Effects of Kangguzengsheng Capsules-containing serum on the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.2017.09.023

Abstract

Abstract:

BACKGROUND: Our previous studies have found that the Kangguzengsheng Capsules can promote fracture healing.
OBJECTIVE: To explore the influence of Kangguzengsheng Capsules-containing serum on the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells.
METHODS: Forty Sprague-Dawley rats were randomly divided into four groups. The capsule powder was resolved into 1 mL natural saline and intragastrically administered into rats according to 1.16 g/100 g, 3.48 g/100 g, 10.44 g/100 g in low, medium and high dose groups. Rats in control group were given equal volume of natural saline. After consecutive administration for 12 days, blood samples from the abdominal aorta were collected and serum samples were isolated and preserved until use. Bone marrow mesenchymal stem cells were isolated and purified by the whole marrow adhesion method, and identified by flow cytometry. Harvested cells were divided into four groups and cultured in the osteogenic culture medium containing different kinds of serum samples as described above. MTT method was adopted to test the cell proliferation at 24, 48 and 72 hours of culture. Alkaline phosphatase activity in cells was detected at 7 and 14 days of culture. Alkaline phosphatase staining was performed at 7 days of culture, and alizarin red staining performed at 14 days of culture.
RESULTS AND CONCLUSION: Compared with the blank control group, bone marrow mesenchymal stem cells showed no significant changes in the proliferative ability and alkaline phosphatase activity in the low dose group, but these two indices were significantly increased in the high and medium dose groups at 48 and 72 hours of culture or at 7 and 14 days of culture, respectively (P < 0.05). Cells positive for alkaline phosphatase and alizarin red staining were observed in the low, medium and high dose groups, and the cell staining was most remarkable in the high dose group. To conclude, Kangguzengsheng Capsules-containing serum can promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells.

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: Stem Cells, Mesenchymal Stem Cells, Cell Proliferation, Tissue Engineering

CLC Number:

R394.2

Song Yong-zhou, Tong Jiu-hui, Liu Hui-ling, Ma Wei, Li Ming. Effects of Kangguzengsheng Capsules-containing serum on the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(9): 1439-1443.

Figures/Tables 2

2.1 BMSCs培养和表型测定原代培养的细胞形状不规则，有长梭形、多角形等，培养的第一二天开始有少量细胞贴壁，半量换液后，细胞团块克隆生长，增殖迅速，7-9 d长满瓶底，呈纺锤形，细胞整体排列呈漩涡状、辐射状样排列；传代后细胞贴壁及生长更加迅速，24 h内即可完全贴壁，六七天可铺满瓶底，细胞形态更均一，整体呈现漩涡状、辐射状或鱼群样排列(图1)。经流式细胞仪检测，CD44、CD29、CD90均为阳性表达，阳性表达率分别为99.3%、99.2%及99.97%；CD14、CD34、CD45均为阴性表达(图2)，符合BMSCs的生物学特性。 2.2 MTT检测细胞增殖结果低剂量组不同时间点的细胞A值与空白组无差异(P > 0.05)，提示对BMSCs增殖无促进作用；除中剂量组24 h外，中、高剂量组其余各时间点细胞A值均高于空白组(P < 0.05)，提示对BMSCs增殖有促进作用；高剂量组不同时间点的A值高于中剂量组(P < 0.05)，见表1，说明高剂量组对BMSCs的增殖促进作用更明显。 2.3 碱性磷酸酶活性测定结果低剂量组不同时间点的A值与空白组无差异(P > 0.05)；中、高剂量组不同时间点的A值高于空白组(P < 0.05)，说明中、高剂量抗骨增生胶囊对BMSCs分泌碱性磷酸酶有促进作用；高剂量组不同时间点的A值高于中剂量组(P < 0.05)，说明高剂量组对BMSCs分泌碱性磷酸酶作用更明显，见表2。 2.4 碱性磷酸酶及茜素红染色结果诱导第7天，碱性磷酸酶染色可见胞浆内被染成红色颗粒状碱性磷酸酶，表明诱导后的细胞有内源性碱性磷酸酶的表达，其中低、中、高剂量组胞浆阳性颗粒数均较空白组增加，且随剂量增加阳性颗粒数增加(图3)。诱导第14天，胞外基质中有大小不等的聚集团状结节，茜素红染色将结节中沉积的钙盐染成红色，低、中、高剂量组钙结节数量及面积均较空白组增多，高剂量组形成的结节更大、更致密(图4)。"

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