【作者】 周佩；

【导师】 董宝玮； 梁萍； 徐迎新；

【作者基本信息】 中国人民解放军军医进修学院 ， 影像医学与核医学， 2009， 博士

【摘要】 研究背景肝癌是临床进展较快的恶性肿瘤,目前主要治疗方法为手术切除、肝移植、局部消融及经动脉介入治疗。局部消融治疗小肝癌的远期疗效与手术切除相当,已被公认为是继手术和肝移植之后的根治性治疗方法之一,然而仍迫切需要探索新的疗法,以解决高转移复发率的难题。近20年来,过继性细胞免疫治疗发展迅速,有望成为肝癌的有效辅助治疗方法之一。目的摸索微波消融治疗时有效灭活肿瘤细胞并释放肿瘤抗原的温控和能量条件;观察肝癌消融后局部注射未成熟树突状细胞对荷瘤小鼠生存期及肿瘤生长的影响;观察肝癌微波消融联合树突状细胞疫苗及效应细胞综合过继免疫治疗的临床安全性,研究微波联合细胞免疫治疗后近期外周血淋巴亚群比例及细胞因子浓度的变化,初步探讨细胞免疫治疗对肝癌微波消融后近期复发率的影响。材料与方法1.模拟肝癌微波治疗中常用消融区边缘的温控和能量条件,消融人肝细胞癌株SMMC-7721细胞悬液,分为五组:A、对照组,B、45℃10min组,C、50℃3min组,D、54℃3min组,E、60℃即刻组,消融后30分钟及2小时进行凋亡检测。2.32只C57小鼠左侧腹股沟皮下接种Hepa1-6鼠肝癌细胞瘤块作为消融瘤,间隔1周,右侧腹股沟皮下接种瘤块作为观察瘤,成瘤后分为四组:A组,对照组,不行治疗;B组,微波组,仅微波消融小鼠消融瘤;C组,成熟DC组（mDC组）,微波消融小鼠消融瘤,并于小鼠双侧腋下注射肿瘤抗原致敏的树突状细胞;D组,未成熟DC组（iDC组）,微波消融小鼠消融瘤,并于消融瘤周注射未成熟树突状细胞。观察瘤均不予处理,荷瘤后每7-8天测量瘤体大小,直至小鼠死亡。3.知情同意原则下,10例行根治性微波消融联合多种免疫细胞过继治疗的原发性肝癌患者纳入联合组,同期符合相同入组条件的121例微波治疗患者纳入微波组。联合组患者免疫治疗方案设计为三个疗程,治疗前抽血45ml用于分离培养DC和效应细胞,在超声造影引导下回输到肝内消融区与充血带交界区或在超声引导下回输到腹股沟淋巴结内及右上腹腹腔内。10例联合组及24例微波组患者还于治疗前及治疗后1、3、6个月进行外周血淋巴细胞亚群及细胞因子检测。结果1.体外实验:受微波热损伤细胞主要表现为死亡,凋亡细胞比例小;在各温控和能量条件下,随温度升高,存活细胞百分比降低,消融后2h与30min比,各消融组存活细胞比例显著降低,其中60℃即刻组94.36%细胞均死亡或凋亡,其它组仍有较多存活细胞。2.体内实验:消融后12天,各治疗组消融瘤最大径均显著小于对照组,但治疗组间无显著差异,观察瘤最大径亦显著小于对照组且iDC组明显小于mDC组（p＜0.05）;各治疗组小鼠观察120天,消融瘤原位灭活率均为37.5%,iDC组（87.5%）及mDC组（75%）观察瘤消失率均高于单纯微波组（62.5%）,iDC组最高;与对照组相比,各治疗组小鼠生存期显著延长,iDC组最长;iDC组及mDC组生命延长率高于单纯微波治疗组,iDC组最高。3.临床研究:微波联合免疫治疗54次细胞回输未出现Ⅱ级以上严重不良反应,治疗后1、3、6个月患者血常规、肝肾功能及凝血功能与治疗前比较均无显著差异。患者经微波消融联合免疫治疗后1、3、6个月,外周血CD8+CD28+淋巴细胞亚群百分比较治疗前升高并显著高于微波组,CD4+/CD8+较治疗前降低并显著低于微波组;治疗后1和3个月,CDS+CD28-效应细胞亚群百分比较治疗前显著升高;治疗后1个月,CD4+CD25^highTreg细胞百分比较治疗前显著降低,并显著低于微波组,治疗后3和6个月时,虽仍低于治疗前但有所回升,且与消融组无差异。治疗后1个月,联合组IL-2浓度及IL-2/IL-4值较治疗前增高,治疗后3和6个月时回落。联合组患者治疗后3-9月内局部肿瘤进展率及肝内复发或肝外转移率均较微波组有降低趋势,但尚无统计学差异。联合治疗后1个月,57.14%患者产生了病毒应答。结论1)微波消融肿瘤细胞达到60℃,静置2小时,94.36%肿瘤细胞死亡或凋亡,推测该温控条件及时间点是消融区边缘未成熟树突状细胞回输治疗的较好选择;2)肝癌微波消融联合消融区边缘注射未成熟DC,可延长小鼠生存期,并可使远处肿瘤灶明显缩小甚至消退;3)微波消融联合树突状细胞和效应细胞过继免疫治疗肝癌在临床实施中是安全的;4)联合治疗改善了机体免疫状态,并可实现较高的病毒应答率,但是持续时间有限,提示现有治疗方案需要缩短时间间隔、增加疗程,并有必要进行抗病毒免疫治疗改善患者整体免疫状况,临床疗效尚需扩大病例数延长随访期后进一步评价。更多还原

【Abstract】 BACKGROUND Hepatocellular carcinoma（HCC）is a malignant tumor with poor prognosis.Current treatment methods include resection,liver transplantation,local thermal ablation and TACE.The long-term results of thermal ablation for HCC are similar with resection.Thermal ablation therapy had been generally acknowledged as one of the radical methods for HCC.However it still has an urgent demand for new methods to solve the problem of high rate of recurrence and metastasis.In the last twenty years,adoptive cellular immunotherapy developed quickly and is expected to be an effective adjuvant therapy for HCC.OBJECTIVE Research the condition of temperature and energy which could kill tumor cells and release neoantigen effectively.Observe the influence of combination therapy with microwave ablation（MWA）and peritumoral injection of immature dendritic cells on survival and tumor growth in mice.Observe clinical safety of combination therapy with MWA and adoptive immunotherapy of dendritic cells and eJector cells.Research the change of percentage of lymphocyte subsets and concentration