人甲床来源的脱细胞支架搭载骨髓间充质干细胞向指甲干细胞分化的研究

Human nail bed-derived decellularized scaffolds serve as vector for bone marrow-derived mesenchymal stem cell differentiation into nail stem cells

目的探究人甲床来源的脱细胞支架搭载骨髓间充质干细胞向指甲干细胞分化的作用。方法收集2022年至2023年在承德医学院附属医院手足外科进行截指术的15例患者的临床废弃甲床组织,制备成脱细胞甲床支架,使用试剂盒检测参照组(未脱细胞组)与脱细胞组(脱细胞甲床支架组)中总胶原蛋白含量、残留DNA含量;将脱细胞甲床支架与人骨髓间充质干细胞(hMSCs)进行共培养14 d。比较对照组(hMSCs组)与共培养组(脱细胞甲床支架+hMSCs组)体外细胞分化能力及指甲干细胞标志物[角蛋白15,角蛋白17,G蛋白偶联受体6(Lgr6)以及β-联蛋白(β-catenin)蛋白]含量。结果脱细胞组总胶原蛋白含量为(189.62±45.45)μg/mg,低于参照组[(196.02±41.93)μg/mg],但两组相比差异无统计学意义(P>0.05);脱细胞组中DNA含量为(0.41±0.15)μg/mg,明显低于参照组[(0.87±0.13)μg/mg],差异有统计学意义(P<0.05)。培养14 d后,共培养组吸光度值为1.09±0.07,对照组吸光度值为1.10±0.5,两组相比差异无统计学意义(P>0.05)。培养14 d后,共培养组角蛋白15、角蛋白17、Lgr6、β-catenin含量分别为(39.56±5.09)、(45.83±4.01)、(5.74±0.99)、(146.79±5.34)pg/mL,均明显高于对照组[(12.10±4.28)、(10.47±3.19)、(0.93±0.67)、(67.28±7.41)pg/mL],差异均有统计学意义(P<0.05)。结论脱细胞甲床支架中的DNA有被清除、保留胶原蛋白,与hMSCs共培养后细胞增殖能力正常,且可诱导hMSCs向指甲干细胞分化,为临床上治疗甲床缺损提供新思路。

Objective To explore the effect of human nail bed-derived decellularized scaffolds serve as vector for bone marrow-derived mesenchymal stem cell differentiation into nail stem cells.Methods Clinically discarded nail bed tissue from 15 patients undergoing finger amputation from 2022 to 2023 at Affiliated Hospital of Chengde Medical College was collected and prepared as decellularized nail bed scaffolds.Total collagen content and residual DNA content in the reference group(undecellularized group)versus decellularized group(decellularized nail bed scaffold group)were measured using the kit.Decellularized nail bed scaffolds were co-cultured with human bone marrow mesenchymal stem cells(hMSCs)for 14 d.The in vitro cell differentiation capacity and nail stem cell markers[keratin 15,keratin 17,leucine-rich repeat-containing G protein-coupled receptor 6(Lgr6),β-catenin protein]were compared between control group(hMSCs group)and co-cultured group(decellularized nail bed scaffolds+hMSCs group).Results The total collagen content in the decellularized group was(189.62±45.45)μg/mg,which was lower than that in the reference group[(196.02±41.93)μg/mg],but there was no statistically significant difference between the two groups(P>0.05);the DNA content in the decellularized group was(0.41±0.15)μg/mg,which was significantly lower than that in the reference group[(0.87±0.13)μg/mg],the difference was statistically significant(P<0.05).After 14 days of cultivation,the absorbance value of the co culture group was 1.09±0.07,while the absorbance value of the control group was 1.10±0.50,there was no statistically significant difference between the two groups(P>0.05).After 14 days of cultivation,the contents of keratin 15,keratin 17,Lgr6,β-catenin in the co-culture group were(39.56±5.09),(45.83±4.01),(5.74±0.99),and(146.79±5.34)pg/mL,which were significantly higher than those in the control group[(12.10±4.28),(10.47±3.19),(0.93±0.67),and(67.28±7.41)pg/mL],and the differences were statistically significant(P<0.05).Conclusion The decellularized nail bed scaffolds have cleared DNA and preserved collagen,and exhibit normal cell proliferation ability after co-culture with hMSCs and induce the differentiation of hMSCs to nail stem cells,which provide a new idea for the treatment of nail bed defects in the clinic.