oHSV1-hTERT-GFP法检测小细胞肺癌循环肿瘤细胞

Capture approach of oHSV1-hTERT-GFP for circulating tumor cells of small cell lung cancer

目的在正常人体细胞中活性被抑制的端粒酶,在肿瘤细胞中处于活化状态,可以特异性示踪循环肿瘤细胞。本研究拟建立基于插入人端粒酶启动子和绿色荧光蛋白(green fluorescent protein,GFP)基因的单纯疱疹病毒(oHSV1-hTERTGFP)检测小细胞肺癌(small cell lung cancer,SCLC)外周血中循环肿瘤细胞的方法。方法观察SCLC细胞在感染病毒oHSV1-hTERT-GFP后的荧光强度来判断GFP的表达,通过建立SCLC循环肿瘤细胞模型,用oHSV1-hTERT-GF法检测CD45/GFP+细胞,分析此法对SCLC细胞的捕获效率和可重复性;通过CD56与GFP进行共定位来验证oHSV1-hTERT-GFP对SCLC细胞示踪的特异性;应用oHSV1-hTERT-GFP法检测50例正常人和10例广泛期SCLC病人治疗前后的外周血中CD45-/GFP+细胞数,分析此法检测正常人与SCLC病人CD45-/GFP+细胞数的差异和SCLC病人治疗前后CD45-/GFP+细胞数变化,并将oHSV1-hTERT-GFP法检测的循环肿瘤细胞数量变化与临床疗效进行对比分析。结果感染病毒oHSV1-hTERT-GFP后24h的SCLC细胞GFP表达量较感染后16h显著增加;模型实验结果显示oHSV1-hTERT-GFP法检测循环肿瘤细胞的效率与其在外周血中的含量无关;oHSV1-hTERT-GFP病毒捕获的细胞与CD56共定位,oHSV1-hTERT-GFP捕获SCLC细胞的特异性高。临床实验中,SCLC病人外周血中的CD45-/GFP+细胞数比健康人显著增加;在病情缓解的病例中,治疗后CD45-/GFP+细胞数较治疗前显著下降。结论基于oHSV1-hTERT-GFP的检测方法能够识别和鉴定出SCLC外周血中的循环肿瘤细胞,对SCLC的复发转移以及疗效评价等有一定临床应用价值。

Objective Telomerase activity can be used to trace circulating tumor cells(CTCs),as it is inhibited in normal human cells but activated in tumor cells.The study is to establish a method for detecting CTCs in the peripheral blood of small cell lung cancer(SCLC) patients based on a herpes simplex virus(oHSV1-hTERTGFP).This virus is engineered to replicate only in cells with telomerase activity and subsequently express green fluorescent protein.Methods The GFP expression at different time points post oHSV1-hTERT-GFP infection of SCLC cell lines was detected by fluorescent microscopy.In CTC mimic models,the capture efficiency and reproducibility were tested by detecting CD45 ^-/GFP^+ cells with oHSVl-hTERT-GFP and fluorescent anti-CD45 monoclonal antibody.The captured cancer cells were validated by CD56 expression.Further,the CD45^-/GFP^+ cells per 4ml blood from 50 healthy donors and 10 SCLC patients were counted.Finally,the association between the CTC number and treatment response for the 10 SCLC patients was investigated.Results In SCLC cell lines,the GFP expression associated with oHSVl-hTERT-GFP infection was observed at 16 h and significantly increased at 24 h.The CTC mimic models showed that the CTC detecting efficiency was not related to the amount of tumor cells in peripheral blood.In the verification experiment,the CD45^-/GFP^+ and CD56 were detected in the same cell.In clinical investigation,the number of CD45 ^-/GFP^+ cells for SCLC patients was significantly higher than that for healthy donors.Paired comparison analysis showed that the CTC number of SCLC patients decreased significantly after treatment.Conclusion This CTC capture method based on oHSVl-hTERT-GFP is capable of detecting circulating tumor cells in small cell lung cancer,and it has clinical application potential concerning SCLC recurrence and metastasis.