Expression of SP7/Osterix and alkaline phosphatase in bone marrow mesenchymal stem cells with Cbfal/RUNX2 gene silencing regulated by the water extracts from Bushen Huoxue Decoction

doi:10.3969/j.issn.2095-4344.0493

Abstract

Abstract:

BACKGROUND: Bushen Huoxue Decoction (BSHXD) can promote osteogenesis of bone marrow mesenchymal stem cells (BMSCs) in vitro. Exploring the molecular mechanisms involved is of clinical benefits.
OBJECTIVE: To discuss the changes in the expression of SP7/Osterix and alkaline phosphatase (ALP) in BMSCs with Cbfal/RUNX2 gene silencing regulated by the water extracts from BSHXD.
METHODS: BMSCs were isolated and cultured by the bone marrow adherent method, and BMSCs at passage 3 were used in the assay. BMSCs were transfected with nothing (blank control group), Cbfal/RUNX2 gene silencing lentivirus (silencing group), and negative viral vector (negative control group), respectively. Then, the cells were cultured in 100 mg/L BSHXD water extract, and 3 days later, the protein and mRNA expression of RUNX2 and Osterix was detected by western blot and qPCR, respectively. Activity of ALP in the BMSCs was also detected in each group.
RESULTS AND CONCLUSION: The transfection efficiency of Cbfal/RUNX2 gene silencing lentivirus was about 90%. The protein and mRNA expressions of RUNX2 and Osterix were significantly decreased in the BMSCs transfected with Cbfal/RUNX2 gene silencing lentivirus as compared with the other two groups, and so was the ALP activity (P < 0.01). After treated with the water extracts from BSHXD, the expression of RUNX2 and Osterix as well as the ALP activity in the BMSCs transfected with Cbfal/RUNX2 gene silencing lentivirus increased significantly (P < 0.01). To conclude, the water extract from the BXHXD can up-regulate the expression of RUNX2 and Osterix and the activity of ALP, thus promoting BMSCs osteogenic differentiation.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Bone Marrow, Mesenchymal Stem Cells, Gene Silencing, Core Binding Factor Alpha 1 Subunit, Drugs, Chinese Herbal, Tissue Engineering

CLC Number:

R394.2

Cheng Ying-xiong, Luo Yi-wen, Wang Bin, Wu Zhi-fang, Shen Wei, Luo Hui, Sun Shi-dong, Huang Wen-qiang. Expression of SP7/Osterix and alkaline phosphatase in bone marrow mesenchymal stem cells with Cbfal/RUNX2 gene silencing regulated by the water extracts from Bushen Huoxue Decoction[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(13): 1987-1992.

Figures/Tables 1

2.1 骨髓间充质干细胞成骨分化鉴定结果见图1。第3代骨髓间充质干细胞经成骨诱导21 d后形成大量的成骨细胞，由长梭形、成纤维样转变成多角形，多层、重叠样排列，鲜红色为钙结节形成。 2.2 第3代骨髓间充质干细胞慢病毒转染后形态及转染效率见图2。转染后第3代骨髓间充质干细胞大部分形态仍呈长梭形、成纤维样，集落成长，融合度为80%-90%；荧光显微镜下观察，带绿色荧光的转染细胞可达90%左右。 2.3 qPCR检测RUNX2及Osterix mRNA表达见图3，4。RUNX2沉默病毒转染骨髓间充质干细胞，与空白对照组、阴性对照组比较，沉默组RUNX2和Osterix的mRNA表达均降低，差异有显著性意义(P < 0.01)；加入补肾活血汤水提物后，空白对照组、阴性对照组和沉默组RUNX2和Osterix的mRNA表达均有升高，差异有显著性意义(P < 0.01)。在补肾活血汤处理前后，阴性对照组RUNX2和Osterix mRNA表达较空白对照组均无明显差异(P > 0.05)。补肾活血汤组处理后，沉默组RUNX2、Osterix mRNA表达仍较空白对照组、阴性对照组低，差异有显著性意义(P < 0.01)。 2.4 Western blot检测RUNX2及Osterix蛋白表达见图5。RUNX2沉默病毒转染骨髓间充质干细胞后，RUNX2、Osterix蛋白表达较空白对照组、阴性对照组均下降(P < 0.01)，而空白对照组与阴性对照组比较，RUNX2和Osterix蛋白表达几乎无明显变化(P > 0.05)。而3组加入补肾活血汤处理后，RUNX2和Osterix蛋白表达均有明显提高(P < 0.01)。补肾活血汤组处理后，沉默组RUNX2、Osterix蛋白表达仍较空白对照组、阴性对照组低，差异有显著性意义(P < 0.01)。 2.5 碱性磷酸酶活性见图6。RUNX2沉默转染后，骨髓间充质干细胞的碱性磷酸酶活性较空白对照组、阴性对照组均下降，差异有显著性意义(P < 0.01)，而空白对照组和阴性对照组的碱性磷酸酶活性差异无显著性意义(P > 0.05)。加入补肾活血汤处理后，3组的碱性磷酸酶活性均有所上升，差异有显著性意义(P < 0.01)。补肾活血汤组处理后，沉默组碱性磷酸酶活性仍较空白对照组、阴性对照组低，差异有显著性意义(P < 0.01)。"

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