【作者】 周霖；

【导师】 王福生； 杨永平；

【作者基本信息】 中国人民解放军军医进修学院 ， 传染病学， 2009， 博士

【摘要】 研究背景:氩氦刀(argon-helium cryosurgical ablation system,CSA)是一种新的冷冻外科(cryosurgery)治疗手段,其基本原理是利用氩气快速制冷产生的超低温(<-140℃)作用于病变组织引起细胞内外冰晶形成、细胞脱水,氦气快速复温(20-40℃),导致肿瘤组织细胞坏死。目前这种微创外科技术是无法切除的中晚期HCC最为主要的局部治疗方法。氩氦刀除了对肿瘤的直接清除作用外,还能增强术后的抗肿瘤免疫。近年研究发现,肿瘤周围有大量CD4~+CD25~+FoxP3~+调节性T细胞(reguolatory T cells.Treg)浸润,通过抑制或杀伤效应性CD8~+或CD4~+T细胞而抑制机体的抗肿瘤免疫;相反,通过去除荷瘤鼠体内的Treg细胞可使抗肿瘤作用增强。我们的前期研究显示,调节性T细胞数量的升高与HCC患者预后较差有关。本研究通过氩氦刀冷冻治疗HCC患者,了解冷冻治疗对HCC患者调节性T细胞表达的影响,同时探索调节性T细胞变化对HCC患者预后的影响及其意义。方法:利用流式细胞仪检测101例HCC患者外周血调节性T细胞频率,同时对34例HCC病人进行氩氦刀治疗,随访治疗前后调节性T细胞频率及功能的变化,分析调节性T细胞频率变化与血清AFP水平之间的相关性,同时在治疗前后取原位肝癌组织进行免疫组化检测CD4、CD8和FoxP3。结果:我们观察到: (1)随着肝癌的进展,外周血中调节性T细胞频率逐渐增加,早期、中期、进展期、终末期HCC患者的Treg频率分别为6.89±1.27%,9.13±1.36%,10.32±2.04%和13.4±2.81%。(2)氩氦冷冻治疗后肿瘤缓解的17例HCC病人,随访结束时其Treg频率较术前降低,其中7例术前AFP阳性(>20ng/mL)的患者,术后Treg频率亦随AFP的降低而降低;对于肿瘤进展的14例HCC病人,随访结束时其Treg频率较术前明显升高,其中9例术前AFP阳性(>20ng/mL)的患者,肿瘤进展时亦伴有AFP的再次升高。(3)在治疗前后HCC患者Treg细胞对于健康人PBMC均有免疫抑制功能,其免疫抑制功能与是否治疗及肿瘤是否进展无关。(4)氩氦冷冻治疗后HCC患者冷冻区域旁肿瘤组织浸润的CD8~+T细胞、CD4~+T细胞和FoxP3~+T细胞的数量均显著低于术前,治疗后的FoxP3~+/CD8~+比值较术前明显降低。结论:氩氦冷冻消融治疗可降低HCC患者Treg频率,Treg频率的变化具有判断HCC病人肿瘤是否进展的意义,尤其是AFP阴性的HCC病人。更多还原

【Abstract】 Background & Objective: Argon-helium cryosurgical ablation (CSA), serving as a new cryosurgical approach, is a kind of mini-invasive surgical therapy, which is able to induce the necrosis of tumor cells through the formation of extracellular and intracellular ice crystals and then cell dehydration on the basis of rapidly freezing (<-140℃) as well as rapidly thawing (20-40℃) tumor tissues with argon/helium gas. Therefore, Argon-helium cryosurgical ablation becomes one of the major therapeutic approach for unresectable primary HCC patients at the middle and advanced satges. Boosting host anti-tumor immune response is alternative way for tumor disease. Recent studies have revealed that large amounts of resident regulatory T cells within tumor can inhibit the anti-tumor role via suppressing or killing effector CD4~+ or CD8~+T cells. Previous study has demonstrated that elimination of regulatory T cells can improve host anti-tumor immunity in tumor-bearing rat. Our findings indicate that high frequency of regulatory T cells correlates with poor prognosis in HCC patients. This study attempts to investigate how Argon-helium cryosurgical ablation influence regulatory T cells in HCC patients for evaluation of the prognosis in these patients .Methods: The frequence of regulatory T cells in 101 HCC patients’ peripheral blood was evaluated with flow cytometery.The frequency and function of regulatory T cells in peripheral blood of 31 HCC patients were longitudinally evaluated before and after cryoablation. Statistical analysis was used to figure out the association of regulatory T cells with serum AFP level. The distribution of resident CD4~+, CD8~+ T cells and FoxP3~+ T cells in liver tissue of HCC patients was examined by using immunochemical staining.Results: Our study found that (1) There was a increased frequency of regulatory T cells in parallel with the disease progression in HCC patients (early stage: 6.89±1.27%; intermediate stage:9.13±1.36%; advanced stage: 10.32±2.04%; terminal stage:13.4±2.81%) . (2) The frequency of regulatory T cells was reduced in 17 responders of 31 HCC patients with cryoablation treatment. In addition, while seven serum AFP positive (>20ng/mL) patients simultaneously exhibited a decrease of serum AFP level. However, we found that there was a increased regulatory T cells in peripheral blood of 14 advanced stage patients following the cryoablation, among them, 9 AFP positive patients were with a increase in serum AFP level. (3) Regulatory T cells of HCC patients were found to suppress proliferation of PBMCs of healthy donors. (4) There is decreased tumor resident CD8~+, CD4~+ T and FoxP3~+ T cells in HCC patients compared with their counterparts at baseline, together with more decrease of FoxP3~+/CD8~+ T cell ratio than those before treatment.Conclusions: Our findings suggest that Argon-helium cryosurgical ablation affect the variation of regulatory T cells, which may favor to boost host anti-tumor immunity and indicate the prognosis of the disease.更多还原