目的 通过构建体外胎盘屏障模型,研究人脐带间充质干细胞(hUC-MSC)对传统胎盘屏障模型功能的影响及其可能的分子机制。方法 将hUC-MSC、人脐静脉内皮细胞(HUVEC)和人滋养层细胞(HTR-8)共同培养于transwell中建立体外胎盘屏障模型,设置对照组(HUVEC+HTR-8)、forskolin组(HUVEC+HTR-8+forskolin)、MSC组(HUVEC+HTR-8+hUC-MSC)、MSC+forskolin组(HUVEC+HTR-8+hUC-MSC+forskolin),通过测量跨膜电阻值、荧光黄CH的渗透性以及P-糖蛋白(P-gp)的活性,评估各组体外胎盘屏障模型的功能;利用实时荧光定量PCR (qRT-PCR)和Westernblotting检测各组体外胎盘屏障模型中P-gp、紧密连接蛋白5(Claudin-5)、闭合小环蛋白-1(ZO-1)、ZO-2、血小板-内皮细胞黏附分子(CD31)的mRNA及蛋白表达水平;利用LC-MS/MS检测阿替洛尔、米诺地尔、美托洛尔3种渗透性工具药透过各组体外胎盘屏障模型的表观渗透系数(P_app),验证模型的功能完整性。结果 与对照组相比,forskolin、MSC、MSC+forskolin组的跨膜电阻值均显著增高(P<0.05、0.001)、P-gp外排功能均增强、荧光黄的P_app值均显著降低(P<0.05、0.01、0.001),且二者联用后作用效果尤为显著,表明该模型具备完整的屏障功能,且MSC+forskolin屏障功能最好;qRT-PCR和Western blotting结果显示,与对照组相比,forskolin组P-gp、ZO-1、ZO-2、CD31的表达显著增加(P<0.05、0.01、0.001),MSC组P-gp、Claudin5、ZO-1和CD31的表达也显著增加(P<0.05、0.01、0.001);联合应用后,各蛋白的mRNA及蛋白表达水平均更显著增加(P<0.01、0.001),表明hUC-MSC增强传统胎盘屏障功能;与对照组相比,各实验组的P_app值均降低,MSC+forskolin组3个工具药均差异显著,美托洛尔差异最显著。结论 hUC-MSC通过增强传统胎盘屏障模型中紧密连接蛋白和外排蛋白的表达,进一步增强了传统胎盘屏障模型的屏障功能。

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 hUCMSC, 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 (qRTPCR) 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 (P_app) 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 P_app 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, P_app 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.

R965.2

中国医学科学院医学与健康科技创新工程资助项目(2019-I2M-5-020);天津市“项目+团队”重点培养专项资助项目(创新类)(XC202030);天津市科技计划项目细胞制品的成药性及转化研究(23ZGCXQY00050);药物成药性评价与系统转化全国重点实验室项目(712023001)