【作者】 郝权； 徐勇； 李文录； 高兴； 邱志强；

【Author】 Hao Quan Xu Yong Li Wen-Lu Gao Xing Qiu Zhi-Qiang Pelvis department Tianjin Cancer Hospital affiliated to Tianjin Medical University, Tianjin 300060 Urological department The Second Hospital affiliated to Tianjin Medical University, Tianjin 300211 Medical Research Centre Medical college,Xian Communication University, Xian 710061 Pathology department Tianjin Cancer Hospital affiliated to Tianjin Medical University, Tianjin 300060

【机构】 天津医科大学附属肿瘤医院盆腔科； 天津医科大学第二医院泌尿科； 西安交通大学医学院医学实验中心； 天津医科大学附属肿瘤医院病理科；

【摘要】 目的:当对有间质细胞混杂的膀胱移行上皮细胞与膀胱移行细胞癌进行基因差异表达分析时,为保证研究结果的准确性、可靠性,必须获取均一的研究目的细胞,激光捕获显微切割技术(laser capture microdissection,LCM)是不可缺少的。本文首次将LCM技术用于膀胱癌相关基因研究,目的是确定一条切实可行的技术路线,将膀胱移行上皮细胞从膀胱粘膜中分离出来,将膀胱癌细胞从肿瘤间质细胞中分离出来,进行RNA提取、纯化、浓缩以备进一步研究。方法: 采用LCM技术分别从正常膀胱粘膜及膀胱癌组织冰冻切片中获取膀胱移行上皮细胞及膀胱癌细胞,提取RNA,并对微量 RNA进行纯化、浓缩。然后用RT—PCR验证Total RNA中β-actin基因表达水平。结果:对照实验I证实经LCM后 RNA完整性较好;经对照实验Ⅱ初步确定设定条件下LCM shooting次数与可获得RNA量间对应关系。从膀胱粘膜种捕获膀胱移行上皮细胞25万shootings;从膀胱癌组织中获取癌细胞20万shootings。经RT—PCR验证β-actin基因表达表达完整。结论:使用LCM技术能成功地获取较为均一的研究目的细胞,RNA完整性较好,能用于进一步研究中。更多还原

【Abstract】 Objective: Laser Capture Microdisection has become indispensable for obtaining the homogeneous objective cells that can ensure the accuracy and reliability of the results when the analysis of the difference of gene expression between human bladder transitional cell and bladder transitional cell carcinoma(BTCC) is performed. The study was designed to determine a feasible technical routine to isolate transitional cells from bladder membrane ,separate carcinoma cells from stromal cells and to purify、 concentrate the RNA from laser-capture microdissected cells. Methods: Bladder transitional cells were obtained from frozen bladder membrane sections applying LCM, by the same taken, BTCC cells from frozen BTCC tissue section. Then RNA was extracted 、 purified and concentrated. The expression level of β - actin in total RNA was detected using RT-PCR. Results: RNA integrity is good after LCM confirmed by contral experiment I, By contral experiment II ,the correlation between the number of LCM-shooting and RNA quantity under arranged conditions was preliminary conformed. β - actin level was integral. Conclusion: Laser Capture Mi-crodissection can be apUied to successfully obtain pure objective cells for research. The integrity of RNA is good and can be used in further research.更多还原