肿瘤抗原负载的树突状细胞免疫过继治疗乳腺癌的研究
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摘要
目的
树突状细胞(dendritic cells，DC)是人体内重要的抗原递呈细胞，其可以把肿瘤抗原特异性地递呈给免疫效应细胞而产生肿瘤特异性免疫反应。它有很强的摄取、处理、递呈抗原的能力以及高表达共刺激分子，自90年代以来已成为免疫治疗研究热点之一。本研究系统地探讨乳腺癌患者外周血中免疫细胞如T、B淋巴细胞、NK细胞等的变化，以及乳腺癌细胞表面分子的改变，进一步明确乳腺癌患者的免疫状况。希望乳腺癌抗原负载后的DC能够弥补乳腺癌患者的免疫缺陷，并探讨肿瘤抗原负载的DC免疫过继治疗乳腺癌动物模型的效果。
材料和方法
1、乳腺癌患者的免疫功能检测：随机抽选2001年3月至10月30例乳腺癌患者的外周血，采用流式细胞技术分析淋巴细胞表面CD3、CD4、CD8、CDl9、CD28的表达，单核细胞表面CD40、CD80、CD86的表达。
2、乳腺癌细胞表面分子的表达：采用流式细胞技术检测5株乳腺癌细月包MCF-7、SK-BR-3、T47D、MDA-MB-435s、ZR-75-30表面MHCⅡ类抗原与共刺激分子CD40、CD80(B7-1)、CD86(B7-2)的表达，并与正常乳腺细胞HBL-100作比较。同时检
1汕k厂村人学博【：学位论义
渺J35仲憎L腺癌肿瘤标本这些分子的表达，并与乳腺良性病变标本
作比较。
3、人外周血中树突状细胞分离、培养与鉴定：采集健康成人外
周血 20人份，乳腺癌患者外周血 30人份，每份 10毫升，肝素
抗凝。用淋巴细胞分离液分离外周血，收集单个核细胞，调节细
J包浓度为 IO‘／ml，然后在 6 JL板上占壁，每JLh。Zml，2，J、时后
吸去悬浮细胞，加入新鲜营养液。每隔1天半量换液一次。用共
聚焦显微镜拍摄所培养的细胞，流式细胞枝术鉴定所培养的细
胞。
4、B：lb儿，J、鼠骨髓DC的培养：元菌操作取，J、鼠后肢股骨，用
注射针将髓腔中的骨髓细胞吹打出，用0．8％TriSNH人溶去红
细月后，PBS洗2次，用含mGM－CSF（．3ng／ml），lL＊20ng／ml）
和 10o，J、牛血清的 yMI 640 Z全培养基重悬至 l．0 xlo‘／ml，
铺于 6孔板上，ZmNL，第 3天轻轻晃动培养板，吸弃全部上清
（去除粒细胞），及时补入含细胞因子浓度相同的完全培养基，
第 6天轻轻吹吸下疏松粘附于培养板底的聚集体，置于新的培养
板中，并加入等量的上述完全培养基，继续培养24小时，用于
免疫标记鉴定、分泌细胞因子检测及生物学活性的研究。
5、B：lb七，J、鼠乳腺癌模型的建立：选择对数生长期的 TM40D，
用 0．25％胰 蛋白酶消 化，PBS洗涤2次；细胞浓度调至 10‘．10‘／ml。
在 Balb七小鼠胸壁第 56肋间或腹壁第 4对乳腺处皮下接种肿瘤
go胞0．1m1，相当于10’1～胞／X小鼠。每天观察肿瘤的生长情
况，用游标卡尺测量肿块的大小。
6、肿瘤抗原负载的DC免疫过继治疗B：lb止。J、鼠乳腺癌模型：
动物分为 3组，每组 10 ．q小鼠，皆于腹壁右侧第 4对乳腺处皮
下接种对数生长期的 TM40D细胞，IX 10‘／只，在肿瘤细胞接种
7
内水厂村人学博卜学位沦义
后第 3天，于同侧腹股沟皮下接种 TM40D负载的 DC（2 XIO‘／
只／次），以后分别于第10，门，24天每组动物接受与第一次同
样的治疗。观察肿瘤的生长、动物存活率以及肿瘤的转移情况。
结 果
l、乳腺癌患者外周血中 CD3、CD4、CDS、CD16、CD19阳性
的淋巴细胞与对照组相比无显著性差异…功刀5八但是CD28阳
性 T i田胞少于对照组（p＜0．05），特别是CD4／CD28双阳性 T兰
胞明显少于对ig组（p功．005）。手术后7天左右CD28阳性T细
胞就恢复到正常，与对照组相比无显著性差异（p＞0刀5），说明
乳腺癌患者免疫细胞减少与肿瘤负荷有关。乳腺癌患者单核细胞
、表达共刺激分子 CD80（p＜0．05）、CD86（p＜0．00L CD40表
达与对照组相比无显著性差异巾叩刀5）。
2、5株乳腺癌细胞MHC H类分子表达都与HB卜mo有显著性差
异（p＜0．05），MCF－7 i田胞表达水平最低，约为 HBL－100勺 1／5。
MDA－MB－4355与 ZR刁5S0细包表达水平是HBLl 的 2倍，
MDA．MB．4355荧光强度也比HBLl0O及其它乳腺癌细胞高。但
MDA－MB－4355 i田包表＠CD40分子表达水平最低，约为 MCF－7
与HBL－mo CD4O分子表达水平的m％。MDA－MB．4355细月
CD80、CD86分子表达水平与 HBL－100才当（p＞0．05），另 4株
乳腺癌细月 CD80、CD86分子表达者比 HBL－100｛（p＜0．05）。
乳腺癌原代细胞 CD40表达水平与对照组相比无显著性差异
（p＞0刀5），＊＊0、＊＊6白
Study of the Adoptive Immunotherapy on Breast Cancer with Dendritic Cells Loaded with Tumor Antigen
Fan Ping (student), Wu Zhengyan (tutor)
Objective
Dendritic cells are important antigen presenting cells in human being. They can specifically present antigen to T lymphocytes and initiate specific antitumor immunity reaction in vitro and in vivo. Their remarkable effectiveness is in large part due to their efficiency in capturing, processing, and presenting antigens along with costimulatory signals. Therefore, dendritic cells have become one of the hot spots in the field of tumor immuno-adoptive therapy since 90s. In order to determine immuno-status of breast cancer patients in this paper, we study the changes of immunocytes such as T lymphocytes, B lymphocytes, and natural killer ect. in the peripheral blood of breast cancer patients. We also study the molecules such as CD80, CD86, and MHC ect. expressing on the breast cancer cell lines and primary breast cancer cells. We expect that tumor antigen loaded dendritic cells could make up for the immunodeficiency of breast cancer patients. For this purpose, we first research on adoptive immunotherapy to breast cancer animal model using tumor antigen loaded dendritic cells.
