Experimental study on effect of complement C3 on bone formation observed by cell culture in vitro

Abstract

To explore effect of lentivirus-mediated complement C3 silencing vector on the osteogenic ability of human B lymphocyte Raji-osteoblast cell line MG63 co-culture system and its mechanism. The lentiviral complement C3 silencing vector was constructed and transfected into human B lymphocyte Raji to establish in vitro Raji-osteoblast cell line MG63 co-culture system. The cells were divided into blank control group (without special treatment), complement C3 silencing group (lentiviral complement C3 silencing vector transfection co-culture system) and model group(lentiviral vector transfection co-culture system). The expression of complement C3 in each group was detected by reverse transcription-polymerase chain reaction(RT-PCR) and Western-Blot at 24 h after culture, proliferation of MG63 cells was detected by CCK-8 at 0, 3, 6, 12, 24, 48 and 72 h, apoptosis of MG63 cells in each group was detected by flow cytometry at 24 h after culture, and alkaline phosphatase(AKP) activity of MG63 cells in each group was detected by AKP detection kit, and osteoprotegerin (OPG ) protein expression of MG63 cells in each group was detected by Western-Blot method. RT-PCR results showed that the expression level of C3 in complement C3 silencing group was lower than that in blank control group and model group, Western-Blot results showed that expression of C3 in complement C3 silencing group was lower than that in blank control group and model group, CCK-8 results showed that there was no difference in proliferation ability of MG63 among complement C3 silencing group and blank control group and model group at 3 and 6 h after culture;at 12 h after culture, proliferation ability of MG63 cells with C3 silencing was higher than that of blank control group and model group;at 24, 48 and 72 h after culture, proliferation ability of MG63 cell line with complement C3 silencing group were higher than that of blank control group and model group. Flow cytometry resluts showed that apoptosis of proliferation ability of MG63 cell line with complement C3 silencing group was lower than that of blank control group and model group;AKP detection kit suggested that AKP activity in complement C3 silencing group was higher than that in blank control group and model group, Western-Blot results showed that expression level of OPG protein in complement C3 silencing groupwas higher than that in blank control groupand model group. Silencing of complement C3 could enhance bone formation ability of osteoblast MG63, and it takes time to accumulate this ability. Complement C3 may affect osteogenesis by altering OPG / RANKL / RANK axis.