| Stem cells transplantation, as a therapy for patients with myocardial infarction, has been one of the research priorities recently. It has huge potential contribution to cardiac regeneration and cardiac founction improvement. However, several meta-anlysis revealed that the outcome of the stem cells transplantation is dissatisfactory. Meanwhile, recent studies showed that combined stem cells and gene therapy may bring more benefit.This study combines transplantation of bone marrow derived mesenchymal stem cells (MSCs) with myocardial transfection of hypoxia inducible factor-la via an adenoviral vector as a treatment for myocardial infarction. The safety, efficacy and mechanism of this new mode of cardiovascular therapy are estimated in this study. This study is aimed at exploring the best way via which the stem cell-based therapy can achieve the best effect.Our study demonstrates the efficacy of the myocardial transfection of hypoxia inducible factor-1α via an adenoviral vector, which makes the cardiomyocytes express high level of HIF-la. We find that HIF-la can improve the homing and the survival rate of MSCs. It also increases the capillary density in the ischeamic area of the heart. 4 weeks after the implantation of MSCs, it’s detacted that MSCs differentiated into endothelial cells in Group HIF-la+MSCs. The combined therapy enhances the expression of certain cytokines such as SDF-la, VEGF, which are induced by high level of HIF-la, as well as the angiogensis in the ischeamic area. Thus, the microenvironment is improved which reduces the apoptosis of cardiomyocytes and improves the homing and the suvivial rate of MSCs. These may be part of the mechanism through which the combined therapy protects the cardiomyocytes and improves the cardiac founction.PART ONE:Investigation on myocardial transfection of hypoxia inducible factor-la via an adenoviral vector in acute infarcted myocardium of ratsObjectives(1)To investigate the efficacy of myocardial transfection of hypoxia inducible factor-1α via an adenoviral vector in acute infarcted myocardium of rats. (2) To detect the expression of HIF-1α 1 week,2 weeks,4 weeks after transfection respectively.Methods27 Sprague Dawley (SD) rats were divided into 3 groups randomly:(1)Group HIF-la (n=9):Myocardial infarction (MI) was induced by ligation of the left anterior descending coronary artery of rats. Ad-HIF-1α-GFP(6×109PFU)was intramyocardial injected into the border zone. (2)Group control (n=9):Ad-null-GFP (6×109FU) was intramyocardial injected into the border zone after MI. (3)Group sham (n=9):the left anterior descending coronary artery was not ligated. Frozen slides of myocardial tissue were detected under the fluorescence microscopy to evaluate the efficacy of Ad-HEF-1α-GFP transfection 1 week after MI. Myocardial expression of HDF-1α was determined by real-time PCR and Western blot 1 week,2 weeks and 4 weeks after MI respectively.Results(1) Successful transfection of the cardiomyocytes by Ad-HIF-la-GFP was detected under the fluorescence microscopy 1 week after MI.(2) The expression of HIF-1α mRNA was declined in both Group HIF-1α and Group control along the time.1 week after MI, the expression of HIF-1α mRNA was higher in Group HIF-la than in Group control or Group sham (p<0.05). The expression of HIF-la mRNA was higher in Group control than in Group sham (p<0.05).2 weeks after MI, the expression of HIF-la mRNA was higher in Group HIF-la than in Group control or Group sham (p<0.05).4 weeks after MI, the expression of HIF-1α mRNA was higher in Group HIF-la than in Group control or Group sham (p<0.05).(3) The expression of HIF-1α protein can’t be detected in Group sham. The expression of HIF-la protein was declined in both Group HIF-1α and Group control along the time. The expression of HIF-1α protein was higher in Group HIF-1αthan in Group control or Group sham 1 week,2 weeks,4 weeks after MI respectively.ConclusionsIscheamic cardiomyocyte can be transfected by Ad-HIF-la-GFP successfully. The expression of HIF-la mRNA and protein was declined in both Group HIF-la and Group control along the time. The expression of HIF-la mRNA and protein was higher in Group HIF-1α than in Group control or Group sham 1 week,2 weeks,4 weeks after MI respectively.PART TWO:Effects of HIF-1α on homing and engraftment of mesenchymal stem cells in ischeamic myocardiumObjectivesTo evaluate the effects of HIF-1α on homing and engrafting of mesenchymal stem cells in ischeamic myocardium.MethodsMSCs of male SD rats were isolated, purified, proliferated and fluorescently labeled with Dir.20 female SD rats were divided into 2 groups and randomly paired: (1) Group HIF-1α+MSCs (n=10):Myocardial infarction (MI) was induced by ligation of the left anterior descending coronary artery of rats. Ad-HIF-1α (6×109PFU) and MSCs(1×106) were intramyocardial injected into the border zone. (2) Group MSCs (n=10):Ad-null (6×109PFU) and MSCs(1×106) were intramyocardial injected into the border zone after MI. UCG was done before and 3 weeks after MI.1 week and 3 weeks after MI, the survival rate of MSCs was measured by "multi-mode imaging system for small animals". The suvial rate was also detected by real-time quantitative PCR via analyzing the sry sequence of Y chromatosome.Results(1) The labeling rate can reach 97±3% when MSCs were labeled with Dir.