JOURNAL ARTICLE

[A experimental study on transfecting human stromal cell-derived factor 1alpha and human vascular endothelial growth factor 165 genes into myoblasts in vitro]

Dengqi He, Xiufa Tang, Chengge Hua
Chinese Journal of Reparative and Reconstructive Surgery 2011, 25 (10): 1239-44
22069983

OBJECTIVE: To explore the human stromal cell-derived factor 1alpha (hSDF-1alpha) and human vascular endothelial growth factor 165 (hVEGF165) mRNA expressions of the transfected cells after hSDF-1alpha gene and hVEGF165 gene were transfected into rat myoblasts in vitro so as to lay a foundation for further study on the synergistic effects of 2 genes on tissue engineered skeletal muscle vascularization.

METHODS: The myoblasts of 1-day-old Sprague Dawley rats were cultured and purified by trypsin digestion assay in vitro and were identified by immunohistochemistry staining of Desmin. Approximately 70%-80% of confluent myoblasts were transfected with enhanced green fluorescent protein (EGFP)-hSDF-1alpha and EGFP-hVEGF165 genes in vitro (transfected group) and were not transfected (control group). The expressions of hSDF-1alpha and hVEGF165 mRNA and protein in the transfected cells were detected by RT-PCR, ELISA, and Western blot respectively.

RESULTS: The cultured cells were identified as myoblasts by immunohistochemistry staining of Desmin. The expression of green fluorescent protein was observed in transfected cells, indicating that hSDF-1alpha and hVEGF165 genes were transfected into myoblasts successfully. The mRNA and protein expressions of the 2 genes were positive in the transfected group by RT-PCR and Western bolt assay at 2, 4, 6, and 8 days after transfection, and were negative in the control group. The expressions of hSDF-1alpha and hVEGF165 showed a stable low level in the control group, but the expressions of the proteins increased at 2 days and then showed gradual downtrend with time in the transfected group by ELISA assay. There were significant differences in the expressions of hSDF-1alpha and hVEGF165 proteins between different time points in the transfected group, and between 2 groups (P < 0.05).

CONCLUSION: hSDF-1alpha and hVEGF165 genes are successfully transfected into myoblasts in vitro, and mRNA and proteins of hSDF-1alpha and hVEGF165 can be expressed in the transfected myoblasts, which may provide the experimental evidence for the expressions of hSDF-1alpha and hVEGF165 mRNA and proteins in vivo successfully.

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