rhBMP-2诱导脂肪成体干细胞向成骨细胞分化的量效作用

Effect of rhBMP-2 in Vitro on Differentiation of Adipose-derived Adult Stem Cells into Osteoblasts

目的探讨体外条件下rhBMP-2诱导脂肪成体干细胞(adipose-derived adult stem cells,ADASCs)向成骨细胞分化和增殖的情况,并观察其量效关系。方法从成年兔颈后部获取脂肪组织,采用酶消化法分离并培养脂肪成体干细胞。稳定传代后,用不同浓度rhBMP-2对ADASCs进行诱导分化,采用碱性磷酸酶染色及Ⅰ型胶原免疫组织化学染色进行成骨细胞鉴定,采用碱性磷酸酶活性测定及MTT比色法进行分化和增殖活性比较。结果碱性磷酸酶及Ⅰ型胶原免疫组织化学染色为阳性;当rhBMP-2浓度在100～400ng/ml时,各组碱性磷酸酶活性提高明显(P<0.05),在400ng/ml以上各组则无明显差异(P>0.05);当rhBMP-2浓度在800ng/ml以下时,各组增殖活性无明显差异(P>0.05),在800ng/ml以上则存在明显的抑制作用,各组存在明显差异(P<0.05)。结论研究表明,在体外条件下rhBMP-2能诱导脂肪来源干细胞向成骨细胞分化;并且当其浓度在400～800ng/ml时,能促进脂肪来源干细胞的增殖和分化。

Objective To investigate the effect of rhBMP - 2 on differentiation and proliferation of cultured adipose derived adult stem cells （ADASCs） in vitro, and observe whether there is a dose - effect. Methods The fat tissue was resected from posterior cervical region of rabbits. Stem cells was isolated by type I collagenase digesting. ADASCs were cultured in vitro, and induced to differentiate into osteoblasts by rhBMP - 2 of different concentrations. Ostcoblasts were identified by ALP staining and type Ⅰ immunohistochemistry stain. The effect of rhBMP - 2 on differentiation and proliferation of the stem cells was compared by determining ALP and MTT activity. Results The cells isolated from rabbit adipose tissue could rapidly expand, ALP staining and type Ⅰ collagen immunohistochemistry staining showed positive; ALP activity increased rapidly with the increase of the dose of rhBMP- 2 between 100 and 400 ng/ml （P〈 0.05） without significant difference among the groups over 400 ng/ml （P 〉 0.05 ） ; there was no significant difference in proliferation activity below 800 ng/ml rhBMP - 2 （P 〉 0.05 ） while over 800 ng/ml of rhBMP - 2 could inhibit proliferation significantly （P 〈 0.05）. Conclusion In vitro, rhBMP - 2 could induce adipose derived stem cells into ostcoblasts. 400 - 800 ng/ml rhBMP - 2 could promote differentiation and proliferation of adipose derived stem cells significantly.