温阳散寒、补气养血法对树突状细胞影响及治疗非小细胞肺癌疗效之研究
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摘要
目的:探讨温阳散寒、补气养血法治疗恶性肿瘤的作用机理,并观察其治疗非小细胞肺癌(NSCLC)的临床疗效。
方法:(1)抗肿瘤作用。复制小鼠S180肉瘤和EAC模型,观察温阳散寒、补气养血法组方的抑瘤和延长荷EAC小鼠生存期的作用。(2)对树突状细胞(DC)数量及成熟的影响。复制小鼠艾氏腹水癌(EAC)模型,分组给药,抽取腹水,分离单个核细胞,流式细胞仪器检测CD11c、CD80阳性率及两者双阳性率;取小鼠骨髓细胞,加入含中药及重组小鼠粒细胞-巨噬细胞集落刺激因子(rmGM-CSF)、重组小鼠白细胞介素-4(rmIL-4)培养液,不同时间点观察培养细胞形态,流式细胞仪检测培养细胞CD11c、CD80阳性率及两者双阳性率;取治疗前后非小细胞肺癌患者外周血,流式细胞仪检测外周血髓系DC数量及与单核细胞比值。(3)对相关细胞因子的影响。采用ELISA方法,测定中药干预后小鼠艾氏腹水癌中IL-12、IL-4、IFN-γ变化;测定含中药体外诱导DC培养液中IL-12水平。(4)对DC介导的细胞毒性T细胞(CTL)杀伤活性影响。收集合中药培养液诱导的小鼠髓源性DC,与小鼠脾脏分离、培养的T淋巴细胞以1:50比例共同培养3天,分离EAC小鼠腹水中肿瘤细胞,效靶比为20:1与DC、T淋巴细胞共同孵育,MTT法测T细胞对肿瘤细胞的杀伤活性。(5)对照观察温阳散寒、补气养血法配合化疗与单纯化疗治疗NSCLC患者的临床疗效。
结果:(1)大、小剂量温阳散寒、补气养血法组方组抑瘤率分别为47.22%、45.78%,生命延长率分别为46.56%、39.69%,较模型组比较差异有显著性(P＜0.01),其中大剂量组生存期为(19.20±1.55)天,较化疗组差异有显著性(P＜0.05)。(2)大、小剂量中药组EAC小鼠腹水单个核细胞CD11c阳性率、CD80阳性率、CD11cCD80双阳性率均较模型组及化疗组高,差异有显著性(P＜0.05)。含药培养液体外诱导骨髓细胞CD11c阳性率、CD80阳性率、CD11cCD80双阳性率较空白对照组高,差异有显著性(P＜0.05)。(3)大、小剂量中药组小鼠腹水中IL-12、IFN-γ水平均较化疗组、模型组高,差异有显著性(P＜0.01),IL-4较化疗组、模型组低,差异有显著性(P＜0.05)。含中药培养液体外诱导DC分泌IL-12含量较空白组比高,差异有显著性(P＜0.05)。(4)高、低浓度中药组DC介导的CTL杀伤活性均较空白组高,差异有显著性(P＜0.05)(5)中药与化疗合用治疗晚期NSCLC,2月后淋巴结转移率为4.8%,较单用化疗组小,差异有显著性(P＜0.05);中药组主症疗效及生活质量提高稳定率分别为76.2%、90.5%,较单用化疗组高,差异有显著性(P＜0.05)。
结论:(1)温阳散寒、补气养血法能明显抑制S180肉瘤的生长,显著延长荷EAC小鼠的生存时间。(2)本法在体外和体内均能明显提高小鼠树突状细胞特异性表面抗原CD11c和共刺激分子CD80的表达,说明该法方能提高DC的数量及成熟比例。(3)本法能提高EAC小鼠腹水中IL-12、Th1类细胞因子IFN-γ水平,降低Th2类细胞因子IL-4水平,使免疫微环境中Th1/Th2向有利于肿瘤免疫的Th1方向漂移,提高肿瘤局部免疫功能。可能与该法提高DC的数量及成熟比例,增加IL-12分泌有关。(4)本法能提高体外诱导DC所介导的CTL杀伤肿瘤细胞活性。(5)本法与化疗合用治疗NSCLC,能降低淋巴结转移率,改善临床症状,提高生活质量。
This study was aimed at exploring the mechanisms of Warming Yang to Dispelling Coldness and Invigorating Qi and Nourishing Blood (WYDCIQNB) decoction on treating malignant tumor and observing the clinical effect on non-small-cell lung cancer and. It was composed of following trials.
