Laser-capture microdissection for analysis of cell type-specific gene expression of muscarinic receptor subtypes in the rat bladder with cyclophosphamide-induced cystitis.
Laser-capture microdissection for analysis of cell type-specific gene expression of muscarinic receptor subtypes in the rat bladder with cyclophosphamide-induced cystitis.
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激光捕获显微切割用于分析环磷酰胺诱导的膀胱炎大鼠膀胱中毒蕈碱受体亚型的细胞类型特异性基因表达。
DOI：
10.1007/s11255-015-0926-z
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发表时间：
2015
影响因子：
2
通讯作者：
Yoshimura,Naoki
中科院分区：
文献类型：
--
作者：
Sugino,Yoshio;O'Malley,KatherineJ;Wang,Zhou;Tyagi,Pradeep;Birder,LoriA;Ogawa,Osamu;Yoshimura,Naoki
目的本研究探讨激光捕获显微切割 (LCM) 方法是否可以使用 α-平滑肌肌动蛋白 (SMA) 和细胞角蛋白 20 (CK20) 实现尿路上皮细胞与逼尿肌细胞的分离，或浅表尿路上皮细胞与中间/基底尿路上皮细胞的分离。此外，我们还研究了慢性膀胱炎大鼠激光捕获的尿路上皮和逼尿肌细胞中毒蕈碱受体表达的变化。方法雌性SD大鼠在第1、4、7和10天腹腔注射环磷酰胺（75 mg/kg）诱导慢性膀胱炎。以相同的方案注射盐水作为对照。将膀胱标本切成8μm厚，固定在70％乙醇中，并用苏木精和伊红轻微染色，然后分别激光捕获浅表尿路上皮、中间/基底尿路上皮和逼尿肌。实时荧光定量PCR检测α-SMA、CK20、毒蕈碱2受体(M2R)和毒蕈碱3受体(M3R)的表达。结果逼尿肌细胞中α-SMA mRNA表达量比对照组尿路上皮细胞高200倍。顶端尿路上皮细胞中的CK20 mRNA表达量是逼尿肌中的55倍，是中间/基底尿路上皮细胞中的4倍。慢性膀胱炎后，尿路上皮细胞中M2R和M3R mRNA的表达增加，而逼尿肌中M2R和M3R mRNA的表达减少。结论LCM可用于逼尿肌和不同层尿路上皮细胞的组织采集，同时其他细胞类型的污染最小，并且可以准确分析细胞类型特异性的分子表达变化。尿路上皮 MR 表达的增加可能会增强尿路上皮传入相互作用，从而诱发与膀胱炎症相关的膀胱过度活动/疼痛状况。
PurposeThis study examined whether the laser-capture microdissection (LCM) method can achieve separation of urothelial cells from detrusor cells or superficial urothelial cells from intermediate/basal urothelial cells, using α-smooth muscle actin (SMA) and cytokeratin 20 (CK20). In addition, we investigated the changes in expression of muscarinic receptors in laser-captured urothelial and detrusor cells in rats with chronic cystitis.MethodsFemale SD rats were injected with cyclophosphamide (75 mg/kg) intraperitoneally at day 1, 4, 7 and 10 to induce chronic cystitis. Saline was injected in the same protocol for controls. Bladder specimens were cut at 8 μm thickness, fixed in 70 % ethanol and lightly stained by hematoxylin and eosin, and then superficial urothelium, intermediate/basal urothelium and detrusor muscles were laser-captured separately. Real-time PCR was performed to examine expressions of α-SMA, CK20, muscarinic 2 receptors (M2R) and muscarinic 3 receptors (M3R).ResultsThe expression of α-SMA mRNA in detrusor muscle cells was 200 times higher than that in urothelial cells in controls. CK20 mRNA expression in apical urothelial cells was 55 times more than that in detrusor muscle and four times more than that in intermediate/basal urothelial cells. Expressions of M2R and M3R mRNA were increased in urothelial cells and decreased in detrusor muscles following chronic cystitis.ConclusionsThe LCM could be useful for tissue collection of detrusor muscle and different layers of urothelial cells with minimal contamination of other cell types, and cell type-specific changes in molecular expression could accurately be analyzed. Increased expression of urothelial MR might enhance urothelial–afferent interactions to induce bladder overactivity/pain conditions associated with bladder inflammation.