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| Effects of Deer (Cervidae) Thymosin β10 Gene Overexpression on Proliferation, Senescence and Migration of Antler Stem Cells In vitro |
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Abstract Overexpression of thymosin beta 10 (Tβ10) gene is detected in many of neoplastic tissues and cell lines compared to the respective normal tissue types. So Tβ10 is considered to be an anticipated marker for tumorigenesis and tumor progression. Deer (Cervidae) antlers are not a cancerous organ with the astonishing growth rate up to 2 cm/d. Tβ10 gene was highly expressed in the growing tips of antlers by using in situ hybridization. Therefore, tests of the effects on biological properties (proliferation, migration and senescence) of endogenous Tβ10 gene expressed in the antler stem cells by using a lentiviral vector were performed in this study. In the experiment, antlerogenic periosteum (AP) tissue from a 8 months old male sika deer was collected by using surgical operation in a relative sterile environment and primary culture for AP cells was carried out by using typeⅡcollagenase. Total RNAs of AP cells were extracted by using PureLinkTM mini RNA kit and Tβ10 gene was cloned using RT-PCR. AP cell lines with the overexpression of Tβ10 gene were successfully established by using lentiviral vector guidance system and determined the effects on proliferation, migration and senescence, which may be influenced by endogenous Tβ10. AP cells could proliferate in vitro, morphology of these cells was bipolar spindle shaped under a phase contrast microscope. Tβ10 gene was cloned accurately based on sequencing and inserted into the lentiviral vector plvx-ires-mCherry-puro successfully. Recombinant lentivirus was packed and produced successfully by using co-transfection of three-plasmid lentiviral vectors and X-treme GENE HP transfection reagents in ERK 293T cells. Transfection efficiency was evaluated through the expression of red fluorescent protein (RFP) by using an inverted fluorescence microscope. The lentivirus from culture medium were collected and concentrated by using Lenti-X concentrator regents, and the titer of lentivirus achieved 4×108 TU/mL detected with 293T cells. AP cells infected by recombinant lentivirus were selected and enriched by applying puromycin (2.5 μg/mL) in culture medium for 10 days. AP cell lines with overexpression of Tβ10 gene were established and identified by qRT-PCR and Western blotting. The results of methyl thiazolyl tetrazolium (MTT) assay showed that Tβ10 gene inhibited proliferation of AP cells significantly (P＜0.01), compared to the control; β-galactosidase staining showed that Tβ10 gene promoted senescence of AP cells significantly (P＜0.01) and the migration assay showed that Tβ10 gene inhibited human (Homo sapiens) umbilical vein endothelial cells (HUVEC) migration significantly (P＜0.05). This study would lay the foundation for revealing the underlying mechanism of rapid growth of deer antlers without becoming cancerous.
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Received: 18 August 2016
Published: 24 December 2016
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