基因沉默组织型转谷氨酰胺酶抑制SaOS-2细胞成骨分化

Knockdown of tissue transglutaminase in SaOS-2 cell line inhibits its osteoblastic differentiation and mineralization

目的研究组织型转谷氨酰胺酶(tissue transglutaminase,TG2)是否参与人SaOS-2细胞系成骨分化过程。方法使用携带短发夹RNA(short hairpin RNA,shRNA)的慢病毒转染SaOS-2细胞以敲减TG2表达,以SaOS-2细胞及转染了含阴性对照shRNA病毒的SaOS-2作为对照组,分别进行体外成骨诱导培养,并进行以下检测:诱导14 d后各组矿化情况(茜素红染色);诱导4、7 d后碱性磷酸酶活性及I型胶原、骨钙素、骨形态发生蛋白-2(BMP-2)的mRNA表达,并与诱导前的表达水平相比较。结果 SaOS-2细胞组及转染阴性对照shRNA组在体外成骨诱导过程中I型胶原、骨钙素、BMP-2的mRNA表达和ALP活性逐渐增加,14 d时形成明显矿化结节,而TG2敲减后的SaOS-2细胞在诱导14 d时矿化水平显著低于对照组,诱导7 d时ALP活性及I型胶原、骨钙素、BMP-2的mRNA表达水平显著低于对照组。结论组织型转谷氨酰胺酶参与SaOS-2细胞体外成骨分化及矿化。

Objective To investigate whether TG2 plays an important role in the osteoblast differentiation and mineralization. Methods TG2 mRNA of SaOS-2 cells was knocked down using a lentivirus stably expressing short-hairpin（ sh） RNA targeting TG2. Then the cells were cultured in osteo-inductive medium for 14 d to measure mineralization and for7 d to measure the levels of osteoblastic differentiation markers including ALP activity and mRNA of collagen I,osteocalcin（ OCN） and BMP-2. The wild-type SaOS-2 cells and scrambled shRNA-transducted SaOS-2 cells served as the controls.Results The controls displayed an increasing trend of the level of ALP activity and mRNA of collagen I,osteocalcin and BMP-2,and notable mineralization at 14 d. When TG2 was knocked down,ALP activity,mRNA of collagen I,osteocalcin and BMP-2 at 7d,and mineralization at 14 d were all significantly lower in comparison with the corresponding values in the controls. Conclusion TG2 is involved in the differentiation and mineralization of osteoblasts in vitro.