【作者】 李小丽；

【导师】 郭志；

【作者基本信息】 天津医科大学 ， 肿瘤学， 2008， 硕士

【摘要】 目的探讨小鼠皮下移植型肝癌经冷冻处理后,机体免疫功能变化情况,为临床冷冻免疫的应用提供免疫学依据。材料方法取Balb/c小鼠72只,随机分三组,A、B、C三组均制成H22皮下移植瘤模型,分别行氩氦冷冻治疗(实验组A,n=24),氩氦冷冻治疗1天后辅以弗氏佐剂(实验组B,n=24),荷瘤对照(对照组C,n=24)。观察小鼠皮下肿瘤生长情况。A、B、C三组于冷冻术前和术后1周、2周、3周,每组各取6只小鼠,分别采用眼球采血法采集外周血,无菌条件下取小鼠脾脏制成脾淋巴细胞,采用ELISA法检测小鼠外周血IFN-γ、IL-4浓度,LDH法检测小鼠脾淋巴细胞杀伤活性。结果接种后5天小鼠右侧腹股沟处可以扪及肿瘤长出。接种后2周,局部可形成一直径0.8-1.0cm的肿块,72只Balb/c小鼠全部接种成功。对照组随肿瘤种植时间的延长,IFN-γ呈逐渐下降趋势,IL-4呈逐渐升高趋势,于对照处理后2、3周IL-4浓度显著高于处理前(P＜0.05);对照处理后3周IFN-γ浓度显著低于处理前(P＜0.05),发生了由Th1向Th2的漂移。实验组A和实验组B IL-4均呈逐渐降低趋势,而IFN-γ均呈逐渐升高趋势。实验组A治疗后IL-4逐渐降低趋势,治疗后1、2、3周均显著低于治疗前和同期对照组(P＜0.05);而IFN-γ逐渐升高,治疗后1、2、3周均显著高于对照组(P＜0.05),但与治疗前比较无显著性差异(P＞0.05)。实验组B治疗后IL-4呈逐渐降低趋势,治疗后1、2、3周均显著低于治疗前和同期对照组(P＜0.05);而IFN-γ逐渐升高,治疗后1、2、3周均显著高于治疗前和对照组(P＜0.05),但治疗后3周与2周比较无显著性差异。实验组A与实验组B比较,实验组B IL-4低于实验组A,IFN-γ高于实验组A,于治疗后3周时有显著性差异(P＜0.05)。各组脾淋巴细胞杀伤活性处理后1周时显著增加,治疗后2、3周呈下降趋势,各组间变化趋势相同。对照组,对照处理后3周显著低于处理前、处理后1周、2周(P＜0.05)。实验组A,治疗后1周、2周脾淋巴细胞杀伤活性均显著高于同期对照组及治疗前(P＜0.05)。实验组B,治疗后1、2周脾淋巴细胞杀伤活性增强,显著高于同期实验组A和对照组(P＜0.05)。结论荷H22肝癌小鼠随肿瘤种植时间延长,机体IFN-γ逐渐降低趋势,IL-4逐渐升高趋势,Th1/Th2逐渐降低,发生Th1向Th2漂移;脾淋巴细胞杀伤活性逐渐减弱,机体抗肿瘤免疫功能逐渐降低。冷冻治疗可使荷瘤小鼠外周血IFN-γ轻度升高,IL-4显著降低,Th1/Th2逐渐升高,使荷瘤机体产生Th2向Th1逆转,脾淋巴细胞的杀伤活性增强,产生抗肿瘤免疫作用。冷冻与弗氏佐剂的联合应用可进一步增强和维持机体抗肿瘤免疫应答。更多还原

【Abstract】 ObjectiveThe purpose of this study was to analyze the effect of Argon-Helium Cryosurgery(AHCS)on the cryoimmunologic responses of mice bearing H22 liver cancer through detecting splenocyte cytotoxicity,and expression pattern of cytokines T helper type 1(Th1)/Th2.Materials and MethodsSixty Balb/c mice were randomized into four group:the AHCS therapy group (group A,n=18),AHCS combined with Freund adjuvant group(group B,n=18),the tumor-bearing control group(group C,n=18)and normal control group(group D, n=6).Peripheral blood samples and the splenocyte of six mice were obtained from group A,B,C at 1w,2w,3w after the treatment,Peripheral blood samples and the splenocyte were obtained from group D before the treatment.The concentrations of interferon-γ(IFN-γ)and interleukin-4(IL-4),were detected by enzyme-linked immunoabsorbent assay(ELISA),the splenocyte cytotoxicity were detected by lactate dehydrogenase(LDH).ResultsH22 liver cancer cell has stable biology characteristics,and th e incadence of cancer was 100%.IFN-γconcentration were lower than that in normal group in three weeks(P＜0.05),but IL-4 was higher than that in normal group in two weeks in tumor-bearing groups(P＜0.05).It was higher than that in tumor-beating group for IFN-γconcentration in one week,two weeks and three weeks after AHCS therapy group,respectively(P＜0.05),but it was no significant differences between the AHCS and that of normal group in one week,two weeks and three weeks.It was lower than that in normal group and that in tumor-bearing group for IL-4 concentration in one week,two weeks and three weeks after AHCS therapy group(P＜0.05).It was higher than that in normal group and that in tumor-bearing group for IFN-γconcentration in one week,two weeks and three weeks after AHCS combined with Freund adjuvant group,respectively(P＜0.05),but it was no significant differences between that of the two and three weeks.It was lower than that in normal group and that in tumor-bearing group for IL-4 concentration in one week,two weeks and three weeks after AHCS combined with Freund adjuvant group(P＜0.05).Remarkably,IFN-γconcentration was higher in the AHCS combined with Freund adjuvant group than that in AHCS therapy group,but it has signify cant differences only in three weeks after the AHCS(P＜0.05).IL -4 was lower in the AHCS combined with Freund adjuvant group than that in AHCS therapy group,but it has signify cant differences only in three weeks after the AHCS(P＜0.05) Splenocyte cytotoxicity increased in the one week significantly,but decreesed in the two and three weeks in the group A,B and C.In the tumor-bearing group, splenocyte cytotoxicity in three weeks was lower than that in normal group and in one and two weeks(P＜0.05).In AHCS therapy group,splenocyte cytotoxicity in one and two weeks were higher than that in normal group and tumor-bearing group(P＜0.05).In the AHCS combined with Freund adjuvant group,splenocyte cytotoxicity in one and two weeks were higher than that in AHCS group and in normal group and tumor-bearing group(P＜0.05).ConclusionCryosurgery can effectively treat mice with H22 liver cancer,enhance splenocyte cytotoxicity to tumor cells,facilitate the secretion of Th1 type cytokines in mice with H22 liver tumors,promote the transformation from Th2 to Th1.Finally, Cryosurgery may increase the immune function of anti-tumor in tumor-bearing mice.The Freund adjuvant could enhandes this effect furtherly.更多还原