骨碎补提取物促小鼠成骨细胞株MC3T3-E1细胞增殖、分化和钙化作用的研究

Effects of traditional chinese medicine Drynaria fortunei smith on promoting the proliferation,differentiation and calcification of mouse osteoblastic MC3T3-E1 cells

目的 :探讨中药骨碎补有无促进成骨细胞增殖、分化和钙化的作用。方法 :制备骨碎补的水、醇提取液。小鼠成骨细胞株MC3T3 E1作为药物筛选的细胞模型 ;用MTT法和流式细胞术测定不同浓度的骨碎补水、醇提取液的促细胞增殖作用 ;采用ALP活性和骨钙素定量检测分别观察上述药物提取液的促细胞分化作用 ;以Vonkossa钙化染色法了解上述药物提取液的促细胞钙化作用。结果 :0 .0 1,1mg·L-1骨碎补水提液能促进MC3T3 E1细胞数量增加 (P <0 .0 5 ) ;0 .0 1,1mg·L-1骨碎补水提液和醇提液能使S期细胞百分率升高、G1期细胞百分率减少 ;1,10 0mg·L-1骨碎补醇提液能使细胞ALP的活性升高 (P <0 .0 5 ) ;10 0mg·L-1骨碎补醇提液能明显促进细胞骨钙素合成和分泌 (P <0 .0 0 1) ;1mg·L-1骨碎补水提液及 0 .0 1mg·L-1骨碎补醇提液均可促进细胞钙化 (P <0 .0 5 )。结论 :骨碎补水相和醇相提取物中分别存在有较高活性的促成骨细胞增殖、分化和钙化的物质。

Objective: To explore the effects of Drynaria fortunei J. Smith (DFS) on promoting the proliferation,differentiation and calcification of cells. Method: Two DFS preparations were extracted with distilled water (DFS aqueous-extract) and 95 % ethanol (DFS ethanol-extract),respectively. A mouse osteoblast cell line MC3T3-E1 was used as a cell model for screening potency of DFS. MTT and Flow cytometry were applied to determine proliferation of the cell promoted by DFS aqueous-and ethanol-extracts at different dosages. Differentiating effects of the two extracts with different concentrations in the cell were evaluated through the examinations of alkali phosphate (ALP) activities and osteocalcin levels. Von kossa staining method was used to understand the effects of the two extracts promoting calcification of the cell. Result: 0.01 mg·L -1 and 1 mg·L -1 of DFS aqueous-extracts showed the proliferative promotion for MC3T3-E1 cell ( P <0.05). Both the 0.01 mg·L -1 and 1 mg·L -1 of the two DFS extracts increased the percentages of S-phase cells whereas the percentages of G 1-phase cells were decreased. 1 and 100 mg·L -1 of the ethanol-extract remarkably increased the synthesis and secretion of osteocalcin of the cell ( P <0.001). 1 mg·L -1 of the aqueous-extract and 0.01 mg·L -1 of the ethanol-extract were able to promote the cell calcification ( P <0.05). Conclusion: In the DFS aqueous-and ethanol-extracts,there are some of the components promoting the proliferation,differentiation and calcification of osteoblast.