The Effects Of Lentivirus Nrf2 On Proliferation,Apoptosis And Differentiation And The Construction Of Osteoarthritis ACLT Model In Mice

Objective We constructed NF-E2 related factor(Nrf2) lentivirus, Nrf2 siRNA lentivirus and ATF4 si RNA lentivirus in order to study the effects of gene Nrf2 modification on proliferation, apoptosis and differentiation during the differentiation of osteogenesis and cartilage cells, and built the ACLT model of osteoarthritis in mice. Methods The plasmid pWPT-GFP-Nrf2 was co-transfected with other two lentivirus packaging plasmids pMD2 G and pSPAX2, into 293 T cells to produce Lv-Nrf2 virus particles, and we got the Lv-siNrf2 and Lv-siATF4 with the same method. Different treatment groups of lentivirus were set under the induction of BMP2(bone morphogenetic protein 2), the proliferation, apoptosis and expression of osteogenic-differentiation-related marker proteins were detected respectively in C2C12 cells and high-density cultured C3H10 cells. Operated, built and evaluated the ACLT model of osteoarthritis in mice. Results The recombinant lentivirus Nrf2, siNrf2 and siATF4 were successfully packaged with titers about 1×108efu/mL; Under the induction of BMP2, Under the induction of BMP2, the FCM analysis showed that the apoptosis rate in BMP2+Lv-Nrf2 group was lower than that in BMP2+Lv-GFP group, the apoptosis rate in BMP2+Lv-si Nrf2 group was higher than that in BMP2+Lv-GFP group, the apoptosis rate in BMP2+Lv-Nrf2+Lv-siATF4 group was lower than that in BMP2+Lv-siATF4 group; the cell immunochemistry staining showed that the expression levels of OCN and ALP in BMP2+Lv-Nrf2 group were higher compared to the control group BMP2+Lv-GFP, the two proteins’ levels in BMP2+Lv-siNrf2 group were lower than that in control group, the two proteins level in combined treatment group BMP2+Lv-Nrf2+Lv-siATF4 were both lower than those in control group and single treatment group BMP2+Lv-Nrf2; Compared with the Lv-GFP group, the apoptosis rate of C3H10 cells was lower and the cell cycling rates of S phase was higher in Lv-Nrf2 group; The symptoms of ACLT model in PGRN-/- mice were more serious than in wildtype mice. Conclusion Under the induction of BMP2, gene Nrf2 could inhibit the apoptosis and promote the osteogenic differentiation of C2C12 cells; additionally, gene Nrf2 could rescue the increased cell apoptosis level, but could not rescue the decreased osteogenic differentiation level both caused by ATF4 deficiency; gene Nrf2 could inhibit the apoptosis and promote the proliferation of C3H10 cells during the cartilage differentiation; gene Nrf2 could promote the growth of the osteogenesis and inhibit protect the cartilage from apoptosis in mice limbs; the symptoms of the osteoarthritis model in mice were most visible in 8 weeks after the ACLT surgery; PGRN probably takes part in the repair of the cartilage damage and the development of arthritis.