壳聚糖／硫酸葡聚糖／重组人骨形态发生蛋白-2微球对大鼠骨髓基质干细胞增殖与分化的影响  

The effect of recombinant human bone morphogenetic protein-2 loaded chiston/dextran sulfate com-posite microspheres on proliferation and differentiation of marrow stromal stem cells in SD rats

作　　者：夏远军[1] 章莹[1] 李丽华[1] 尹庆水[1] 余翔[1] 黄显华[1] 谢会斌[1] 夏虹[1] 

机构地区：[1]广州军区广州总医院骨科医院创伤骨科、广州中医药大学,510010

出　　处：《中华创伤骨科杂志》2014年第5期421-426,共6页Chinese Journal of Orthopaedic Trauma

基　　金：国家自然科学基金（51303031）;广东省自然科学基金项目（S2012010009743）;广东省应用基础研究计划项目（2013J4100120）

摘　　要：目的 探讨壳聚糖（CS）/硫酸葡聚糖（DS）/重组人骨形态发生蛋白-2（rhBMP-2）微球对大鼠骨髓基质干细胞（BMSCs）增殖与分化的作用. 方法 采用离子交联法制备CS/DS/rhBMP-2微球.取SD大鼠体外培养传至第3代的BMSCs-C57.实验分为4组（n=4）：CS/DS/rhBMP-2微球组加入CS/DS/rhBMP-2微球,rhBMP-2组加入rhBMP-2,CS/DS组加入CS/DS微球,空白对照组.分别于培养2、4、6d应用四甲基偶氮唑盐比色法测定各组细胞的增殖情况;于成骨诱导培养1、3、5、7、9、11、14d采用碱性磷酸酶（ALP）检测试剂盒测定细胞ALP活性;于成骨诱导培养7、14d应用茜素红染色法检测各组细胞钙结节的生长情况.结果 培养2、4、6d,各组之间BMSCs-C57的OD值比较差异均无统计学意义（P＞0.05）.成骨诱导培养3d时,CS/DS/rhBMP-2微球组BMSCs-C57的ALP活性低于rhBMP-2组,差异有统计学意义（P＜0.05）,但是培养5d后,CS/DS/rhBMP-2微球组BMSCs-C57的ALP活性明显高于rhBMP-2组、CS/DS组和空白对照组,差异均有统计学意义（P＜0.05）.成骨诱导培养7d,CS/DS/rhBMP-2微球组和rhBMP-2组有钙结节形成,而CS/DS组和空白对照组无钙结节形成;培养14d,CS/DS/rhBMP-2微球组和rhBMP-2组钙结节形成明显,而CS/DS组和空白对照组虽然出现了钙结节,但不明显. 结论 CS/DS/rhBMP-2微球对BMSCs-C57的增殖无明显作用,但具备良好的BMSCs-C57成骨诱导及分化作用.Objective To observe the effect of composite microspheres of recombinant human bone morphogenetic protein-2 （rhBMP-2）/chitosan （CS）/dextran sulfate （DS） on the proliferation and differentiation of bone marrow stromal stem cells （BMSCs） in SD rats.Methods rhBMP-2/CS/DS nanometer microspheres were prepared using ionic crosslinking.The third passage of BMSCs-C57 cultured from the SD rats in vitro was harvested for the experiment which was divided into 4 groups （n =4）.Group A was added with CS/DS/rhBMP-2 microspheres,group B with rhBMP-2,group C with DS/CS microspheres,and group D served as blank control.The cellular proliferation in every group was determined by MTT （thiazole blue） colorimetric method on days 2,4 and 6 after culture.The alkaline phosphatase （ALP） activity was detected on days 1,3,5,7,9,11 and 14 after osteoinduction culture.The formation of calcium nodules was observed by Von Kossa staining on days 7 and 14 after osteoinduction culture.Results The OD values of BMSCs-C57 on days 2,4 and 6 showed no significant between-group differences （P 〉 0.05）.The cellular ALP activity in group A was significantly lower than in group B on day 3 after osteoinduction （P 〈 0.05）,but was significantly higher on day 5 than in groups B,C and D （P 〈 0.05）.On day 7 after osteoinduction,calcium nodules formed in groups A and B,but did not in groups C and D.On day 14,calcium nodules formed obviously in groups A and B but slightly in groups C and D.Conclusions CS/DS/rhBMP-2 microspheres have no significant effect on the proliferation of BMSCs-C57,but show a fine effect on the osteoinduction and differentiation of BMSCs-C57.

关 键 词：骨形态发生蛋白质类 硫酸葡聚糖 壳聚糖 骨髓 间质干细胞 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]