原发性肺腺癌患者外周血端粒酶逆转录酶阳性白细胞检测方法的建立

doi:10.3969/j.issn.1004-616x.2024.06.002

摘要/Abstract

摘要： 目的： 建立一种基于端粒酶逆转录酶(TERT)活性的外周血白细胞亚群检测方法，以评估恶性肿瘤患者外周血中白细胞的增殖潜能。方法： 改造的重组I型单纯疱疹病毒(HSV1-hTERTp-eGFP)能够特异性地在TERT启动子高转录活性的白细胞中复制，并表达绿色荧光蛋白(GFP)，通过观察绿色荧光识别具有高增殖潜力的TERT阳性白细胞，评估机体的免疫增殖潜能。分别将100、500、1 000个经HSV1-hTERTp-eGFP病毒感染24 h后的Jurkat阳性细胞，加入到未经病毒感染的2.5×10⁶个外周血白细胞中，作为模拟样品进行检测，设置3个平行组，并对阳性细胞的回收率、重复性、线性回归系数等指标进行检验。同时，采集5例良性肺结节患者外周血，设置3个平行组，检测每组1 mL外周血中的TERT阳性白细胞比例，通过计算组内变异系数评估外周血样本检测技术的可靠性。此外，利用本检测技术，对80例良性肺结节患者及80例原发性肺腺癌患者外周血TERT阳性白细胞百分比进行检测和分析。结果： Jurkat细胞系能够被HSV1-hTERTp-eGFP病毒感染并启动GFP的表达，可以作为模拟样本进行技术验证。100、500、1 000个Jurkat细胞的模拟样品平均检出阳性细胞数分别为80.3、448.2、920.6个，回收率分别为80.3%、89.6%和92.1%；3次重复实验的批间变异系数分别为11.3%、7.2%、4.4%，而3个平行实验组内的批内变异系数均小于10%；5例良性肺结节患者外周血TERT阳性细胞百分比的组内变异系数也均在10%以内。说明本检测技术准确度高，且具有良好的稳定性。在80例良性肺结节患者中，随着年龄的增长，TERT阳性白细胞的百分比呈下降趋势(r=-0.61；P<0.01)。与良性肺结节患者对照组相比，早期肺腺癌患者的TERT阳性白细胞百分比降低(P<0.01)；与非浸润性肺腺癌患者相比，浸润性肺腺癌患者的TERT阳性白细胞的比例显著降低(P<0.01)。结论： 肺腺癌患者的外周血存在免疫细胞增殖能力下降的现象，且在浸润性肺腺癌患者中表现更明显。这一发现可能与年龄相关的免疫衰老有密切联系，为理解浸润性肺腺癌患者较差的预后提供了新视角，并为深入探索免疫衰老机制指明了方向。

关键词: 肺腺癌, 端粒, 端粒酶逆转录酶, 免疫衰老

Abstract: OBJECTIVE： This study aimed to develop a method for detecting white blood cell subsets with high telomerase reverse transcriptase (TERT) activity to evaluate proliferative potential of white blood cells in patients with malignant tumors. METHODS： A modified recombinant type I herpes simplex virus (HSV1-hTERTp-eGFP) was able to replicate in white blood cells with high TERT promoter transcriptional activity and to express green fluorescent protein (GFP). By observing green fluorescence，TERT positive white blood cells with high proliferation potential was identified，and the immune proliferation potential of individuals was evaluated. The addition of 100，500 and 1 000 GFP positive Jurkat cells that were infected with HSV1-hTERTp-eGFP virus for 24 hours to 2.5×10⁶ peripheral blood white blood cells that have not been infected with the virus were used as simulated samples for detection. Three parallel groups were set up and were tested for recovery rate，repeatability，linear regression coefficient，and other indicators. At the same time，peripheral blood samples were collected from 5 patients with benign pulmonary nodules，and 3 parallel groups were set up to detect the proportion of TERT positive white blood cells in 1 mL of peripheral blood in each group. The technical reliability of detecting peripheral blood samples was evaluated by calculating the coefficient of variation within each group. Using this detection technology，the percentage of TERT positive white blood cells in peripheral blood of 80 patients with benign pulmonary nodules and 80 patients with primary lung adenocarcinoma were detected and analyzed. RESULTS： In the simulated samples with Jurkat cells，the average number of positive cells were 80.3，448.2 and 920.6，respectively，with recovery rates of 80.3%，89.6% and 92.1%，respectively；The inter batch coefficients of variation for the three repeated experiments were 11.3%，7.2% and 4.4%，respectively，while the intra batch coefficients of variation within the three parallel experimental groups were less than 10%；The intra group coefficient of variation for the percentage of TERT positive cells in peripheral blood of 5 patients with benign pulmonary nodules was also within 10%. This detection technology showed high accuracy and stability. In 80 patients with benign pulmonary nodules，the percentage of TERT positive white blood cells showed a decreasing trend with age (r=-0.61；P<0.01). Compared with the control group of benign pulmonary nodule patients，the percentage of TERT positive white blood cells in early lung adenocarcinoma patients decreased (P<0.01)；Compared with non-invasive lung adenocarcinoma patients，the proportion of TERT positive white blood cells in invasive lung adenocarcinoma patients was significantly reduced (P<0.01). CONCLUSION： The peripheral blood immune cells of patients with lung adenocarcinoma showed a decrease in proliferation ability，which was more pronounced in patients with invasive lung adenocarcinoma. The observation was reminiscent to immunosenescence，and would provide a better understanding of poor prognosis among patients with invasive lung adenocarcinoma.

Key words: lung adenocarcinoma, telomeres, telomerase reverse transcriptase, immunosenescence

中图分类号:

R734.2

谢佩佩, 张颀, 张小利, 万朵, 张文, 程书钧, 张开泰. 原发性肺腺癌患者外周血端粒酶逆转录酶阳性白细胞检测方法的建立[J]. 癌变·畸变·突变, 2024, 36(6): 431-437,443.

XIE Peipei, ZHANG Qi, ZHANG Xiaoli, WAN Duo, ZHANG Wen, CHENG Shujun, ZHANG Kaitai. Development of a detection method for TERT positive white blood cells in patients with primary lung adenocarcinoma[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2024, 36(6): 431-437,443.

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