骨唾液酸蛋白在体外培养人骨髓间充质干细胞向成骨细胞分化中的角色

Role of bone sialoprotein in human bone marrow-derived mesenchymal stem cells differentiating into osteoblasts in vitro

目的:骨唾液酸蛋白在骨矿化形成方面扮演重要角色,实验观察其是否能诱导体外培养的人骨髓间充质干细胞向成骨细胞分化。方法:实验于2005-10/2006-12在解放军广州军区广州总医院医学实验科完成。①材料来源:选取在本院体检的健康志愿者2人,对本实验均知情同意。采用Ni-NTA亲和纯化技术,从本室构建的毕赤酵母GS115/pPICZaA-hbsp发酵上清中纯化重组人骨唾液酸蛋白。②实验方法:对健康志愿者进行髂骨穿刺抽取骨髓液,采用贴壁法培养得到骨髓间充质干细胞。设立4组:骨唾液酸蛋白组添加0.1nmol/L骨唾液酸蛋白;成骨诱导液组添加10nmol/L地塞米松、10mmol/L磷酸甘油、50mg/L抗坏血酸;联合组添加上述两组的所有试剂;空白对照组不添加任何处理因素;各组均处理细胞12d。③实验评估:光镜及电镜观察培养的细胞形态;以细胞计数法测定生长曲线,应用流式细胞仪分析细胞周期,采用免疫荧光细胞化学法和流式细胞分析检测干细胞标志物STRO-1的表达;生化试剂盒测定碱性磷酸酶活性;von Kossa染色法检测钙沉积。结果:①单个骨髓间充质干细胞为长梭形,经骨唾液酸蛋白处理后细胞大而扁平,原来密集的克隆分散开。②与空白对照组比较,骨唾液酸蛋白组引起细胞生长曲线右移,细胞G0/G1期比例平均增加12.09%(P<0.01),S期比例减少65.92%(P<0.01),STRO-1阳性细胞百分率下降26.54%(P<0.01)。③与空白对照组比较,骨唾液酸蛋白组细胞碱性磷酸酶活性增加50.0%,成骨诱导液组增加59.5%,联合组增加71.43%,并且随着处理时间的延长,活性增加越显著。④空白对照组细胞von Kossa染色呈阴性,其余各组均呈阳性。其中联合组的黑色矿化结节体积最大、数目最多;骨唾液酸蛋白组的结节体积较小、数目较少;成骨诱导液组居中。结论:骨唾液酸蛋白对人骨髓间充质干细胞有促进成骨分化和矿化作用,且与成骨诱导液联用效果更佳。

AIM： Bone sialoprotein （BSP） plays an important role in bone mineralization. Whether to enhance osteogenic differentiation of human bone marrow-derived mesenchymal stem cell （hBMSC） cultured in vitro is a sticking point. METHODS： The experiment was performed in Department of Medical Research of Guangzhou General Hospital of Guangzhou Area Military Command of Chinese PLA from October 2005 to December 2006. ①Materials： Two healthy volunteers in this hospital knew the fact and agreed to participate the study. By Ni-NTA affinity purification method, recombinant human BSP was obtained from zymolytic supernatant of Pichia pastoris GS115/pPICZaA-hbsp established in this lab.②Methods： hBMSCs were obtained from iliac bone marrow of healthy volunteers by adhesive culture method. There were 4 groups in this study： BSP group was added in 0.1 nmol/L BSP; osteogenic supplements group was added in 10 nmol/L dexamethasone, 10 mmoi/L β-glycerophosphate, and 50 mg/L ascorbic acid; combined group was added in all chemicals of the above two groups; control group received no treatment factors; Each group was treated for 12 days. ③Evaluations： Cell morphology was observed the light microscope and electron microscopy; Cell growth curves were tested by cell number counting method, cell cycle was examined with flow cytometry. Expression of stem cell marker STRO-1 was analyzed by immunofluorescence cytochemistry and flow cytometry, alkaline phosphatase （ALP） activity was tested by a specific biochemistry kit, and hydroxyapatite crystals were showed by von Kossa staining. RESULTS：①After treatment with BSP, shuttle-like hBMSCs became fiat, wide and scattered.②Compared with control, cell growth curve of BSP group moved rightward, fluorescence at G0/G1 phase increased 12.09% （P 〈 0.01） and that at S phase decreased 65.92% （P 〈 0.01）; STRO-1-positive cell number decreased 26.54% （P 〈 0.01）.③Compared with control, ALP activity of BSP group increased 50.0%, that of osteogenic supplements group increased 59.5%, that of combined group increased 71.43%, and with the treatment time longer, the activity became more higher.④The cells in control group were von Kossa staining-negative, while those in the other groups were all positive. Among.the other three groups, the black mineralized hydroxyapatite crystals in combined group were the most and the largest; those in BSP group was the least and the smallest; and those in osteogenic supplements group were placed in the middle. CONCLUSION： BSP can improve the differentiation and mineralization of hBMSCs, and the effects become stronger when it is applied with classic osteogenic supplements together.