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[目的]体外分离培养非人类灵长类动物恒河猴骨髓MSCs,研究其生长、扩增及诱导分化为成骨细胞的特性。[方法]梯度离心法分离、纯化恒河猴骨髓源MSCs,观察不同接种密度和换液时间对细胞生长的影响;在体外应用成骨添加剂(含地塞米松10-7mol/L、β-甘油磷酸钠10 mmol/L、维生素C 50 mg/L)或rhBMP-2(100 ng/L)定向诱导分化,对分化后的细胞进行免疫组化染色和ALP、BGP定量测定以鉴定成骨细胞,并比较两种定向诱导分化方法的效果。[结果]梯度离心法分离、纯化得到的恒河猴骨髓源MSCs具有分裂和克隆增殖的能力,种植细胞密度为10×104~30×104/cm2,48 h后半量换液,以后每3 d全量换液的方法较合理,MSCs能少量表达ALP活性,分泌的钙量较少;诱导分化后的细胞具有和体内成骨细胞相同的形态学特征,ALP染色阳性,能表达Ⅰ型胶原,不表达Ⅱ型胶原,细胞融合后3 d,ALP和BGP分泌明显增加,融合后14 d细胞ALP、BGP的分泌水平略有升高,但无明显增加;采用成骨添加剂和rhBMP-2诱导分化MSCs后相同时期成骨细胞ALP活性、BGP含量无明显差异。[结论]密度梯度离心法分离得到的非人类灵长类动物骨髓源MSCs,体外培养具有分裂、克隆增殖的能力,在加有成骨细胞诱导剂或rhBMP-2的培养基里能诱导分化为成骨细胞,化学药物在定向诱导分化过程中具有和生长因子相一致的作用。
[Objective] The research aimed to isolate and culture the non-human primate rhesus monkey bone marrow MSCs in vitro and study their growth, expansion and differentiation into osteoblasts. [Method] The MSCs of bone marrow derived from rhesus monkey were isolated and purified by gradient centrifugation. The effects of different inoculation density and liquid exchange time on the growth of MSCs were observed. The effects of osteogenic additives (including dexamethasone 10-7mol / L, β-glycerol (10 mmol / L sodium phosphate, 50 mg / L vitamin C) or rhBMP-2 (100 ng / L). The differentiated cells were harvested for immunohistochemical staining and ALP and BGP quantification to identify osteoblasts, And compare the effects of two methods of directed differentiation. [Result] The MSCs isolated and purified by gradient centrifugation had the ability to divide and clone, the cell density was 10 × 104 ~ 30 × 104 / cm2 and the volume was changed after 48 h, and every 3 d The method of full volume fluid exchange is more reasonable. MSCs can express ALP activity in a small amount and less calcium is secreted. The differentiated cells have the same morphological characteristics as osteoblasts in vivo. Positive ALP staining can express type I collagen. Expression of collagen type Ⅱ, ALP and BGP secretion increased significantly 3 days after cell fusion, ALP and BGP secretion slightly increased 14 days after fusion, but no significant increase; using osteogenic additives and rhBMP-2 induced differentiation of MSCs After the same period of osteoblast ALP activity, BGP content no significant difference. [Conclusion] The MSCs derived from non-human primates isolated by density gradient centrifugation have the ability of dividing and cloning in vitro, and can differentiate into medium containing osteoblast inducer or rhBMP-2 Osteoblasts and chemotherapeutic drugs have the same function as growth factors in the process of directional differentiation.