Objective: To explore the role and mechanism of mesenchymal stem cell (MSC) in modulating human pulmonary microvascular endothelial cell (HPMECs) permeability via hepatocyte growth factor (HGF). Methods: The study introduced a co-cultured model between HPMECs and human mesenchymal stem cell conditioned media (MSC-CM) collected from MSCs after 24 h hypoxia culture, and meanwhile HGF was neutralized in MSC-CM by anti-HGF antibody respectively, followed by lipopolysaccharide (LPS) stimulation. Finally, the following measurements were performed: the permeability of HPMECs, the protein expression of vascular endothelial cadherin (VE-cadherin), Occludin in HPMECs by Western blot, HPMECs apoptosis by Annexin V-FITC/PI and HPMECs proliferation by 3-(4, 5)-dimethylthiahiazo (-z-y1)-3, 5-di- phenytetrazoliumromide(MTT). Results: Compared to LPS group (4.15±0.88), MSC-CM reduced endothelial paracellular permeability injured by LPS(1.56±0.36, P<0.01), however, the MSC-CM effect was significantly blocked by anti-HGF antibody(3.11±0.74, P<0.05). Furthermore MSC-CM significantly increased the expression of VE-cadherin(0.71±0.05 vs. 0.38±0.19, P<0.05)and Occludin protein(0.96±0.05 vs. 0.51±0.02, P<0.05) in HPMECs, which was significantly blocked by anti-HGF antibody (P<0.05). MSC-CM significantly reduced the number of early apoptotic cells (6.82±1.80 vs. 17.09±1.89, P<0.05). However, the effect of MSC-CM was significantly blocked by neutralizing HGF (12.07±0.98, P<0.01). The cell viability results by MTT assay confirmed that MSC-CM(6.82±1.80, P<0.05)restored cell viability to a greater extent than LPS stimulation only(0.47±0.09), and meanwhile the MSC-CM effect was significantly inhibited by neutralizing HGF from MSC-CM with anti-HGF antibody (0.69±0.29, P<0.05). Conclusion: HGF secreted by MSCs reduces endothelial cell paracelluar permeability induced by LPS, and the possible mechanisms include remodelling of endothelial intercellular adherence junction, promoting endothelial cell proliferation and restraining endothelial cell apoptosis.
目的： 研究间充质干细胞(MSC)旁分泌肝细胞生长因子(HGF)对肺微血管内皮细胞(HPMECs)通透性的作用及机制。 方法： 将MSC缺氧培养24 h，收集MSC培养液，加入HGF抗体。将5×10(4)个HPMECs种植到transwell小室上层，培养2～3 d形成内皮细胞单层，与MSC培养液共培养24 h，再加入脂多糖。将HPMECs分为空白对照组、脂多糖组、MSC组及抗HGF抗体组。采用酶联免疫吸附(ELISA)法检测MSC培养液中HGF浓度，采用异硫氰酸荧光素(FITC)－葡聚糖法检测HPMECs通透性，采用western blot法检测内皮细胞连接血管内皮钙黏蛋白和闭锁蛋白，观察内皮细胞通透性，采用膜连蛋白V－异硫氰酸荧光素/碘化丙啶(Annexin V－FITC/PI)法和3－(4，5－二甲基噻唑－2)－2，5－二苯基四氮唑溴盐(MTT)法检测内皮细胞凋亡及增殖。 结果： 与脂多糖组(4.15±0.88)比较，MSC组HPMECs通透性降低(1.56±0.36，P<0.01)；抗HGF抗体组HPMECs通透性较MSC组升高(3.11±0.74，P<0.05)。MSC组血管内皮钙黏蛋白(0.71±0.05)及闭锁蛋白(0.96±0.05)的表达明显高于脂多糖组(0.38±0.19，0.51±0.02，均P<0.05)，抗HGF抗体组中这种作用被明显抑制(P<0.05)。与脂多糖组(17.09±1.89)比较，MSC组内皮细胞凋亡减少(6.82±1.80，P<0.05)，抗HGF抗体组中减少内皮细胞凋亡的作用被抑制(12.07±0.98，P<0.01)。与脂多糖组(0.47±0.09)比较，MSC组血管内皮细胞增殖活力增强(0.94±0.09，P<0.05)；抗HGF抗体组中增殖作用受到抑制(0.69±0.29，P<0.05)。 结论： MSC旁分泌HGF可降低肺微血管内皮细胞通透性，其可能机制为保护肺血管内皮细胞间连接、促进血管内皮细胞增殖及抑制内皮细胞凋亡。.
Keywords: Endothelial cell; Hepatocyte growth factor; Mesenchymal stem cells; Permeability.