CAJ | 학술논문

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.
목적 탐토각취당(CS)/류산포취당(DS)/중조인골형태발생단백-2(rhBMP-2)미구대대서골수기질간세포(BMSCs)증식여분화적작용. 방법 채용리자교련법제비CS/DS/rhBMP-2미구.취SD대서체외배양전지제3대적BMSCs-C57.실험분위4조(n=4):CS/DS/rhBMP-2미구조가입CS/DS/rhBMP-2미구,rhBMP-2조가입rhBMP-2,CS/DS조가입CS/DS미구,공백대조조.분별우배양2、4、6d응용사갑기우담서염비색법측정각조세포적증식정황;우성골유도배양1、3、5、7、9、11、14d채용감성린산매(ALP)검측시제합측정세포ALP활성;우성골유도배양7、14d응용천소홍염색법검측각조세포개결절적생장정황.결과 배양2、4、6d,각조지간BMSCs-C57적OD치비교차이균무통계학의의(P＞0.05).성골유도배양3d시,CS/DS/rhBMP-2미구조BMSCs-C57적ALP활성저우rhBMP-2조,차이유통계학의의(P＜0.05),단시배양5d후,CS/DS/rhBMP-2미구조BMSCs-C57적ALP활성명현고우rhBMP-2조、CS/DS조화공백대조조,차이균유통계학의의(P＜0.05).성골유도배양7d,CS/DS/rhBMP-2미구조화rhBMP-2조유개결절형성,이CS/DS조화공백대조조무개결절형성;배양14d,CS/DS/rhBMP-2미구조화rhBMP-2조개결절형성명현,이CS/DS조화공백대조조수연출현료개결절,단불명현. 결론 CS/DS/rhBMP-2미구대BMSCs-C57적증식무명현작용,단구비량호적BMSCs-C57성골유도급분화작용.
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.