of cytokine in peripheral blood after combination therapy.Initially observe the short-term result of combination therapy.MATERIAL AND METHODS 1.Simulate the condition of temperature and energy control in percutanous MWA for HCC to ablate SMMC-7721 cell strain suspension.There were five experiment groups:A.control group,B.45℃10 min group,C.50℃3min group,D.54℃3min group and E.60℃instant group.Detection for apoptosis was performed at 30 minutes and 2 hours after ablation.2.Thirty-two C57 mice were undertaken hypodermic inoculation of Hepal-6 tumor piece at right groin as ablation tumor and at left groin as observation tumor one week later.The mice with double tumors were separated into four groups:A.control group with no treatment,B.MWA group treated with MWA only,C.mDC group treated with MWA and subcutaneous injection of mature dendritic cells sensitized by neoantigen of Hepal-6 cell strain,D.iDC group treated with MWA and peritumoral injection of immature dendritic cells without neoantigen sensitization.The observation tumors were not treated.Tumors were measured every 7-8 days after ablation until mice died.3.With informed consent,10 HCC patients accepting combination treatment of radical MWA and adoptive immunotherapy with multiple immunocytes were included in the combination group.121 patients undertaken MWA alone with similar indication were included in the MWA group.The immunotherapy was designed as three treatment courses.Dendritic cells and effector cells separated from peripheral blood of patient were injected into the junctional zone of ablation region and hyperemia band with the guidance of contrast-enhanced sonography or into gorin lymph nodes or abdominal cavity of right upper quadrant under sonography guidance. 10 patients in the combination group and 24 patients in the MWA group accepted detection of lymphocyte subset and cytokine in peripheral blood before and 1,3,6 months after therapy.RESULT 1.In vitro experiment,the major state of tumor cells was dead and fewer cells were apoptotic after MWA.The percentage of live cells decreased in groups with higher temperature and energy.The percentage of live cells decreased markedly at 2h after ablation comparing with 30min after ablation.94.36%tumor cells were dead or apoptotic in 60℃instant group while many live cells were detected in other groups at 2h after ablation.2.In vivo experiments,the mean maximum diameter of ablation tumors in three treatment groups was markedly smaller than that of control group however had no statistical difference among three treatment groups at the 12^th day after ablation.The mean maximum diameter of observation tumors in three treatment groups was markedly smaller than that of control group and the size of observation tumors in iDC group was markedly smaller than that of mDC group （p＜0.05）at the 12^thday after ablation.After 120 days observation,the rates of complete response of ablation tumors were 37.5%in all treatment groups;the rate of disappearance of observation tumors was 87.5%,75%and 62.5%in iDC,mDC and MWA group