【作者】 黄香；

【导师】 陈龙邦；

【作者基本信息】 南京大学 ， 内科学， 2011， 博士

【摘要】 [背景与目的]传统化疗药物的免疫调节作用已逐渐受到人们的重视,特定剂量特定模式的给药方式可通过多种机制重建机体免疫内环境,调变肿瘤细胞免疫原性。将其作为预处理方案联合后续的过继性细胞免疫治疗,可有效动员机体的免疫系统发挥抗肿瘤作用。本研究旨在观察预处理化疗在小鼠动物肿瘤模型(Lewis(?)市癌、CT-26结肠癌、B16黑色素瘤)中对细胞因子诱导的杀伤细胞(cytokine-induced killer cells, CIK cells)的抗肿瘤活性的增强作用,并从机体免疫内环境的调节以及肿瘤细胞免疫原性的调变两方面探讨介导预处理化疗增效作用的机制。[材料与方法]1.建立C57BL/6小鼠Lewis肺癌模型,选用紫杉醇(Paclitaxel, PTX)联合顺铂(Cisplatin, DDP)作为预处理方案(TP方案),荷瘤小鼠随机分为六组：对照组(给予生理盐水,Normal Saline, NS)、NS-3-CIK组(NS后3天给予CIK细胞)、NS-7-CIK组(NS后7天给予CIK细胞)、TP组(给予TP方案)、TP-3-CIK组(TP方案预处理后3天联合CIK细胞)及TP-7-CIK组(TP方案预处理后7天联合CIK细胞)。隔日测量肿瘤长短径监测肿瘤体积,观察各组治疗方案对Lewis肿瘤的抑制作用。2.分别建立BALB/c野生鼠或BALB/c nu/nu裸鼠CT-26结肠癌模型,随机分为四组：NS组(给予NS)、CIK组(给予CIK细胞)、DDP组(给予DDP)、DDP-CIK组(DDP预处理3天后联合CIK细胞)。隔日测量肿瘤长短径监测肿瘤体积,观察各组治疗方案对CT-26结肠癌的抑制作用,并比较联合方案在两种动物模型中的抗肿瘤作用。3.建立C57BL/6小鼠B16黑色素瘤模型,随机分为四组：NS组(给予NS)、CIK组(给予CIK细胞)、DDP组(给予DDP)、DDP-CIK组(DDP预处理3天后联合CIK细胞)。隔日测量肿瘤长短径监测肿瘤体积,观察各组治疗方案对B16黑色素瘤的抑制作用。4.利用绿色荧光蛋白(Green Fluorescence Protein, GFP)转基因小鼠制备GFP+CIK细胞,荧光显微镜追踪其体内迁移分布,观察预处理化疗对CIK细胞体内归巢功能的影响。5.分离Lewis(?)市癌模型及CT-26结肠癌模型中的肿瘤组织,分别行CD3、FoxP3(Forkhead boxP3)、CD31分子免疫组化染色以评估肿瘤组织局灶T淋巴细胞、Treg细胞的浸润情况以及肿瘤微血管密度的变化。6.流式细胞术观察预处理化疗后荷瘤鼠各组织中内源性T淋巴细胞、树突状细胞(Dendritic cells, DCs)、髓系来源抑制细胞(Myeloid-derived suppressor cells, MDSCs)、调节性T细胞(Regulatory T cells, Treg cells)的动态变化。7.MTT法筛选化疗药物5-氟尿嘧啶(5-Fluorouracil,5-FU)、DDP、PTX、多西紫杉醇(Docetaxel,DTX)的体外高、中、低毒性浓度,将这些浓度的化疗药物处理人肺腺癌细胞(A549、SPC-A1、SPC-A1/DTX), WST-1法比较肿瘤细胞在化疗药物预处理前后对CIK细胞体外杀伤作用的敏感性。8.RT-PCR检测肿瘤细胞经化疗药物处理后各时间点免疫原性分子mRNA水平表达的改变：NKG2D配体(Natural killer group 2, member D ligands, NKG2DL)、Fas受体分子、细胞间黏附分子-1 (Intercellular adhesion molecule-1, ICAM-1)、高迁移率族蛋白1 (High-mobility group box-1, HMGB-1)、钙网织蛋白(Calreticulin, CRT)、DNAM分子(DNAX accessory molecule-1)、Clr-b分子(C-type lectin-related molecule b)。[结果]1.在C57BL/6小鼠Lewis肺癌模型中,TP预处理化疗后不同时间点给予CIK细胞免疫治疗均能明显抑制(?)Lewis肺癌的生长(P<0.05),且两时间点间无统计学差异；而单独CIK免疫治疗或TP化疗均不能抑制Lewis肿瘤的生长(P>0.05)。实验结束时两联合治疗组肿瘤体积分别为3377.82±1603.43mm3 (TP-3-CIK组),3183.38±806.08mm3 (TP-7-CIK组);单独CIK治疗组及TP化疗组肿瘤体积分别为5997.46±1372.90mm3 (NS-3-CIK组),6206.70±1700.61mm3 (NS-7-CIK组)及6387.09±1019.48mm3 (TP组),NS对照组肿瘤体积为7087.57±1103.37mm3。2.在BALB/c野生鼠CT-26结肠癌模型中,单独CIK免疫治疗或DDP化疗均可明显抑制CT-26结肠癌的生长(P<0.05),而DDP预处理化疗联合CIK细胞免疫治疗与单独治疗组相比,则可诱导更为显著的抗肿瘤作用(P<0.05)。实验结束时DDP-CIK组肿瘤体积为2115.62±436.61mm3,CIK组肿瘤体积为2937.44±773.78mm3,DDP组肿瘤体积为2885.26±318.88mm3,NS对照组肿瘤体积为3867.55±207.56mm3。在BALB/c裸鼠CT-26结肠癌模型中,DDP预处理化疗联合CIK细胞免疫治疗、单独CIK治疗及单独DDP化疗均不能抑制CT-26结肠癌的生长(P>0.05)。实验结束时DDP-CIK组肿瘤体积为3198.15±863.57mm3,CIK组肿瘤体积为3445.42±786.73mm3,DDP组肿瘤体积为3298.26±788.13mm3,NS对照组肿瘤体积为3778.65±819.22mm3。3.在C57BL/6小鼠B16黑色素瘤模型中,DDP预处理化疗联合CIK细胞免疫治疗可显著抑制B16黑色素瘤的生长(P<0.05),单独的CIK免疫治疗或单独的DDP化疗均不能抑制B16黑色素瘤的生长。实验结束时DDP-CIK组肿瘤体积为2644.41±910.8mm3,CIK组肿瘤体积为5634.08±486.89mm3,DDP组肿瘤体积为5215.31±1118.87mm3,NS对照组肿瘤体积为5587.62±1390.01mm3。