Materials and Methods
1.We selected 30 admission patients with breast cancer between March and October in 2001 at random . Using flow cytometery analysis, we examined CD3, CD4, CD8, CD 16, CD 19 and CD28 molecules expressing on the lymphocytes and CD40, CD80 and CD86 molecules expressing on the monocytes in the peripheral blood samples from breast cancer patients.
2.We detected these molecules expressing on the five breast cancer cell lines using flow cytometery analysis. The cell lines include MCF-7, SK-BR-3, T47D, MDA-MB-435s, and ZR-75-30. The expression levels were compared with that of normal mammary cell line HBL-100 . At the same time, we examined those molecules expressing on the 35 primary breast cancer cells and compared with that of benign breast diseases.
3.Fifty cases peripheral blood were drawn from 20 healthy adult persons and 30 breast cancer patients. Every person was drawn 10 milliliter blood and the blood was anticoagulated by heparin (5unit per milliliter). We use Ficoll Hypaque centrifugation to isolate the mononuclear cells in the peripheral blood. These cells are plated in six-well culture plates(106/ml,2ml/well) in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, 100ng/ml GM-CSF, 20ng/ml IL-4, and/or 20ng/ml TNF- a .After 2 hours, nonadherent cells were gently removed and fresh medium was added. Culture medium was refreshed every other day. Cultured cells were taken a picture using focus scanning microscope (Zeiss LSM510)
and analyzed by flow cytometry with labeled monoclonal antibodies. 4.Under sterile condition, lower limbers of Balb/c mice were cut with scissors. In the dish, the marrow was flushed out using 2ml of RPMI 1640 with a syringe. The tissue was suspended and red cells were lysed with 0.8% ammonium chloride. After washed twice with PBS, these cells are plated in six-well culture plates(106/ml,2ml/well) in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, 3.3ng/ml GM-CSF, 20ng/ml IL-4. Three days later, the cultures were fed by gently swirling the plates, aspirating all of the medium, and adding back fresh medium with the same concentration cytokines as before. An object of this wash was to remove nonadherent granulocytes. On 6th day, the aggregates of loosely attaching to culture plate cells were dislodged with pipettes. Those cells were removed to a new culture plate and cultured 24 hours. Cultured cells were analyzed by flow cytometry with labeled monoclonal antibodies, cytokines secretion was detected with RT-PCR, and immunological activity was searched with mixed lymphocytes reaction.
