犬骨髓基质干细胞诱导为成骨细胞的实验研究

Experimental Study of the Inducing BMSCs into Osteogenesis

目的研究骨髓基质干细胞(Bonemarrowstromalcells,BMSCs)在体外培养、诱导后的成骨活性,探讨BMSCs作为骨组织工程种子细胞的可能性和可行性。方法抽取成年犬骨髓,用梯度离心法获取单核细胞,经条件培养液体外诱导培养后,进行组织化学、免疫组化、原位杂交检测,并在透射电镜下观察其成骨活性。结果第1代细胞Alkalinephosphatase(AKP)染色、Core-bindingfactoralphasubunit1(Osf2/Cbfa1)、Osteopotin(OPN)、Ⅰ型胶原的免疫细胞化学检测和I型胶原的原位杂交结果开始呈现阳性,第3代细胞BoneSailoplain(BSP)、Osteocalcin(OCN)免疫细胞化学染色开始呈现阳性,透射电镜显示细胞外有胶原分泌和钙盐沉积。结论BMSCs不仅能大量扩增,且在一定条件下可向成骨细胞分化,是骨组织工程理想的种子细胞。

Objective To study the bone tissue osteogenesis ability of bone marrow stromal cells (BMSCs) in vitro and in vivo. Methods The BMSCs were separated by gradient centrifugation on Percoll, culture-expanded and induced with a conditional medium. The cells were examined by transmission electron microscopy and histochemistry or immunochemistry stains technique including AKP,Cbfa1,OCN, OPN, BSP and collagen type Ⅰ. The mRNA of collagen type I in cultured cell was also examined by in situ hybridization. Results Histochemical studies showed positive stain of AKP,Cbfa1, OCN, OPN,BSP and collagen type Ⅰ,and in situ hybridization also showed the positive express of mRNA of collagen type I. The excretion of collagen and deposition of calcium salt were observed under transmission electron microscopy. Conclusion BMSCs cultured in conditional medium shows osteoblast-like functions in vitro, which provides an ideal seed cells for bone tissue engineering.