激光捕获技术在心房细胞定位分离中的应用

An Application of Laser Capture Microdissection to Isolate the Pure Atrial Cells

目的探索激光捕获显微切割技术（laser capture microdissection，LCM）定位分离心房细胞，并对提取的微量RNA进行β-actin及GAPDH基因扩增。方法首先验证预做LCM样品的完整性，然后常规制备冰冻切片，采用改进的HE快速染色脱水法染色，提取刮片组织的RNA验证其质量，确保在切片染色过程中RNA的完整性．最后进行LCM分离纯化心房细胞，通过逆转录-聚合酶链反应（RT—PCR）扩增β-actin及GAPDH基因．结果经快速HE染色心房细胞形态学清晰；预做LCM的样品和染色后组织刮片可提取出高质量的RNA，紫外分光光度计测定吸光度值A260／A80为1．9～2．0之间。甲醛变性凝胶电泳显示清晰的28SrRNA和18SrRNA条带，捕获细胞提取的微量RNA经RT—PCR后进行凝胶电泳显示有300bp及357bp的扩增条带．结论LCM可以分离出纯度较高的心房细胞，提取的微量RNA可以扩增出管家基因β-actin和GAPDH．

Objective To use the laser capture microdissection（LCM） to isolate the pure atrial cells from heart. To extract tiny amount RNA and amplify the genes of β-actin and GAPDH. Methods First, the prepared sample RNA was extracted and verified. Second, the frozen sections were scraped after haematoxylin-eosin staining, and then the RNA of scraped tissues was extracted and verified. Last, the tiny amount RNA was extracted from atrial cells captured by LCM, and the genes of β-actin and GAPDH were amplified by reverse transcriptase polymerase chain reaction （RT-PCR）. Results The atrial cells isolated with LCM had clear morphology after quick staining. The high quality RNA was extracted from LCM sample and scraped tissues, of which A60/A280 was 1.9-2. 0. And 18S rRNA and 28S rRNA were seen distinctly on the gel of RNA formaldehyde denaturing gel electrophoresis, which indicated that RNA didn＇t degrade before cells captured. Too small amount of RNA extracted from captured cells was below the detectable limit of gel electrophoresis or ultraviolet spectrophotometer. Bands of 300 bp and 357 bp could be amplified by RT-PCR from tiny amount RNA. Conclusion Highly pure atrial cells could be obtained by LCM. Tiny amount RNA of captured atrial cells could be extracted and amplified with genes of β-actin and GAPDH.