| Heart failure is a final developmental stage of many chronic heart diseases. The incidence and prevalence of heart failure is increasing, the end-stage heart failure with two-year mortality rates of 70-80% affects over 60,000 patients in the US each year. The treatment of heart failure continues to be a challenge. Current therapeutic modalities for the treatment of heart failure, especially end-stage cardiac failure are limited. There is a well-recognized urgent need for development of novel alternative therapeutic approaches. As the newly developed strategy, cell transplantation and cell-based therapeutic maneuver, which aim to introduce healthy myogenic cells and therapeutic genes into the myocardium of damaged heart, are showing bright future in this field. To provide a new and optimized strategy for heart failure, especially for end-stage heart failure, this research investigated the possibility of MSCs as an effective platform for the target gene delivery, compared the therapeutic effects with traditional gene therapy, or cell therapy. Furthermore, we explored the underlined mechanisms.Part one was to transfer of adeno-virus vector into different passages of bone marrow stem cell of rats. The efficiency and stability of adenovirus-medicated gene transfer into different passages of MSCs were investigated. MSCs were obtained from bone marrow of SD rat and cultured. Then MSCs from passage 3 (P3) or passage 8 (P8) were transfected with Ad-CMV-GFP, respectively. The transfection efficiency was evaluated by flow cytometer. At the same time the expressions of Coxsackie and Ad receptor (CAR) were estimated by RT-PCR and Western blot in transfected cells. The green fluorescence could be observed 24 hour after transfection, while the intensity of fluorescence was rising with time and peaked at 7 days. It was seen that the transfection ratio was over 80% and there was no difference between P3 and P8 MSCs (P>0.05). Flow cytometeranalysis by different gating showed the transfection ratio was high in active metabolic MSCs. The mRNA expression level of CAR in P3, P6 and P8 was similar, and the same change was seen on protein expression of CAR in P3 and P8 MSCs. Ad-CMV-GFP could be transferred to MSCs effectively and sustained for about 28 days. The results suggested that MSCs in mitotic phase were easy to be transferred by Ad-CMV-GFP and MSCs from P3 to P8 could serve as a high efficient gene vehicle.Part two was to explore the expression of SERCA2a gene mediated by adeno-virus vector in bone marrow stem cells of rats. MSCs were transfected with Ad-CMV-SERCA2a and the expression of SERCA2a were evaluated to provide the experimental foundation of gene therapy treating heart failure. MSCs were obtained from bone marrow of SD rats and transfected with Ad-CMV-SERCA2a, then the expression of SERCA2a at both mRNA and protein levels were determined by RT-PCR, Western blotting respectively. The transduction ratio of Ad-CMV-SERCA2a to MSCs was 80% and there was no obvious cell toxic reaction. SERCA2a were successfully expressed in MSCs, which were confirmed by FACS and RT-PCR analysis. These in vitro data provide an experimental basis for cell-based gene therapy of heart failure.Part Three researched combined bone marrow stem cells transplantation and SERCA2a gene therapy for heart failure after acute myocardium infarct. The possibility of using MSCs for target delivery of therapeutic gene was investigated and the therapeutic effects among gene therapy, MSCs transplantation and combination of gene and cell therapy were evaluated in this study. Furthermore, the mechanism of cell-based therapy for heart failure after myocardium infarct were investigated. MSCs were infected with an adenoviral expression vector carrying SERCA2a. SD female rats were used to make an animal model with heart failure after AMI and divided into 4 groups randomly. Group I (n=7) received SERCA2a gene therapy, group II (n=7) received MSCs transplantation, group III (n=8) received MSCs infected with SERCA2a gene transplantation, andgroup IV (n=7) received empty adenoviral vector. Cardiac function was evaluated by echocardiography and physiological recorder. SERCA2a expression and function were evaluated. Compared to group IV, EF and FS of group I, group II and group III were elevated significantly on 14 days after therapy (EF: 67.71 ± 3.87,62.57 ± 4.01,67.88 ± 3.71 versus 45.00 ± 2.24; FS: 33.86 ± 1.93,31.14 ± 2.01, 33.88 ± 1.88 versus 22.50 ± 1.12, P<0.05). While the elevation values of EF and FS began to reduce in group 114 days after, it continued to increase in both group II and group III. EF 14D and FS 14D in group I, group II and group III were 65 %, 49 %, 61 % and 65 %, 48 %, 56 %, while EF 21D and FS 21D were 48 %, 51 %, 63 % and 48 %, 51 %, 59% (P<0.05) . LVEDP at 21 days after treatment was decreased in group I, group II and group III compared to group IV (-5.34 ± 1.23 mmHg, -5.97 ± 1.29 mmHg,-6.20 ± 1.24 mmHg versus 3.89 ± 5.14 mmHg, P<0.05), as well as dP/dtmin (-4756.24 ± 270.00 mmHg/s, -5028.25 ± 253.39 mmHg/s, -5282.62 ± 362.74 mmHg/s versus -2789.53 ± 624.13 mmHg/s, P<0.05). DP/dtmax at 21 days after treatment increased in group I, group II and group III