IGF-Ⅰ联合BMP-2促MC 3T3-E1和NIH 3T3细胞的增殖和分化及钙化效应

Effects of IGF-Ⅰ and BMP-2 combined application on promoting proliferation,differentiation and calcification of MC 3T3-E1 and NIH 3T3 cells

目的：探讨重组人胰岛素样生长因子-Ⅰ（rhIGF—Ⅰ）和重组人骨形成蛋白-2（rhBMP-2）单独或联合应用对MC3T3-E1和NIH 3T3细胞增殖、分化和钙化的影响。方法：单独或联合应用不同浓度rhIGF—Ⅰ和rhBMP-2作用于小鼠成骨样细胞株MC3T3-E1和小鼠成纤维细胞株NIH 3T3，采用四唑盐比色法（MTT）和流式细胞技术检测细胞增殖，碱性磷酸酶（ALP）试剂盒检测细胞分化，放射免疫法检测细胞分泌的骨钙素水平（OC），以及Von kossa钙化染色法观察细胞的钙化。结果：1～50ng／ml rhIGF—Ⅰ作用于MC 3T3-E1细胞或5～75ng／ml rhIGF—Ⅰ作用于NIH 3T3细胞后，均能显示明显的促细胞增殖作用（P〈0．01），使S期细胞百分率升高、G1期细胞百分率减少，以及使胞内ALP活性、钙化面积百分比增高（P〈0．05）。10～100ng／ml rhBMP-2也可促进MC 3T3-E1和NIH 3T3细胞的增殖作用（P〈0．01），使S期细胞百分率升高、G1期细胞百分率减少，并使2种细胞ALP活性、钙化面积百分比升高（P〈0．05）。各浓度rhIGF—Ⅰ或rhBMP-2对MC 3T3-E1及NIH 3T3细胞作用8h、24h和48h的MTT检测结果相似。rhIGF—Ⅰ和rhBMP-2联合作用后，对2种细胞的促增殖、增强ALP活性、促钙化的作用均较各自单独作用更为明显（P〈0．05）。单独或联合应用不同浓度rhIGF—Ⅰ和rhBMP-2对细胞分泌的OC水平均无显著性差异（P〉0．05）。结论：rhIGF—Ⅰ和rhBMP-2具有明显的促进细胞增殖、早期分化及钙化的协同作用，但对成骨末期细胞分化影响不大，而其活性主要与使用剂量有关。

Objective： To demonstrate the effects of recombinant human insulin-like growth factor- I （rhIGF- I ） and/or recombinant human bone morphogenetic protein-2 （rhBMP-2） on proliferation,differentiation and calcification of MC 3T3-E1 cells and NIH 3T3 cells. Methods： Mouse osteoblast-like cell line MC 3T3-E1 and mouse fibroblast cell line NIH 3T3 were treated with different dosages of rhIGF- I or rhBMP-2 and rhIGF- I plus rhBMP-2. Cell proliferation was measured by methylthiazol tetrazolium （ MTT ） method and flow cytometry. Cell differentiation was examined by using alkaline phosphatase （ ALP ） measurement kit. Radioimmunoassay was applied to detect levels of osteocalcin （OC） secreted by cultured cells. Von kossa staining method was used to study the calcification effects. Results： MC 3T3-E1 cells treated with 1 -50 ng/ml rhIGF-I and NIH 3T3 cells treated with 5- 75 ng/ml rhIGF-I showed marked effects of promoting proliferation （P〈0.01）,increasing the percentages of S- phase cells,decreasing the percentages of G1-phase cells and increasing activities of cellular ALP and percentages of calcification area （P〈0. 05）. 10-100 ng/ml rhBMP-2 was also able to promote proliferation （P〈0. 01 ）, increase the percentages of S-phase cells, decrease the percentages of G1-phase cells and enhancing cellular ALP activities and percentages of calcification area for both the two cells （P〈0.05）. The results of MTT were similar,when the same different concentrations of rhIGF-I and rhBMP-2 treated for 8, 24, 48 h, respectively. rhIGF- I plus rhBMP-2 showed stronger effects on promoting proliferation, increasing cellular ALP activities and promoting calcification for both cell lines than using any one of the two cytokines （P〈0.05）. However,no significant changes in levels of OC secreted by the two cell lines were found when using different dosages of rhIGF-I or rhBMP-2 and rhIGF-I plus rhBMP-2（P〈0. 05）. Conclusion： rhIGF- I and rhBMP-2 have synergistical effects on promoting cell proliferation,early cell differentiation and calcification depending on the used dosages ,but no significant effects on promoting advanced cell differentiation.