新型荧光探针组合用于循环肿瘤细胞中鉴定的研究

A novel combined fluorescent probe staining method for CTCs identification

目的验证新型荧光探针组合鉴定循环肿瘤细胞(CTCs)的可行性,建立一种新的鉴定CTCs的方法。方法选取Eca-109和HeLa两种肿瘤细胞系,将肿瘤细胞系制作成单细胞悬液代替CTCs,对其进行分离、荧光探针组合染色和瑞氏吉姆萨染色,分别观察染色的肿瘤细胞的形态,对比测量到的肿瘤细胞形态学特征,包括细胞长径、细胞核长径、细胞表面积和核质比,计算新型荧光探针组合与瑞氏吉姆萨分别染色肿瘤细胞系的符合率。在此基础上,课题组选取5例次肿瘤患者,按照上述实验方法进行临床验证,观察新型荧光探针组合染色CTCs的情况。结果分析瑞氏吉姆萨染色和荧光探针组合染色Eca-109和HeLa的细胞形态学结果,发现细胞长径、细胞核长径、细胞表面积和核质比均无差异;对两种肿瘤细胞系的细胞形态学结果的均数分析发现也无差异。因此,课题组认为瑞氏吉姆萨染色和荧光探针组合染色的同一肿瘤细胞系在形态学上是一致的。在5例次临床验证实验中,新型荧光探针组合染色检出2例次。结论建立的新型荧光探针组合可用于CTCs的检测。

Objective To verify the feasibility of a novel combination of fluorescent probes to identify circulating tumor cells(CTCs),and to establish a new method for identifying CTCs.Methods Two types of tumor cell lines(Eca-109 and HeLa)were selected by the research group.The tumor cell lines were made into single-cell suspensions instead of CTCs.The new fluorescent probe combination SOP was developed and compared.Morphological characteristics of tumor cells,including cell diameter,nucleus diameter,cell surface area,and nuclear-to-cytoplasmic ratio,were measured by fluorescent probe and Wright’s Gemsa staining.The coincidence rate of the new fluorescent probe combination with Wright’s Gimsa staining tumor cell lines was calculated with preliminary determination of the new fluorescent probe combination to identify the SOPs of CTCs.On this basis,5 tumor patients were selected according to the above experimental methods and CTCs were observed by the combination of new fluorescent probes staining.Results The morphological results of two tumor cell lines(Eca-109 and HeLa)stained by Wright’s Giemsa staining and fluorescent probes were analyzed.It was found that the cell diameter,nucleus diameter,cell surface area,and nuclear-to-cytoplasm ratio were all no difference.Therefore,after morphological analysis,there was no difference in the cell morphological results of the two tumor cell lines(Eca-109 and HeLa)stained by Wright’s Giemsa staining and fluorescent probe combination.The mean of cell morphology results of the two tumor cell lines stained by Wright’s Giemsa staining and fluorescent probes was analyzed,and no difference was found in cell diameter,nucleus diameter,cell area,and cytoplasmic ratio.Therefore,after morphological analysis,the same tumor cell line stained by Wright’s Giemsa staining and fluorescent probe combination was morphologically consistent.In 5 clinical verification experiments,2 cases were detected by combined staining of new fluorescent probes.Therefore,the SOP for identification of CTCs by combined staining of new fluorescent probes was feasible.Conclusions The new fluorescent probe combination can be used for the detection of CTCs,and the research team has established a new method for identifying CTCs.