参与调控人牙髓干细胞黏附能力的相关整合素分析

Integrins involved in the regulation of adherence of human dental pulp stem cells

目的探究体外模拟微重力环境下参与调控人牙髓干细胞黏附能力的相关整合素。方法以聚乳酸-羟基乙酸共聚物(PLGA)支架为载体,将原代培养的人牙髓干细胞接种于PLGA支架上,分为模拟重力组(所受重力保持在10-2g)与常规培养对照组,每组各12个支架;用阳离子脂质体介导的细胞转染构建整合素α5寡核苷酸序列转染组,同时设置只加入siRNA-mate的空转染组、空白对照组。Western blot检测细胞支架复合物中整合素α5、αV、β1以及粘附斑激酶、磷酸化粘附斑激酶的表达水平;使用整合素α5寡核苷酸序列转染人牙髓干细胞,抑制整合素α5表达。荧光染色检测转染后人牙髓干细胞的粘附能力; Western blot检测整合素β1及下游信号分子(粘附斑激酶、磷酸化粘附斑激酶)的表达水平。结果模拟重力组整合素α5、粘附斑激酶、磷酸化粘附斑激酶相对表达量明显高于对照组(P <0. 05);整合素αV、整合素β1相对表达量在模拟重力组与对照组之间差异无统计学意义(P> 0. 05)。整合素α5寡核苷酸序列转染的人牙髓干细胞中整合素α5蛋白表达水平明显低于空白对照组和空转染组(P <0. 01)。整合素α5寡核苷酸序列转染组人牙髓干细胞在PLGA支架上的粘附细胞数明显低于空白对照组、空转染组(P <0. 01);整合素α5寡核苷酸序列转染组粘附斑激酶、磷酸化粘附斑激酶的相对表达量明显低于空白对照组(P <0. 05);整合素β1在整合素α5寡核苷酸序列转染组与对照组之间差异无统计学意义(P>0. 05)。结论体外模拟微重力环境下,人牙髓干细胞在PLGA支架上的粘附能力增强与整合素α5及其下游信号分子粘附斑激酶、磷酸化粘附斑激酶的表达水平上调有关。

Objective To explore the integrins involved in the regulation of adherence of human dental pulp stem cells under simulated microgravity in vitro.Methods The primary cultured human dental pulp stem cells were inoculated on PLGA scaffolds and divided into simulated gravity group and conventional culture control group.Western blot was used to detect the expression levels of integrinα5,αV,β1,focal adhesion kinase and phosphorylated focal adhesion kinase in the cell scaffold complex.Human dental pulp stem cells were transfected with integrinα5 oligonucleotide sequence to inhibit integration.The adhesion ability of human dental pulp stem cells after transfection was detected by fluorescence staining,and the expression levels of integrinβ1 and downstream signal molecules(focal adhesion kinase and phosphorylated focal adhesion kinase)were detected by Western blot.Results The relative expressions of integrinα5,focal adhesion kinase and phosphorylated focal adhesion kinase in simulated gravity group were significantly higher than those in control group(P<0.05),and the relative expressions of integrinαV and integrinβ1 were not significantly different between simulated gravity group and control group(P>0.05).The expression level of integrinα5 protein in human dental pulp stem cells transfected with integrinα5 oligonucleotide sequence was significantly lower than that in blank control group and blank transfection group(P<0.01).The number of adherent cells of human dental pulp stem cells on PLGA scaffolds in integrinα5 oligonucleotide sequence transfection group was significantly lower than that in blank control group and blank transfection group(P<0.01),and the rela-tive expression of focal adhesion kinase and phosphorylated focal adhesion kinase in integrinα5 oligonucleotide sequence transfection group was significantly lower than that in blank control group(P<0.05).There was no significant difference in integrinβ1 between integrinα5 oligonucleotide sequence transfection group and control group(P>0.05).Conclusion The enhanced adhesion of human dental pulp stem cells to PLGA scaffolds under simulated microgravity in vitro is related to the up-regulation of integrinα5 and its downstream signal molecules,focal adhesion kinase and phosphorylated focal adhesion kinase.