The Regulatory Mechanism Of IGFs Gene Silencing On Growth And Development Of Sika Deer Antler Chondrocytes And Mesenchymal Cells

In order to understand the mechanism of IGF-Ⅰ and IGF-ⅡII in deer antler growth, the expressions of IGF-Ⅰ and IGF-Ⅱ in chondrocytes and mesenchymal cells were interfere by RNAi-Ready SIREN-RetroQZsGreen vector. For each gene, three RNAi plasmids were constructed respectively, named pshRNA1-1, pshRNA1-2, pshRNA1-3, pshRNA2-1, pshRNA2-2and pshRNA2-3. After tranfection for48h, the interfere efficiency to the IGF-I and IGF-II of these vectors were analysed by RT-PCR and Western Blot, we studied action mechanism of IGFs on development of chondrocytes and mesenchymal cells proliferation、 apoptosis and cycle.The results were listed as follow:(1) IGF-I RNAi could significantly reduce the expression of the gene for the antler chondrocytes and mesenchymal cells. pshRNA1-1, pshRNAl-2and pshRNA1-3could significantly reduced the expression of IGF-ⅠI in both mRNA and protein levels, and the pshRNAl-2recombinant plasmid had the best interference efficiency, for chondrocytes and mesenchymal cells, inhibition of IGF-ⅠI mRNA was60%;(2) IGF-II RNAi could also significantly reduce the expression of the gene for the antler chondrocytes and mesenchymal cells. pshRNA2-1, pshRNA2-2and pshRNA2-3could significantly reduced the expression of IGF-II in both mRNA and protein levels, and the pshRNA2-2recombinant plasmid had the best interference efficiency, for chondrocytes and mesenchymal cells, inhibition of IGF-ⅡII mRNA was60%;(3) Proliferation was assessed using thiazolyl blue tetrazolium bromide (MTT). The results prove that proliferation of chondrocytes and mesenchymal cells were inhibited after transfection for48h. The experimental group compared with control group and negative control group in chondrocytes, appreciation rates were16.97±9.83%,19.13±8.11%,24.59±10.22%,22.11±10.19%; The experimental group compared with the control group and negative control group in mesenchymal cells, appreciation rates were22.63±11.22%,23.46±12.77%,29.46±12.88%,28.02±15.69%; (4) Apoptosis (V-APC/propidiumiodide) was analyzed by flow cytometry. The results showed that in the chondrocytes and mesenchymal cells, transfection can induce significant apoptosis. After transfection for48h, chondrocytes in the control group, pshRNAl-2, pshRNA2-2experimental group, pshRNA-negative control apoptotic rates were:0.46±0.39%,14.45±3.39%,23.64±2.81%,0.67±0.24%(p<0.01); After transfection for48h, mesenchymal cells in the control group, pshRNAl-2, pshRNA2-2experimental group, pshRNA-negativecontrol apoptotic rates were:4.40±2.05%,25.78±5.87%,38.15±16.66%,6.32±3.03%(p<0.01);(5) Cell cycle (propidium iodide) was analyzed by flow cytometry. The results showed that pshRNAl-2, pshRNA2-2was significant effectd on the cell cycle, both in chondrocytes or in mesenchymal cells, pshRNA1-2, pshRNA2-2would reduced the number of the cells in the S phase. S phase in blank control groups and pshRNA1-2, pshRNA2-2experimental group and pshRNA-negative control of chondrocytes:12.59±0.04%,7.19±0.03%,7.12±0.05%,8.70±0.03%. S phase in blank control groups and pshRNAl-2, pshRNA2-2experimental group and pshRNA-negative control of mesenchymal cells:9.47±3.56%,4.95±0.48%,3.48±4.74%,8.28±2.98%.The results show that IGF-Ⅰ and IGF-Ⅱ plays an important role in regulating antler growth, especially for antler chondrocytes and mesenchymal cell proliferation, apoptosis, followed by regulation cell cycle of chondrocytes and mesenchymal cells (G1to S phase of the transition period), affecting their growth and development, moreover, IGFs have a significant impact on the regulation of mRNA and protein expression levels.