摘要
背景与目的：肿瘤免疫治疗是肿瘤治疗的重要手段之一。调节性T细胞(regulatory T, Treg)是一种免疫抑制性细胞,在肿瘤免疫中起重要作用。明确免疫抑制性细胞与肿瘤患者的临床病理特点及治疗效果的关系。有效地体外扩增获得杀伤广谱的效应细胞,一直是肿瘤免疫治疗中亟待解决的主要问题。本研究通过检测外周血Treg表达水平,探讨胰腺癌患者外周血Treg细胞水平的变化及其临床意义；同时通过RetroNectin法培养高效的效应细胞,探讨其在体外对于胰腺癌细胞的杀伤效应。
材料和方法：收集2006年10月至2009年3月我院胰腺癌患者外周血标本62例,健康人志愿者外周血标本30例。用流式细胞仪检测CD4+CD25high调节性T细胞比率变化差异,并分层分析探讨Treg细胞表达与胰腺癌分期和预后的关系；以免疫磁珠法获取外周血CD56+T细胞后用RetroNectin法培养获得NK效应细胞,并用MTS法对于JF-305人胰腺癌细胞株进行体外杀伤实验。通过表型检测、增殖测定以及杀伤率测定,分析RetroNectin法获得的NK细胞对于胰腺癌细胞杀伤作用的影响。
结果：胰腺癌患者外周血CD4+CD25+Foxp3调节性T细胞富含于CD4+CD25high调节性T细胞群中,其占CD4+T细胞的比率为(15.16±13.97)%,明显高于健康人群(3.38±1.70)%(P<0.001)。胰腺癌Ⅰ-Ⅱ期患者,其外CD4+CD25high调节性T细胞占CD4+T细胞的比率为(14.53±6.95)%(n=13)：III期为(17.75±18.66)%(n=31)；Ⅳ期为(18.48±21.27)%(n=18)。各期CD4+CD25high调节性T细胞占CD4+T细胞的比率均显著高于健康人群水平(P<0.001)；进一步分析显示其与患者的年龄、性别及胆红素水平、CA199水平、TNM分期无关；同时将其术前、术后外周血的水平进行比较结果显示：治疗性手术后比率下降具有统计学差异；生存分析显示其与预后无关,TNM分期为独立预后因素。
RetroNectin法培养效应细胞结果显示：RetroNectin法协同抗CD3抗体培养可以更高效的获得的效应细胞,2周时的增殖倍数可以达到371倍。表型的检测发生明显变化,RetroNectin法获取的NK效应细胞明显高于其他两组。体外杀伤实验结果显示：RetroNectin组培养获得的效应细胞在各效靶比杀伤下,杀伤活力明显高于另外两组。
结论：对CD4+CD25high调节T细胞的鉴定显示其富含Foxp3+细胞,本实验条件下的CD4+CD25high调节T细胞可以代表功能性的Treg细胞水平。胰腺癌患者外周血CD4+CD25high调节T细胞水平较健康对照者明显升高。非脾切除的治疗性手术后外周血treg细胞水平较术前明显下降。胰腺癌患者术前外周血Treg水平与预后无关。进一步的实验显体外实验显示RetroNectin法培养效应细胞NK细胞表型发生改变。联合CD3抗体培养增殖活性明显增强。RetroNectin法培养的NK细胞对于胰腺癌JF305细胞株的杀伤效率明显提高。因而如果能去除Treg细胞并联合应用高效的RetroNectin法培养的效应细胞,通过这两种免疫影响方式对胰腺癌进行治疗,未来可能成为胰腺癌免疫治疗新模式。
[English Abstract]
Research o f Detecting Regulatory T Cell in the Peripheral Blood of Patients and adoptive transfer RetroNectin activated NK cells in vitro in Pancreatic cancer M.D. Student:zhou jian guo Supervisor:zhao ping
Abdominal Surgery Department, Cancer Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100021, China
[Background and Purpose]
Immunotherapy is an important mode of treatment for cancer. Regulatory T cells (Treg) is an immunosuppressive cell, which plays an important role in tumor immunity. How to define immunosuppressed state of the tumor-bearing individuals associated with clinicopathologic features and therapeutic efficacy, and How to effectively acquire proliferation and broad-spectrum effector cells in vitro have been the main problems in cancer immunotherapy. In this study, the impact and significance of immunosuppressed state of pancreatic cancer is discussed by detecting the expression level of Treg in peripheral blood of pancreatic cancer and inhibitory effect of the highly efficient natural killer cells cultured by RetroNectin method in vitro.
[Materials and Methods]
62 peripheral blood samples of pancreatic cancer patients and 30 samples of healthy volunteers were collected from October 2006 to March 2009 in our hospital. Ratio difference of CD4+CD25high regulatory T cells was determined by flow cytometry. The relationship between the prevalence of Treg cells and pancreatic cancer staging and prognosis were Analysed. CD56+T cells of peripheral blood were acquired by immunomagnetic bead separation, and then NK cell were cultured by RetroNectin method. killing assay in vitro with JF-305 human pancreatic cancer cells was detected by MTS method. The impact of RetroNectin method for the killing of effector cells was Analysed by detecting phenotype, proliferation and anti-determined kill rate.
[Results]
The prevalence of CD4+CD25high regulatory T cells in CD4+T cells in the peripheral blood of patients with pancreatic carcinoma was(15.16±13.97)%, which was significantly higher than the level of healthy controls (3.38±1.70)%(P＜0.001).The proportions of Treg cells in the patients with pancreatic carcinoma in stageⅠ-Ⅱ(14.53±6.95)%(n=13) stageⅢ(17.75±18.66)%(n=31), stageⅣ(18.48±21.27)% (n=18), were all significantly higher when compared with healthy controls (P<0.01). Univariate analysis showed that there was no relationship between the prevalence of Treg cells with age, sex, CA199, bilirubin level and TNM staging. The proportion of CD4+CD25high regulatory T cells at the the 7th day after operation was signigicanfly lower than that level before operation with radical resection or palliation treatment. Univariate analysis didn't showed that it's the relationship with prognosis. Multivariate analysis indicated that only TNM stage was independent prognostic factor that influenced survival.
Results in vitro showed that Proliferative activity of effector cells increased 371-folds, which cultured by Anti-CD3 antibody and RetroNectin method. while the detection changed significantly in phenotype. CD56+T/CD56+CD16+T cell ratio was significantly higher than control group and Anti-CD3 antibody+RetroNectin group. Killing assay was observed with effector:target ratios from 10 to 40.The results of showed that RetroNectin group's kill rate were significantly different compared with other groups on every different effector target ratio.
[Conclusion]
Prevalence of Treg cells in peripheral blood of patients with pancreatic cancer significantly increased when compared with normal donors. Immunosuppression in patients with pancreatic cancer may be closely related to the increase in Treg cells. In compare with the proportion before resection and at first week after operation,the proportion of CD4+CD25high regulatory T cells descends by radical resection or palliation treatment. The proportion of CD4+CD25high regulatory T in peripheral blood was not related to prognosis.Further experiments showed that the immune killing effect of the NK cells cultured by RetroNectin method.be enhanced significantly. When added Anti-CD3 antibody, Proliferative activity of effector cells increased. RetroNectin method can be more efficient access to effector cells. This study shows good prospect of Culturing effector cells by combining removal of Treg cells and efficiently RetroNectin method for immunotherapy is a feasible idea. It may provide theoretical and experimental basis for building a new adoptive immunotherapy model for pancreatic cancer in future.
引文
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