热休克法制备高效树突状细胞肿瘤疫苗治疗小鼠乳腺癌
本文选题:树突状细胞 + 乳腺癌 ; 参考:《泰山医学院》2010年硕士论文
【摘要】:背景 乳腺癌是女性最常见的恶性肿瘤,且发病率呈逐年上升趋势,当前,乳腺癌的治疗主要治疗手段包括手术治疗、化疗、放疗及内分泌治疗,即便如此,术后仍有很高的复发率,预后仍不理想。近年来,生物治疗逐渐受到人们重视,人们开始利用制备肿瘤疫苗的方法治疗恶性肿瘤。肿瘤疫苗因其以较低的抗原刺激就能激发强烈的免疫反应、肿瘤特异性、自体耐受和安全性以及长时间的免疫记忆能避免肿瘤复发等优点被越来越多的用来治疗各种恶性肿瘤,并且取得了良好的效果。同样,以树突状细胞(dendritic cells,DC)为基础构建的乳腺癌治疗疫苗也成为乳腺癌生物治疗的研究热点;其中,直接利用裂解的肿瘤细胞抗原负载树突状细胞制备的肿瘤疫苗因肿瘤抗原易于制备和疗效确切,被广泛应用;人们利用裂解肿瘤细胞抗原负载树突状细胞制备的肿瘤疫苗治疗非霍奇金淋巴瘤、骨髓瘤、恶性黑色素瘤、非小细胞肺癌、前列腺癌、结肠直肠癌等,在临床试验中均取得了一定效果,但是,因为多数肿瘤的免疫原性很弱,使得树突状细胞不能正常呈递抗原以有效激活特异性细胞毒T淋巴细胞(cytotoxic T cell s,CTL),从而限制了树突状细胞肿瘤疫苗的疗效不能进一步提高,为了提高肿瘤的免疫原性,提高树突状细胞肿瘤疫苗的效价,人们想到了在抗原中加入佐剂(如热休克蛋白、绿脓杆菌菌毛等)的方法,并且已经在体外实验中取得了可喜的成果。热休克蛋白(HSPs)又称分子伴侣,是机体细胞受到各种应急刺激时产生的一类保护性蛋白,参与有关蛋白的折叠、装配、细胞内运输及蛋白质降解等过程,调节这些蛋白的活性和功能,具有免疫佐剂和激活DC的功能。本研究我们分别采用热休克法和冻融法制备小鼠乳腺癌树突状细胞肿瘤疫苗,并对采用不同方法制备的肿瘤疫苗治疗小鼠乳腺癌进行疗效对比。目的 1.研究利用粒-巨噬细胞集落刺激因子(Granulocyte-Macrophage Clone stimulating factor, GM-CSF)、白细胞介素-4(Interleukin-4,IL-4)和肿瘤坏死因子-a(Tumor necrosis factor-a, TNF-a)联合培养小鼠骨髓来源的成熟树突状细胞。 2.分别利用热休克和冻融法处理小鼠EMT6乳腺癌细胞,研究所获得的乳腺癌细胞裂解物当中热休克蛋白-70(Hot Shock Protein-70,HSP70)的表达情况。 3.将获得全细胞抗原与培养的树突状细胞混合培养,制备小鼠树突状细胞乳腺癌肿瘤疫苗。分别利用以上制备的不同的小鼠乳腺癌树突状细胞肿瘤疫苗治疗小鼠移植瘤,对比热休克法和冻融法制备的小鼠乳腺癌树突状细胞肿瘤疫苗对荷瘤小鼠的疗效。 4.21天时处死小鼠,称量小鼠肿瘤体积及质量,并取血比较不同治疗组小鼠血清中细胞因子(cytokine)干扰素-γ(Interferon-γ,INF-γ)、白细胞介素-2(Interleukin-2,IL-2)和白细胞介素-12(P70)(Interleukin-12,IL-12(P70))的水平。 方法 1.定期通过光学显微镜观察培养的树突状细胞的形态,利用流式细胞仪对树突状细胞的表型进行分析。 2.用免疫组织化学法(immunohistochemical method)检测不同方法制备的小鼠乳腺癌肿瘤细胞中HSP70的表达量的不同。 3.建立小鼠乳腺癌动物模型并分组,应用不同的树突状细胞肿瘤疫苗进行治疗。 4.至21天,,处死小鼠,称量小鼠肿瘤体积和质量,并取血利用酶联免疫吸附测定(enzyme linked immunosorbent assay,ELISA)法测定各组小鼠血清中细胞因子INF-γ、IL-2、IL-12(P70)的水平。 结果 1.通过对骨髓源性树突状细胞形态的观察及流式细胞仪(flow cytometry)对细胞表面表型分析,利用GM-CSF、IL-4和TNF-a联合培养的小鼠骨髓来源的树突状细胞为典型的成熟树突状细胞。 2.经免疫组织化学法检测,热休克法制备的小鼠EMT6乳腺癌细胞裂解物中HSP70的表达量远远大于冻融法制备的小鼠EMT6乳腺癌细胞裂解物中HSP70的表达量。 3.与冻融法疫苗治疗组小鼠肿瘤相比较,热休克法疫苗治疗组小鼠的肿瘤质量及体积均受到明显抑制(p0.05)。 4.用ELISA法测得的热休克法疫苗治疗组小鼠体内INF-γ、IL-2和IL-12(P70)的水平均显著高于冻融法疫苗治疗组(p0.05)和空白对照组(p0.05)。 结论 由于热休克蛋白的免疫佐剂作用,热休克法制备的DC肿瘤疫苗较冻融法制备的DC肿瘤疫苗能在小鼠体内激发更强的免疫反应,对小鼠乳腺癌有更好的治疗效果。3
[Abstract]:background
