| Antler is the secondary sexual characteristic of sika deer or red deer. It is unique and special in mammals because of shedding and regeneration in periodic every year, at the same time, it can growth thirty times than the tumor cell’s in the most vigorous stage, this is unmatched by other mammalian organs. These features make Velvet antler become the research focus in the cancer medicine and regenerative medicine. Current research has shown that velvet antler regeneration is a complex process based on stem cells and be regulated by the various factors. The purpose of this study are to explore the molecular mechanism of velvet antler regeneration process, to reveal the role of igf-1, miR-a and miR-18b in the velvet antler regeneration process and to provide the groundwork for further research.In this study, the telomerase activity in different parts of top tissue of velvet antler and the change of telomerase activity in mesenchymal layer cells which was cultured after different generation in vitro was detected by TRAP assay. On the basis of this, a length of914bp of TERT gene was cloned. The mRNA expression of TERT gene in different parts of top tissue of velvet antler was detected by fluorescence quantitative assay. Four sequences of Pre-miRNA was designed and synthesized according to the mRNA sequence of sika deer. The mRNA expression of igf-1gene was detected by fluorescence quantitative PCR after four Pre-miRNA were transfected respectively, the best Pre-miRNA sequence was selected by its silence effect, What’s more, the silence effect of igf-1was detected by western blotting, the effect of igf-1gene against the proliferation of velvet antler cell was detected by MTT assay, the vary of cell cycle of velvet antler cell was detected by Flow cytometry and the changes of telomerase activity in velvet antler cell was detected by TRAP assay.On the basis of above experiment, the mimics and inhibitor of endogenous miR-18a and miR-18b was designed and synthesized by the methods of gene chip and software prediction, the expression level of mimics and inhibitor in cartilage cells after transfection were detected by fluorescent quantitative PCR and protein expression level were detected by Western blotting, the Acting site of miRNA was verified through transfect miRNA and vector which was inserted the igf-13’UTR sequence of luciferin enzyme report gene, the proliferation of chondrocytes in velvet antler was determined by MTT assay; the changes of cell cycle in velvet antler were detected by Flow cytometry.Results show that telomerase activity could be detected in different parts of top tissue of velvet antler and the telomerase activity in mesenchymal is the highest. The telomerase activity in Ectomesenchymal cells were cultured after different generation in vitro changed little. A length of914bp TERT gene sequence has be cloned, its nucleotide sequence homology with cattle and sheep were94.89%and94.31%respectively and Amino acid sequence homology were92.16%and92.11%respectively. The expression of TERT gene in mesenchymal cells of velvet antler was the highest detected by fluorescence quantitative PCR. Four pre-miRNA sequences of igf-1have designed and synthesized and they were all have silence effects detected by fluorescence quantitative and the igf-l-miR-2was the best one. Western blotting results show that the igf-1protein expression after the transfection was decreased compared with the control group, the results of MTT assay show that the proliferation of chondrocytes cells in velvet antler was restrained. The results of cell cycle detected by flow cytometry show that the percentage of S phase cells decreased, the number of cells in G0/G1phase increased. After miR-18a and miR-18b be selected transfected into velvet cells, the expression of miRNA increased which has detected by fluorescence quantitative PCR assay, this illustrate that miRNA has success transfected into antler cells. The results of western blotting show that the igf-1protein expression levels in cells be transfected with mimics have decline, while the igf-1protein expression levels in cells be transfected with inhibitor have increase. The results of MTT assay show that the proliferation of cells was transfected with mimics were restrained, while the cells were transfected with inhibitor increased compared with controls. The results of cell cycle detected by Flow cytometry show that the percentage of cell transfected with mimics in S phase declined and in G0/G1phase increased, while the percentage of cell transfected with mimics in S phase increased and in G0/G1phase declined compared with controls. |
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