以CFTR基因敲除大鼠的原代鼻上皮细胞培养物作为囊性纤维化鼻窦炎疾病模型的特征分析。
Characterization of primary rat nasal epithelial cultures in CFTR knockout rats as a model for CF sinus disease.
作者信息
Tipirneni Kiranya E, Cho Do-Yeon, Skinner Daniel F, Zhang Shaoyan, Mackey Calvin, Lim Dong-Jin, Woodworth Bradford A
机构信息
Department of Otolaryngology, University of Alabama at Birmingham, Birmingham, Alabama, U.S.A.
Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama, U.S.A.
出版信息
Laryngoscope. 2017 Nov;127(11):E384-E391. doi: 10.1002/lary.26720. Epub 2017 Aug 3.
OBJECTIVE
The objectives of the current experiments were to develop and characterize primary rat nasal epithelial cultures and evaluate their usefulness as a model of cystic fibrosis (CF) sinonasal transepithelial transport and CF transmembrane conductance regulator (CFTR) function.
STUDY DESIGN
Laboratory in vitro and animal studies.
METHODS
CFTR and CFTR rat nasal septal epithelia (RNSE) were cultured on semipermeable supports at an air-liquid interface to confluence and full differentiation. Monolayers were mounted in Ussing chambers for pharmacologic manipulation of ion transport and compared to similar filters containing murine (MNSE) and human (HSNE) epithelia. Histology and scanning electron microscopy (SEM) were completed. Real-time polymerase chain reaction of CFTR RNSE, MNSE, and HSNE was performed to evaluate relative CFTR gene expression.
RESULTS
Forskolin-stimulated anion transport (ΔIsc in μA/cm ) was significantly greater in epithelia derived from CFTR when compared to CFTR animals (100.9 ± 3.7 vs. 10.5 ± 0.9; P < 0.0001). Amiloride-sensitive I was equivalent (-42.3 ± 2.8 vs. -46.1 ± 2.3; P = 0.524). No inhibition of CFTR-mediated chloride (Cl ) secretion was exhibited in CFTR epithelia with the addition of the specific CFTR inhibitor, CFTR -172. However, calcium-activated Cl secretion (UTP) was significantly increased in CFTR RNSE (CFTR -106.8 ± 1.6 vs. CFTR -32.2 ± 3.1; P < 0.0001). All responses were larger in RNSE when compared to CFTR and CFTR (or F508del/F508del) murine and human cells (P < 0.0001). Scanning electron microscopy demonstrated 80% to 90% ciliation in all RNSE cultures. There was no evidence of infection in CFTR rats at 4 months. CFTR expression was similar among species.
CONCLUSION
The successful development of the CFTR rat enables improved evaluation of CF sinus disease based on characteristic abnormalities of ion transport.
LEVEL OF EVIDENCE
NA. Laryngoscope, 127:E384-E391, 2017.
目的
当前实验的目的是建立并鉴定原代大鼠鼻上皮细胞培养体系，并评估其作为囊性纤维化（CF）鼻窦跨上皮转运及CF跨膜电导调节因子（CFTR）功能模型的效用。
研究设计
实验室体外研究及动物研究。
方法
将CFTR和CFTR大鼠鼻中隔上皮（RNSE）在半透性支持物上于气液界面培养至汇合及完全分化。将单层细胞置于尤斯灌流小室中进行离子转运的药理学操作，并与含有小鼠（MNSE）和人类（HSNE）上皮的类似滤膜进行比较。完成组织学和扫描电子显微镜（SEM）检查。对CFTR RNSE、MNSE和HSNE进行实时聚合酶链反应以评估CFTR基因的相对表达。
结果
与CFTR动物来源的上皮相比，佛司可林刺激的阴离子转运（ΔIsc，单位为μA/cm²）在CFTR来源的上皮中显著更高（100.9±3.7对10.5±0.9；P<0.0001）。氨氯地平敏感电流相当（-42.3±2.8对-46.1±2.3；P = 0.524）。在添加特异性CFTR抑制剂CFTR -172后，CFTR上皮中未表现出对CFTR介导的氯离子（Cl⁻）分泌的抑制。然而，CFTR RNSE中钙激活的Cl⁻分泌（UTP）显著增加（CFTR -106.8±1.6对CFTR -32.2±3.1；P<0.0001）。与CFTR和CFTR（或F508del/F508del）小鼠及人类细胞相比，所有反应在RNSE中都更大（P<0.0001）。扫描电子显微镜显示所有RNSE培养物中有80%至90%的纤毛。4个月时CFTR大鼠无感染迹象。CFTR表达在各物种间相似。
结论
CFTR大鼠的成功建立使得基于离子转运特征性异常对CF鼻窦疾病进行更好的评估成为可能。
证据水平
无。《喉镜》，2017年，第127卷：E38-E391页。
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