Study On Proliferation, Cycle And Apoptosis Of Physical Activity Of Sika Deer Antler Chondrocyte Cells With IGF1R Gene Silenced

Deer velvet is a significant secondary sexual characteristics with periodic regeneration properties. In the most vigorous growth stage, its growth spead may rise up to 2cm/d, which is very alarming. The recent study showed velvet antler regeneration is based on stem cells in its forehead, a complex process that is regulated by multiple factors. This study was designed to explore the IGF1 R functions from the perspective of antler chondrocyte proliferation and differentiation, in order to further study the molecular mechanism of antler growth and development to provide a new perspective. In this experiment, RNAi technology interferences IGF1 R m RNA expression in antler chondrocyte, verify its effect on chondrocyte from different ways.In this experimnet, RNAi-Ready p SIREN-Retro QZs Green plasmid as a vector, is used to transfect cartilage cell of antler in order to explore mechanisms of IGF1 R gene.Two successfully recombinant plasmids-RNAi constructed were named psh RNA-1 and psh RNA-2. m RNA expression and protein of IGF1 R were tested by the tool of Real-Time PCR and Western blot technique, after 48 h transfection with recombinant plasmids-RNAi. In order to understand the function of IGF1 R gene, we performed sh RNA-mediated knockdown experiments in cartilage cell and examined the effect on the apoptosis,cell-cycle and proliferation.Research results are as follows:(1) Compared to the psh RNA-negative group for 48 h transfection, the result showed that m RNA expression residue rates of cartilage cells treated by psh RNA-1 was 51% and treated by psh RNA-2 was 75%;The expression of the protein was also decreased significantly. Indicating that interfere effect was obvious.(2) Cell proliferation of cartilage, treated by two recombinant plasmids-RNAi(psh RNA-1 and psh RNA-2), were tested by cck-8 in the 24 h, 48 h, 72 h. we found cell proliferation were inhibited, specially psh RNA-1 recombinant plasmid(1.09 ± 0.10 vs1.16 ± 0.09(P <0.01), 1.04 ± 0.02 vs1.10 ± 0.06(P<0.05), 0.79 ± 0.06 vs 1.01 ± 0.07(P <0.01)), compared to the growth rate of the negative control group.(3) compared to the psh RNA-negative group for 48 h transfection, the result showed that most cells in the experimental group were in G1-phrase, and significantly higher than that of psh RNA-negative group(79.47±0.11 vs 82.27±0.35(P<0.01)); while the number of cells in S phase was significantly decreased(16.56 ± 0.10 vs 14.82 ± 0.014(P <0.05)).(4) apoptosis of cartilage cell were detected by the V-APC apoptosis detection kit and flow cytometry after transfection for 48 h.The result showed that early apoptosis rates of the experimental group treated by psh RNA-1 was significantly higher than that the negative control group of psh-negative(22.12 ± 2.72% vs 8.56 ± 0.16%, P <0.01); however, living cells rates of the experimental group treated by psh RNA-1 was significantly lower than that the psh RNA-negative group(62.85 ± 5.97 VS 77.39 ± 4.46, P <0.05), Relate apoptotic-regulated gene expression of p53(P<0.01), bcl-2(P<0.05) increased, bax expression was significantly decreased(P<0.01). the result showed recombinant plasmid-RNAi-psh RNA-1 can induce apoptosis of cartilage cell.The results showed that the gene of IGF-1R plays an important role in the growth process of antler, especially for antler cartilage cell proliferation, apoptosis and their growth and development through the cell cycle regulation of chondrocytes.