Breast cancer is the most common malignant tumor in women, and the incidence of the disease is increasing year by year. At present, the main treatment methods of breast cancer are surgical treatment, chemotherapy, radiotherapy and endocrine therapy. Even so, there are still high recurrence rates and poor prognosis after operation. In recent years, biological therapy has gradually been paid attention to, and people have begun to use it. The tumor vaccine is used to treat malignant tumor. The tumor vaccine can stimulate strong immune response with lower antigen stimulation, tumor specificity, self tolerance and safety, and long time immune memory can avoid tumor recurrence. It has been used more and more to treat various malignant tumors and has achieved good effect. Also, the treatment vaccine of breast cancer, based on dendritic cells (DC), has also become a research hotspot in the biological treatment of breast cancer. The tumor cell antigen loaded dendritic cells are used to treat non Hodgkin's lymphoma, myeloma, malignant melanoma, non small cell lung cancer, prostate cancer, colorectal cancer and other clinical trials. However, because the immunogenicity of most of the tumor is weak, the dendritic cells can not be normal. In order to effectively activate the specific cytotoxic T lymphocyte (cytotoxic T cell s, CTL), the efficacy of the dendritic cell tumor vaccine can not be further improved. In order to improve the immunogenicity of the tumor and increase the titer of the dendritic cell tumor vaccine, an adjuvant (such as heat shock protein, green pus) is thought to be added to the antigen. HSPs, also known as molecular chaperone, is a kind of protective protein produced by various emergency stimuli, and participates in the process of protein folding, assembly, intracellular transport and protein degradation, and regulates the activity of these proteins. In this study, we used heat shock and freeze-thaw method to prepare mouse breast cancer dendritic cell tumor vaccine, and to compare the effect of tumor vaccine prepared by different methods in the treatment of breast cancer in mice.
1. the study used Granulocyte-Macrophage Clone stimulating factor (GM-CSF), interleukin -4 (Interleukin-4, IL-4) and tumor necrosis factor -a (Tumor necrosis factor-A) to cultivate the mature dendritic cells from the bone marrow of mice.
2. the mice EMT6 breast cancer cells were treated with heat shock and freeze-thaw respectively. The expression of heat shock protein -70 (Hot Shock Protein-70, HSP70) in the lysates of breast cancer cells was studied.
