应用单细胞cDNA扩增技术鉴定循环肿瘤细胞

Identifi cation of circulating tumor cells by single-cell c DNA amplifi cation techniques

目的建立单细胞cDNA扩增技术,实现在分子水平鉴定循环肿瘤细胞。方法建立结肠癌循环肿瘤细胞模型,通过免疫荧光染色和显微切割技术分离出单个血细胞和肿瘤细胞;裂解细胞后利用碱基修饰的P1(dT)_(24)引物反转录出cDNA第一链,并给第一链cDNA加上PolyA尾后利用碱基修饰的P2(dT)_(24)引物合成第二链cDNA,然后利用P1(dT)_(24)和P2(dT)_(24)引物扩增分别得到血细胞和肿瘤细胞的全长cDNA;最后通过PCR检测细胞特异的标记物CD45、EpCAM和CK19。结果改良后的单细胞cDNA扩增方法能够获得高质量的全长cDNA,并能显著增加PCR检测效率;利用扩增得到的cDNA通过PCR检测细胞表面标记物CD45、EpCA M和CK19的表达,能有效鉴定出外周血中混杂的肿瘤细胞。结论通过单细胞cDNA扩增技术在基因水平识别肿瘤细胞,建立了一种新的循环肿瘤细胞的鉴定方法。

Objective To identify circulating tumor cells by single-cell cDNA amplification techniques.Methods Individual tumor cells from blood cells were separated by immunofluorescence staining,and then the cells were lysed and total RNA was protected by the lysate.First strand cDNA was transcripted using modified primer P1（dT）24 and then tailed with poly dA.Second-strand cDNA was synthesized using modified primer P2（dT）24.Then the double-strand cDNA was amplificated using primer P1（dT）24 and P2（dT）24.At last,the amplificated cDNA was used to detect cell specific marker of CD45,EpCAM and CK19 by PCR.Results Our results showed thatthe high-quality full-length cDNA was produced bythe single-cell cDNA amplification techniqueswhich could improve the effect of PCR significantly.The expression of CD45,EpCAM and CK19 detected by cDNA amplification techniques combined with gene specific PCR could identify tumor cells from blood cells effectively.Conclusion Single-cell cDNA amplification techniques can identify tumor cells from blood cells.