Transfection of calcitonin gene-related peptide mediated by lentivirus vector in vitro and its effects on cardiac stem cell viability |
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| Authors: | RONG Ji-dong LI Ling LONG Xian-ping DENG Wen-wen SHI Bei |
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| Institution: | Department of Cardiology, The First Affiliated Hospital, Zunyi Medical College, Zunyi 563003, China |
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| Abstract: | AIM: To investigate the effect of calcitonin gene-related peptide (CGRP)transfection into c-kitpos cardiac stem cells (c-kit+CSCs) on the cell viability. METHODS: Under the sterile condition, the auricles of SD rats were taken out,and then c-kit+CSCs were collected through enzyme digestion and immunomagnetic bead separation (MACS). The cells were identified by flow cytometry. c-kit+CSCs were transfected with enhanced green fluorescent protein CGRP lentiviral vector (Lv-EGFP-CGRP) or enhanced green fluorescent protein lentiviral vector (Lv-EGFP). The cells were randomly divided into Lv-EGFP-CGRP-CSCs group, Lv-EGFP-CSCs group and CSCs group. The transfection was observed under the fluorescence microscope. The transfection efficiency was detected by flow cytometry. The CGRP protein secretion in the cell culture supernatants was detected by ELISA. The viability of c-kit+CSCs transfected with Lv-EGFP-CGRP or Lv-EGFP was measured by CCK-8 assay. RESULTS: c-kit+CSCs were isolated and cultured successfully. The expression positive rate of c-kit was 91.0% and the expression positive rates of CD45 and CD34 were 4.5% and 4.0%, respectively. After transfected with lentivirus for 48 h, the stable fluorescence in c-kit+CSCs was observed under fluorescence microscope. The transfection efficiency were 80% when MOI was 20. The level of CGRP was significantly increased in Lv-ECFP-CGRP-CSCs group compared with Lv-EGFP-CSCs group and CSCs group (P<0.05). Meanwhile, transfection with lentiviral vector in each group did not affect the viability of c-kit+CSCs. CONCLUSION: Transfection of Lv-EGFP-CGRP into c-kit+CSCs was successful. The secretion of CGRP was found in the transfected c-kit+CSCs and the viability was not changed after transfection. CGRP-modified c-kit+CSCs may play a role in treating myocardial infarction. |
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| Keywords: | Calcitonin gene-related peptide Lentiviral vector Cardiac stem cell Cell viability |
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