趋化因子CCL5在食管癌组织中的表达及意义

Expression and clinical significance of CCL5 in patients with esophageal carcinoma

目的 探讨趋化因子CCL5在食管癌组织中的表达及意义.方法 采用逆转录聚合酶链反应（RT-PCR）检测食管癌组织和癌旁组织中CCL5、CD8和CD8^＋T细胞杀伤功能相关细胞因子穿孔素（perforin）和颗粒酶B（granzyme B）的表达水平.采用流式细胞术检测食管癌患者外周血单核细胞（PBMC）和肿瘤浸润性淋巴细胞（TIL）中CD8^＋T细胞和CCR5^＋CD8^＋T细胞比例.采用Transwell实验检测CCL5对T细胞运动的影响.结果 食管癌组织中CCL5和perforin mRNA的表达水平分别为0.348 2±0.300 1和0.181 9±0.118 6,癌旁组织中CCL5和perforin mRNA的表达水平分别为0.279 6±0.138 0和0.118 0±0.1098,但差异均无统计学意义（均P＞0.05）.食管癌组织中CD8和granzyme B mRNA的表达水平分别为0.4649±0.3008和0.6487±0.5160,癌旁组织中CD8和granzyme B mRNA的表达水平分别为0.279 0±0.173 4和0.469 7±0.259 1,差异有统计学意义（均P ＜0.05）.食管癌患者中CCL5与CD8、perforin和granzyme B表达呈正相关（rCD8=0.272,P=0.034;rerforin=0.305,P=0.026;rgranzymeB=0.108,P=0.012）.流式细胞术结果显示,TIL和PBMC中CD8^＋T细胞比例分别为（45.86±16.09）％和（34.05±15.07）％,差异有统计学意义（P =0.022）;TIL和PBMC中CCR5^＋ CD8^＋T细胞比例分别为（48.12±26.75）％和（19.53±13.67）％,差异有统计学意义（P＜0.001）.Transwell实验结果显示,CCL5显著增强T细胞趋化运动.CCL5表达与患者性别、年龄和淋巴结转移无关,但CCL5在早期食管癌组织中的相对表达水平为0.319 9±0.161 7,在晚期食管癌组织中的相对表达水平为0.232 8±0.121 0,差异有统计学意义（P=0.008）.早期食管癌患者TIL中CD8^＋T细胞和CCR5^＋ CD8^＋T细胞比例分别为（48.86±15.87）％和（56.23±26.23）％,而在晚期食管癌患者TIL中CD8^＋T细胞和CCR5^＋ CD8^＋T细胞比例分别为（33.25±16.49）％和（33.53±21.03）％,差异均有统计学意义（PCD8 =0.007,PccR5=0.010）.结论 CD8^＋T细胞能够在CCL5的诱导下向肿瘤部位运动,并影响患者疾病进展,CCL5可作为食管癌治疗的新靶点。

Objective To investigate the expression and significance of CCL5 in patients with esophageal carcinoma.Methods Using reverse transcriptase polymerase chain reaction （RT-PCR）,the expressions of CCL5/CD8/granzyme B/perforin in tumor and corresponding adjacent tissues from esophageal carcinoma patients were examined.Flow cytometry （FACS） was used to detect the percentages of CD8 ＋ T cells and CCR5 ＋ CD8 ＋ T cells in TIL and PBMC from the patients.Transwell assay was performed to study the effect of CCL5 on the migration of T cells in vitro.T test and Spearman correlation analysis were performed.Results The mRNA expressions of CCL5 and perforin were 0.348 2 ±0.300 1 and 0.181 9 ± 0.118 6,respectively,in the tumor samples,while their expressions in adjacent samples were 0.279 6 ± 0.138 0 and 0.118 0 ± 0.109 8,respectively,with no statistically significant differences between them （P 〉 0.05 for both）.The mRNA expressions of CD8 and granzyme B were significantly higher in the tumor tissues than in adjacent tissues （0.464 9 ± 0.300 8 vs.0.279 0 ± 0.173 4,0.648 7 ± 0.516 0 vs.0.469 7 ± 0.259 1 ; P 〈 0.05 for both）.The relative expression of CCL5 was positively correlated with that of CD8,perforin and granzyme B （rCD8 =0.272,P =0.034; rperforin =0.305,P =0.026; rgranzymeB =0.108,P=0.012） in the tumor sites.FACS data revealed that the proportions of CD8 ＋ T cells in TIL and PBMC were （45.86 ± 16.09） % and （34.05 ± 15.07） %,respectively,showing a significant difference （P =0.022）.Similarly,CCR5 ＋ CD8^＋ T cells fraction in TIL （48.12 ± 26.75）% was much higher than that in PBMC（19.53 ± 13.67） % （P 〈 0.001）.Transwell assay showed that CCL5 protein enhanced the migration of T cells,supporting that CCL5 is crucial for CD8 ＋ T cells recruitment in vivo.Intriguingly,CCL5 expression was down-regulated in advanced patients （stage Ⅱb-Ⅳ）.The accumulation of CD8 ＋ T cells and CCR5 ＋ CD8 ＋ T cells was strongly reduced in advanced patients,suggesting that CCL5 expression may be involved in the local control of the disease and its reduction may be involved in disease progression.Conclusions The current data indicate the involvement of CCL5 in the regulation of CD8 ＋ T cell entry into tumor lesions in esophageal carcinoma patients.This process may affect the disease status and potentially as a prognostic factor for cancer patients.Enhancing local CCL5 expression in tumor lesions may represent a novel strategy in esophageal cancer therapy.