改良“彗星”分析法检测实体肿瘤放射敏感性的方法学研究

A methodological study on modified comet assay in predicting solid tumor radiosensitivity

目的改进和完善改良“彗星”分析法以更好地用于检测实体肿瘤的放射敏感性。方法门诊活检标本制成单细胞悬液，冰上照射后立即铺胶制板，在裂解液中裂解50min，漂洗后进行细胞电泳20min。PI染色后在荧光显微镜下采图并用专用软件进行图像分析。每例样品均设对照（包括外参对照和自身对照）和5Gy照射，并对活检样品的正常细胞和肿瘤细胞进行分类采图。观察指标为细胞DNA含量和尾力矩。结果影响实验室测定结果与临床肿瘤放射反应相关性的因素主要有：（1）取材较表浅样品中仅有淋巴细胞而无肿瘤细胞，从而导致实验室测定结果与临床肿瘤放射敏感性的吻合性下降；（2）样品中坏死细胞较多，使本底增高，是影响放射敏感性分析的重要因素；（3）增设淋巴瘤细胞作为外参对照可对实验系统误差进行校正，是质量控制不可缺少的环节；（4）对活检样品中正常细胞和肿瘤细胞进行分类采图能提高临床与实验室检测结果的吻合性。结论实验同时设置内、外参对照和对样品中肿瘤细胞和正常组织细胞进行分类采图，能不同程度提高实验室检测结果与临床肿瘤实际放射反应的吻和性。

Objective To improve the method of “modified comet assay” in predicting the radiosen- sitivity of solid tumor. Methods A single cell suspension from biopsy sample was irradiated on ice with a dose of 5 Gy. The microscope slide was spread with agarose, lysed for 50 minutes, rinsed 3 times in rinse solution, and given electrophoresis for 20 minutes. After being stained with PI, cell images were collected through the microscope and analyzed with Lucia G software （ Version 4.6）. In order to check system/back-ground errors, every sample was made into control slide and irradiation slide. The end-points were cell DNA contents and tail moment. Results The factors influencing the results included： （1） Sample was faulty for the biopsy taken from mucosa and no tumor cells were contained. （2） The slides with a high background （induced by necrosis） disturbed the measurement of comet assay. （3） Setting lymphecytes as control to check system errors was very important. （4） To separately collect images of the normal tissue cells and tumor cells from the biopsy sample improved the conformity between the clinical observation and the lab resuit. Conclusions To increase the correlation between comet assay and clinical response, it is very help-ful to set double control for checking system/background errors and to collect images of the normal tissue cells and tumor cells through the microscope, respectively.