The Effect Of ATF4 Lentivirus On Proliferation And Apoptosis Of Osteogenesis And Chondrogenesis And Preliminary Cells Study On Bone Healing Of PGRN^-/- Mice

Objective To construct the lentivirus vector targeting human activating transcriptional factor4(ATF4) gene,and to study the effect of lentivirus modification of human ATF4 gene on cell apoptosis and cycle in osteogenesis and cartilage cells.And preliminary study on bone healing of PGRN-/- mice.Methods Primer pairs of ATF4 gene were designed and synthesized.Hunman ATF4 gene was amplified by PCR from the expression plasmid pCDNA3.1(-)-ATF4 and was cloned into pWPT-GFP-V5 lentivirus vector.Then the recombinant ATF4 lentivirus were produced in 293T cells following to co-transfection between pWPT-GFP-ATF4 and the lentivirus packaging plasmids pMD2G,pSPAX2.The supernatants were collected 48h after transfection and myoblast C2C12 were infected in vitro. Infection efficiency of green fluorescent protein(GFP) was observed.The effect of recombinant lentivirus ATF4 on proliferation and apoptosis in C2C12 cells and C3H10 cells was detected by flow cytometry(FCM) in ER stress.Western blotting analysis was used to examine the expression of apoptosis-ralated proteins Cleaved Caspase-3,Chop and p-JNK.Results the recombinant lenticirus ATF4 was successfully constructed,with a titer of 1×108efu/mL.FCM analysis showed that under BMP2-induced differentiation condion,ATF4 can promot the apoptosis in C2C12 cells and C3H10 cells.Western blotting analysis showed that the expression of Cleaved Caspase-3,Chop and p-JNK was consistent with results of FCM. Successful build the fracture model and bone defect model in WT mice and PGRN-/- mice. Conclusion LV-ATF4 infection can promot the apoptosis in the diffrentiation of osteogenesis and cartilage cells.Compared with WT mice,the bone injury healing ability in PGRN-/- mice is declining.