【作者】 段浩清；

【导师】 王远东；

【作者基本信息】 广州医学院 ， 外科学， 2009， 硕士

【摘要】 目前恶性肿瘤主要的治疗方法有手术、化学治疗、放射治疗和生物治疗,热疗是继上述方法后出现的第五种肿瘤治疗方法。高热对肿瘤细胞有直接的不可逆性损伤,对肿瘤细胞的杀伤作用表现为抑制RNA、DNA、蛋白质的合成,细胞酶活性的改变以及对细胞周期的影响等。高热对肿瘤细胞还有间接杀伤作用。热疗可刺激肿瘤患者的免疫应答,增强患者的抗肿瘤免疫反应。全身热疗(41.8一42.2℃)时,24小时内淋巴细胞亚群和相应细胞因子出现应激性可逆性改变,表现为自然杀伤细胞上升, CD4+T细胞、白细胞介素-12、肿瘤坏死因子-γ、白细胞介素-10显著下降,因此认为高热最初在肿瘤患者体内诱导了一个可逆的抗炎症反应,刺激机体免疫应答。热疗过程中,肿瘤细胞死亡率增加,血管破坏增多,放射敏感度增高,同时肿瘤局部血流量增加,化疗敏感度增加,氧化作用增强。血管内皮生长因子(VEGF)又称血管通透因子(VPF)或促血管因子(VAS)。VEGF是一种特异作用于内皮细胞的糖蛋白,具有促进内皮细胞增殖,增加微血管通透性、诱导血管生成等功能。目前,血管内皮生长因子作为肿瘤血管新生中具有特异性的关键性调节因子之一,得以广泛的研究。大量研究表明,在绝大多数恶性肿瘤中均呈现VEGF及其受体的高表达,从而形成旁/自分泌作用方式,促进内皮细胞分裂、增殖、诱导血管新生,并直接作用于肿瘤细胞,刺激肿瘤细胞生长。VEGF甚至是某些肿瘤的独立预后指标,而VEGF是肺癌血管生成过程中最重要的的促血管生成因子。目的:探讨41.8℃全身热化疗对晚期肺癌患者外周血清VEGF, T淋巴细胞,NK细胞,B淋巴细胞的影响。方法:1.研究对象恶性肿瘤患者均为广州医学院附属肿瘤医院2006年7月至2009年1月期间的住院的肺癌病人,其中腺癌26例,鳞癌19例,腺鳞癌3例,大细胞癌1例。男性28例,女性21例。热化疗组29例,病人均接受41.8℃全身热疗,并与热疗同步给予含铂类药物化疗。化疗组20例,只单纯给予含铂类药物化疗。2.热疗过程及采血时间全身热疗过程大约需6小时,包括4小时加热期,60～90分钟41.8℃的平台期,30～60分钟的降温期。分别于热疗前或化疗前(bef)、热疗平台期末或化疗中(mid)、热疗或化疗结束时(end)、热疗后24小时或化疗后24小时(24h)、热疗后48小时或化疗后48小时(48h)抽取患者外周静脉血3ml,其中1ml置于肝素抗凝管即送流式细胞仪检测,2ml置于无抗凝管即行血清提取后-70℃保存备用。3.血清VEGF测定及淋巴细胞亚群分析汲取患者外周血清加入VEGF试剂盒中测出VEGF浓度;用直接免疫荧光技术测定样本淋巴细胞各亚群细胞百分率,经流式细胞仪分析结果。将经肝素抗凝的外周静脉血混匀后,加入各种小鼠抗人单克隆抗体:FITC-CD3/PE-CD16,56、FITC-CD4/PE-CD8双标记单抗和FITC-CD19单标记单抗,置室温暗处反应20分钟后再加入红细胞溶解剂,充分混匀,室温暗处反应30分钟后上机。经流式细胞计数仪分析CD3+(全T细胞)、CD4+CD8-( Th细胞)、CD4-CD8+( Ts细胞)、CD16+56+ (NK细胞)和CD19+(全B细胞)细胞百分率。结果:1.热化疗组患者热疗后血清中VEGF浓度在热疗结束时,热疗后24、48小时均低于热疗前,有统计学意义(p﹤0.05);而化疗组患者化疗前后血清中VEGF浓度无明显差异(p﹥0.05)。热化疗组患者热疗后血清VEGF浓度较单纯化疗组血清VEGF浓度降低,差异有统计学意义(P<0.05)。2.淋巴细胞亚群的变化热化疗组病人在热疗期间T淋巴细胞含量包括Th细胞和Ts细胞持续下降,从起始52.1%下降至热疗结束时最低值42.3%,较热疗前有显著差异性(P<0.05),热疗后24小时全面恢复接近起始水平,其后的24小时又显著下降,其中Th细胞与Ts细胞的变化趋势相类似,但Th/Ts比值在热疗期间持续上升,至热疗结束时达到高峰1.20,热疗后48小时内逐渐下降至起始水平;化疗组病人在化疗期间Th细胞和Ts细胞含量均有下降,Th细胞在化疗后逐渐上升,至化疗后48小时已高于起始水平, Th/Ts比值在化疗时升至1.14,无统计学差异(P>0.05)。热疗作用使热化疗组病人的NK细胞含量由起始的19.3%陡升至51.6%,显著高于其他时间点(P<0.05),并随着体温的下降而恢复至起始水平,热疗后仍有缓慢上升;化疗组病人NK细胞含量变化不大,在各时间点没有显著差异性(P>0.05)。两组病人的B细胞含量受热化疗或化疗的影响不大,在各时间点没有显著差异性(P>0.05)。结论:1. 41.8℃全身热疗可以降低晚期肺癌患者血清中VEGF的浓度;全身热化疗组和单纯化疗组治疗后VEGF水平有明显差异。2、41.8℃全身热疗发挥的是短期而显著的效应;有助于解除恶性肿瘤患者的免疫抑制,增强机体抗肿瘤免疫作用;而对B淋巴细胞影响不大;全身热疗联合化疗和单纯化疗比较,患者免疫功能状态的变化趋势有明显差异。更多还原

【Abstract】 At present, the main malignant tumor treatment have surgery, chemotherapy, radiation therapy and biological therapy, the whole body hyperthermia (WBH), following the above-mentioned methods are the fifth treatment. Hyperthermia on the tumor cells have a direct irreversible damage, the killing effect on tumor cell for inhibiting the performance of RNA, DNA, protein synthesis, cellular enzyme activity, cell cycle and so on. Hyperthermia on the tumor cell killing effect also indirectly. Hyperthermia may stimulate the patient’s immune response to tumor, and enhance anti-tumor patients with immune response. WBH (41.8- 42.2℃) , the 24-hour period and the corresponding lymphocyte subsets and cytokines appears reversible stress-induced changes, Manifested