5. Female Balb/c mice were 4-6 weeks old. Breast cancer cell line TM40D derived from Balb/c mice. Digested by 0.25% trypsin , washed twice with PBS, exponentially growing TM40D cells (
树突状细胞(dendritic cells，DC)是人体内重要的抗原递呈细胞，其可以把肿瘤抗原特异性地递呈给免疫效应细胞而产生肿瘤特异性免疫反应。它有很强的摄取、处理、递呈抗原的能力以及高表达共刺激分子，自90年代以来已成为免疫治疗研究热点之一。本研究系统地探讨乳腺癌患者外周血中免疫细胞如T、B淋巴细胞、NK细胞等的变化，以及乳腺癌细胞表面分子的改变，进一步明确乳腺癌患者的免疫状况。希望乳腺癌抗原负载后的DC能够弥补乳腺癌患者的免疫缺陷，并探讨肿瘤抗原负载的DC免疫过继治疗乳腺癌动物模型的效果。
材料和方法
1、乳腺癌患者的免疫功能检测：随机抽选2001年3月至10月30例乳腺癌患者的外周血，采用流式细胞技术分析淋巴细胞表面CD3、CD4、CD8、CDl9、CD28的表达，单核细胞表面CD40、CD80、CD86的表达。
2、乳腺癌细胞表面分子的表达：采用流式细胞技术检测5株乳腺癌细月包MCF-7、SK-BR-3、T47D、MDA-MB-435s、ZR-75-30表面MHCⅡ类抗原与共刺激分子CD40、CD80(B7-1)、CD86(B7-2)的表达，并与正常乳腺细胞HBL-100作比较。同时检
1汕k厂村人学博【：学位论义
渺J35仲憎L腺癌肿瘤标本这些分子的表达，并与乳腺良性病变标本
作比较。
3、人外周血中树突状细胞分离、培养与鉴定：采集健康成人外
周血 20人份，乳腺癌患者外周血 30人份，每份 10毫升，肝素
抗凝。用淋巴细胞分离液分离外周血，收集单个核细胞，调节细
J包浓度为 IO‘／ml，然后在 6 JL板上占壁，每JLh。Zml，2，J、时后
吸去悬浮细胞，加入新鲜营养液。每隔1天半量换液一次。用共
聚焦显微镜拍摄所培养的细胞，流式细胞枝术鉴定所培养的细
胞。
4、B：lb儿，J、鼠骨髓DC的培养：元菌操作取，J、鼠后肢股骨，用
注射针将髓腔中的骨髓细胞吹打出，用0．8％TriSNH人溶去红
细月后，PBS洗2次，用含mGM－CSF（．3ng／ml），lL＊20ng／ml）
和 10o，J、牛血清的 yMI 640 Z全培养基重悬至 l．0 xlo‘／ml，
铺于 6孔板上，ZmNL，第 3天轻轻晃动培养板，吸弃全部上清
（去除粒细胞），及时补入含细胞因子浓度相同的完全培养基，
第 6天轻轻吹吸下疏松粘附于培养板底的聚集体，置于新的培养
板中，并加入等量的上述完全培养基，继续培养24小时，用于
免疫标记鉴定、分泌细胞因子检测及生物学活性的研究。
5、B：lb七，J、鼠乳腺癌模型的建立：选择对数生长期的 TM40D，
用 0．25％胰 蛋白酶消 化，PBS洗涤2次；细胞浓度调至 10‘．10‘／ml。
在 Balb七小鼠胸壁第 56肋间或腹壁第 4对乳腺处皮下接种肿瘤
go胞0．1m1，相当于10’1～胞／X小鼠。每天观察肿瘤的生长情
况，用游标卡尺测量肿块的大小。
6、肿瘤抗原负载的DC免疫过继治疗B：lb止。J、鼠乳腺癌模型：
动物分为 3组，每组 10 ．q小鼠，皆于腹壁右侧第 4对乳腺处皮
下接种对数生长期的 TM40D细胞，IX 10‘／只，在肿瘤细胞接种
7
内水厂村人学博卜学位沦义
后第 3天，于同侧腹股沟皮下接种 TM40D负载的 DC（2 XIO‘／
只／次），以后分别于第10，门，24天每组动物接受与第一次同
样的治疗。观察肿瘤的生长、动物存活率以及肿瘤的转移情况。
结 果
l、乳腺癌患者外周血中 CD3、CD4、CDS、CD16、CD19阳性
的淋巴细胞与对照组相比无显著性差异…功刀5八但是CD28阳
性 T i田胞少于对照组（p＜0．05），特别是CD4／CD28双阳性 T兰
胞明显少于对ig组（p功．005）。手术后7天左右CD28阳性T细
胞就恢复到正常，与对照组相比无显著性差异（p＞0刀5），说明
乳腺癌患者免疫细胞减少与肿瘤负荷有关。乳腺癌患者单核细胞
、表达共刺激分子 CD80（p＜0．05）、CD86（p＜0．00L CD40表
达与对照组相比无显著性差异巾叩刀5）。
2、5株乳腺癌细胞MHC H类分子表达都与HB卜mo有显著性差
异（p＜0．05），MCF－7 i田胞表达水平最低，约为 HBL－100勺 1／5。
MDA－MB－4355与 ZR刁5S0细包表达水平是HBLl 的 2倍，
MDA．MB．4355荧光强度也比HBLl0O及其它乳腺癌细胞高。但
MDA－MB－4355 i田包表＠CD40分子表达水平最低，约为 MCF－7
与HBL－mo CD4O分子表达水平的m％。MDA－MB．4355细月
CD80、CD86分子表达水平与 HBL－100才当（p＞0．05），另 4株
乳腺癌细月 CD80、CD86分子表达者比 HBL－100｛（p＜0．05）。
乳腺癌原代细胞 CD40表达水平与对照组相比无显著性差异
（p＞0刀5），＊＊0、＊＊6白
Study of the Adoptive Immunotherapy on Breast Cancer with Dendritic Cells Loaded with Tumor Antigen
Fan Ping (student), Wu Zhengyan (tutor)
Objective
Dendritic cells are important antigen presenting cells in human being. They can specifically present antigen to T lymphocytes and initiate specific antitumor immunity reaction in vitro and in vivo. Their remarkable effectiveness is in large part due to their efficiency in capturing, processing, and presenting antigens along with costimulatory signals. Therefore, dendritic cells have become one of the hot spots in the field of tumor immuno-adoptive therapy since 90s. In order to determine immuno-status of breast cancer patients in this paper, we study the changes of immunocytes such as T lymphocytes, B lymphocytes, and natural killer ect. in the peripheral blood of breast cancer patients. We also study the molecules such as CD80, CD86, and MHC ect. expressing on the breast cancer cell lines and primary breast cancer cells. We expect that tumor antigen loaded dendritic cells could make up for the immunodeficiency of breast cancer patients. For this purpose, we first research on adoptive immunotherapy to breast cancer animal model using tumor antigen loaded dendritic cells.
