【作者】 付晶鑫；

【导师】 沈建根； 夏大静；

【作者基本信息】 浙江大学 ， 免疫学， 2007， 硕士

【摘要】 目的：研究新型乳腺癌生长抑制因子BDGI治疗4T1高转移乳腺癌的抗肿瘤作用，通过比较各组细胞因子及病理学变化，观察BDGI基因对4T1乳腺癌的治疗效果并探讨其可能机制，为BDGI基因治疗乳腺癌提供实验依据。方法：一、动物实验将处于对数生长期的4T1细胞皮下注射于54只小鼠体内建立乳腺癌实验动物模型，24h后随机分组为治疗组Ⅰ、治疗组Ⅱ、空质粒治疗组、对照组、CTX组、联合组建模后第3天，以不同剂量的药物皮下注射各组小鼠，共3次(间隔3天)。观察荷瘤小鼠健康、生存状况、肿瘤生长情况。在未次治疗后第7天脱颈椎处死部分小鼠(3只／组)，取脾脏制备淋巴细胞悬液，用MTT法测NK杀伤活性；T淋巴细胞悬液铺板、收集培养上清，-20℃保存，待检测细胞因子；取肿瘤组织称重，10％甲醛固定，待进行病理观察；取肺组织，10％甲醛固定，待进行病理观察肿瘤组织转移情况；剩余小鼠留做生存期观察；二、NK杀伤活性测定将已处死的小鼠在无菌条件下摘取脾脏，放入研钵中轻轻研磨，经120目网过滤去除脂肪组织，制取细胞悬液，计数后，调整其浓度为2×10~6ml，直接用做效应细胞，靶细胞为YAC-1，浓度为2×10~5／ml。采用MTT法测定NK细胞杀伤活性。三、病理观察肿瘤组织、肺组织依次经过10％甲醛固定、EDTA脱钙、乙醇梯度脱水、常规包埋及切片，苏木素-伊红(HE)染色，镜检观察组织病理变化，比较各组间差异。四、细胞因子检测收集T淋巴细胞悬液培养上清，细胞因子IL-2、TNF-α、IFN-γ的测定均按照EIA试剂盒说明书要求进行，即将待测培养上清和不同浓度的标准品加入相应孔中(100ul／孔)，37℃孵育120分钟，洗板5次，在每孔加入第一抗体工作液50ul，37℃孵育60分钟，洗板5次，每孔加入酶标抗体工作液100ul，37℃孵育60分钟，沈板5次，每孔加入底物工作液100ul，37℃暗处反应5分钟，每孔加入50ul终止液混匀，上机490nm波长检测吸光值，绘制标准曲线计算检测结果。结果：一、病理切片显示肿瘤组织：各组肿瘤细胞大小不等、形态多样，有较多核碎片分布，癌细胞异型性明显。与对照组、空质粒治疗组相比，治疗组Ⅰ、治疗组Ⅱ、CTX组、联合组有一定程度的炎性细胞和淋巴细胞浸润，伴有部分肿瘤组织的坏死，但组间差异并不显著。肺组织：对照组、空质粒治疗组、治疗组Ⅰ、治疗组Ⅱ可见明显的肺转移灶，肿瘤细胞大小不一、形似瓜子或燕麦，部分呈条索状排列，间有血窦、伴有一定程度的炎性细胞和淋巴细胞浸润。CTX组、联合组未见转移灶出现，肿瘤转移受到明显抑制。二、NK杀伤活性测定与对照组相比各治疗组小鼠NK细胞杀伤活性显著增高(P＜0.05)；与空质粒治疗组相比，各治疗组小鼠NK细胞的杀伤活性显著增高(P＜0.05)；与CTX组相比，CTX组NK细胞杀伤活性高于组治疗组Ⅰ、治疗组Ⅱ(P＜0.05)，但与联合组无显著差异(P＞0.05)；组间比较，治疗组ⅡNK细胞杀伤活性高于治疗治疗组Ⅰ(P＜0.05)，联合组高于治疗组Ⅱ(P＜0.05)。三、细胞因子检测1、重组pcDNA3.1-BDGI对荷瘤小鼠IFN-γ的影响：与对照组相比空质粒治疗组无显著差异，其它各治疗组小鼠IFN-γ显著增高(P＜0.05)；与空质粒治疗组相比各治疗组小鼠IFN-γ显著增高(P＜0.05)；与CTX组相比，CTX组IFN-γ显著高于组治疗组Ⅰ、治疗组Ⅱ(P＜0.05)，但与联合组无显著差异(P＞0.05)；组间比较，治疗组ⅡIFN-γ显著高于治疗治疗组Ⅰ(P＜0.05)，联合组显著高于治疗组Ⅱ(P＜0.05)。2、重组pcDNA3.1-BDGI质粒治疗对荷瘤小鼠IL-2的影响与对照组相比，空质粒治疗组无显著差异，其它各治疗组小鼠IL-2显著增高(P＜0.05)；与空质粒治疗组相比各治疗组小鼠IL-2显著增高(P＜0.05)；与CTX组相比，CTX组IL-2显著高于组治疗组Ⅰ、治疗组Ⅱ(P＜0.05)，但与联合组无显著差异(P＞0.05)；组间比较，治疗组ⅡIL-2显著高于治疗治疗组Ⅰ(P＜0.05)，联合组显著高于治疗组Ⅱ(P＜0.05)。3、重组pcDNA3.1-BDGI质粒治疗对荷瘤小鼠TNF-α的影响与对照组相比，空质粒治疗组无显著差异，各治疗组小鼠TNF-α显著增高(P＜0.05)；与空质粒治疗组相比各治疗组小鼠TNF-α显著增高(P＜0.05)；与CTX组相比，CTX组TNF-α显著高于组治疗组Ⅰ、治疗组Ⅱ(P＜0.05)，但与联合组无显著差异(P＞0.05)；组间比较，治疗组ⅡTNF-α显著高于治疗治疗组Ⅰ(P＜0.05)，联合组显著高于治疗组Ⅱ(P＜0.05)。结论：1．在小鼠4T1乳腺癌模型中，联合BDGI和CTX或提高BDGI剂量，可抑制肿瘤的生长。治疗组Ⅱ和联合治疗组的小鼠生存期延长。2．联合BDGI和CTX或提高BDGI剂量治疗增强了脾细胞NK杀伤活性，并诱导了更多的Th1细胞因子(IL-2、IFN-γ)及TNF-α的分泌，诱导机体产生特异性和非特异性的抗肿瘤免疫效应。3．联合BDGI和CTX进行治疗可有效抑制了肿瘤的肺转移。4．联合治疗组和高剂量治疗组小鼠肿瘤周围有更多的炎性细胞浸润，肿瘤组织出现坏死，但组间差异并不显著。5．BDGI对乳腺癌的治疗存在一定的剂量依赖关系。上述结果提示BDGI在恶性肿瘤的基因治疗中具有潜在的应用价值，为开辟治疗恶性肿瘤的新方法和新途径提供了新思路。更多还原

【Abstract】 Objective :To further investigate the antitumor responses of a novel human bone marrow stromal cell-derived growth inhibitor(BDGI), and apply the recombination plasmid pcDNA3.1-BDGI to 4T1 murine mammary carcinoma model by intratumoral injection and evaluate its antitumor efficacy and possible antitumor mechanisms in tumor bearingmice.Methods :1. Animal experiment54 mice were inoculated s.c. with 1×10~6 4T1 mammary tumor cells to establish experimental animal model of breast cancer. After 24 hours of injection,mice were randomly divided into 6 groups: the control group, theblank plasmid group, the treatment group I, the treatment group II, the CTX group, the Joint Unit group. Different doses of the drug were injected s.c. in mice and treated for three times (the interval of 3 days). Surveied bearing mice health, living conditions, the growth of tumors. On the seven day after the last treatment, executed from cervical of mice (3/group) for the spleen lymphocytes, MTT measured NK cytotoxicity assay. Collect the supernatant, store at -20°C, to be detected cytokines; Weighing in tumor tissue from 10% formalin-fixed, pending pathological observation; The lungs, 10% formalin -fixed, pending pathology observed tumor metastasis; the remaining miceretained for survival observation. 