as increased natural killer cells, CD4 + T cells, interleukin -12, tumor necrosis factor-γ, interleukin--10 significantly decreased , Therefore consider the initial heat inducing a reversible Anti-inflammatory response in tumor patients, stimulating immune response. In hyperthermia process, the tumor cell death rate increased, the increase of vascular damage, radiation sensitivity increased, while increasing of blood flow local tumor and chemotherapy to increase sensitivity, enhance oxidation.Vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF) or angiogenesis factor (VAS), which is a potent mitogen for vascular endothelial cells. It has been associated with angiogennsis, vasopersbility and vascularization. Now ,VEGF is investigated widely as one of the specific and criticality regulatory factor in tumor vessel neogenesis. According to the generous statistics, Overexpression of VEGFis associated with increased angiogenesis, growth, invasion and metastasis in solid tumor through paracrine/autocrine action, irritation tumor cellsgrowth directly. VEGF is even a independence prognostic indicator in certain tumors. and VEGF is the most important factor in promoting angiogenesis of the lung cancer .OBJECTIVE: To probe with influence of WBH(41.8℃) for VEGF, T lymphocyte, B lymphocyte and NK cells in patients with advanced lung cancer.Methods1. Research object and groups: all the research objects are chosen from the lung cancer patients of Guangzhou medical college affiliated tumor hospital from July 2006 to Jan 2009,which 26 cases are adenocarcinoma of them、19 cases are squamous cell carcinoma、3 cases are adenosquamous carcinoma、and large cell carcinoma in 1 case. 28 cases are male, 21 cases are female. The thermochemotherapy group are 29 cases which are received whole body hyperthermia at 41.8℃,and received chemotherapy with platinum drugs simultaneously.The chemotherapy group that are given platinum drugs chemotherapy only.2. WBH procedure and colleting sample time: WBH will experience about 6 hours including 4 hours warm-up period, 60~90 minutes steady period and 30~60 minutes cooling period. Before WBH or chemotherapy(pre), in the middle of WBH or chemotherapy(mid), at the end of WBH or chemotherapy(end), 24 hours after WBH or chemotherapy(24h) and 48 hours WBH or chemotherapy(48h) 3ml peripheral venous blood will be drown from patients, among them 1ml will be put in anticoagulation tube containing heparin to detect by FCM and 2ml will be put in non-anticoagulation tube to extract serum and conserve at 70℃below zero.3. VEGF density detection and lymphocyte subsets analysis by FCM: To draw some peripheral blood serum of patients and add to VEGF-kit ,then detect the concentration of VEGF.we will detect the percents of lymphocyte subsets by direct immunofluorescence technic and analyze the result by FCM. Misce bene the anticoagulant samples and mix with various