兰克尔
分子生物学
化学
基因敲除
鱼腥草素骨
重组DNA
HEK 293细胞
转染
激活剂(遗传学)
病毒载体
骨钙素
细胞培养
同源重组
骨保护素
生物
基因
生物化学
碱性磷酸酶
遗传学
酶
作者
Junxiong Cheng,Dan Guo,Xi Chen,Ting Wang,Jiayi Huang
出处
期刊:PubMed
日期:2016-11-01
卷期号:32 (11): 1481-1485
摘要
Objective To construct a recombinant adenovirus expressing siRNA targeting human sclerostin (SOST) gene, and test the function of MG-63 cells while co-cultured with MDA-MB-231 cells infected by Ad-siSOST. MethodsAccording to the RNA sequence of SOST gene, two pairs of primers which contained 3 siRNA sequences were designed, and a pB2B plasmid was taken as template to amplify 2 DNA sequences. Both of the 2 DNA sequences were ligated to pAdTrace-OK by Gibson DNA Assembly way. After homologous recombination between recombinant shuttle plasmid and adenovirus vector plasmid, the adenovirus was packaged in HEK-293 cells. PCR and ELISA were used to test the expression of SOST in MDA-MB-231 cells which were infected with Ad-siSOST. In the co-culture system of MG-63 cells and MDA-MB-231 cells infected Ad-siSOST, osteoprotegerin (OPG), osteocalcin (OCN), integrin binding sialoprotein (IBSP) and receptor activator of nuclear factor-κB ligand (RANKL) were tested by quantitative real-time PCR. Results Recombinant shuttle plasmid which contained 3 interfering fragments was constructed successfully, and Ad-siSOST was obtained after homologous recombination and packaging. SOST expression in MDA-MB-231 cells was downregulated significantly after infeceted with Ad-siSOST. The mRNA levels of OPG, OCN, IBSP in MG-63 cells increased significantly, while the level of RANKL mRNA decreased significantly, and all 4 gene expressions were reversed after the infection of Ad-siSOST. Conclusion Knockdown of SOST in MG-63 cells increases osteogenesis and ratio of OPG/RANKL in vitro.
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