摘要
目的 探讨膀胱平滑肌细胞是否存在自噬及其在环磷酰胺诱导的大鼠膀胱炎中的作用及意义.方法 30只SD大鼠完全随机法分为对照组和实验组（4 h组、48 h组、72 h组、96 h组）,每组6只.实验组腹腔给予环磷酰胺150 mg/kg处理,对照组腹腔给予等体积生理盐水0.6 ml处理.Western印迹检测膀胱肌层自噬标志蛋白微管相关蛋白1轻链3（LC3）、白细胞介素（IL）-6、IL-1β和肿瘤坏死因子（TNF）-α蛋白表达变化.LC3和平滑肌肌动蛋白α（α-SMA）免疫荧光双标膀胱平滑肌细胞自噬斑点共定位.透射电镜观察膀胱平滑肌细胞自噬小体.HE染色行膀胱病理组织评分.结果 电镜、免疫荧光双标以及Western 印迹证实实验组和对照组膀胱平滑肌细胞均存在自噬.Western印迹结果显示实验4 h组膀胱肌层LC3表达显著低于对照组（0.08±0.01比0.13±0.01,P〈0.05）;实验48、72及96 h组膀胱肌层LC3表达高于对照组（0.29±0.04比0.13±0.01,P〈0.05;0.23±0.03比0.13±0.01,P〈0.05;0.16±0.03比0.13±0.01,P〉0.05）,分别为对照组的2.19、1.75、1.20倍.电镜和免疫荧光双标的结果支持该变化趋势.此外,实验组膀胱肌层炎症因子（IL-6、IL-1β和TNF-α）以及膀胱病理组织评分均显著高于对照组（均P〈0.05）.结论 膀胱平滑肌细胞存在自噬.在环磷酰胺诱导的大鼠膀胱炎中膀胱平滑肌自噬的激活不充分,未能保护大鼠膀胱肌层.
Objective To explore the existence of bladder smooth muscle autophagy and its function in eyelophosphamide（CYP） -induced cystitis. Methods A total of 30 Sprague-Dawley rats were randomized into control（ n = 6 ） and experimental groups （ 4 h, 48 h, 72 h, 96 h, n = 24 ）. The experimental group received a single intraperitoneal injection of 150 mg/kg CYP in 0. 6 In] saline while the control group had the same volume of saline injection. The changes of microtubule-assoeiated protein light chain 3 （LC3）, interleukin-6 （IL-6）, IL-113 and tumor necrosis factor-alpha （TNF-ct） proteins in bladder muscular layer were measured with Western blot. Co-location of LC3 and alpha-small muscle action （oL-SMA） was detected with double-labeled immunofluorescence. Autophagie vacuoles were observed with electron microscopy. The changes in bladder inflammation were measured by hematoxylin and eosin staining. Results Autophagy was confirmed in detrusor myoeytes with electron microscope, LC3 Western blot and double-labeled immunofluoreseenee. As compared with the control group, the expression level of LC3 in experimental groups decreased （0. 08 ±0. 01 vs 0. 13 ±0. 01 ,P 〈0. 05） at 4 h and increased 1.19, 0. 75 and 0. 20 fold（0. 29 ± 0.04 vs 0. 13 ±0.01,P 〈0.05;0. 23 ±0.03 vs 0. 13 ±0.01,P 〈0.05;0. 16 ±0.03 vs 0. 13 ±0.01,P 〉 0. 05 ） at 48, 72 and 96 h respectively. Electron microscopy and double-labeled immunofluorescent also supported the above change of LC3 by Western blot. In addition, as compared with the control groups, IL-6, IL-113 and TNF-ot and bladder histological scores of bladder muscular layer significantly increased in experimental groups （ all P 〈 0. 05 ） . Conclusions Autophagy exists in bladder smooth muscle. In eyelophosphamide-induced cystitis, bladder smooth muscle autophagy activation may not be sufficient to protect bladder muscle layer inflammation in rats.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2013年第42期3333-3337,共5页
National Medical Journal of China
关键词
自噬
膀胱
肌
平滑
膀胱炎
Autophagy
Urinary bladder
Muscle, smooth
Cystitis
