Effect of Cathelicidin LL-37 on Transmembrane Barrier Function of Urothelial Cells
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摘要:
目的 探索LL-37对膀胱尿路上皮细胞跨膜屏障功能破坏的具体作用，构建适于体外研究间质性膀胱炎（interstitial cystitis，IC）的细胞实验模型。 方法 分别用不同浓度的LL-37处理人尿路上皮永生化细胞SV-HUC-1，通过跨上皮电阻测量仪测量跨上皮细胞电阻，CCK-8检测细胞增殖活力，流式细胞仪检测细胞周期和钙离子浓度，RT-qPCR和WB检测葡萄糖胺聚糖（GAGs）关键成分硫酸乙酰肝素的表达水平。 结果 体外细胞跨上皮电阻测量显示，100和200 μg/mL LL-37可显著抑制细胞的跨上皮电阻，破坏SV-HUC-1细胞的跨膜屏障功能。CCK-8和PI染色流式细胞检测显示，100 μg/mL 以上的LL-37可显著降低SV-HUC-1细胞的增殖活力，提高SV-HUC-1细胞处于G0/G1期的比例（P < 0.05），提示细胞增值受到显著抑制。进一步流式细胞术检测SV-HUC-1细胞中的钙离子浓度显示，LL-37处理后的尿路上皮细胞内钙离子浓度显著升高（P < 0.05），且增高量与LL-37浓度相关。RT-qPCR和WB检测证实经LL-37处理后SV-HUC-1细胞的GAGs关键成分硫酸乙酰肝素表达显著上调（P < 0.05）。 结论 抗菌肽LL-37可抑制SV-HUC-1细胞内GAGs关键成分硫酸乙酰肝素的表达，并破坏SV-HUC-1的细胞增殖和跨膜屏障功能。 Abstract:Objective To explore the specific effect of LL-37 on the destruction of the transmembrane barrier function of bladder urothelial cells and develop a cellular experimental model suitable for studying interstitial cystitis （IC） in vitro. Methods Human urothelial immortalized cells SV-HUC-1 were treated with LL-37 at different concentrations. The transepithelial electrical resistance （TEER） was measured by a transepithelial resistor, and the cell proliferative activity was detected by CCK-8 kit. Cell cycle and calcium ion concentration were detected by flow cytometry. The expression levels of heparan sulfate, a key component of glycosaminoglycan （GAGs）, were detected by RT-qPCR and WB. Results In vitro measurement showed that 100 and 200 μg/mL of LL-37 significantly inhibited the TEER and destroyed the transmembrane barrier function of SV-HUC-1. The CCK-8 test and PI staining flow cytometry results showed that LL-37 above 100 μg/mL could significantly reduce the proliferation activity of SV-HUC-1 and increase the proportion of SV-HUC-1 in the G0/G1 phase （P < 0.05）, suggesting that cell proliferation was significantly inhibited. Further flow cytometry analysis showed that the calcium ion concentration in SV-HUC-1treated with LL-37 was significantly increased （P < 0.05）, and the increase was related to LL-37 concentration. RT-qPCR and WB assays confirmed significant up-regulation of heparan sulfate, a key GAGs component, in the LL-37-treated SV-HUC-1 （P < 0.05）. Conclusion The cathelicidin LL-37 can inhibit the expression of GAGs and destroy the cell proliferation and transmembrane barrier function of SV-HUC-1. -
图 1 LL-37显著降低SV-HUC-1细胞的增殖活力和跨膜电阻
A：跨膜电阻测量仪检测SV-HUC-1细胞跨膜电阻；B：CCK-8检测细胞增殖活力；C、D：流式细胞仪检测细胞周期。*P < 0.05。
Figure 1. LL-37 significantly reduced the proliferative activity and transmembrane resistance of SV-HUC-1 cells
图 2 LL-37调控SV-HUC-1细胞内钙离子浓度和GAGs关键成分硫酸乙酰肝素的表达
A、B：流式细胞术检测细胞中的钙离子；C：RT-qPCR检测GAGs关键成分硫酸乙酰肝素的表达；D：WB检测GAGs关键成分硫酸乙酰肝素的表达。*P < 0.05，**P < 0.01。
Figure 2. LL-37 Regulating the Intracellular Calcium Ion Concentration of SV-HUC-1 and the Expression of heparan sulfate，a key component of GAGs
表 1 引物序列
Table 1. Primer sequences
目的基因 引物序列（F：正向序列；R：反向序列；5′-3′） HS2ST1 F: CAGCGGCGTTGGTGATAGCG R: AGAGAGCGACAGCGAGAGAACC HS3ST1 F: CCAAGTGTTCTACAACCACATG R: CTTTAGGAACCTCTCGACCTTT HS3ST2 F: CTTACCTGTGTTACAGCTTCCT R: CTTCTGGAGAAGTTTCTGGCC HS3ST3A1 F: AACCTGAACTCGGGCGGAGAG R: GTGGCGAGCGACAGTGACTTC HS6ST1 F: GAAGACGTCGTTGCATATGTG R: GATGAAGGACAGGTTGTAGCAG 
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