Construction Of Human Perk And ATF4 Gene SiRNA Adeno Virus And Effect On The Apoptosis Of Chondrocytes

Objective:To investigate the effects of small interfering RNA targeting protein kinase-like endoplasmic reticulum kinase gene and activiting transcription factor 4 gene adenovirus on apoptosis of chondrocytes.Methods:By using AdEasyTM adenovirus vector system,to build and pack recombinant adenovirus siPERK and siATF4. The cells were infected with siPERK and siATF4 and the expression levels of PERK and ATF4 mRNA and protein were detected by reverse transcription-PCR and western blot. Endoplasmic reticulum stress model was induced by tunicamycin, study the effects of proliferation and apoptosis of ATDC5 cells when ATF4 gene knockdown. Investigate proliferation and apoptosis of SW1353 cells when the recombinant adenovirus siPERK was infected into the cells. Micro mass cultures of ATDC5 and C3H10T1/2 cells infected with either Ad-RFP or recombinant adenovirus siPERK and siATF4, incubated with BMP2 at different time points of 1/3/5 days, study on the apoptosis of cartilage cells by FCM,TUNEL,western blot and immunohistochemistry.Results:The recombinant adenovirus siPERK and siATF4 were constructed successfully. After 48h of treatment by the recombinant adenovirus,the expression of ATF4 and PERK gene in ATDC5 cells were significantly inhibited. Under the ERS condition,compared with the control group, the apoptosis rate of ATDC5 cells after infected with siATF4 was decreased(P<0.05), and the proliferation rate was increased significantly(P<0.05). The proliferation rate was increased significantly (P<0.05)and the apoptosis rate was decreased(P<0.05) after SW1353 cells were infected with the recombinant adenovirus siPERK. When the cartilage cells were under differentiation codition induced by BMP2, FCM analysis showed that the apoptosis rate of ATDC5 cells was incresed gradually after infected with both siPERK and siATF4, and it was also higher than the control groups(P<0.05).Western blot and immunohistochemistry were in accordance with what FCM showed.Conclusion:ATF4 knockdown can promote the proliferation of the cartilage cells and inhibit the apoptosis under ERS condition. PERK gene knockdown can promote the proliferation of human chondrosarcoma cells and inhibit the apoptosis when SW1353 cells in endoplasmic reticulum stress. Under differtiation condition induced by BMP2, both PERK and ATF4 gene knockdown can promote the apoptosis of the cartilage cells.