2. NK cytotoxicity assayNK cytotoxicity assay was measured by the MTT colorimetry assay.Executed the mice in sterile conditions and removed the spleen, after 120 Mesh filters remove adipose tissue, from cell suspension system, breaking red. adjust its concentration of 2×10~6/ml, directly used as effector cells. YAC-1 as target cells in the concentration of 2×10~5/ml, MTT measured NK cell samples. In briefly, YAC-1 cells (2×10~5/ml) as target cells were seeded in 96-well plastic plate. Spleen cells were prepared from the mice as effector cells and simultaneously seeded with YAC-1 (2×10~6/ml) at 10:1 ratios of effector to target (E:T) and total of volume was 200μl. After the plate was incubated at 37°C in 5% CO2 for 8 hours, the supernatant was discarded, and then each well was added with10μl MTT for additional 4 hours incubation. The supernatants were discarded and 150μl dimethylsulfoxide was added and shaken for a minute, then optical densities were measured with the plate reader (Bio-rad) at OD 570.3. pathology observed In tumor tissue, lung tissue followed after 10% formalin-fixed, decalcified EDTA, ethanol dehydration gradient, conventionalembedded and sectioned at HE staining and microscopy observation of histopathologic changes, as compared to the differences between the groups. 4. Dectection of Cytokines Cytokines collection of T lymphocytes were cultured medium. IL-2, TNF-α and IFN-γ were dectected in accordance with the EIA requirements kit brochures. Put culture supernatant or the analyte concentration of the standard 100 ul/well, 37°C for 120 minutes, wash five times, in each well to work with the first antibody 50ul. 37°C for 60 minutes, wash five times, each well ELISA antibody fluid 100ul, 37°C for 60 minutes, wash five times, each well to work with the substrate, while the reaction 100 ul, 37°C five minutes, 50 ul per well to terminate the reaction, 490 nm absorbance detection wavelength, drawing standard curve calculation results.Results :1. Pathological change of the tumor and the lungTumor tissue:The tumor cells vary in size and shape, there was much more spread of nuclear debris, markedly abnormal cells. Compare with the control group, the blank plasmid group, each drug treatment group was a certain degree of inflammatory cells(neutrophils,lymphocytes) infiltration accompanied by some of the tumor necrosis, but the difference among the groups was not significant. Lung tissue:The control group, the blank plasmid group, the treatment group I and the treatment group II was observed in lung metastases. Tumor cell sizes, the shape of a melon or oats, was part of the cords arranged between sinusoids,accompanied by a certain degree of inflammatory cell infiltration. The CTX group, the joint group appeared no metastases, tumor metastasis were significantly inhibited, the control group, the blank plasmid group, the treatment group I, the treatment group II, the CTX group, the Joint Unitgroup.2. Improvement activities of NK in tumor-bearing mice treated with recombinant pcDNA3.1-BDGICompared with the control group the treated mice NK cytotoxic activity was significantly increased (P<0.05); With the blank plasmid group, NK cytotoxic activity of the other treatment groups were significantly increased (P<0.05); Compared with the CTX group, the CTX group NK