恶性肿瘤HIFU治疗后免疫抑制因子变化的研究
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摘要
目的：在前期研究的基础上，本课题提出高强度聚焦超声（high intensity focused ultrasound，HIFU）治疗恶性肿瘤病人后宿主抗肿瘤免疫功能增强可能与肿瘤局部免疫抑制因子失去活性，肿瘤对宿主免疫功能的直接抑制作用减弱有关这一假设。本研究通过观察HIFU治疗人恶性肿瘤后靶区、转移淋巴结和外周血免疫抑制因子血管内皮生长因子(vascular endothelial growth facter, VEGF) 、转化生长因子-β1 ( transforming growth facter-β1 , TGF-β1 )、转化生长因子-β2 ( transforming growth facter-β2 , TGF-β2)、白细胞介素6 (interleukin 6, IL-6) 和白细胞介素10 ( interleukin 10, IL-10 ) 的变化，了解HIFU治疗对肿瘤细胞合成和分泌免疫抑制因子的影响，探讨HIFU后宿主抗肿瘤免疫功能增强的机制。
材料与方法：（1）48例经细针穿刺活检证实的女性乳腺癌病人，分为HIFU组（23例）和对照组（25例），两组均接受乳腺癌改良根治术，其中HIFU组于术前1～2周先行HIFU治疗；术中收集每例病人乳腺癌肿瘤局部及同侧腋窝淋巴结；采用SP免疫组织化学染色法对两组病人肿瘤局部及有肿瘤转移的阳性腋窝淋巴结（HIFU组11例，对照组12例）分别进行肿瘤免疫抑制因子VEGF、TGF-β1、TGF-β2、IL-6和IL-10的染色，记录两组病人肿瘤免疫抑制因子的
阳性表达率和计数单位面积內有阳性表达的乳腺癌细胞数，两组计数和计量资料分别采用X2检验和t检验进行统计学处理。（2）应用双抗体夹心ELISA法检测15例恶性肿瘤病人HIFU治疗前后外周血上述免疫抑制因子水平的变化，统计学处理采用自身配对t检验。
结果：（1）对照组病人肿瘤局部VEGF、TGF-β1、TGF-β2、IL-6和IL-10的阳性率分别为72%、72%、80%、68%和84%; 而HIFU组的阳性率分别为30.4%、56.5%、69.6%、47.8%和60.9%；对照组同时表达上述五个指标的为48%，HIFU组为27.3%。与对照组比较，HIFU组治疗局部乳腺癌细胞VEGF、IL-6和IL-10的阳性率明显下降，有显著性差异（P<0.05）。（2）对照组病人肿瘤局部阳性表达VEGF、TGF-β1、TGF-β2、IL-6和IL-10的乳腺癌细胞数分别为344.5±90.6/mm2、355.7±111.4/mm2、391.5±87.3/mm2、399.1±95.8/mm2和 327.2±140.8/mm2; HIFU组病人肿瘤局部分别为235.9±93.8/mm2、243.7±69.2/mm2、209.8±77.1/mm2、303.1±99.4/mm2和 238.1±79.8/mm2；两组比较，HIFU组治疗局部阳性表达上述免疫抑制因子的乳腺癌细胞数明显减少，差异有显著性 (P<0.05)。（3）HIFU组病人腋窝淋巴结内乳腺癌细胞VEGF、TGF-β1、TGF-β2、IL-6和IL-10的阳性率下降，但两组比较仅TGF-β2的差异有统计学意义 (P<0.05)。（4）对照组病人腋窝淋巴结内表达VEGF、TGF-β1、TGF-β2、IL-6和IL-10的阳性肿瘤细胞数分别为410.6±36.9/mm2、 324.0±43.6/mm2、366.4±130.2/mm2、300.5±60.2/mm2和 328.7±
60.0/mm2；HIFU组分别为315.3±51.4/mm2、243.1±52.0/mm2、 249.1±102.7/mm2、217.9±57.7/mm2和 212.0±49.5/mm2；与对照组比较，HIFU组腋窝淋巴结内表达VEGF、TGF-β1、IL-6和IL-10的阳性肿瘤细胞数明显减少，差异有显著性 (P<0.05)。(5) 15例恶性肿瘤病人HIFU治疗前外周血VEGF、TGF-β1、TGF-β2、IL-6和IL-10水平分别为594.6±354.5 pg/ml、43.1±17.8 ng/ml、27.4±12.1ng/ml、23.0±8.7pg/ml和55.8±22.4 pg/ml；治疗后1周分别为546.1±352.6pg/ml、39.8±19.1ng/ml、24.5±11.1ng/ml、20.6±7.5pg/ml和54.5±23.7pg/ml；治疗后外周血免疫抑制因子水平下降，其中VEGF、TGF-β1和TGF-β2水平明显下降，差异有显著性 (P<0.05)。
结论：（1）HIFU治疗乳腺癌原发灶后靶区、转移淋巴结内肿瘤免疫抑制因子VEGF、TGF-β1、TGF-β2、IL-6和IL-10的阳性表达明显减少，表明HIFU治疗可以削弱和消除肿瘤局部、区域淋巴结内肿瘤细胞分泌免疫抑制因子的能力，这将有利于肿瘤局部、转移灶内免疫细胞发挥抗肿瘤的活性，改善宿主局部的免疫功能抑制状态。(2) HIFU治疗后恶性肿瘤病人外周血免疫抑制因子VEGF、TGF-β1和TGF-β2水平明显下降，削减了肿瘤对全身抗肿瘤免疫功能的负向调节作用，有助于恢复宿主抗肿瘤免疫平衡，改善病人的预后。
Objective: Based on previous research results, in this study we hypothesized that the enhanced antitumor immunity induced by high intensity focused ultrasound (HIFU) in the treatment of malignancies would correlate with the reduced secretion of tumor-origin immunosuppressive factors, which can directly inhibit host immune function against tumor. The purpose of this study was to investigate the mechanism of the reversal of tumor-induced immunosuppression after HIFU. For assessing the effects of HIFU on immunosuppressive factors in patients with malignancies, vascular endothelial growth factor（VEGF）, transforming growth factor-β1（TGF-β1）, transforming growth factor-β2（TGF-β2）, interleukin 6（IL-6）, and interleukin 10 (IL-10) were examined in primary tumor, in metastasis-positive axillary lymph nodes, and in peripheral blood samples before and after HIFU treatment.
