内参基因GAPDH和β-actin在BMP9诱导的骨髓间充质干细胞成骨分化过程中的表达情况

Expression of GAPDH and β-actin during osteogenic differentiation of BMP9-induced mesenchymal stem cell

摘要:

目的：观察常用内参基因甘油醛-3-磷酸脱氢酶（glceraldehyde-3-phosphate dehydrogenase，GAPDH）和β肌动蛋白（β-actin）在骨形态发生蛋白9（bone morphogenetic proteins 9，BMP9）诱导的骨髓间充质干细胞（mesenchymal stem cells，MSCs）成骨分化过程中表达情况，以确定相关研究的内参基因。方法：检测碱性磷酸酶（alkaline phosphatase，ALP）确定BMP9诱导的MSCs（包括C3H10、C2C12和MEF）向成骨分化；以real-time PCR分别检测0、1、3、5、7 d GAPDH和β-actin的mRNA表达水平；以Western blot检测GAPDH和β-actin蛋白。结果：（1）β-actin在BMP9诱导的MSCs成骨分化过程中mRNA表达水平逐渐升高，0 d与5 dβ-actin的mRNA表达量存在统计学差异（C3H10、C2C12和MEF的P值均为0.000，GAPDH 的mRNA表达水平未见明显改变（P ＞0.05）；β-actin蛋白表达量随成骨分化逐渐增加，而GAPDH蛋白表达未见明显变化。结论：在BMP9诱导的MSCs成骨分化过程中，GAPDH比β-actin更适合做内参。

Abstract:

Objective：To observe the changes of glceraldehyde-3-phosphate dehydrogenase（GAPDH） and β-actin gene expression during bone marrow mesenchymal stem cells（MSCs） differentiation to the osteogenic lineage induced by bone morphogenetic proteins 9 in order to select more suitable reference genes. Methods：Alkaline phosphatase（ALP） was used as a marker to identify BMP9-in－duced MSCs’ osteogenic differentiation；and mRNA and protein of GAPDH and β-actin were detected by real-time PCR and West－ern blot respectively on 0，1，3，5，7 d during MSCs’ osteogenic differentiation. Results：（1）mRNA expression of β-actin was signifi－cantly increased during osteogenic differentiation；there were significant differences in mRNA expression of β-actin between d0 and d5；no apparent change was observed in GAPDH. Results showed that the protein level of β-actin was increased gradually espe－cially on d5 and d7，while no apparent change was observed in GAPDH. Conclusion：During BMP9 induced MSCs’ osteogenic dif－ferentiation，GAPDH is more suitable than β-actin as internal reference gene.