【作者】 夏纪筑；

【导师】 伍烽；

【作者基本信息】 重庆医科大学 ， 超声医学， 2010， 博士

【摘要】 目的在前期研究基础上,本研究进一步观察高强度聚焦超声(high intensity focused ultrasound,HIFU)治疗H22移植性肝癌荷瘤鼠后,外周血抗肿瘤细胞免疫功能的变化,过继免疫后荷瘤鼠外周血淋巴细胞在肿瘤局部的特异性表达和抗肿瘤免疫功能的变化,为阐明HIFU治疗肿瘤所诱发的抗肿瘤免疫效应机制提供实验室依据。方法1、HIFU治疗后荷瘤鼠抗肿瘤细胞免疫功能的变化72只成年健康C57BL/6J系小鼠,随机分为三组:HIFU治疗组(n=24)、HIFU假照组(n=24)和对照组(n=24)。前两组小鼠背部皮下接种H22肝癌细胞株,成瘤后(接种7天时)分别接受HIFU治疗或HIFU假照;对照组为正常小鼠,不接种肿瘤也不接受HIFU照射。三组小鼠在HIFU治疗后第14天采取外周血,淋巴细胞密度梯度离心法分离外周血淋巴细胞,MHC-肽五聚体特异性荧光染色,流式细胞术分析CTL细胞数量的变化;乳酸脱氢酶释放法检测淋巴细胞对同种肿瘤细胞的特异性杀伤活性;酶联免疫吸附法检测淋巴细胞与H22细胞共培养24小时后上清液IFN-γ、TNF-α的含量。2、过继免疫HIFU活化的CTL在荷瘤鼠肿瘤局部的特异性表达24只背部皮下接种H22肝癌细胞株成功的C57BL/6J小鼠,在接种7天时随机分为三组:HIFU治疗组(n=8)、HIFU假照组(n=8)和对照组(n=8)。三组小鼠分别接受尾静脉注射HIFU治疗、HIFU假照后荷瘤鼠和正常小鼠经MHC-肽五聚体荧光染色的外周血淋巴细胞1×107个/只。输注24小时后处死小鼠,手术取出各组小鼠接种部位肿瘤组织块作冰冻切片,激光共聚焦显微镜观察荧光染色阳性的CTLs在肿瘤局部的表达,图像分析软件分析CTLs的数量, HE染色,光镜下观察、比较CTLs的分布情况。3、过继免疫HIFU活化的CTL在荷瘤鼠肿瘤局部的功能变化30只背部皮下接种H22肝癌细胞株成功的C57BL/6J小鼠,在接种7天时随机分为三组:HIFU治疗组(n=10)、HIFU假照组(n=10)和对照组(n=10)。三组小鼠分别接受尾静脉注射HIFU治疗、HIFU假照荷瘤鼠和正常小鼠外周血淋巴细胞1×107个/只。输注7天后处死小鼠,手术取出各组小鼠接种部位肿瘤组织块,制备成单细胞悬液,酶联免疫斑点法检测肿瘤局部淋巴细胞功能的变化。结果1、HIFU治疗后荷瘤鼠抗肿瘤细胞免疫功能的变化HIFU治疗后第14天,HIFU组、假照组和对照组淋巴细胞中CTL细胞表达率(%)分别为1.9±0.08、1.42±0.09和对照组1.27±0.05,与假照组或对照组比较,HIFU组CTL细胞明显增加,差异有显著的统计学意义(P<0.01)。HIFU组、假照组、对照组小鼠外周血淋巴细胞对H22肿瘤细胞的杀伤率随效靶比的增加而升高,HIFU组在1:1、2.5:1、5:1、10:1效靶比时的淋巴细胞杀伤率(%)分别为3.01±1.02、13.76±2.21、33.38±3.68、51.59±3.74;与假照组或对照组比较,效靶比在2.5:1、5:1、10:1时HIFU组淋巴细胞的细胞毒活性明显增加,差异有显著的统计学意义(P<0.01)。HIFU组、假照组、对照组小鼠外周血淋巴细胞与H22肿瘤细胞共培养24h时上清液INF-γ和TNF-α浓度随效靶比的增加而升高,HIFU组在1:1、10:1、20:1、40:1效靶比时,上清液INF-γ含量(pg/ml)分别为141.17±19.24、178.46±16.56、443.98±13.26、743.43±18.18;TNF-α含量(pg/ml)分别为242.41±21.74、510.07±16.63、764.86±15.86、1212.83±89.21;与假照组或对照组比较,效靶比在1:10、1:20、1:40时HIFU组上清液TNF-α和INF-γ含量明显增加,差异有显著的统计学意义(P<0.01)。2、过继免疫HIFU活化的CTL在荷瘤鼠肿瘤局部的特异性表达MHC-肽五聚体荧光染色的淋巴细胞过继免疫治疗后1天,HIFU组7只小鼠,假照组6只小鼠,对照组2只小鼠肿瘤局部有呈PE标记的MHC-肽五聚体红色荧光、FITC标记的CD8分子绿色荧光的阳性细胞和MHC-肽五聚体和CD8双重染色阳性的呈黄色荧光的淋巴细胞出现;CTL细胞主要分布在肿瘤与正常组织交界区,肿瘤中央区未见表达;HIFU组CTL细胞数每个视野17.13±8.29个,假照组10.37±6.47个,对照组0.83±0.74个,与假照组及对照组比较,HIFU组CTL细胞数量明显增多,差异有显著的统计学意义(P<0.05)。3、过继免疫HIFU活化的CTL在荷瘤鼠肿瘤局部的功能变化淋巴细胞过继免疫治疗后7天,HIFU组、假照组和对照组能分泌INF-γ的淋巴细胞数(个/孔)分别为110±18.04、38±9.41和23±1.87,与假照组或对照组比较,HIFU组能分泌INF-γ淋巴细胞数量明显增加,差异有显著的统计学意义(P <0.01)。结论1、HIFU治疗H22移植性肝癌后,荷瘤鼠外周血淋巴细胞受到活化,CTL细胞数量明显增多,对同种肿瘤细胞的特异性杀伤活性明显增强。2、HIFU活化的淋巴细胞过继免疫治疗同种肿瘤荷瘤鼠后,CTL细胞在肿瘤局部的特异性表达明显增加,表明活化的CTL细胞能从外周血迁移到肿瘤局部。3、HIFU活化的淋巴细胞过继免疫治疗同种肿瘤荷瘤鼠后,在肿瘤局部表达的CTL细胞特异性抗肿瘤免疫功能明显活跃。更多还原

【Abstract】 OBJECTIVEBased on the previous results, this study was designed to investigate the functional changes in peripheral blood lymphocytes after high intensity focused ultrasound (HIFU) treatment of H22 implanted tumor in mice, and to determine the local expression and specific antitumor immunity of HIFU-activated cytotoxic T lymphocytes (CTLs) within the implanted tumor region after adoptive immunotherapy for the syngeneic tumor-bearing mice. It will provide more evidences involved in HIFU-triggered antitumor immune response against the homogeneous tumor cells.MATERIALS AND METHODS1. Changes in the cellular immune functions of peripheral blood lymphocytes after HIFU treatment in tumor-bearing miceSeventy-two normal C57BL/6J mice were randomly divided into three groups: HIFU group (n =24), sham-HIFU group (n =24), and control group (n=24). The mice in both HIFU group and sham-HIFU group received H22 hepatocellular carcinoma implantation using the hypodermic injection of H22 cells suspension in the back of each mouse. Seven days after the tumor implantation, tumor mass was