Materials and Methods: (1) A total of 48 women with biopsy-proven
breast cancer were divided into HIFU group (n=23) and control group (n=25). All patients were treated by modified radical mastectomy, while the patients in HIFU group received HIFU treatment 1～2 weeks before surgery. After the operation, the specimens of tumor lesions and axillary lymph nodes were collected in both groups. SP immunohistochemical stain was used to detect the tumor expression of VEGF，TGF-β1，TGF-β2，IL-6, and IL-10 in primary tumor and in metastasis-positive axillary lymph nodes (HIFU, n=11; Control, n=12), respectively. The positive rate was qualitatively assessed in both groups, and the number of immunostained positive tumor cells was counted in a microscopic grid. Statistical significance of the data obtained was analyzed by the Fisher exact test and the Student t test. (2) Using sandwich ELISA technique, serum level of VEGF，TGF-β1，TGF-β2，IL-6, and IL-10 was measured in 15 patients with malignancies before and one week after HIFU treatment. Statistical difference was analyzed by using the Student t test.
Results: (1) The positive rate of tumor-origin VEGF, TGF-β1, TGF-β2, IL-6, and IL -10 in primary tumor was 72%, 72%, 80%, 68%, and 84% in control group, respectively; while in HIFU group the positive rate was 30.4%, 56.5%, 69.6%, 47.8%, and 60.9%, respectively. The simultaneous positivity of the five immunosuppressive factors was 48% in control group, and 27.3% in HIFU group. The positivities of these
immunosuppressive factors were lower in HIFU group than those in control group, and there were significant differences of VEGF, IL-6, and IL-10 between two groups (P<0.05). (2) The number of positive tumor cells of VEGF, TGF-β1, TGF-β2, IL-6, and IL-10 in primary tumor was 344.5±90.6/mm2, 355.7±111.4/mm2, 391.5±87.3/mm2, 399.1±95.8/mm2, and 327.2±140.8/mm2 in control group, respectively; while in HIFU group the number was 235.9±93.8/mm2, 243.7±69.2/mm2, 209.8±77.1/mm2, 303.1±99.4/mm2, and 238.1±79.8/mm2, respectively. The expressions of these immunosuppressive factors were significantly lower in HIFU group than those in control group (P<0.05). (3) The positive rate of tumor-origin VEGF, TGF-β1, TGF-β2, IL-6, and IL-10 in metastasis-positive axillary lymph nodes was lower in HIFU group than those in control group, and there was a significant difference of TGF-β2 between two groups (P<0.05). (4) The number of positive tumor cells of VEGF, TGF-β1, TGF-β2, IL-6, and IL-10 in metastasis-positive axillary lymph nodes was 410.6±36.9/mm2, 324.0±43.6/mm2, 366.4±130.2/mm2, 300.5±60.2/mm2, and 328.7±60.0/mm2 in control group, respectively; while in HIFU group, the number was 315.3±51.4/mm2, 243.1±52.0/mm2, 249.1±102.7/mm2, 217.9±57.7/mm2, and 212.0±49.5/mm2, respectively. The expressions of these immunosuppressive factors were lower in HIFU group than those in
control group, and there were significant differences of VEGF, TGF-β1, IL-6, and IL-10 between two groups (P<0.05). (5) Fifteen patients with malignant tumors received one-session HIFU treatment. Before HIFU the serum level of VEGF, TGF-β1, TGF-?