Materials and Methods
1.We selected 30 admission patients with breast cancer between March and October in 2001 at random . Using flow cytometery analysis, we examined CD3, CD4, CD8, CD 16, CD 19 and CD28 molecules expressing on the lymphocytes and CD40, CD80 and CD86 molecules expressing on the monocytes in the peripheral blood samples from breast cancer patients.
2.We detected these molecules expressing on the five breast cancer cell lines using flow cytometery analysis. The cell lines include MCF-7, SK-BR-3, T47D, MDA-MB-435s, and ZR-75-30. The expression levels were compared with that of normal mammary cell line HBL-100 . At the same time, we examined those molecules expressing on the 35 primary breast cancer cells and compared with that of benign breast diseases.
3.Fifty cases peripheral blood were drawn from 20 healthy adult persons and 30 breast cancer patients. Every person was drawn 10 milliliter blood and the blood was anticoagulated by heparin (5unit per milliliter). We use Ficoll Hypaque centrifugation to isolate the mononuclear cells in the peripheral blood. These cells are plated in six-well culture plates(106/ml,2ml/well) in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, 100ng/ml GM-CSF, 20ng/ml IL-4, and/or 20ng/ml TNF- a .After 2 hours, nonadherent cells were gently removed and fresh medium was added. Culture medium was refreshed every other day. Cultured cells were taken a picture using focus scanning microscope (Zeiss LSM510)
and analyzed by flow cytometry with labeled monoclonal antibodies. 4.Under sterile condition, lower limbers of Balb/c mice were cut with scissors. In the dish, the marrow was flushed out using 2ml of RPMI 1640 with a syringe. The tissue was suspended and red cells were lysed with 0.8% ammonium chloride. After washed twice with PBS, these cells are plated in six-well culture plates(106/ml,2ml/well) in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, 3.3ng/ml GM-CSF, 20ng/ml IL-4. Three days later, the cultures were fed by gently swirling the plates, aspirating all of the medium, and adding back fresh medium with the same concentration cytokines as before. An object of this wash was to remove nonadherent granulocytes. On 6th day, the aggregates of loosely attaching to culture plate cells were dislodged with pipettes. Those cells were removed to a new culture plate and cultured 24 hours. Cultured cells were analyzed by flow cytometry with labeled monoclonal antibodies, cytokines secretion was detected with RT-PCR, and immunological activity was searched with mixed lymphocytes reaction.
5. Female Balb/c mice were 4-6 weeks old. Breast cancer cell line TM40D derived from Balb/c mice. Digested by 0.25% trypsin , washed twice with PBS, exponentially growing TM40D cells (
引文
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