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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,激光捕获显微切割技术(lasercap-turemicrodissection,LCM)是不可缺少的,然而从LCM所得的细胞中获取足够RNA量相当困难。本文首次将LCM与RNA线性扩增结合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,并对其进行RNA体外线性扩增,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进行体外线性扩增。然后用RT-PCR验证扩增前、后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LCMshooting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。产物RNA扩增后获得片段大小为0.5-2.5kh的aRNA,且β-actin基因表达表达完整。结论LCM结合RNA体外线性扩增技术能成功地获取较为单一的研究目的细胞,扩增后RNA完整性较好,能用于进一步研究中。
OBJECTIVE: Laser capture-microdissection (LCM) is an indispensable technique for the differential gene expression analysis of transitional cell carcinoma of the bladder between mesenchymal cells and bladder transitional cell carcinoma. However, Getting enough amount of RNA is quite difficult. This paper, for the first time, combined the linear amplification of LCM and RNA for the study of bladder cancer-associated genes in order to determine a viable technical route to separate bladder transitional epithelial cells from the bladder mucosa and to separate bladder cancer cells from tumor stromal cells Isolated, and its RNA in vitro linear amplification for further study. Methods The bladder transitional epithelial cells and bladder cancer cells were obtained from the frozen sections of normal bladder mucosa and bladder cancer tissue respectively by LCM technique. The RNA was extracted and the microRNAs were linear amplified in vitro. RT-PCR was then used to verify the β-actin gene expression level before and after amplification. The results of the control experiment I confirmed that the integrity of the RNA after LCM better; control experiment Ⅱ initially determine the set conditions LCMshooting the number of times and available RNA correspondence between the amount. Capture 250,000 shootings of bladder transitional epithelial cells from bladder mucosa; 200,000 shootings of cancerous cells from bladder cancer tissues. After amplification of the product RNA, aRNA with a fragment size of 0.5-2.5 kh was obtained, and the expression of β-actin gene was complete. Conclusion LCM combined with RNA in vitro linear amplification technology can successfully obtain a single target cell for study. The RNA integrity after amplification is good and can be used for further research.