4. Lewis(?)市癌模型中的TP预处理化疗可促进过继性输注的CIK细胞至肿瘤局灶及脾脏组织的归巢。5. Lewis(?)市癌模型中的TP预处理化疗及CT-26结肠癌模型中的DDP预处理化疗均可促进CD3+T淋巴细胞至肿瘤局灶的浸润,而对肿瘤组织的微血管密度无明显作用。6.预处理化疗可以诱导肿瘤引流淋巴结(Tumor-draining lymph nodes, TDLNs)及肿瘤组织中内源性T淋巴细胞比例的一过性升高；增加骨髓、外周血及脾脏组织中DCs的比例；降低外周血、骨髓、脾脏组织及TDLNs中MDSCs的比例；下调肿瘤局灶及脾脏组织中Treg细胞的比例。7.化疗药物预处理可增加人肺腺癌细胞对CIK细胞体外杀伤作用的敏感性。8.化疗药物预处理可上调人肺腺癌细胞ULBP、Fas、ICAM-1及DNAM分子1mRNA水平的表达,下调Clr-b分子mRNA水平的表达。[结论与意义]1.预处理化疗可在多种动物肿瘤模型中增强CIK细胞免疫治疗的疗效,且此种增效作用与宿主的内源性T淋巴细胞相关。2.预处理化疗可通过调节机体的免疫内环境以促进CIK细胞至肿瘤及脾脏组织的归巢,增强CIK细胞免疫治疗的疗效。3.预处理化疗可能通过调节肿瘤细胞免疫原性相关分子的表达以调变肿瘤细胞的免疫原性,进而增加肿瘤细胞对CIK细胞体外杀伤作用的敏感性。4.综上所述,本研究首次证实预处理化疗可增强CIK细胞免疫治疗在多种动物肿瘤模型中的抗肿瘤作用,并发现这种增强作用与预处理化疗可调节机体免疫微环境及增强肿瘤细胞的免疫原性有关。5.上述结果为预处理化疗联合CIK细胞免疫治疗的临床应用提供了实验基础与理论依据,也为晚期恶性肿瘤的临床治疗提供了一种安全有效的联合治疗模式。更多还原

【Abstract】 Background and ObjectiveIt is now clear that chemotherapy, in addition to its direct cytotoxic effects on tumor cells, could exert a profound influence on antitumor immune response. A number of studies have demonstrated that some chemotherapeutic agents could enhance the antitumor activity of subsequent adoptive cell transfer when used as a preconditioning regimen. The purpose of this study was to investigate the enhancement of the antitumor effect of cytokine-induced killer (CIK) cells induced by preconditioning chemotherapy and to elucidate the underlying mechanisms by which chemotherapeutic agents break down the immunosuppressive barriers to release the full potential of the CIK cell therapy.Materials and Methods1. C57BL/6 mice were inoculated with Lewis cells to establish the murine lung carcinoma model and then randomly divided into six groups. (a) Group Control:treated with normal saline (NS); (b) Group NS-3-CIK:treated with NS followed 3 days by CIK cells; (c) Group NS-7-CIK:treated with NS followed 7 days by CIK cells; (d) Group TP:treated with TP regimen:paclitaxel (PTX) plus cisplatin (DDP); (e) Group TP-3-CIK:preconditioned with TP regimen followed 3 days by CIK cells; (f) Group TP-7-CIK:preconditioned with TP regimen followed 7 days by CIK cells. Tumor size was monitored as indicator of therapeutic response.2. BALB/c wild type and BALB/c nu/nu mice were challenged with CT-26 cells to establish the colon adenocarcinoma model and then randomly divided into four groups. (a) Group NS: administered with NS; (b) Group CIK:administered with CIK cells; (c) Group DDP: administered with DDP; (d) Group DDP-CIK:administered with DDP followed 3 days by CIK cells. Tumor size and weight were used as indicators of therapeutic response.3. C57BL/6 mice were injected with B16 cells to establish the murine melanoma model and then randomly divided into four groups. (a) Group NS:administered with NS; (b) Group