obviously detected in the local region. Subsequently, the mice in HIFU group underwent HIFU ablation for the implanted tumor, but those in sham-HIFU group received a sham therapy procedure with no acoustic energy deposition in the tumor. Those in control group were normal mice, without either tumor implantation or HIFU treatment. Fourteen days after HIFU, the peripheral blood was collected in each mouse, and flow cytometry technique was employed to observe changes in the positive expression of CTLs stained with recombinant MHC peptide fluorescence in each group, respectively. Using lymphocyte density gradient centrifugation, lymphocytes were extracted and purified from the peripheral blood of each mouse, and LDH assay was performed to measure the cytotoxic activity of the lymphocytes against H22 tumor cells in vitro in the three groups respectively. In addition, both IFN-γand TNF-αlevels were determined by the ELISA method in the supernatant after a 24-hour culture of the lymphocytes with H22 cells. 2. Local expression of HIFU-activated CTLs after adoptive immunotherapy in the homogeneous tumor-bearing miceSeven days after after the H22 tumor implantation, 24 tumor-bearing C57BL/6J mice were randomly divided into three groups: HIFU group (n =8), sham-HIFU group (n =8), and control group (n=8). They underwent the intravenous injection of 1×107 lymphocytes stained with recombinant MHC peptide fluorescence via the tail vein. The lymphocytes were obtained from the syngeneic tumor-bearing mice treated by either HIFU or sham-HIFU procedure, and the normal syngeneic mice, respectively. Twenty-four hours after the injection, the tumor and normal tissues surrounding the tumor were harvested in each mouse, and then sliced into histological sections using a freeze section cutter. The local expression of the positive-stained CTLs was examined under a laser confocal microscope in the tumor, and Software Image-Pro Version 5.0 was used to analyze the amount of the positive-stained CTLs in each group. Furthermore, the slices were stained with H & E method, and were observed to determine the distribution characteristic of the CTLs using optical microscopy.3. The functional change of HIFU-activated CTLs after adoptive immunotherapy in the homogeneous tumor-bearing miceSeven days after H22 tumor implantation, 30 tumor-bearing C57BL/6J mice were randomly divided into three groups: HIFU group (n =10), sham-HIFU group (n =10), and control group (n=10). They received the intravenous injection of 1×107 lymphocytes via the tail vein. The lymphocytes were obtained from the syngeneic tumor-bearing mice treated by either HIFU or sham-HIFU procedure, and the normal syngeneic mice, respectively. Seven days after adoptive immunotherapy, surgical procedure was performed to harvest the implanted tumor in each mouse. Viable tumor tissue was minced and dispersed to a single-cell suspension by grinding in a loose-fitting ground glass homogenizer and filtrating through a sterile steel mesh. The ELISpot assay was used to measure the number of IFNγ-secreting lymphocytes.RESULTS1. Changes in the cellular immune functions of peripheral blood lymphocytes after HIFU treatment in the tumor-bearing miceFourteen days after HIFU treatment, the positive expression rates (%) of CTLs in the HIFU group, sham-HIFU group and control group were 1.9±0.08, 1.42±0.09 and 1.27±0.05 respectively. Compared to the values in the sham-HIFU and control