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

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RNA...

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Published in西安交通大学学报（医学版） Vol. 35; no. 2; pp. 175 - 181
Main Author 王伟卓 程一钊 郭雄 王坤正 袁普卫
Format Journal Article
LanguageChinese
Published 西安交通大学医学院第二附属医院骨科,陕西西安,710004%西安交通大学医学院环境与疾病相关基因教育部重点实验室及卫生部微量元素与地方病重点实验室,陕西西安,710061%陕西中医学院附属医院骨科,陕西咸阳,712000 2014
Subjects
PAPSS2
基因功能
大骨节病
成骨细胞
碱性磷酸酶
细胞矿化
逆转录病毒
骨关节炎
成骨细胞
骨关节炎
基因功能
细胞矿化
PAPSS2
大骨节病
碱性磷酸酶
逆转录病毒
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ISSN1671-8259
DOI10.7652/jdyxb201402007

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Summary: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.
Bibliography: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 progres
ISSN:1671-8259
DOI:10.7652/jdyxb201402007