CIK: administered with CIK cells; (c) Group DDP:administered with DDP; (d) Group DDP-CIK: administered with DDP followed 3 days by CIK cells. Tumor size was used as indicators of therapeutic response.4. The in vivo trafficking and homing ability of GFP+ CIK cells were observed by fluorescent microscopy.5. Immunohistochemistry was performed to observe the intratumoral infiltration of CD3+ T lymphocytes and FoxP3+ regulatory T (Treg) cells and tumor microvessel density.6. The dynamic changes of endogenous T lymphocytes, dendritic cells (DCs), myeloid-derived suppressor cells (MDSCs) and Treg cells in various tissues after preconditioning chemotherapy were analyzed through flow cytometry.7. The in vitro high, moderate and low toxic concentrations of chemotherapeutic agents were determined by MTT assay and WST-1 assay was used to evaluate the in vitro cytotoxity of CIK cells against drug-treated or untreated tumor cells.8. RT-PCR was employed to detect the expression of immunogenicity-related molecules of tumor cells in mRNA level after treatment with chemotherapeutic agents.Results1. In Lewis lung carcinoma model, CIK cells following TP preconditioning chemotherapy could significantly inhibit the growth of Lewis lung carcinoma (P<0.05), whereas CIK therapy alone or DDP chemotherapy alone failed to induced evident inhibition of tumor growth (P >0.05). At the end of the experiment, the tumor volumes were 3377.82±1603.43mm3 in Group TP-3-CIK,3183.38±806.08mm3 in Group TP-7-CIK,5997.46±1372.90mm3 in Group NS-3-CIK,6206.70±1700.61mm3 in Group NS-7-CIK,6387.09±1019.48mm3 in Group TP and 7087.57±1103.37mm3 in Group NS, respectively.2. In CT-26 colon adenocarcinoma model, CIK cell therapy alone or DDP alone could markedly inhibit the growth of CT-26 colon adenocarcinoma in BALB/c wild type mice (P<0.05), however the combination of DDP preconditioning chemotherapy and CIK cells could induce more significant tumor growth retardation compared with single therapy (P<0.05). At the end of the experiment in BALB/c wild type mice, the tumor volumes were 2115.62±436.61mm3 in Group DDP-CIK,2937.44±773.78mm3 in Group CIK, 2885.26±318.88mm3 in Group DDP and 3867.55±207.56mm3 in Group NS, respectively. In BALB/c nu/nu mice, the combination therapy and both of the single therapies failed to induce significant tumor growth inhibition (P>0.05). At the end of the experiment in BALB/c nu/nu mice, the tumor volumes were 3198.15±863.57mm3 in Group DDP-CIK, 3445.42±786.73mm3 in Group CIK,3298.26±788.13mm3 in Group DDP and 3778.65±819.22mm3 in Group NS, respectively.3. In B16 melanoma model, the combination of DDP preconditioning chemotherapy and CIK cells could significantly inhibit the growth of