Regulation of hypoxic response elements to expression of hVEGF165 gene transferred to cardiomyocytes under anoxic and normoxic conditions |
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| Authors: | DONG Hong-yan ZHANG Zhong-ming YAN Ying-qun |
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| Institution: | 1.Center of Neurobiological Research,2 Department of Throcic Cardiovascular Surgery,The Affiliated Hospital,Xuzhou Medical College,Xuzhou 221002,China,E-mail: zhang-zhongming@yahoo.com.cn |
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| Abstract: | AIM:To investigate effects of hypoxic response elements (HRE) on expression of hVEGF165 gene transferred to myocardiocyte under anoxic and normoxic conditions in vitro.METHODS:Myocardiocytes from neonatal SD rats were isolated and cultured in serum-free medium.The r-AAV vector packaged with 293T cells was used to transfer cultured myocardiocytes.Myocardiocytes were divided into eight groups: Group Ⅰ: myocardiocytes were cultured for 24 hours under normoxic conditions as control;Group Ⅱ: cells was cultured under hypoxia for 8 hours;Group Ⅲ: infected cells were cultured for 24 hours under normoxic conditions;Group Ⅳ: infected cells were cultured under hypoxia for 8 hours;GroupⅤ: infected cells were cultured under hypoxia for 8 hours and normoxia for 4 hours;GroupⅥ: infected cells were cultured under hypoxia for 8 hours and normoxia for 8 hours;GroupⅦ: infected cells were cultured under hypoxia for 8 hours and normoxia for 12 hours;Group Ⅷ: infected cells were cultured under hypoxia only for 20 hours.hVEGF165 protein was quantified from culture medium using ELISA,hVEGF165 protein in myocardiocytes was detected with immunofluorescence and expression of hVEGF165 mRNA was also determined by RT-PCR.RESULTS:95% myocardiocytes showed beating rhythmically with 86% of cTn-I positive staining.87% virual transferred efficiency was achieved.The contents of hVEGF165 protein in group Ⅳ,Ⅴ and Ⅷ were higher than those in other groups (P<0.01).Immunofluorescence positive cells were observed in group Ⅳ,Ⅴ and group Ⅷ.RT-PCR revealed that a 484 bp strip was also found in the same groups.CONCLUSION:rAAV-HRE9-hVEGF165 vector infected cultured myocardiocyte successfully.Under hypoxia,expression of hVEGF165mRNA and hVEGF165 protein were regulated by HRE,whereas under normoxic conditions the expression of hVEGF165 mRNA and hVEGF165 protein were ceased. |
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| Keywords: | Anoxia Endothelial growth factors Genes hVEGF Cardiomyocytes |
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