抗菌肽LL-37对尿路上皮细胞跨膜屏障功能破坏的作用  

Effect of Cathelicidin LL-37 on Transmembrane Barrier Function of Urothelial Cells

作　　者：杨童欣 王光[1] 徐蕊 苏思维 方克伟[1] 刘建和[1] 李炯明[1] YANG Tongxin;WANG Guang;XU Rui;SU Siwei;FANG Kewei;LIU Jianhe;LI Jiongming(Dept.of Urology,The 2nd Affiliated Hospital of Kunming Medical University,Kunming Yunnan 650101,China)

机构地区：[1]昆明医科大学第二附属医院泌尿外科,云南昆明650101

出　　处：《昆明医科大学学报》2022年第8期34-40,共7页Journal of Kunming Medical University

基　　金：国家自然科学基金资助项目(82160150);云南省科技厅-昆明医科大学应用基础联合专项青年博士基金资助项目(2019FE0001-149);云南省科技厅-昆明医科大学联合专项基金资助项目(202001AY070001-062);云南省医学学科后备人才培养基金资助项目(H-2017045)。

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SVHUC-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200μg/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,100μg/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SVHUC-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。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 200μg/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 100μg/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.

关 键 词：间质性膀胱炎 抗菌肽LL-37 尿路上皮细胞 跨膜屏障 葡萄糖胺聚糖 

分 类 号：R694[医药卫生—泌尿科学]