1. Experiments on the Anti-tumor Effects of Warming Yang to Dispelling Coldness and Invigorating Qi and Nourishing Blood decoction Here, cells of S180 sarcoma and EAC cell line were transplanted to Kunming line mice respectively to produce transplanted tumors among the animals. Following transplantation of tumor cells, the animals were fed with the solution of WYDCIQNB herb. In this experiment, investigated in vivo were the effect of tumor-inhibiting on mice with transplanted tumors of S180 sarcoma and the effect of survival duration-extending on mice with ascites due to transplanted EAC.
2. Experiments on the Effects of Warming Yang to Dispelling Coldness and Invigorating Qi and Nourishing Blood on the Dendritic Cell In these experiments, the EAC mouse was produced, giving the solution of WYDCIQNB, separated the mononuclearcell from the ascites of mice with EAC transplated. Investigated the CD11c, CD80 and both of them positive cell by flow cytometry. The marow cell were get from mouse, culture the cells in media with WYDCIQNB herb solution and rm GM-CSF, rm IL-4. Observe the cells in different time, Investigated the CD11c, CD80 and both of them positive cell by flow cytometry. The peripheral blood were get from the NSCLC patients before and after treatment. Investigated the quantitation of DC and ratio of DC/PBMC(peripheral blood mononuclear cell) by flow cytometry.
3. Experiments on the Effects of WYDCIQNB on the Levels of Cytokines Enzyme-linked immunosorbent assay (ELISA) was taken to determine the levels of IL-4, IL-12, INF-γin the ascites of mice with EAC transplated after taken WYDCIQNB solution. The same method to determine the level of IL-12 in media of culture marow-derived DC with WYDCIQNB solution.
4. Observation the Effects of WYDCIQNB on the Killing Activity of Cytotoxic T-lymphocyte (CTL) mediated by DC Here, get the marow-derived DC which cultured by the media with WYDCIQNB solution. Co-cultured with T cells separated and culture from mouse spleen at the ratio of 1:50 for 3 days. Separated the tumor cell from the escites of mice with EAC transplated. Distributed at an effector-target raio of 1:20 for DC, T cell to tumor cell. Investigating the killing activity of T cell by MTT.
5. Clinical Trial to investigate the Therapeutic Effect of WYDCIQNB on Patients with Non-small-cell Lung Cancer In this trial, two groups of cases were included, one group given with combined WYDCIQNB therapy as treating one and the other with chemotherapy only as controlling one. Then, the therapeutic effects of different therapies were compared among these cases to see the clinical response to this medicine.
From the experiments done as stated as above, the results obtained were shown as follows.
1. Tumor-Inhibiting Effects of WYDCIQNB These experiments showed that the tumor-inhibiting rates for the animals with the cells of S180 sarcoma transplanted were 47.22% and 45.78%, the survival duration-extending rates were 46.56% and 36.69% for those with EAC cells transplanted respectively, when they were treated with high and low dose WYDCIQNB herb. There were statistically significant differences present among them when compared with that of other groups (P<0.05).
2. The Effects of WYDCIQNB on the Dendritic These experiments showed that in the cells of separated from escited of mice with EAC transplanted, in the treating group taken orally by WYDCIQNB herb were significantly higher than modelling and chemotherapy group(P<0.05). On the other hand, The ratio of CD11c positive, CD80 postive, both CD11c and CD80 positive in the marow cell cultured with WYDCIQNB were significantly higher than blank contrl group(P<0.05).