kinds of mouse anti-human MA: FITC-CD3/PE-CD16,56、FITC-CD4/PE-CD8 double marker MA and FITC-CD19 single marker MA, after reacting 20 minutes in dark place at room temperature mix with erythrocyte lytic agent thoroughly and reacting 30 minutes in dark place at room temperature once more, then analyze the respective percent of CD3+cell ( T cell ) , CD4+CD8-cell(Th cell), CD4-CD8+cell(Ts cell), CD16+56+cell(NK cell) and CD19+cell(B cell) by FCM.Result1. The concentration of VEGF in serum from thermochemotherapy group Patients after thermotherapy 24 and 48 hours are lower than before,it has statistical significance (p <0.05);and the concentration of VEGF in serum of chemotherapy group patients before and after chemotherapy have no significant difference(p﹥0.05)。The concentration of VEGF in serum from thermochemotherapy group patients after thermotherapy are lower than chemotherapy group patients,the difference has statistical significance (p <0.05).2. Change of lymphocyte subset in different time point: As to patients in group WBH-CT, T lymphocytes including Th cell and Ts cell percentage is continuing decreasing during thermotherapy which descends from beginning 52.1% to WBH ending 42.3%nadir with the statistical significance (P<0.05) and completely recovers to the original level in 24 hours after WBH and then significantly descends once more in the second 24 hours after WBH. The variation tendencies of Th cell and Ts cell are similar, but the ratio of Th/Ts is continuing rising up to the peak 1.20 in the end of WBH and falling off to the original level within 48 hours after WBH. As to patients in group CT, the percentages of Th cell and Ts cell are both decreased. In 48 after CT the content of Th cell works up to the level higher than the level before CT, The radio Th/Ts is slightly ascending to 1.14, which has no significant difference ( P>0.05).The effect of thermotherapy make the content of NK cell suddenly ascending from 19.3% to 51.6% in the group of WTH-CT. significantly higher than the level in other time point(P<0.05), and then recover to initiative level with the descensus of body temperature, and still go up slowly after thermotherapy. In the group of CT the content of NK cell has little variance, which has no significant difference comparing with the level of other time point(P>0.05).Conclusion:1. 41.8℃WBH can degrade VEGF in serum of advanced lung cancer, there was conspicuous different of VEGF density in the malignant tumor patients who accepted chemotherapy or 41.8℃WBH combined with chemotherapy.2. 41.8℃WBH could show short term and conspicuous effest,aontributed to disengage immune suppression and enhance organism anti-tumor immunity effect, but had little influence on Blymphocyte. there was conspicuous different of functional status change tendency in the malignant tumor patients who accepted chemotherapy or 41.8℃WBH combined with chemotherapy.更多还原