大骨节病差异表达基因PAPSS2对成骨细胞矿化及碱性磷酸酶活性的影响

Effect of Kashin-Beck disease differentially expressed gene PAPSS2 on osteoblast mineralization and alkaline phosphatase activity

目的探讨大骨节病差异表达基因PAPSS2在MC3T3-E1细胞成骨样分化过程中的表达及其对碱性磷酸酶(ALP)活性和细胞矿化的影响。方法培养MC3T3-E1细胞并诱导成骨分化,观察PAPSS2在成骨细胞分化过程中的基因及蛋白表达情况;用慢病毒介导的RNA干扰(RNAi)技术或逆转录病毒转染高表达载体,分别构建PAPSS2低表达慢病毒及逆转录病毒高表达载体包装,滴度测定,验证细胞转染效率;分别转染MC3T3-E1细胞并设空白对照组,观察PAPSS2沉默或高表达后ALP活性的变化及细胞成骨矿化的影响。结果 PAPSS2在成骨细胞分化过程中mRNA及蛋白均随着矿化过程表达增加。用慢病毒介导的RNAi技术,成功降低了MC3T3-E1成骨细胞中PAPSS2的表达,使用逆转录病毒转染高表达载体显著增加PAPSS2基因的表达。PAPSS2沉默表达时,ALP活性和细胞矿化作用显著降低,高表达后其作用相反。结论 PAPSS2可调节成骨细胞ALP活性和细胞矿化作用,其异常表达可能与大骨节病骨骼病变相关。

Objective To investigate the expression of differentially expressed gene PAPSS2 in Kashin-Beck disease during the differentiation process of osteoblast-like cells MC3T3-E1 osteoblast and its effect on the cell mineralization and alkaline phosphatase （ALP） activity. Methods We cultured MC3T3-E1 cells and induced osteogenic differentiation, and then observed PAPSS2 expression during osteoblast differentiation process at the gene and the protein levels. PAPSS2 low expression shRNA lentivirus packaging and PAPSS2 retroviral vector were constructed, tittered, and checked for cell transfection efficiency. MC3T3-E1 was transfected and blank control group was set up. We observed changes of ALP activity and mineralization of osteoblasts after silence or high expression of PAPSS2. High expression vector was transfected with lentivirus-mediated RNA interference （RNAi） technology or retrovirus. Results The mRNA and protein expressions of PAPSS2 increased during differentiation of osteoblasts as the mineralization progressed. Our lentivirus-mediated RNAi technology successfully reduced the expression of PAPSS2 in MC3T3-E1 osteoblasts. And the retroviral transfection high expression vector significantly increased PAPSS2 gene expression. Silence PAPSS2 expression significantly decreased ALP activity and cell mineralization, contrary to its role in the high expression. Conclusion PAPSS2 can regulate ALP activity of osteoblasts and cell mineralization. Its abnormal expression may be related to bone lesion in Kashin-Beck disease.