groups, a statistical increase of the positive CTLs was significantly observed in the HIFU group (P < 0.01). The cytotoxic activity of the lymphocytes against H22 tumor cells gradually enhanced with an increase of effector/target cell ratios in each group. The cytotoxicity of the lyphocytes was 3.01±1.02, 13.76±2.21, 33.38±3.68 and 51.59±3.74 in the HIFU group while effector/target cell ratios were 1:1, 2.5:1, 5:1 and 10:1 respectively. Compared to the sham-HIFU and control groups, the cytotoxic activity was significantly increased in the HIFU group, and statistical differences were observed among them while effector/target cell ratios were 2.5:1, 5:1 and 10:1. The concentration of the IFN-γand TNF-αwas increased in the supernatant with an increase of effector/target cell ratios in each group. The IFN-γlevels were 141.17±19.24, 178.46±16.56, 443.98±13.26 and 743.43±18.18 pg/ml in the HIFU group respectively while effector/target cell ratios were 1:1, 10:1, 20:1 and 40:1. The TNF-αlevels were 242.41±21.74, 510.07±16.63, 764.86±15.86 and 1212.83±89.21 pg/ml in the HIFU group respectively while effector/target cell ratios were 1:1, 10:1, 20:1 and 40:1. Compared to the sham-HIFU and control groups, there were significant increases of the IFN-γand TNF-αlevels in the HIFU group while effector/target cell ratios were 1:10, 1:20 and 1:40 (P <0.01).2. Local expression of HIFU-activated CTLs after adoptive immunotherapy in the homogeneous tumor-bearing miceOne day after adoptive immunotherapy with the HIFU-activated lymphocytes stained by Pro5 Recombinant MHC peptide fluorescence, the positive-stained CTLs were observed in 7 mice in the HIFU group, 6 in the sham-HIFU group, and 2 in the control group. These positive cells mainly distributed along the marginal region between the tumor and normal tissues, with no appearance in the central tumor. The number of the positive-stained CTL was 17.13±8.29 in the HIFU group, 10.37±6.47 in the sham-HIFU group, and 0.83±0.74 in the control group. Compared to the values in the sham-HIFU group and control groups, there was a significant increase of the activated-CTL infiltration within the local tumor in the HIFU group (P < 0.05). 3. The functional change of HIFU-activated CTLs after adoptive immunotherapy in the homogeneous tumor-bearing miceSeven days after adoptive immunotherapy with the HIFU-activated lymphocytes, the number of INF-γsereting lymphocyte frequencies was 110±18.04 in the HIFU group, 38±9.41 in the sham-HIFU group, and 23±1.87 in the control group respectively. Compared to the sham-HIFU and control groups, there was a significant increase of INF-γsereting lymphocytes in the HIFU group (P < 0.01).CONCLUSION1. Peripheral blood lymphocytes were significantly activated after HIFU treatment of implanted H22 hepatocellualr carcinoma in mice, with an increase of the positive CTLs. The activated lymphocytes had a stronger cytotoxicity against H22 tumor cells in vitro.2. After adoptive immunotherapy with the HIFU-activated lymphocytes, the local expression of the HIFU-activated CTLs was significantly increased within the implanted tumor in the homogeneous tumor-bearing mice, suggesting that the activated CTLs could move from peripheral blood into the local tumor.3. After adoptive immunotherapy with the HIFU-activated lymphocytes, local CTLs presented a stronger specific antitumor immune response within the tumor in the homogeneous tumor-bearing mice.更多还原