目的 通過(guò)構(gòu)建體外胎盤(pán)屏障模型,研究人臍帶間充質(zhì)干細(xì)胞(hUC-MSC)對(duì)傳統(tǒng)胎盤(pán)屏障模型功能的影響及其可能的分子機(jī)制。方法 將hUC-MSC、人臍靜脈內(nèi)皮細(xì)胞(HUVEC)和人滋養(yǎng)層細(xì)胞(HTR-8)共同培養(yǎng)于transwell中建立體外胎盤(pán)屏障模型,設(shè)置對(duì)照組(HUVEC+HTR-8)、forskolin組(HUVEC+HTR-8+forskolin)、MSC組(HUVEC+HTR-8+hUC-MSC)、MSC+forskolin組(HUVEC+HTR-8+hUC-MSC+forskolin),通過(guò)測(cè)量跨膜電阻值、熒光黃CH的滲透性以及P-糖蛋白(P-gp)的活性,評(píng)估各組體外胎盤(pán)屏障模型的功能;利用實(shí)時(shí)熒光定量PCR (qRT-PCR)和Westernblotting檢測(cè)各組體外胎盤(pán)屏障模型中P-gp、緊密連接蛋白5(Claudin-5)、閉合小環(huán)蛋白-1(ZO-1)、ZO-2、血小板-內(nèi)皮細(xì)胞黏附分子(CD31)的mRNA及蛋白表達(dá)水平;利用LC-MS/MS檢測(cè)阿替洛爾、米諾地爾、美托洛爾3種滲透性工具藥透過(guò)各組體外胎盤(pán)屏障模型的表觀(guān)滲透系數(shù)(P_app),驗(yàn)證模型的功能完整性。結(jié)果 與對(duì)照組相比,forskolin、MSC、MSC+forskolin組的跨膜電阻值均顯著增高(P<0.05、0.001)、P-gp外排功能均增強(qiáng)、熒光黃的P_app值均顯著降低(P<0.05、0.01、0.001),且二者聯(lián)用后作用效果尤為顯著,表明該模型具備完整的屏障功能,且MSC+forskolin屏障功能最好;qRT-PCR和Western blotting結(jié)果顯示,與對(duì)照組相比,forskolin組P-gp、ZO-1、ZO-2、CD31的表達(dá)顯著增加(P<0.05、0.01、0.001),MSC組P-gp、Claudin5、ZO-1和CD31的表達(dá)也顯著增加(P<0.05、0.01、0.001);聯(lián)合應(yīng)用后,各蛋白的mRNA及蛋白表達(dá)水平均更顯著增加(P<0.01、0.001),表明hUC-MSC增強(qiáng)傳統(tǒng)胎盤(pán)屏障功能;與對(duì)照組相比,各實(shí)驗(yàn)組的P_app值均降低,MSC+forskolin組3個(gè)工具藥均差異顯著,美托洛爾差異最顯著。結(jié)論 hUC-MSC通過(guò)增強(qiáng)傳統(tǒng)胎盤(pán)屏障模型中緊密連接蛋白和外排蛋白的表達(dá),進(jìn)一步增強(qiáng)了傳統(tǒng)胎盤(pán)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

中國(guó)醫(yī)學(xué)科學(xué)院醫(yī)學(xué)與健康科技創(chuàng)新工程資助項(xiàng)目(2019-I2M-5-020);天津市“項(xiàng)目+團(tuán)隊(duì)”重點(diǎn)培養(yǎng)專(zhuān)項(xiàng)資助項(xiàng)目(創(chuàng)新類(lèi))(XC202030);天津市科技計(jì)劃項(xiàng)目細(xì)胞制品的成藥性及轉(zhuǎn)化研究(23ZGCXQY00050);藥物成藥性評(píng)價(jià)與系統(tǒng)轉(zhuǎn)化全國(guó)重點(diǎn)實(shí)驗(yàn)室項(xiàng)目(712023001)