FnCBD64增强骨髓基质干细胞黏附力的实验研究

Study on Effect of carboxy-terminal cell-binding domain of recombination fibronectin on adhesion of bone marrow stromal cells: Experimental study

目的观察预涂蛋白对细胞黏附的影响,以寻找增强细胞黏附力的有效方法。方法①取四个96孔板分为四组:Fn、FnCBD64(重组纤维连接蛋白羧基端细胞结合域)、牛血清白蛋白BSA及未预涂组,前三种蛋白分别预涂孔底,未预涂组为对照组。分八个时点,间隔30min,每孔接种骨髓基质干细胞悬液,细胞数为5×104个,MTT检测;②无菌条件下将脱细胞瓣叶圆片置入12孔板中(心室面向上)别给,分予Fn、FnCBD64、牛血清白蛋白BSA预先包埋,未预包埋作为对照组。接种细胞悬液,每只瓣叶种植细胞数为3×10个。在开始种植细胞的第8天取瓣叶进行细胞计数和MTT检测。结果各组随着时间的延长5MTT的OD吸光值增加,组内各时点比较有显著性差异。经Fn和FnCBD64预涂组其MTT的OD吸570570光值在各时点均明显高于预涂BSA和对照组。Fn组与FnCBD64组相比无显著性差异。脱细胞瓣叶进行预处理后再种植骨髓基质干细胞,经Fn和FnCBD64预处理组,其细胞计数、MTT的OD570明显高于预涂牛血清白蛋白及对照组。结论Fn和FnCBD64促进细胞的聚集、黏附及增殖,是制备组织工程瓣的有用方法。

Objective To study the methods for promoting the adhesion of bone marrow stromal cells(BMSCs).Methods (1)Four 96-well plates were precoated with fibronectin(Fn) and carboxy-terminal cell-binding domain of fibronectin(FnCBD64),bovine serum albumin, respectively; the control group were not precoated. The bone marrow stromal cells were seeded in them at different timepoint, the intervals were 30min.After four hours, the cell numbers were observed in per well by MTT assay.(2) Four groups decellular porcine heart valve leaflets were used as scaffold. They were pre-conditioned by Fn, FnCBD64, BSA for 12h, then BMSCs were seeded onto them at 24h intervalfor 3 times. Later 7 days after seeding,the adhesion of BMSCs were observed by cell counting and MTT.Results (1)the MTT absorbency value(OD570)of each group were increasing along with time and the values was of significant difference at different timepoint. The values of Fn group and FnCBD64 group were more than BSA group and control group, there was no significant difference Fn group vs FnCBD64group,BSA group vs control,respectively. (2) The adhesioned cell numbers of Fn group and FnCBD64 group were found more than BSA group and control group by cell counting,MTT(P<0.01).There was no significant difference,Fn group vs FnCBD64 group, BSA group vs control group, respectively.Conclusion Fn and FnCBD64 can promote the BMSCs adhesion obviously and pro1iferation on the scafo1d.These methods are helpful to creating tissue-engineered heart valve in vitro.