BMP4促进人骨膜来源细胞体外成软骨细胞分化

BMP4 promote chondrogenic differentiation of human periosteum-derived cells in vitro

目的探讨细胞因子BMP4在骨膜来源细胞体外培养中的定向成软骨分化作用。方法采用人胫骨骨膜为材料，体外组织块法分离骨膜来源细胞，置于DMEM／F12完全培养基培养，倒置显微镜观察细胞形态，台盼蓝染色细胞计数法检测第3、9代骨膜来源细胞和BMP4诱导后细胞的增殖活性，绘制细胞生长曲线图。随机分为对照组、软骨诱导组和实验组，分别加入完全培养基、软骨诱导液和BMP4加软骨诱导液，第14和21天分别采用甲苯胺蓝染色和Ⅱ型胶原免疫组化染色检测细胞的蛋白多糖和Ⅱ型胶原；定量PCR检测软骨基因Aggrecan、Ⅱ型胶原和SOX9mRNA表达。结果（1）骨膜来源细胞在体外贴壁生长，经成软骨诱导1周后可见类圆形细胞形成，2周后细胞呈多角形，甲苯胺蓝染色呈阳性；细胞生长曲线证实诱导分化后细胞与第3代至第9代细胞增殖能力相似。（2）第14和21天软骨细胞标志物阳性率（％）实验组蛋白多糖（分别为40．29±4．29、56．74±5．12）和Ⅱ型胶原（分别为19．27±3．71、38．31±4．25）较软骨诱导组（第14和21天蛋白多糖分别为28．12±3．25、41．23±4．18，11型胶原分别为10．24±1．21、15．28±2．23）明显升高（P〈0．05）。（3）定量PCR：实验组Aggrecan（25．76-t-0．57）、Ⅱ型胶原（6．48±0．48）、SOX9（2．91±0．18）mRNA表达均明显高于软骨诱导组（11．12±0．38、2．24±0．41、1．54±0．35）和对照组（2．37±0．24、1．12±0．31、1．07±0．22）（P〈0．05）。结论骨膜来源细胞体外增殖能力强，经诱导可定向成软骨分化，BMP4在体外具有明显促进骨膜来源细胞分化为软骨细胞的作用。

Objective To explore biological characteristics of chondrogenic differentiation of human periosteum-derived cells and the role of BMP4 in chondrogenic differentiation of these cells. Methods From October 2009 to September 2012, periosteum was obtained from tibia of patients undergoing leg amputation surgery, and isolated periosteum-derived cells by tissue culture method. Cells were cultured in DMEM/F12 containing 10% fetal bovine serum, and morphology of cells were observed under inverted microscope. Periosteum-derived cells growth and the effect of BMP4 on cells growth examined by cell count using trypan blue, and cells growth curve was made. Experiment was divided into control group, chondrogenic differentiation group and BMP4 group, cells were expanded and differentiated in the presence or absence of BMP4 and complete medium. Then toluidine and immunohistoehemical staining analyzed proteoglycan and collagen Ⅱ expression of these cells after 14 and 21 days. The expression of aggrecan, collagen Ⅱ and SOX9 mRNA of these cells using real-time PCR. Results （ 1 ） Periosteum- derived cells adhered to growth in vitro, the shape of cell presented fibroblast-like morphology changing into polygonal after 1 week and round cell formation after 2 weeks chondrrogenic differerntiation. Growth curve showed that the passage 3 and 9 cells had similar reproductive activity. The passage 3 cells were positive for CD90 （ 21.07% ） and CDI05 （25.84%）. （2）Toluidine bule staining and type Ⅱ collagen immunohistoehemieal staining showed BMP4 group （40. 29 ± 4. 29, 56. 74 ±5.12 ） and chondrogenic differentiated group （ 19. 27± 3.71, 38.31 ±4. 25 ） ccould secrete proteoglycan and collagen Ⅱ , control group were negative （ 10. 24 ±1.21, 15.28 ± 2. 23 ） , BMP4 group were significantly than chondrogenic differentiated group. （ 3 ） The expression of aggrecan, collagen Ⅱ and SOX9 mRNA of BMP4 group（25.76±0. 57, 6.48±0.48, 2. 91 ±0. 18）were significantly higher than that of control group（2. 37 ±0. 24, 1. 12 ± 0. 31, 1.07 ± 0. 22）and chondrogenic differentiated group （ 11.12 ± 0. 38, 2. 24 ± 0. 41, 1.54 ± 0. 35 ） using real-time PCR. Conclusion Periosteum-derived cells have strong proliferative, and have good potentials of differentiating into chondroblasts like mesenchymal stem cells. BMP4 can promote chondrogenic differentiation of periosteum-derived cells in vitro cultures.