of MSCs via the Akt pathways.6、Trimetazidine promoted the paracrine functions of UC-MSCs. We used the protein chip to compare the differences in the protein expression levels between the H/SD group and the H/SD+TMZ group. The results showed that Trimetazidine promoted the paracrine functions, which secreted many anti apoptosis, anti-inflammatory and regeneration factor, etc. It suggested that Trimetazidine promoted MSCs survival and secreted more paracrine factor.Part II investigation demonstrated that H/SD induced the MSCs apoptosis; Trimetazidine enhanced MSCs survival and reduced the H/SD induced apoptosis by activation of Akt and Inhibition of Caspase-3 activity. It indicated that Trimetazidine might be an effective medication to promote MSCs survival after transplanting into myocardium.Part III Human umbilical cord blood mesenchymal stem cells conditioned media inhibits hypoxia and serum deprivation induced apoptosis in H9c2 cells1 s Hypoxia/Serum deprivation induced apoptosis of h9c2 cells. H9c2 cells were exposed to 24 hours H/SD for simulating myocardial infarction model. Apoptosis of h9c2 cells were measured by the Hoechst33342 and Annexin V/PI staining, we found that H/SD induced the h9c2 cells apoptosis.2n MSCs-conditioned medium have protective effect on H/SD induced h9c2 cells apoptosis. H9c2 cells were exposed to 24 hours H/SD with MSCs-conditioned medium; Apoptosis of MSCs were measured by using FACS analysis after staining with Annexin V/PI. We found that MSCs-conditioned medium could suppress the H/SD induced h9c2 cells apoptosis, which also promoted the survival of h9c2 cells, suggested that MSCs-conditioned medium had a significant protective effect against H/SD-induced apoptosis.3、MSCs-conditioned medium up-regulated the expression of p-Akt under H/SD environmet. The expresion of p-Akt was assessed in protein level by Western-blot. We found that H/SD decreased p-Akt expression, MSCs-conditioned medium increased p-Akt expression, and Akt inhibitor, LY294002, decreased p-Akt exprseeion and increased the h9c2 cells apoptosis. It suggested that MSCs-conditioned medium might exerts protective effects on the apoptosis of h9c2 cells via the Akt pathways.4、MSCs-conditioned medium contains more paracrine protective factor. Protein chip were used to compare the differences in the protein expression levels between the DMEM medium and the MSCs-conditioned medium. The results showed that MSCs-conditioned medium contains much more paracrine protective factor, such as anti-apoptotic, anti-inflammatory factor, et al, which suggested the protective effect of UCB-MSCs conditioned medium.Part III data suggested that H/SD induced the h9c2 cells apoptosis, MSCs-conditioned medium enhanced h9c2 cells survival and reduced the H/SD induced apoptosis by activation of Akt. Further protein chip detection suggested that MSCs-conditioned medium contains more paracrine protective factor, which may be the reason for the above functions. All these indicated that MSCs-conditioned medium embodies the paracrine mechanism to promote h9c2 cells survival under H/SD environment.Part IV The effect and mechanism of Human umbilical cord blood derived mesenchymal stem cells for the treatment of cTnTR141w transgenic mouse of dilated cardiomyopathy1、UCB-MSCs were transplanted into the myocardium of transgenic mice of dilated cardiomyopathy. The male mice were randomly divided into four groups:(A) Control: Transgene-negative mice; (B) DCM; (C) DCM+PBS:Myocardial injection of PBS; (D) DCM+MSCs:Myocardial injection of MSCs. n=6 animals in each group,1.5×106 MSCs (15ul cell suspension) were injected, with a microsyringe, into the left ventricular anterior free wall in three spots. To track the transplanted cells in the recipient myocardium, the MSCs were labeled with eGFP (enhanced green fluorescence protein) using lentiviral vectors before transplantation.2、No implanted MSCs differentiated into cardiomyocytes after transplanting. By day one month following MSCs injection, the mouse was sacrificed for identifying implanted MSCs in LV myocardium. Based on histological analyses, numerous eGFP-stained undifferentiated MSCs were found to have engrafted in the DCM+MSCs group. However, cellular staining of eGFP could not be identified by troponin I. It suggested that few MSCs survive after transplanting, but fail to participate in the myocardial cell regeneration.3^ MSCs transplantation improved cardiac function. Cardiac function was analysed at one month after MSCs transplantation. Systolic function deteriorated in the transgenic heart, which exhibited an enlarged ventricular chamber and decreased EF, FS. One month after the MSCs injection, there was an increase in EF (56.96±3.54 vs 47.40±6.64%) and FS (32.26±2.93 vs 23.68±4.00%).4、Reduction of myocardial cell apoptosis and myocardial fibrosis by MSCs transplantation. Apoptotic nuclei in heart sections were identified by TUNEL staining. Compare with DCM and DCM+PBS group, MSCs significantly decreased myocardial apoptosis. Moreover, the masson staining showed that fibrosis was reduced following MSCs transplantation.5、MSC transplantation promted the expression of regeneration factor and angiogenesis. We extracted the supernatant of myocardium tissue, vascular endothelial growth factor (VEGF) and Insulin-like growth factor-1 (IGF-1) were measured by ELISA, and the results showed that MSCs treated hearts have significant increased levels of VEGF and IGF-1. Morover, the increased angiogenesis were found by a-sma and CD31 staining.6、UCM inhibits h9c2 cells apoptosis by a paracrine protective effect in vitro. The UCM was used to treat h9c2 cells in order to determine the effects on hypoxia and serum deprivation induced apoptosis. We found that the UCM inhibits h9c2 cells apoptosis; more important, UCM-H further decreased cell apoptosis compared the UCM-N. It verified the paracrine effects of MSCs in vitro.Part Ⅳ results shown that UCB-MSCs preserve cardiac function after intramyocardial transplantation in a DCM mouse model, which may be associated with inhibition of cellular apoptosis, inflammatory, hypertrophy and myocardial fibrosis, up-regulated expressions of Akt, VEGF, IGF-1 and enhanced angiogenesis.In summary, our present study analysed the following problems:(1) We simulated the microenvironment of myocardial infarction to observe the anti-apoptosis effect of MSC-conditioned medium on the h9c2 cells, It suggested the mechanism of paracrine effect. (2) We also found Trimetazidine enhanced MSCs survival and reduced the H/SD induced apoptosis. (3) UCB-MSCs preserve cardiac function after intramyocardial transplantation in cTnTR141w transgenic mouse of dilated cardiomyopathy, which provide the experimental basis for the MSCs in the treatment of dilated cardiomyopathy. For the dvantages of UCB-MSCs (noninvasive, strong ability of proliferation and differentiation, no ethical restriction), UCB-MSCs have a wide application prospect to treat cardiovascular disease.