骨形态发生蛋白-9对兔骨髓间充质干细胞诱导分化作用

Differentiation induced by bone morphogenetic protein-9 of rabbit bone marrow mesenchymal stem cells

目的 分离、培养并鉴定新西兰兔骨髓间充质干细胞（BMSCs）,观察BMSCs经腺病毒重组人骨形态发生蛋白-9（AdBMP-9）诱导后的成骨分化及其在明胶海绵上的生长.方法 采用全骨髓培养分离提取兔BMSCs,噻唑蓝（MTT）检测第2、3、4、5代细胞的增殖.流式细胞仪检测兔BMSCs表面抗原CD44和CD34.利用AdBMP-9转染第3代BMSCs,分别于诱导后7、14 d行碱性磷酸酶（ALP）染色、茜素红S染色和免疫荧光染色检测早期和晚期成骨标志物碱性磷酸酶、钙结节及骨钙素（OC）的表达.同时在14 d行油红O染色观察其成脂分化能力,荧光显微镜下观察BMSCs与明胶海绵复合生长.结果 成功分离提取兔BMSCs,经传代,细胞由长梭形变为短梭形,第3代形态、大小趋于稳定.MTT检测发现第2、3、4、5代细胞均呈对数生长,生长曲线近似“S”型,第2代细胞生长最慢,第4代生长最快（P＜0.05）.流式细胞仪检测显示,CD44和CD34的阳性率分别为94.38％和2.63％.AdBMP-9诱导后,可检测到早期和晚期成骨标志物及脂滴的生成,转染后的BMSCs可较好地与明胶海绵复合生长.结论 采用全骨髓培养法可分离得到较纯的兔BMSCs,在AdBMP-9诱导下,其可向成骨、成脂分化,且能较好地与明胶海绵复合生长.

Objective To isolate,culture and purify the New Zealand rabbit bone marrow mesenchymal stem cells （BMSCs）,research the osteogenic differentiation ability of BMSCs induced by recombinant adenovirus bone morphogenetic protein-9 （AdBMP-9） and the growth of BMSCs on absorbable gelatin sponge,expect to provide cytology basis for the study of bone tissue engineering.Methods Separated and obtained BMSCs by whole bone marrow culture method,the proliferation of the 2nd,3rd,4th,and 5th cell passages were analyzed by 3-（4,5-dimethyl-2-thiazolyl）-2,5-diphenyl-2-H-tetrazolium bromide （MTT）.Flow cytometry was used to confirm the expression of surface antigen marker CD34 and CD44.Take the third generation cell after 7 and 14 days inducing by AdBMP-9,use alkaline phosphatase staining,alizarin red S staining and immunofluorescence staining to detect the expression of early and late osteogenic markers alkaline phosphatase （ALP）,calcium nodules and osteocalcin,respectively.Rathonum red staining carried out at the 21th day to observe the adipogenic differentiation of BMSCs.Finally,observe the cell＇ s growth and expression in the gelatin sponge by fluorescent microscope.Results Successfully isolated BMSCs from New Zealand rabbit,after passaging constantly,the long fusiform shaped primary cell became short fusiform shaped,when passaged to the third generation,morphology and size of the cells tends to be stable and consistent.MTT showed that the 2nd,3rd,4th and 5th generation cells approximately proliferate like a logarithmic pattern,owning a growth curve of ＂S＂ type,the 2nd and 4th generation cells have the slowest and fastest proliferation rate,respectively （P ＜ 0.05）.Flow cytometry was used to quantify BMSCs percentages,CD44-positive cells were 94.38％,and the CD34-positive cells were 2.63％.After AdBMP-9 inducing,there can be detected early and late osteoblast markers and lipid droplets,and it showed that BMSCs can growth well in the gelatin sponge.Conclusion By using the wbole bone marrow culture method,we can separate and obtain purified BMSCs,which can be induced by AdBMP-9 to differentiate into osteoblasts and adipocytes,and it can also grow well with gelatin sponge,expect to use in the further study of bone tissue engineering.