separately which the iDC group was the highest.Comparing with control group,the mean survival time was markedly prolonged in all treatment groups which the iDC group was the longest.The ratio of life extension in iDC and mDC group was higher than MWA group which the iDC group was the highest.3.In clinical research,there were no serious adverse effects higher than gradeⅡafter fitly-four times of cellular injection and the results of hepatic,renal and blood coagulation function and blood routine had no statistical significance at 1,3,6 months after treatment comparing with those before treatment in the combination group.At 1,3,6 months after combination therapy,the percentage of CD8+CD28+ subset of peripheral blood lymphocytes in the combination group increased which was markedly higher（p＜0.05）than that of MWA group and CD4+/CD8+ decreased which was markedly lower（p＜0.05）than that of MWA group.The percentage of CD8+CD28-subset of peripheral blood lymphocytes in the combination group at 1 and 3 months after therapy markedly increased comparing with that of before treatment.The percentage of CD4+CD25high Treg subset of peripheral blood lymphocytes in the combination group markedly decreased（p＜0.05）which was remarkably lower than that of MWA group（p＜0.05）at 1 month after treatment, however increased which had no significant difference with that of MWA group at 3 and 6 months after treatment although was still lower than that of before treatment. The concentration of IL-2 and IL-2/IL-4 in peripheral blood of the combination group were increased at 1 month after treatment but returned at 3 and 6 months after treatment.The rate of local tumor progression or the rate of intrahepatic recurrence and extrahepatic metastasis in the combination group were lower than that of MWA group although had no statistical difference.57.14%patients obtained virological response at 1 month after combination therapy.CONCLUSIONS 1)94.36%tumor cells were dead or apoptotic at the second hours after microwave ablation under ablation temperature control of 60℃.The condition of ablation temperature control of 60℃and time control of 2 hours after ablation was presumed to be a good choice for combination therapy with microwave ablation and iDC injection at the marginal area of ablation.2)Combination therapy with microwave ablation and iDC injection at the marginal area of ablation could prolong the survival of HCC mice and the distance tumors were contracted or vanished.3)Combination therapy with microwave ablation and adoptive immunotherapy was safe in clinical application.4)Combination therapy improved the immune state of patients and obtained high rate of virological response,however could not last long duration.The duration between immunotherapy courses could be shortened and the number of treatment course be increased to upgrade current program.Antiviral immunotherapy would be needed after ablation to ameliorate whole immune state.Clinical result should be further evaluated after expanding the number of cases and extending the duration of follow-up.更多还原