cytotoxic activity was significantly increased than the treatment group I, the treatment group II (P<0.05), but here was no significant difference with the Joint group (P>0.05); Compared with the treatment group I, the CTX group、 the Joint group 、 the treatment group II NK cytotoxic activity were significantly increased (P<0.05).3. Level of cytokine3.1 Improvement activities IFN-γ level in tumor-bearing mice treated with recombinant pcDNA3.1-BDGICompared with the control group, the blank plasmid group IFN-γ level was no significant difference(P>0.05), other groups IFN-γ level were significantly increased (P<0.05); Compared with the blank plasmid group, other groups IFN-γ level were significantly increased (P<0.05); Compared with the CTX group, the CTX group IFN-γ level was significantly increased than the treatment group I, the treatment group II (P<0.05), but here was no significant difference with the Joint group (P>0.05); Compared with the treatment group I, the CTX group 、 the Joint group、 the treatment group II IFN-γ level weresignificantly increased (P<0.05).3.2 Improvement activities IL-2 level in tumor-bearing mice treated with recombinant pcDNA3.1-BDGICompared with the control group, the blank plasmid group was no significant difference, while other groups IL-2 level were significantly increased (P<0.05); Compared with the blank plasmid group, other groups IL-2 level were significantly increased (P<0.05); Compared with the CTX group,the CTX group IL-2 level was significantly increased than the treatment group I, the treatment group II (P<0.05), but here was no significant difference with the Joint group (P>0.05); Compared with the treatment group I, the CTX group、 the Joint group 、 the treatment group II IL-2 level were significantly increased(P<0.05).3.3 Improvement activities TNF-αlevel in tumor-bearing mice treated with recombinant pcDNA3.1-BDGICompared with the control group, the blank plasmid group TNF-αlevel was no significant difference, while other groups TNF-αlevel were significantly increased (P<0.05); Compared with the blank plasmid group, other groups TNF-αlevel were significantly increased (P<0.05); Compared with the CTX group, the CTX group TNF-αlevel was significantly increased than the treatment group I, the treatment group II (P<0.05), but here was no significant difference with the Joint group (P>0.05); Compared with the treatment group I, the CTX group、 the Joint group、 the treatment group II TNF-αlevel were significantly increased (P<0.05).Conclusions :1. In 4T1 mouse model of breast cancer, the companied BDGI with CTX treatment, improvement of BDGI dose treatment, can inhibit tumor growth.The treatment group II and the Joint group were extend the survival of mice.2. company BDGI with CTX or raise BDGI dose can increase splenic NK cytotoxic activity, and induce more Th1 cytokines (IL-2, IFN-γ、 the secretion of TNF-α, induce specific and non-specific anti-tumor immune response.3. Company BDGI with CTX can effectively inhibit tumor metastasis to the lung.4. Combined therapy and high-dose treatment of tumor-bearing mice can induce more inflammatory cell infiltration, tumor tissue necrosis, However, the difference between the groups was not significant.5. The BDGI treatment of breast cancer is a certain dose-dependent relationship. These results suggest BDGI may have potential application of cancer gene therapy. Maybe explore a new cancer treatment method and new way to provide new ideas.更多还原