3. The Effect of WYDCIQNB on the Levels of Cytokines The level of IL-12, INF-γin escites of mice with EAC transplanted in treating group with taken orally by WYDCIQNB herb were significantly higher than modelling and chemotherapy group(P<0.01). In the same time, the level of IL-4 in treating group were significantly lower(P<0.01). On the other hand, The level of IL-12 in culture media with WYDCIQNB solution is significantly higher than blank control group(P<0.05).
4. The Effects of WYDCIQNB on the Killing Activity of Cytotoxic T-lymphocyte (CTL) mediated by DC The killing activity of CTL mediated by DC which cultured with WYDCIQNB solution were significantly higher than blank control group(P<0.05).
5. Clinical Therapeutic Effect of WYDCIQNB on Patients with Non-small- cell Lung Cancer The ratio of lymph node metastasis after 2 months of treating group was 4.71%, was significantly lower than that of controlling one treated with chemotherapy only(P<0.05). Furthermore, the effect of main symptoms were also significantly improved among the patients of treating group; with a very significant difference (P<0.05) when compared with that of controlling one.
Based on an analysis on these results, following conclusions could be made.
1. WYDCIQNB holds very strong inhibitory effect on transplanted tumors of S180 sarcoma as tested in vivo and significantly extending effect on the survival duration of animals with transplanted EAC cells in situ.
2. WYDCIQNB can improve the expression of CD11c, a specific surface antigen of DC, and CD80, a kind of costimulatory molecules, in vitro and vivo. It's means WYDCIQNB can improve the number of DC and the ratio of mature.
3. WYDCIQNB can elevate the level of IL-12, IFN-γ,the cytokine of Th1, and decrease the level of IL-4,the cytokine of Th2, in the ascites of mice with EAC transplanted. These effects are able to improve the local immunologic microenviroment of cases with tumor and benefial for Th1/Th2 shifting in the direction towards Th1. The mechanism maybe has some relationship with that WYDCIQNB can improve the number of DC and the ratio of mature.
4. WYDCIQNB can improve the killing activity of CTL mediated which by DC.
5. WYDCIQNB can decrease the ratio of lymph node metastasis, improve effect on the main symptoms seen among the patients with NSCLC when it combined with chemotherapy.
方法:(1)抗肿瘤作用。复制小鼠S180肉瘤和EAC模型,观察温阳散寒、补气养血法组方的抑瘤和延长荷EAC小鼠生存期的作用。(2)对树突状细胞(DC)数量及成熟的影响。复制小鼠艾氏腹水癌(EAC)模型,分组给药,抽取腹水,分离单个核细胞,流式细胞仪器检测CD11c、CD80阳性率及两者双阳性率;取小鼠骨髓细胞,加入含中药及重组小鼠粒细胞-巨噬细胞集落刺激因子(rmGM-CSF)、重组小鼠白细胞介素-4(rmIL-4)培养液,不同时间点观察培养细胞形态,流式细胞仪检测培养细胞CD11c、CD80阳性率及两者双阳性率;取治疗前后非小细胞肺癌患者外周血,流式细胞仪检测外周血髓系DC数量及与单核细胞比值。(3)对相关细胞因子的影响。采用ELISA方法,测定中药干预后小鼠艾氏腹水癌中IL-12、IL-4、IFN-γ变化;测定含中药体外诱导DC培养液中IL-12水平。(4)对DC介导的细胞毒性T细胞(CTL)杀伤活性影响。收集合中药培养液诱导的小鼠髓源性DC,与小鼠脾脏分离、培养的T淋巴细胞以1:50比例共同培养3天,分离EAC小鼠腹水中肿瘤细胞,效靶比为20:1与DC、T淋巴细胞共同孵育,MTT法测T细胞对肿瘤细胞的杀伤活性。(5)对照观察温阳散寒、补气养血法配合化疗与单纯化疗治疗NSCLC患者的临床疗效。
