CD44分子岩藻糖基化修饰对兔BMSCs流体黏附力的影响

The effects of CD44 fucosylation on fluid adhesion force of rabbit bone marrow mesenchymal stem cells

目的探讨CD44分子岩藻糖基化修饰对兔BMSCs流体黏附力的影响。方法采用密度梯度离心结合贴壁培养的方法体外分离纯化获得兔BMSCs,倒置显微镜观察细胞生长状况,流式细胞仪检测细胞表面MSCs相关抗原CD44、CD34、CD29、CD105的表达。使用α-1,3-岩藻糖转移酶Ⅵ(α-1,3-fucosyltransferaseⅥ,FTⅥ)对兔BMSCs进行体外岩藻糖基化处理作为实验组,以未经岩藻糖基化处理的BMSCs作为对照组,采用流式细胞仪检测唾液酸化Lewis X(sialyl-Lewis X,sLe^X)阳性率及E-选择素结合率。使用Hank平衡盐溶液重悬的岩藻糖基化BMSCs作为实验组(A组),未经岩藻糖基化修饰的BMSCs作为研究对照组(B组),添加EDTA冲洗的岩藻糖基化兔BMSCs作为阴性对照组(C组),利用平行板流室黏附试验检测兔BMSCs流体黏附力。结果兔BMSCs形态以长梭形为主,生长旺盛;第3代BMSCs CD44、CD29、CD105呈阳性表达,而CD34呈阴性表达。实验组sLe^X阳性率为32.52%±1.76%,显著高于对照组的1.48%±0.51%,差异有统计学意义(t=29.277,P=0.000);实验组E-选择素结合率为41.05%±1.84%,显著高于对照组的4.33%±0.92%,差异有统计学意义(t=35.674,P=0.000)。随着流体剪切力的增加,A组人脐静脉内皮细胞(human umbilical vascular endothelial cells,HUVEC)表面黏附的BMSCs数量先升高后降低,而B、C组HUVEC表面几乎无BMSCs黏附。各剪切力作用下,A组HUVEC表面黏附的BMSCs细胞数均显著多于B、C组,差异有统计学意义(P<0.05),B、C组间差异无统计学意义(P>0.05)。结论 CD44分子岩藻糖基化修饰能够增强兔BMSCs的流体黏附力。

Objective To investigate the effect of CD44 fucosylation on fluid adhesion force of rabbit bone marrow mesenchymal stem cells（BMSCs）. Methods The rabbit BMSCs were isolated and purified by density gradient centrifugation combined with adherent culture method. The morphology of cells were observed by inverted microscope,and the cell surface markers of CD44, CD34, CD29, and CD105 were assessed by flow cytometry. BMSCs fucosylated by alpha-（1, 3）-fucosyltransferase Ⅵ（FTⅥ） were as the experimental group, and the non-fucosylated BMSCs were as the control group, and then the positive rate of sialyl-Lewis X（sLe^X） and the binding rate of E-selectin were detected by flow cytometry. The fucosylated BMSCs resuspended in Hank balanced salt solution（HBSS） were assigned as the experimental group（group A）, at same time, the non-fucosylated BMSCs resuspended in HBSS solution as the study control group（group B）, and the fucosylated BMSCs resuspended in HBSS solution which was added EDTA as negative control group（group C）. The fluid adhesion force of rabbit BMSCs were detected by the parallel flow chamber adhesion test. Results Primary BMSCs mainly shaped as spindle and kept strong growth. The third generation BMSCs were negative for CD34, but positive for CD44, CD29, and CD105. After fucosylation, the positive rate of sLe^X in the experimental group was 32.52%±1.76%, which was significantly higher than that in the control group（1.48%±0.51%）（t=29.277, P=0.000）. The binding rate of E-selectin in the experimental group was 41.05%±1.84%, which was also significantly higher than that in the control group（4.33%±0.92%）（t=35.674, P=0.000）. With the increase of fluid shear force, the number of BMSCs adhering to the surface of human umbilical vascular endothelial cells（HUVEC） in group A was increased at first and then decreased, while there was few BMSCs adhering to the surface of HUVEC in groups B and C. Under the different fluid shear stress, the number of BMSCs adhered to the surface of HUVEC in group A was significantly higher than that in groups B and C（P0.05）, and there was no significant difference between groups B and C（P0.05）. Conclusion CD44 fucosylation on BMSCs can enhance the fluid adhesion force of rabbit BMSCs.