3. the murine dendritic cell breast cancer vaccine was prepared by mixed culture of total cell antigen and cultured dendritic cells. The mouse breast cancer dendritic cell tumor vaccine was used to treat mice transplanted tumor, and the mouse breast cancer dendritic cell tumor vaccine was compared with the heat shock method and the freeze thawing method. The effect of tumor bearing mice.
Mice were killed at 4.21 days, and the tumor volume and quality of mice were weighed and the serum levels of cytokines (cytokine) interferon gamma (Interferon- gamma, INF- gamma), interleukin -2 (Interleukin-2, IL-2) and interleukin -12 (P70) (Interleukin-12, IL-12 (P70)) were compared in the serum of different treatment groups.
Method
1. the morphology of cultured dendritic cells was observed by optical microscope regularly, and the phenotype of dendritic cells was analyzed by flow cytometry.
2. immunohistochemical method (immunohistochemical method) was used to detect the expression of HSP70 in mouse breast cancer cells prepared by different methods.
3. establish mouse mammary cancer animal models and divide them into different dendritic cell tumor vaccines.
4. to 21 days, mice were killed, the volume and quality of the mice were weighed, and the serum levels of cytokine INF- gamma, IL-2, IL-12 (P70) were measured by enzyme linked immunosorbent assay (ELISA).
Result
1. through the observation of the morphology of bone marrow derived dendritic cells and the analysis of cell surface phenotypes by flow cytometry (flow cytometry), the dendritic cells from bone marrow derived from GM-CSF, IL-4 and TNF-a were typical mature dendritic cells.
2. the expression of HSP70 in EMT6 breast cancer cell lysates prepared by heat shock method was far greater than the expression of HSP70 in EMT6 breast cancer cell lysates prepared by freezing and thawing method.
3. compared with the frozen thawed vaccine treatment group, the tumor mass and volume of the mice in the heat shock vaccine treatment group were significantly inhibited (P0.05).
4. the levels of INF- gamma, IL-2 and IL-12 (P70) in the mice treated with ELISA method were significantly higher than those of the freeze-thaw vaccine treatment group (P0.05) and the blank control group (P0.05).
conclusion
Due to the immune adjuvant of heat shock protein, DC tumor vaccine prepared by heat shock method can stimulate more immune response in mice than DC tumor vaccine prepared by freeze-thaw method, and has a better therapeutic effect on mouse breast cancer,.3
【学位授予单位】:泰山医学院
【学位级别】:硕士
【学位授予年份】:2010
【分类号】:R737.9
【参考文献】
相关期刊论文 前7条
1 刘俊峰;何志旭;沈冬;黄瑾;;小鼠骨髓树突状细胞体外扩增与培养[J];中华肿瘤防治杂志;2009年04期
2 魏云涛;徐阳;姜晓峰;郭大伟;王学范;梁健;;肝癌细胞对重组人HSP70联合肝癌组织冻融抗原修饰树突状细胞的免疫应答[J];中华肿瘤防治杂志;2009年08期
3 马全;武正炎;查小明;郑伟;;Mage-A3抗原肽负载DC过继免疫治疗乳腺癌的实验研究[J];实用临床医药杂志;2006年03期
4 叶欣;刘丽霞;杨雪;徐兴鲁;侯刚;邹玉红;宋文刚;魏绪庭;葛忠民;李毅;;高强度聚焦超声制备树突状细胞瘤苗对BALB/c小鼠肿瘤主动免疫的实验研究[J];中华医学超声杂志(电子版);2007年01期
5 尤长宣 ,罗荣城 ,苏瑾 ,张军一 ,廖旺军 ,郑航 ,Yong Liu ,Paul L Hermonat;BA46基因转染树突状细胞治疗乳腺癌的研究[J];中华微生物学和免疫学杂志;2005年07期
6 贾鑫;李荣;徐迎新;李力;;树突状细胞肿瘤疫苗联合免疫佐剂治疗小鼠乳腺癌的初步探讨[J];中国康复理论与实践;2006年05期
7 ;Uric acid enhances T cell immune responses to hepatitis B surface antigen-pulsed-dendritic cells in mice[J];World Journal of Gastroenterology;2007年07期
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