Konishi H, Steinbach G, Hittelman W N, Fujita K, Lee J J, Glober G A, Levin B, Andreeff M, Goodacre A M, Terry N H
Department of Gastrointestinal Medical Oncology and Digestive Diseases, University of Texas M. D. Anderson Cancer Center, Houston, USA.
Gastroenterology. 1996 Dec;111(6):1493-500. doi: 10.1016/s0016-5085(96)70010-0.
BACKGROUND & AIMS: Precise quantitative and spatial analysis of cell cycle-related biomarkers in colonic crypts is often vital for studies of colon carcinogenesis and cancer prevention. To overcome the limitations of histology, confocal laser microscopy of microdissected whole crypts was used to quantitate S phase and mitotic cells.
Microdissected distal colonic crypts were studied in a modified rat starvation refeeding model. S phase cells were labeled in vivo with 5-bromodeoxyuridine. Mitotic cells were labeled with MPM2 (antibody to mitosis-specific epitope) and also assessed for chromatin morphology with propidium iodide. Sequential optical crypt sections, produced by confocal microscopy, were digitally imaged. S phase labeling indices per whole crypt were also compared with those derived by conventional immunohistochemistry.
S phase and mitotic cells were clearly discriminated without background staining. The labeled S phase cell number and fraction per whole crypt were significantly decreased with starvation and increased with refeeding. Variability in the labeling index between whole crypts analyzed by confocal microscopy was significantly smaller than between histological crypt sections. Consequently, the intervention contributed to 92.2% of the total variability of the labeling index in whole crypts but only to 59% of the variability in histological sections.
Major limitations of histology are overcome by crypt microdissection and confocal microscopic analysis. The total crypt cell population as well as labeled M phase and S phase cells can be imaged, localized, and quantitated with improved precision.
结肠隐窝中细胞周期相关生物标志物的精确定量和空间分析对于结肠癌发生和癌症预防研究通常至关重要。为克服组织学的局限性，采用对显微切割的完整隐窝进行共聚焦激光显微镜检查来定量S期和有丝分裂细胞。
在改良的大鼠饥饿再喂养模型中研究显微切割的远端结肠隐窝。S期细胞在体内用5-溴脱氧尿苷标记。有丝分裂细胞用MPM2（有丝分裂特异性表位抗体）标记，并用碘化丙啶评估染色质形态。通过共聚焦显微镜产生的连续光学隐窝切片进行数字成像。还将每个完整隐窝的S期标记指数与传统免疫组织化学得出的指数进行比较。
S期和有丝分裂细胞清晰可辨，无背景染色。每个完整隐窝的标记S期细胞数量和比例在饥饿时显著减少，再喂养时增加。通过共聚焦显微镜分析的完整隐窝之间标记指数的变异性明显小于组织学隐窝切片之间的变异性。因此，干预对完整隐窝中标记指数总变异性的贡献为92.2%，而对组织学切片变异性的贡献仅为59%。
隐窝显微切割和共聚焦显微镜分析克服了组织学的主要局限性。完整隐窝细胞群体以及标记的M期和S期细胞可以更精确地成像、定位和定量。
