N钙黏蛋白融合蛋白改性表面增强细胞活性与细胞募集能力的研究

Study on surface modification with N-cadherin fusion protein to enhance cell activity and cell recruitment capability

目的:探究N钙黏蛋白融合蛋白改性表面培养人脐带间充质干细胞(HUCMSCs)对细胞活性与细胞募集能力的影响。方法:对聚苯乙烯(TCP)培养皿表面分别行磷酸缓冲盐溶液(PBS)浸润、N钙黏蛋白融合蛋白改性处理,通过水接触角实验检测表面亲水性,将HUCMSCs分别接种于PBS浸润(对照组)和N钙黏蛋白融合蛋白改性(改性组)的培养皿,利用CCK-8评估细胞活性,通过划痕实验、迁移实验评估N钙黏蛋白融合蛋白改性表面培养HUCMSCs对细胞募集能力的影响,通过RT-qPCR和ELISA评估基质细胞衍生因子-1(SDF-1)的基因表达和旁分泌变化。结果:水接触角检测显示对照组培养皿表面水接触角高于改性组培养皿表面水接触角(t=41.99,P<0.01);CCK-8实验结果显示,培养1 d改性组细胞活性明显高于对照组(t=2.907,P<0.05);划痕实验结果显示体外培养12、24 h后,改性组划痕愈合面积均高于对照组(t=4.144、17.75,均P<0.05);迁移实验结果显示改性组募集到上室下层的细胞数量高于对照组(t=23.14,P<0.01);RT-qPCR结果显示,改性组SDF-1基因表达上调(t=29.01,P<0.01);ELISA结果显示,改性组SDF-1因子旁分泌高于对照组(t=4.681,P<0.01)。结论:利用N钙黏蛋白融合蛋白改性表面培养HUCMSCs,可增强细胞活性及细胞募集能力。

Objective:To investigate the effect of surface-modified culture of human umbilical cord mesenchymal stem cells(HUCMSCs)using N-calmodulin fusion proteins on cell activity and cell recruitment capacity.Methods:Polystyrene(TCP)culture dishes were treated with phosphate-buffered saline(PBS)infiltration and N-cadherin fusion protein modification,respectively.Surface hydrophilicity was detected by water contact angle test.HUCMSCs were inoculated onto PBS-infiltrated(control group)and N-cadherin fusion protein-modified(modified group)culture dishes.The CCK-8 assay was used to evaluate cell viability,while wound healing assay and migration assay were used to evaluate the effect of N-cadherin fusion protein modified surfaces on the cell recruitment capability of HUCMSCs.RT-qPCR and ELISA were used to assess the gene expression and paracrine changes of the stromal cell-derived factor-1(SDF-1).Results:Water contact angle measurements showed that the water contact angle of the control tissue culture dish was higher than that of the modified group(t=41.99,P<0.01).The CCK-8 assay results showed that the cell viability of the aggregates in the modified group was significantly higher than that in the control group on day 1(t=2.907,P<0.05).Scratch assay results demonstrated that after 12 hours and 24 hours of in vitro culturing,the modified group showed larger scratch healing areas compared with the control group(t=4.144,17.75,both P<0.05).The results of the migration assay showed that the number of cells recruited to the lower layer of the upper chamber was higher in the modified group than in the control group(t=23.14,P<0.01).RT-qPCR results showed SDF-1 gene expression was up-regulated in the modified group(t=29.01,P<0.01),while ELISA results showed that the paracrine secretion of SDF-1 factor in the modified group was higher than that in the control group(t=4.681,P<0.01).Conclusion:Modifying the surface of HUCMSCs with N-cadherin fusion protein can enhance cell viability and cell recruitment capability.