compared to group IV (6026.31 ± 280.89 mmHg/s, 6278.29 ±318.81 mmHg/s, 7056.59 ± 388.99 mmHg/s versus 5521.90 ± 267.85 mmHg/s, P<0.05). SERCA2a expressions and enzyme activity were significantly stronger in group I and group III than in group II and group IV.It showed that all MSCs transplantation, SERCA2a gene therapy and combined therapy could enhance cardiac function. The recovered heart function continued to improve in MSC transplantation group and MSCs-based gene therapy group up to 21 days, however slowed down in single gene therapy group in 21 days. Such therapeutic tendency of MSCs-based gene therapy was stronger than that of MSCs transplantation. Thus, MSC proved an effective platform for the targeted delivery of therapeutic gene.Part four discussed the mechanism of bone marrow stem cells transplantation on heart failure.Experiment one: Bone marrow stem cells transplantation reduced leftventricular remodeling in heart failure following acute myocardial infarction. MSCs transplantation has been shown to improve heart function but the mechanism or mechanisms through which its effects are exerted are unclear. We tested the hypothesis that MSC transplantation reduces left ventricular remodeling through the MMP/TIMP system in heart failure following acute myocardial infarction. Female SD rats underwent coronary artery ligation to induce myocardial infarction. Four weeks later, the rats were divided into 2 groups: rats in the test group (n=7) were transplanted with donor MSCs harvested and expanded from male SD rats (5×10~6 in 50μl PBS) into the ischemic myocardium, while group C received the same volume of PBS. Left ventricular morphology was then evaluated in the two groups through staining with H&E and Masson's trichrome. Immunohistochemical staining was used to examine the expression of MMP2 and TIMP1, as well as type I and type III collagens, in the scar zones. The expression of MMP2 and TIMP1 at protein level was determined by Western blotting. MSCs differentiated into fibroblast-like cells 21 days after transplantation in the test group. In addition, many inflammatory cells infiltrated and aggregated in the scar area but this effect was reduced by day 7 after transplantation. The following changes were noted in the group which received MSC transplantation compared to the control group: the left ventricular thinning ratio was significantly higher (76.34±2.66% versus 64.37±2.36%, P<0.05); the infarct size was smaller (36.19±0.83% versus 42.12±1.88%, P<0.05); type I collagen expression in the scar area was much higher; type III collagen expression was much lower; MMP2 expression was reduced and TIMP1 expression was increased.MSCs transplantation led to dynamic changes in the collagen network through regulation of MMP2/TIMP1 and consequently interrupted the progress of adverse LV remodeling in heart failure following acute myocardial infarction.Experiment two: To elucidate effect of MSCs transplantation on border and remote region around scar zone with heart failure (CHF) due to myocardiuminfarct. SD rats with post-ischaemic congestive heart failure were divided randomly into 2 groups, group M (n=7) and group C (n=7). Group M animals were transplanted with donor MSCs harvested and expanded from male SD rats (5×10~6 in 50μl PBS) into the ischemic myocardium, while group C received the same volume of PBS. All rats underwent Doppler tissue imaging (DTI) echocardiographic study to assess LV recovery of function and ventricle motion. Apoptosis in border and remote zone were examed by TUNEL. Bax and bcl2 expression were evaluated by RT-PCR, immunohistochemistry and Western blotting.4 weeks after ligation of coronary artery, all rats had abnormal regional contratile. The endocardial, epicardial velocities and the myocardial velocity of the systolic stage and diastolic stage from short-axis view decreased. After MSCs transplantation, E, A and S peak velocity were all significantly increased at posterior wall, anterior wall and anterior interventricular septum of left ventricle from short-axis view, mitral annulus from long-axis view. Ischemia myocardium size around scar zone reduced in group M compared to group C. Meanwhile, we observed neoangiogenesis in scar zone in group M but not in group C. Some MSCs transplanted into scar zone were visualized up to express VIII. Histology revealed a regional raise in myocyte nuclear density in border zone (440.1±136.7nuclei/mm~2 versus 177.75±40.4nuclei/mm~2, P<0.001). Myocardium from MSC transplantation group ameliorated regional myocyte hypertrophy in remote zone (myocyte size per nucleus, 29.49±5.5 um2 in the MSC transplantation group versus 78.49±13.0 urn2 in the control group, P<0.001). Apoptosis cell number was decreased (9.22± 1.1 count/mm2 versus 28.45±3.6 count/mm2, P<0.001). Western blot analysis and immunohistochemistry demonstrated significantly higher level expression of Bcl2 in group M compared with group C, while the expression of bax was significantly lower in group M.These results provide evidences for efficacy of treatment of heart failure with MSC transplantation, based on functional recovery of ischemia and hibernating...
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