Laser microdissection as a new tool to investigate site-specific gene expression in enteric ganglia of the human intestine
Laser microdissection as a new tool to investigate site-specific gene expression in enteric ganglia of the human intestine
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DOI：
10.1111/j.1365-2982.2009.01424.x
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发表时间：
2010-02-01
影响因子：
3.5
通讯作者：
Wedel, T.
中科院分区：
文献类型：
--
作者：
Boettner, M.;Baer, F.;Wedel, T.
背景肌神经节是控制肠道运动的关键结构，其mRNA表达谱在病理条件下可能发生改变。传统的全层标本RT-PCR的一个缺点是基因表达分析是基于异质组成的组织。为了克服这一问题，激光显微解剖结合实时RT-PCR可以检测和量化离体肠神经节中低水平的基因表达。方法对8例与肠动力障碍无关的疾病患者采集新鲜乙状结肠未固定全层标本。10 μ m冷冻切片装在膜包覆的载玻片上，用甲苯胺蓝超快速染色。采用激光显微解剖和弹射法分离心肌神经节mRNA。选择生长因子、神经递质受体和特定细胞类型标记物进行实时RT-PCR检测。收集0.5 mm2的神经节组织足以获得RT-PCR阳性结果。收集4 mm2的ct值可对基因表达水平进行可靠的定量比较。mRNA分析显示，神经营养生长因子、神经营养因子-3、血清素受体3A、PGP 9.5和S100 β在人结肠肌肠神经节特异性表达。结论与推论激光显微解剖联合实时RT-PCR是一种可靠地检测和定量与人肠道神经系统相关的低丰度mrna（如生长因子、神经递质受体）位点特异性表达的新技术。这种技术方法扩大了可用工具的范围，在分子生物学水平上表征人类胃肠运动障碍的肠道神经病理学。
BackgroundMyenteric ganglia are key-structures for the control of intestinal motility and their mRNA expression profiles might be altered under pathological conditions. A drawback of conventional RT-PCR from full-thickness specimens is that gene expression analysis is based on heterogeneously composed tissues. To overcome this problem, laser microdissection combined with real-time RT-PCR can be used to detect and quantify low levels of gene expression in isolated enteric ganglia.MethodsFresh unfixed full-thickness specimens of sigmoid colon were obtained from patients (n = 8) with diseases unrelated to intestinal motility disorders. 10 mu m cryo-sections were mounted on membrane-coated slides and ultra-rapidly stained with toluidine blue. Myenteric ganglia were isolated by laser microdissection and catapulting for mRNA isolation. Real-time RT-PCR was performed for selected growth factors, neurotransmitter receptors and specific cell type markers.Key ResultsCollection of 0.5 mm2 of ganglionic tissue was sufficient to obtain positive RT-PCR results. Collection of 4 mm2 resulted in ct-values allowing a reliable quantitative comparison of gene expression levels. mRNA analysis revealed that neurotrophic growth factor, neurotrophin-3, serotonin receptor 3A, PGP 9.5 and S100 beta are specifically expressed in myenteric ganglia of the human colon.Conclusions & InferencesLaser microdissection combined with real-time RT-PCR is a novel technique to reliably detect and quantify site-specific expression of low-abundance mRNAs (e.g. growth factors, neurotransmitter receptors) related to the human enteric nervous system. This technical approach expands the spectrum of available tools to characterize enteric neuropathologies underlying human gastrointestinal motility disorders at the molecular biological level.