(2) The difference of left ventricular ejection fraction (LVEF) between 2 groups was not statistically significant(88.7±7.5% vs 87.4±7.4%,p>0.05) before MI. The LVEF was higher in Group HIF-la+MSCs than in Group MSCs (35.5±8.4% vs 29.8± 7.6%,p<0.05) 3 weeks after MI.(3) Though MSCs mainly engrafted in the originally ejected area, some of them migrated to the surrounding area.3 weeks after transplantation, MSCs can be detected in the infarcted area.(4) 1 week after transplantation, "multi-mode imaging system for small animals" demonstrated that the optical density of Group HIF-1α+MSCs was higher than that of Group MSCs (8.34±0.81×1012 P/s/mm2 vs 7.63±0.82×1012 P/s/mm2, P<0.01). The optical density of Group HIF-1α+MSCs was also higher than that of Group MSCs 3 weeks after transplantation (10.35±2.34×1012 P/s/mm2 vs 8.46± 2.98×1012 P/s/mm2, p<0.05).(5) 1 week after transplantation, the survival rate of MSCs detected by real-time quantitative PCR was higher in Group HIF-la+MSCs than in Group MSCs (7.4% vs 6.6%, p<0.05).3 weeeks after transplantation, the survival rate of MSCs was also higher in group HIF-1α+MSCs than in Group MSCs (1.5% vs 0.7%,p<0.01).Conclusions(1) Rats’MSCs can be successfully and efficiently labeled with Dir. (2) The LVEF was higher in Group HIF-1α+MSCs than in Group MSCs 3 weeks after MI. (3) MSCs can migrate to the surrounding area. (4) HIF-1α can raise the survivial rate of the engrafted MSCs.PART THREE:Investigation of the safety, efficacy and mechanism of combined imtramyocardial mesenchymal stem cells and HIF-la gene therapy after MIObjectivesTo investigate the safety, efficacy and mechanism of combined intramyocardial mesenchymal stem cells and HIF-1α gene therapy after MI.Methods55 SD rats were divided into 5 groups randomly:(1) Group HIF-la+MSCs (n=11):MI was induced by ligation of the left anterior descending coronary artery. 40μl of Ad-HIF-1α (6×109PFU) and 40μ1 of MSCs (1×106) were intramyocardial injected into the border zone. (2) Group HIF-1α (n=11):40μ1 of Ad-HIF-la (6× 109PFU) and 40μ1 of DMEM were intramyocardial injected into the border zone after MI. (3) Group MSCs:40μ1 of Ad-null (6×109PFU) and 40μ1 of MSCs (1×106) were intramyocardial injected in to the border zone after MI. (4) Group control (n=11): 40μ1 of Ad-null-GFP(6×109PFU)and 40μ1 of DMEM were intramyocardial injected in to the border zone after MI. (5) Group sham (n=11):the left anterior descending coronary artery was not ligated. UCG was done 1 week,2 weeks and 4 weeks after MI respectively. The capillary density, apoptosis index of cardiomyocytes, expression of HIF-1α, SDF-1α,VEGF were detected. The safety of the therapy was determined by pathological and histological examination.Results(1) The difference of LVEF among Group HIF-la+MSCs, Group HIF-la, Group MSCs and Group control was not statistically significant (37.18±10.07% vs 34.97± 5.87% vs 35.57±6.82% vs 32.16±5.05%, p>0.05) 1 week after MI.2 weeks after MI, the difference of LVEF among groups was not statistically significant (38.25± 5.72% vs 32.94±7.38% vs 32.23±8.52% vs 30.72±3.19%, p>0.05).4 weeks after MI, The LVEF was higher in Group HIF-1α+MSCs than in other groups (40.96± 8.91% vs 30.99±9.00% 27.26±6.38% vs 25.87±5.58%, p<0.05). The difference of LVEF among Group HIF-la, Group MSCs and Group control was not statistically significant (p>0.05).(2) 4 weeks after MI, it’s detected that MSCs differentiated into endothelial cells in the HIF-1α+MSCs group.(3) 4 weeks after MI, the capillary density in the border zone was higer in Group HIF-1α+MSCs than in other groups (1164±110/mm2 vs 1046±120/mm2 vs 725± 95/mm2 vs 592±97/mm2 vs 166±27/mm2,p<0.05 when compared to Group HIF-1α, p<0.01 when compared to other groups). In the infarcted zone, the result was similar (978±114/mm2 vs 812±91/mm2 vs 573±82/mm2 vs 469±53/mm2, p<0.01).(4) Apoptosis index (AI) was lower in Group HIF-la+MSCs than in Group MSCs or in Group control (15.6±2.3% vs 20.5±4.4%, p<0.05 when compared to Group MSCs,p<0.01 when compared to Group control). No significant difference was detected between Group HIF-1α+MSCs and Group HIF-la (9.1±2.4% vs 13.4± 1.9%,p>0.05).(5) The expression of HIF-la, SDF-la, VEGF was higher in Group HIF-la+MSCs than in Group MSCs or in Group control (p<0.05). No significant difference was detected between Group HIF-1α+MSCs and Group HIF-1α(p>0.05).(6) No neoplasma and no sign of excessive inflammatory response were detected.ConclusionsCombined imtramyocardial mesenchymal stem cells and HIF-la gene therapy after MI are safe and efficient. The combined therapy enhances the expression of certain cytokines such as SDF-1α, VEGF, which are induced by high level of HIF-la, as well as the angiogensis in the ischeamic area. Thus, the microenvironment is improved which reduces the apoptosis of the cardiomyocytes and improves the homing and the suvivial rate of MSCs. These may be part of the mechanism through which the combined therapy protects the cardiomyocytes and improves the cardiac founction. |
PDF Full Text Request