Raji-MG63细胞共培养体系中心肌营养蛋白样细胞因子1(CLCF1)对RANKL/OPG比值及成骨细胞分化的影响

目的探讨Raji-MG63细胞共培养体系中,心肌营养蛋白样细胞因子1(cardiotrophin-like cytokine factor 1,CLCF1)基因对骨保护素(osteoprotegerin,OPG)、核因子κB受体活化因子配体(receptor activator of NF-κB ligand,RANKL)和成骨细胞分化的影响。方法通过CRISPR/Cas9基因编辑技术构建CLCF1基因敲除Raji细胞系,实验分2组:对照组(Control组)和CLCF1敲除组(CLCF1-KO组)。应用Transwell技术建立Raji-MG63细胞共培养体系,通过蛋白质印迹法(western blot,WB)检测CLCF1、OPG、RANKL及JAK2/STAT3蛋白的变化。运用碱性磷酸酶(ALP)活性检测和茜素红染色观察成骨细胞分化能力。结果WB结果显示,与对照组比较,CLCF1敲除组OPG(P<0.01)蛋白明显下调,RANKL/OPG比值升高(P<0.001),磷酸化JAK2(P<0.001)和STAT3(P<0.01)蛋白明显下调,差异具有统计学意义;ALP活性检测和茜素红染色结果显示,Raji-MG63细胞共培养体系中,CLCF1敲除组MG63细胞ALP活性和矿化形成能力降低,与对照组比较差异具有统计学意义(P<0.001)。结论CLCF1基因能通过调控RANKL/OPG比值和JAK2/STAT3通路影响成骨细胞分化。

The effect of cardiotrophin-like cytokine 1 (CLCF1) on the RANKL/OPG ratio and osteoblast differentiation in Raji-MG63 cell co-culture system

Objective To explore the effect of inhibiting the expression of Cardiotrophin-Like Cytokine Factor 1(CLCF1)gene in Raji-MG63 cell co-culture system on osteoprotegerin(OPG)and nuclear factor-κB receptor activator factor ligand(receptor activator of NF-κB ligand,RANKL)and osteoblast differentiation.Methods The CLCF1 knockout Raji cell line was constructed by CRISPR/Cas9 gene editing technology.The experiment was divided into 2 groups:control group(Ctrl group)and CLCF1 knockout group(CLCF1-KO group).Transwell technology was used to establish a Raji-MG63 cell co-culture system,and Western blot(WB)was used to detect the changes of CLCF1,OPG,RANKL and JAK2/STAT3 proteins.Alkaline phosphatase(ALP)activity detection and alizarin red staining were used to observe the differentiation ability of osteoblasts.Results Western blot result showed that compared with the control group,the OPG(P<0.01)protein in the CLCF1 knockout group was significantly down-regulated,the RANKL/OPG ratio increased(P<0.001),and JAK2(P<0.001)and STAT3(P<0.01)phosphorylated The protein was significantly down-regulated,and the difference was statistically significant;the result of ALP activity detection and Alizarin Red staining showed that the Raji-MG63 cell co-culture system,the ALP activity and mineralization ability of MG63 cells in the CLCF1 knockout group were reduced,which was different from the control Statistically significant(P<0.001).ConclusionCLCF1 gene can affect osteoblast differentiation by regulating RANKL/OPG ratio and JAK2/STAT3 pathway.