الفهرس 
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Abstract
The current approach was tailored to address the therapeutic potency of ADMSCs in combating MI induced by ISO in rats. Also, the study was extended to appraise the competence of differentiated ADMSCs into cardiomyocytes and to explore the advantages of growing ADMSCs and differentiated ADMSCs on NF plates in the treatment of the experimental of MI.
For ADMSCs isolation and propagation, ADMSCs were obtained from abdominal fats of male albino Wistar rats. Then, the isolated cells were grown and propagated in the conventional culture medium till confluence. To confirm that the cells in the culture were ADMSCs, they were characterized morphologically by inverted microscope examination, and by the detection of their specific cell surface markers (CD105, CD44, CD45, and CD34) by flow cytometry.
The differentiation process of ADMSCs into cardiomyocytes was carried out by culturing the ADMSCs in the differentiation medium containing specific growth factors (IGF-1, BMP-2 and FGF-2). To confirm their differentiation into cardiomyocytes, genetic analysis for MEF2C and Actn genes expression was done in vitro.
The in vivo study was conducted on 60 adult male albino Wistar rats which were assigned into 6 groups (10 rats/group). group (1): Control, group (2): MI; in which rats received a subcutaneous dose of ISO (100 mg/ kg), group (3): ADMSCs, in which MI challenged rats received a single intravenous dose of undifferentiated ADMSCs (3 x 106 cells/rat), Groups (4): ADMSCs on NF, in which MI challanged rats treated with a single intravenous dose of undifferentiated ADMSCs seeded onto NF (3 x 106 cells/rat), Groups (5): diff. ADMSCs, in
which MI challenged rats received a single intravenous dose of differentiated ADMSCs into cardiomyocytes (3 x 106 cells/rat) and Groups (6): diff. ADMSCs on NF, in which MI challenged rats received a single intravenous dose of differentiated ADMSCs into cardiomyocytes seeded onto NF (3 x 106 cells/rat). The experiment was lasted for two months. To confirm the ability of the administered ADMSCs to migrate into the injured heart, labeling of ADMSCs with sterile aqueous colloid of dextran-coated super paramagnetic iron oxide nanoparticles was implemented. Prussian blue staining was performed to visualize the iron particles in Ferumoxides-labeled ADMSCs.
To evaluate the therapeutic efficiency of ADMSCs as well as differentiated ADMSCs either on NF or without NF against MI in vivo, ECG measurements were achieved. Heart rate per minute, was procured from ECG recordings of average one minute, RR, PR, QTc and QRS intervals as well as P duration and ST height were calculated from ECG recordings using a computer program. Additionally, biochemical determinations including serum LDH and CK-MB enzymes activity was assayed by spectrophotometric kinetic method. Serum and cardiac cTnT, Cx43 and Actn levels were quantified by ELISA. Molecular analysis of GATA4 and NKX2.5 genes expression level in cardiac tissue was done. Finally, histopathological investigation of heart tissue sections was carried out for the different studied groups.
The findings of the in vitro study clarified that the isolated ADMSCs manifested a spindly, fibroblast-like shape under the inverted microscope. Besides, the flow cytometry analysis demonstrated that the derived cells were positive for CD44
(79%) and CD105 (96.8%), while they were negative for CD34 (4.22 %) and CD45 (9.11%). Furthermore, the ADMSCs succeeded to differentiate into cardiomyocytes as proved by the significant upregulation in the gene expression level of MEF2C and Actn.
On the other hand, the results of the in vivo study demonstrated that the treatment of MI challenged rats with ADMSCs as well as differentiated ADMSCs either on NF or without NF modulate ST height, heart rate, RR, PR, QTc, QRS intervals and P duration as manifested in the ECG recordings. The biochemical fingings appreciated the ECG outcomes as a significant inhibition in serum LDH and CK-MB enzymes activity as well as a significant suppression in serum and cardiac cTnT levels paralleled by a significant regression in serum Cx43 level in association with a significant elevation in cardiac Cx43 level were evinced. Meanwhile, serum and cardiac Actn levels showed a significant enhancement in the treated groups versus the untreated MI group. Molecular analysis of cardiac GATA4 and NKX2.5 gene expression levels also confirmed the ECG and biochemical outputs as a significant downregulation of the expression levels of the two genes was detected in the treated groups as compared to the MI group. Histological description of cardiac tissue sections disclosed great renovation in the cardiac microarchitecture upon treatment with ADMSCs with the superior improvement in the group treated with the differentiated ADMSCs into cardiomyocytes seeded onto NF. Thusly, the histomorpho- logical feedback ascertained the ECG, biochemical and molecular determinations.
In conclusion, this study gains insight into the underlying mechanisms of the therapeutic action of ADMSCs categories in combating MI induced by ISO in rats. The underlying mechanisms for this action could be ascribed to their capability to repress the (i) oxidative stress which is most probably, recognized as one of the fundamental mechanisms by which ISO provokes intense stress in the myocardium, leading to an infarct like necrosis of the heart muscle in the experimental animals. (ii) The differentiated ADMSCs showed more convenient action than the undifferentiated ones in alleviating MI in tats. Moreover, the neovascularization functionality of cardiomyocytes yielded from ADMSCs seeded onto NF has been shown to be superior to that of cardiomyocytes donated from ADMSCs without NF. This has been documented from the enhanced cardiac muscle contractility, the improvement of cardiac biomarkers and in turn cardiac functions and the renovation of histological cardiac configuration close to normal. Therefore, this study provides a clear experimental evidence for the prominence of myocardial engineering including cardiomyocytes transplantation as a realistic strategy for treatment of MI which may be beneficaial to proceed for
clinical trial studies.