Mechanisms of human umbilical cord mesenchymal stem cells enhancing barrier function of traditional placental barrier models
Objective To investigate the effect of human umbilical cord mesenchymal stem cells(hUC-MSC)on the function of the traditional placental barrier model and its molecular mechanism by constructing an in vitro placental barrier model.Methods hUC-MSC,human umbilical vein endothelial cells(HUVEC),and human trophoblast cells(HTR-8)were co-cultured in a transwell to establish an in vitro placental barrier model.The experiment included the following groups:HUVEC+HTR-8(control group),HUVEC+HTR-8+forskolin(forskolin group),HUVEC+HTR-8+MSC(MSC group),and HUVEC+HTR-8+MSC+forskolin(MSC+forskolin group).By measuring the transmembrane resistance,permeability of fluorescent dye CH,and the activity of P-gp,we assessed the functionality of the in vitro placental barrier models in each group.Using real-time fluorescence quantitative PCR(qRT-PCR)and Western blotting,we detected the mRNA and protein expression levels of the efflux protein P-gp,as well as the tight junction proteins Claudin5,ZO-1,ZO-2,and CD31 in the in vitro placental barrier model of each group.Additionally,LC-MS/MS was used to detect the apparent permeability coefficient(Papp)of atenolol,minoxidil,and metoprolol,three drugs with different permeabilities,to validate the integrity of the model.Results Compared with control group,the transmembrane resistance values of forskolin,MSC and MSC+forskolin groups were significantly increased(P＜0.05,0.001),the effection function of P-gp was enhanced,and the Papp value of fluorescence yellow was significantly decreased(P＜0.05,0.01,0.001).The combined effect of the two was particularly significant,indicating that the model had complete barrier function,and MSC+forskolin had the best barrier function.qRT-PCR and Western blotting results showed that compared with control group,the expressions of P-gp,ZO-1,ZO-2 and CD31 in forskolin group were significantly increased(P＜0.05,0.01,0.001),and the expressions of P-GP,Claudin5,ZO-1 and CD31 in MSC group were also significantly increased(P＜0.05,0.01,0.001),and after combined application,the mRNA and protein expression levels of each protein were significantly increased(P＜0.01,0.001),indicating that hUC-MSC enhanced the traditional placental barrier function.Compared with control group,Papp values of all experimental groups were decreased,and there were significant differences among the three instrumental drugs in MSC+forskolin group,the most significant difference was metoprolol.Conclusion This study revealed the molecular mechanisms by which MSC enhance the expression of tight junction proteins and efflux proteins in the traditional placental barrier model,thereby enhancing the barrier function of the traditional placental barrier model.
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