材料与方法：（1）48例经细针穿刺活检证实的女性乳腺癌病人，分为HIFU组（23例）和对照组（25例），两组均接受乳腺癌改良根治术，其中HIFU组于术前1～2周先行HIFU治疗；术中收集每例病人乳腺癌肿瘤局部及同侧腋窝淋巴结；采用SP免疫组织化学染色法对两组病人肿瘤局部及有肿瘤转移的阳性腋窝淋巴结（HIFU组11例，对照组12例）分别进行肿瘤免疫抑制因子VEGF、TGF-β1、TGF-β2、IL-6和IL-10的染色，记录两组病人肿瘤免疫抑制因子的
阳性表达率和计数单位面积內有阳性表达的乳腺癌细胞数，两组计数和计量资料分别采用X2检验和t检验进行统计学处理。（2）应用双抗体夹心ELISA法检测15例恶性肿瘤病人HIFU治疗前后外周血上述免疫抑制因子水平的变化，统计学处理采用自身配对t检验。
结果：（1）对照组病人肿瘤局部VEGF、TGF-β1、TGF-β2、IL-6和IL-10的阳性率分别为72%、72%、80%、68%和84%; 而HIFU组的阳性率分别为30.4%、56.5%、69.6%、47.8%和60.9%；对照组同时表达上述五个指标的为48%，HIFU组为27.3%。与对照组比较，HIFU组治疗局部乳腺癌细胞VEGF、IL-6和IL-10的阳性率明显下降，有显著性差异（P<0.05）。（2）对照组病人肿瘤局部阳性表达VEGF、TGF-β1、TGF-β2、IL-6和IL-10的乳腺癌细胞数分别为344.5±90.6/mm2、355.7±111.4/mm2、391.5±87.3/mm2、399.1±95.8/mm2和 327.2±140.8/mm2; HIFU组病人肿瘤局部分别为235.9±93.8/mm2、243.7±69.2/mm2、209.8±77.1/mm2、303.1±99.4/mm2和 238.1±79.8/mm2；两组比较，HIFU组治疗局部阳性表达上述免疫抑制因子的乳腺癌细胞数明显减少，差异有显著性 (P<0.05)。（3）HIFU组病人腋窝淋巴结内乳腺癌细胞VEGF、TGF-β1、TGF-β2、IL-6和IL-10的阳性率下降，但两组比较仅TGF-β2的差异有统计学意义 (P<0.05)。（4）对照组病人腋窝淋巴结内表达VEGF、TGF-β1、TGF-β2、IL-6和IL-10的阳性肿瘤细胞数分别为410.6±36.9/mm2、 324.0±43.6/mm2、366.4±130.2/mm2、300.5±60.2/mm2和 328.7±
60.0/mm2；HIFU组分别为315.3±51.4/mm2、243.1±52.0/mm2、 249.1±102.7/mm2、217.9±57.7/mm2和 212.0±49.5/mm2；与对照组比较，HIFU组腋窝淋巴结内表达VEGF、TGF-β1、IL-6和IL-10的阳性肿瘤细胞数明显减少，差异有显著性 (P<0.05)。(5) 15例恶性肿瘤病人HIFU治疗前外周血VEGF、TGF-β1、TGF-β2、IL-6和IL-10水平分别为594.6±354.5 pg/ml、43.1±17.8 ng/ml、27.4±12.1ng/ml、23.0±8.7pg/ml和55.8±22.4 pg/ml；治疗后1周分别为546.1±352.6pg/ml、39.8±19.1ng/ml、24.5±11.1ng/ml、20.6±7.5pg/ml和54.5±23.7pg/ml；治疗后外周血免疫抑制因子水平下降，其中VEGF、TGF-β1和TGF-β2水平明显下降，差异有显著性 (P<0.05)。
结论：（1）HIFU治疗乳腺癌原发灶后靶区、转移淋巴结内肿瘤免疫抑制因子VEGF、TGF-β1、TGF-β2、IL-6和IL-10的阳性表达明显减少，表明HIFU治疗可以削弱和消除肿瘤局部、区域淋巴结内肿瘤细胞分泌免疫抑制因子的能力，这将有利于肿瘤局部、转移灶内免疫细胞发挥抗肿瘤的活性，改善宿主局部的免疫功能抑制状态。(2) HIFU治疗后恶性肿瘤病人外周血免疫抑制因子VEGF、TGF-β1和TGF-β2水平明显下降，削减了肿瘤对全身抗肿瘤免疫功能的负向调节作用，有助于恢复宿主抗肿瘤免疫平衡，改善病人的预后。
Objective: Based on previous research results, in this study we hypothesized that the enhanced antitumor immunity induced by high intensity focused ultrasound (HIFU) in the treatment of malignancies would correlate with the reduced secretion of tumor-origin immunosuppressive factors, which can directly inhibit host immune function against tumor. The purpose of this study was to investigate the mechanism of the reversal of tumor-induced immunosuppression after HIFU. For assessing the effects of HIFU on immunosuppressive factors in patients with malignancies, vascular endothelial growth factor（VEGF）, transforming growth factor-β1（TGF-β1）, transforming growth factor-β2（TGF-β2）, interleukin 6（IL-6）, and interleukin 10 (IL-10) were examined in primary tumor, in metastasis-positive axillary lymph nodes, and in peripheral blood samples before and after HIFU treatment.