B16 melanoma (P<0.05), whereas CIK therapy alone or DDP chemotherapy alone failed to induced evident inhibition of tumor growth (P >0.05). At the end of the experiment, the tumor volumes were 2644.41±910.8mm3 in Group DDP-CIK,5634.08±486.89mm3 in Group CIK,5215.31±1118.87mm3 in Group DDP and 5587.62±1390.01mm3 in Group NS, respectively.4. The TP regimen could augment the homing ability of infused CIK cells into tumor and spleen tissues in Lewis lung carcinoma model.5. The TP regimen in Lewis lung carcinoma model could increase the level of T lymphocytes in local tumor tissues, and DDP preconditioning chemotherapy in CT-26 colon adenocarcinoma model could increase the percentages of endogenous T lymphocytes in tumor tissue and tumor-draining lymph nodes (TDLNs). Neither of the precondition exerted influence on tumor microvessel density.6. The DDP pretreatment could up-regulate the percentages of DCs in bone marrows, peripheral bloods and spleen tissues, diminish the levels of MDSCs in bone marrows, peripheral bloods, spleen tissues and TDLNs, and decrease the percentages of Treg cells in tumor and spleen tissues.7. The human lung adenocarcinoma cells displayed an increased sensitivity to the lyses of CIK cells after treatment with chemotherapeutic drugs in vitro.8. Treatment with chemotherapeutic agents could up-regulate the expression of ULBP, Fas, ICAM-1 and DNAM, and down-regulate the expression of Clr-b in mRNA level in human lung adenocarcinoma cells.Conclusions1. Our data indicate that the preconditioning chemotherapy could enhance the antitumor activity of CIK cell therapy in various murine tumor models, and the endogenous T lymphocytes were involved in this enhancement.2. The underlying mechanisms mediating the chemotherapy-induced enhancement of CIK cells’ efficacy may involve the augmentation of homing ability of CIK cells to tumor and spleen tissues, and the modulation of percentages of endogenous immune cells in various tissues.3. Treatment of chemotherapeutic agents may sensitize tumor cells to the lyses of CIK cells in vitro through up-regulating in the transcriptional expression of immunogenic molecules of target cells.4. To our knowledge, this is the first study to evaluate the efficacy-enhancing effect of preconditioning chemotherapy on the subsequent adoptive CIK transfer. And this efficacy-enhancing effect may be associated with the modulation of the host immune microenvironment and the immunogenicity of tumor cells induced by chemotherapy.5. This combined chemo-immunotherapy strategy could be easily translated into a clinical setting and is therefore an attractive modality of combined chemo-immunotherapy applicable to patients with malignant cancer.更多还原