Department of Urology, Radboud University Medical Center, Nijmegen, The Netherlands.
Neurourol Urodyn. 2020 Feb;39(2):558-564. doi: 10.1002/nau.24240. Epub 2019 Nov 27.
To determine whether glycosaminoglycan (GAG) replenishment is able to improve recovery of a deficient urothelial barrier, chondroitin sulfate (CS) instillations were tested using an in vitro model. Porcine urothelial cells (Ucells) were terminally differentiated in culture conditions to construct a urothelial layer with a functional barrier. This layer was damaged to compromise barrier function to simulate a key characteristic of bladder pain syndrome/interstitial cystitis. The functional effect of subsequent treatment with CS was evaluated.
Primary porcine Ucells were isolated and cultured on inserts. Differentiation of cells was evaluated with immunohistochemical analysis for the presence of umbrella cells, tight junctions and CS. Transepithelial electrical resistance (TEER) measurements were performed to evaluate barrier function. Protamine was used to simulate mild urothelial damage. CS 0.2% (vol/vol), a GAG, was subsequently instilled in the treatment group. The recovery of barrier function was further evaluated with TEER measurements. The Student t test was used for the analysis of results.
After induction of differentiation, the Ucells expressed barrier markers and a functional barrier was established (measured by high TEER). TEER decreased significantly after instillation with protamine. CS instillation improved recovery of TEER significantly measured after 7 hours (84% vs 22% in controls). After 24 hours; however, the TEER was comparable in both experimental groups.
CS instillation improves the recovery of the urothelial barrier after damage in vitro. This functional experiment shows that CS improves recovery of damaged urothelial function, which supports the hypothesis behind the mechanism of action of GAG-replenishment therapy.
为了确定糖胺聚糖（GAG）补充是否能够改善受损尿路上皮屏障的恢复，我们使用体外模型测试了硫酸软骨素（CS）的灌注。将猪尿路上皮细胞（Ucells）在培养条件下终末分化，以构建具有功能屏障的尿路上皮层。该层被破坏以损害屏障功能，模拟膀胱疼痛综合征/间质性膀胱炎的一个关键特征。随后用 CS 进行治疗的功能效果进行了评估。
原代猪 Ucells 被分离并培养在插入物上。通过免疫组织化学分析伞细胞、紧密连接和 CS 的存在来评估细胞的分化。通过跨上皮电阻（TEER）测量来评估屏障功能。鱼精蛋白用于模拟轻度尿路上皮损伤。随后在治疗组中灌注 0.2%（体积/体积）的 CS，一种 GAG。进一步通过 TEER 测量来评估屏障功能的恢复。使用学生 t 检验对结果进行分析。
在诱导分化后，Ucells 表达了屏障标志物，并建立了功能性屏障（通过高 TEER 测量）。灌注鱼精蛋白后 TEER 显著下降。CS 灌注显著改善了 TEER 的恢复，在 7 小时后测量（对照组为 22%，实验组为 84%）。然而，在 24 小时后，两组的 TEER 相当。
CS 灌注可改善体外损伤后尿路上皮屏障的恢复。这项功能实验表明，CS 可改善受损尿路上皮功能的恢复，这支持了 GAG 补充治疗作用机制背后的假设。