结果:(1)大、小剂量温阳散寒、补气养血法组方组抑瘤率分别为47.22%、45.78%,生命延长率分别为46.56%、39.69%,较模型组比较差异有显著性(P＜0.01),其中大剂量组生存期为(19.20±1.55)天,较化疗组差异有显著性(P＜0.05)。(2)大、小剂量中药组EAC小鼠腹水单个核细胞CD11c阳性率、CD80阳性率、CD11cCD80双阳性率均较模型组及化疗组高,差异有显著性(P＜0.05)。含药培养液体外诱导骨髓细胞CD11c阳性率、CD80阳性率、CD11cCD80双阳性率较空白对照组高,差异有显著性(P＜0.05)。(3)大、小剂量中药组小鼠腹水中IL-12、IFN-γ水平均较化疗组、模型组高,差异有显著性(P＜0.01),IL-4较化疗组、模型组低,差异有显著性(P＜0.05)。含中药培养液体外诱导DC分泌IL-12含量较空白组比高,差异有显著性(P＜0.05)。(4)高、低浓度中药组DC介导的CTL杀伤活性均较空白组高,差异有显著性(P＜0.05)(5)中药与化疗合用治疗晚期NSCLC,2月后淋巴结转移率为4.8%,较单用化疗组小,差异有显著性(P＜0.05);中药组主症疗效及生活质量提高稳定率分别为76.2%、90.5%,较单用化疗组高,差异有显著性(P＜0.05)。
结论:(1)温阳散寒、补气养血法能明显抑制S180肉瘤的生长,显著延长荷EAC小鼠的生存时间。(2)本法在体外和体内均能明显提高小鼠树突状细胞特异性表面抗原CD11c和共刺激分子CD80的表达,说明该法方能提高DC的数量及成熟比例。(3)本法能提高EAC小鼠腹水中IL-12、Th1类细胞因子IFN-γ水平,降低Th2类细胞因子IL-4水平,使免疫微环境中Th1/Th2向有利于肿瘤免疫的Th1方向漂移,提高肿瘤局部免疫功能。可能与该法提高DC的数量及成熟比例,增加IL-12分泌有关。(4)本法能提高体外诱导DC所介导的CTL杀伤肿瘤细胞活性。(5)本法与化疗合用治疗NSCLC,能降低淋巴结转移率,改善临床症状,提高生活质量。
This study was aimed at exploring the mechanisms of Warming Yang to Dispelling Coldness and Invigorating Qi and Nourishing Blood (WYDCIQNB) decoction on treating malignant tumor and observing the clinical effect on non-small-cell lung cancer and. It was composed of following trials.
1. Experiments on the Anti-tumor Effects of Warming Yang to Dispelling Coldness and Invigorating Qi and Nourishing Blood decoction Here, cells of S180 sarcoma and EAC cell line were transplanted to Kunming line mice respectively to produce transplanted tumors among the animals. Following transplantation of tumor cells, the animals were fed with the solution of WYDCIQNB herb. In this experiment, investigated in vivo were the effect of tumor-inhibiting on mice with transplanted tumors of S180 sarcoma and the effect of survival duration-extending on mice with ascites due to transplanted EAC.
2. Experiments on the Effects of Warming Yang to Dispelling Coldness and Invigorating Qi and Nourishing Blood on the Dendritic Cell In these experiments, the EAC mouse was produced, giving the solution of WYDCIQNB, separated the mononuclearcell from the ascites of mice with EAC transplated. Investigated the CD11c, CD80 and both of them positive cell by flow cytometry. The marow cell were get from mouse, culture the cells in media with WYDCIQNB herb solution and rm GM-CSF, rm IL-4. Observe the cells in different time, Investigated the CD11c, CD80 and both of them positive cell by flow cytometry. The peripheral blood were get from the NSCLC patients before and after treatment. Investigated the quantitation of DC and ratio of DC/PBMC(peripheral blood mononuclear cell) by flow cytometry.
3. Experiments on the Effects of WYDCIQNB on the Levels of Cytokines Enzyme-linked immunosorbent assay (ELISA) was taken to determine the levels of IL-4, IL-12, INF-γin the ascites of mice with EAC transplated after taken WYDCIQNB solution. The same method to determine the level of IL-12 in media of culture marow-derived DC with WYDCIQNB solution.