Materials and Methods: (1) A total of 48 women with biopsy-proven
breast cancer were divided into HIFU group (n=23) and control group (n=25). All patients were treated by modified radical mastectomy, while the patients in HIFU group received HIFU treatment 1～2 weeks before surgery. After the operation, the specimens of tumor lesions and axillary lymph nodes were collected in both groups. SP immunohistochemical stain was used to detect the tumor expression of VEGF，TGF-β1，TGF-β2，IL-6, and IL-10 in primary tumor and in metastasis-positive axillary lymph nodes (HIFU, n=11; Control, n=12), respectively. The positive rate was qualitatively assessed in both groups, and the number of immunostained positive tumor cells was counted in a microscopic grid. Statistical significance of the data obtained was analyzed by the Fisher exact test and the Student t test. (2) Using sandwich ELISA technique, serum level of VEGF，TGF-β1，TGF-β2，IL-6, and IL-10 was measured in 15 patients with malignancies before and one week after HIFU treatment. Statistical difference was analyzed by using the Student t test.
Results: (1) The positive rate of tumor-origin VEGF, TGF-β1, TGF-β2, IL-6, and IL -10 in primary tumor was 72%, 72%, 80%, 68%, and 84% in control group, respectively; while in HIFU group the positive rate was 30.4%, 56.5%, 69.6%, 47.8%, and 60.9%, respectively. The simultaneous positivity of the five immunosuppressive factors was 48% in control group, and 27.3% in HIFU group. The positivities of these
immunosuppressive factors were lower in HIFU group than those in control group, and there were significant differences of VEGF, IL-6, and IL-10 between two groups (P<0.05). (2) The number of positive tumor cells of VEGF, TGF-β1, TGF-β2, IL-6, and IL-10 in primary tumor was 344.5±90.6/mm2, 355.7±111.4/mm2, 391.5±87.3/mm2, 399.1±95.8/mm2, and 327.2±140.8/mm2 in control group, respectively; while in HIFU group the number was 235.9±93.8/mm2, 243.7±69.2/mm2, 209.8±77.1/mm2, 303.1±99.4/mm2, and 238.1±79.8/mm2, respectively. The expressions of these immunosuppressive factors were significantly lower in HIFU group than those in control group (P<0.05). (3) The positive rate of tumor-origin VEGF, TGF-β1, TGF-β2, IL-6, and IL-10 in metastasis-positive axillary lymph nodes was lower in HIFU group than those in control group, and there was a significant difference of TGF-β2 between two groups (P<0.05). (4) The number of positive tumor cells of VEGF, TGF-β1, TGF-β2, IL-6, and IL-10 in metastasis-positive axillary lymph nodes was 410.6±36.9/mm2, 324.0±43.6/mm2, 366.4±130.2/mm2, 300.5±60.2/mm2, and 328.7±60.0/mm2 in control group, respectively; while in HIFU group, the number was 315.3±51.4/mm2, 243.1±52.0/mm2, 249.1±102.7/mm2, 217.9±57.7/mm2, and 212.0±49.5/mm2, respectively. The expressions of these immunosuppressive factors were lower in HIFU group than those in
control group, and there were significant differences of VEGF, TGF-β1, IL-6, and IL-10 between two groups (P<0.05). (5) Fifteen patients with malignant tumors received one-session HIFU treatment. Before HIFU the serum level of VEGF, TGF-β1, TGF-?
引文
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