الفهرس 
TitleOnly 14 pages are availabe for public view
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Abstract
Mesenchymal stem cells (MSCs) are defined as self-renewing and multipotent cells capable of differentiating into multiple cell types, including osteocytes, chondrocytes, adipocytes, hepatocytes, myocytes, neurons, and cardiomyocytes. Representing approximately 0.001–0.01% of the bone marrow nucleated cells. Possessing self-renewal and multilineage differentiation capacity and immunomodulation ability so they are attractive for cell-based therapies ranging from regenerative medicine to tissue engineering including diabetes mellitus disease through compensation of degraded β-cells or genetically forming insulin producing cells. The present work aimed to investigate the ability of BM-MSCs transfected with pcDNA3-EGF-INS to express insulin gene in vitro. The current study first part of work was BM-MSCs isolation, expansion, and morphological characterization and exploring immunophenotypic features of the MSCs. BM-MSCs isolated form tibia and femur of male albino rat (age was 2-3month/weight about 250 g) and incubated with complete medium for 10 days as primary culture then splitted every 3 days until reach passage 3(p=3) growth rate, morphology and cell confluence were examined and documented daily using IPCM. Undifferentiated BM-MSCs at passage 3 were cultured on cover slips overnight, followed by fixation. To investigate the morphological features, fixed coverslips have been stained with haematoxylin and eosin (H&E) showed the typical fibroblastic shape cells with central distinct nucleus. In addition, the immunocytochemical staining technique was used to detect the expression of CD44 and CD105 markers, The results of were positive appeared as brown color diffused throughout the cytoplasm of the cells that considered as classic markers for Undifferentiated BM-MSCs.
On the other hand, pcDNA3-EGF-INS construct was prepared; at the first mammalian expressing plasmid pcDNA3-EGF was extracted from bacteria, purified, concentration was measured using NanoDROP and Gel electrophoresis (0.8%) was prepared to determine the exact size 6160 pb and integrity of the extracted plasmid ,stored in -20 or -80. In the same time total genomic DNA was extracted from fresh spleen to be used as template in PCR technique to amplify the preproinsulin gene using predesigned primers the forward primer sequence was5`TTGGATCCACCATTGATGGCCCTGTGGATGCGC-3`and the reverse primer
sequence was 5`GTGAATTCGCCTGTTGCAGTAGTTCTCCAG-3`with restriction sites BamHI and EcoRI underlined respectively. The PCR product was confirmed on 2% gel electrophoresis that was 330 pb. Also the present study, cloning of amplified preproinsulin gene into pcDNA3-EGF was carried using restriction enzyme technique both pcDNA3-EGF and preproinsulin gene were double cutted using BamHI and EcoRI restriction enzymes. The pcDNA3-EGF and preproinsulin was ligated using ligase enzyme followed by purification of the ligated product that the transformed into NEB 10-beta Competent E. coli and incubated at 37OC overnight on ampicillin selection LB Agar plates. Randomly 30 colonies were picked and used for plasmid extraction to explore if any of them was positive for pcDNA3-EGF-INS construct. The results indicated that 3 of the 30 picked colony was pcDNA3-EGF-INS positive. As a confirmation step double digestion using BamHI and EcoRI restriction enzymes and colony PCR were performed. 0.8% gel electrophoresis was prepared to
Summary & Conclusion
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investigate the confirmation step results where BamHI and EcoRI double digestion plasmid showed on gel two bands one at the plasmid size 6160 pb and the second was at the preproinsulin size 330 pb.
Gel prepared for colony PCR results was showed only one band at the size of preproinsulin size 330 pb.
At this point, we were having 3 positive pcDNA3-EGF-INS construct that used with turbofect transfection medium to transfect Undifferentiated BM-MSCs at passage 3 cultured on coverslips.
The transfected cells were examined using fluorescent microscope after 24 hour of transfection. The present results showed EGFP proteins label as fluorescent green color of successful transfected cells.
The immunocytochemical staining technique was used to detect the expression of insulin antibody in transfected cells; the positive results showed diffused brown color throughout the cytoplasm of BM-MSCs indicated that pcDNA3-EGF-INS transfected cells to express insulin in vitro.