4. Observation the Effects of WYDCIQNB on the Killing Activity of Cytotoxic T-lymphocyte (CTL) mediated by DC Here, get the marow-derived DC which cultured by the media with WYDCIQNB solution. Co-cultured with T cells separated and culture from mouse spleen at the ratio of 1:50 for 3 days. Separated the tumor cell from the escites of mice with EAC transplated. Distributed at an effector-target raio of 1:20 for DC, T cell to tumor cell. Investigating the killing activity of T cell by MTT.
5. Clinical Trial to investigate the Therapeutic Effect of WYDCIQNB on Patients with Non-small-cell Lung Cancer In this trial, two groups of cases were included, one group given with combined WYDCIQNB therapy as treating one and the other with chemotherapy only as controlling one. Then, the therapeutic effects of different therapies were compared among these cases to see the clinical response to this medicine.
From the experiments done as stated as above, the results obtained were shown as follows.
1. Tumor-Inhibiting Effects of WYDCIQNB These experiments showed that the tumor-inhibiting rates for the animals with the cells of S180 sarcoma transplanted were 47.22% and 45.78%, the survival duration-extending rates were 46.56% and 36.69% for those with EAC cells transplanted respectively, when they were treated with high and low dose WYDCIQNB herb. There were statistically significant differences present among them when compared with that of other groups (P<0.05).
2. The Effects of WYDCIQNB on the Dendritic These experiments showed that in the cells of separated from escited of mice with EAC transplanted, in the treating group taken orally by WYDCIQNB herb were significantly higher than modelling and chemotherapy group(P<0.05). On the other hand, The ratio of CD11c positive, CD80 postive, both CD11c and CD80 positive in the marow cell cultured with WYDCIQNB were significantly higher than blank contrl group(P<0.05).
3. The Effect of WYDCIQNB on the Levels of Cytokines The level of IL-12, INF-γin escites of mice with EAC transplanted in treating group with taken orally by WYDCIQNB herb were significantly higher than modelling and chemotherapy group(P<0.01). In the same time, the level of IL-4 in treating group were significantly lower(P<0.01). On the other hand, The level of IL-12 in culture media with WYDCIQNB solution is significantly higher than blank control group(P<0.05).
4. The Effects of WYDCIQNB on the Killing Activity of Cytotoxic T-lymphocyte (CTL) mediated by DC The killing activity of CTL mediated by DC which cultured with WYDCIQNB solution were significantly higher than blank control group(P<0.05).
5. Clinical Therapeutic Effect of WYDCIQNB on Patients with Non-small- cell Lung Cancer The ratio of lymph node metastasis after 2 months of treating group was 4.71%, was significantly lower than that of controlling one treated with chemotherapy only(P<0.05). Furthermore, the effect of main symptoms were also significantly improved among the patients of treating group; with a very significant difference (P<0.05) when compared with that of controlling one.
Based on an analysis on these results, following conclusions could be made.
1. WYDCIQNB holds very strong inhibitory effect on transplanted tumors of S180 sarcoma as tested in vivo and significantly extending effect on the survival duration of animals with transplanted EAC cells in situ.
2. WYDCIQNB can improve the expression of CD11c, a specific surface antigen of DC, and CD80, a kind of costimulatory molecules, in vitro and vivo. It's means WYDCIQNB can improve the number of DC and the ratio of mature.
3. WYDCIQNB can elevate the level of IL-12, IFN-γ,the cytokine of Th1, and decrease the level of IL-4,the cytokine of Th2, in the ascites of mice with EAC transplanted. These effects are able to improve the local immunologic microenviroment of cases with tumor and benefial for Th1/Th2 shifting in the direction towards Th1. The mechanism maybe has some relationship with that WYDCIQNB can improve the number of DC and the ratio of mature.
4. WYDCIQNB can improve the killing activity of CTL mediated which by DC.
5. WYDCIQNB can decrease the ratio of lymph node metastasis, improve effect on the main symptoms seen among the patients with NSCLC when it combined with chemotherapy.
引文
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