联体共生模型对荷瘤小鼠肿瘤免疫的影响

发布时间：2018-03-11 00:06

  本文选题：联体共生模型　切入点：血液交互　出处：《河北医科大学》2015年博士论文　论文类型：学位论文

【摘要】：联体共生(parabiosis)是医学实验中的一种造模方法,此动物模型可很好的体现两只动物经联体手术后形成血液、体液交互,达到共生目的,观察联体动物之间从血液到免疫等各个层面对机体的影响。联体共生动物常以小鼠作为模型动物,在医学实验研究中应用超过了150年,其中以美国、英国、瑞士及日本应用联体共生动物模型的研究较多,我国对这一实验方法应用较少。2013年,Francesco S.L等应用联体共生小鼠模型在cell杂志上发表了一篇循环因子GDF11 (Growth Differentiation Factor-11,GDF11)逆转年龄相关性心肌肥厚的文章,引起轰动,随后science杂志在2014年相继发表两篇文章佐证这一观点,证实GDF11有改善衰老小鼠的大脑和骨骼肌的功能,使这一实验造模方法再次引发关注。肿瘤是由逃避了机体的免疫监视而生长繁殖的恶性细胞组成。R.DSchreiber提出的肿瘤免疫编辑理论认为,免疫系统不但具有排除肿瘤细胞的能力,也有促进肿瘤生长的作用。癌细胞在机体内发生、发展是一个免疫系统与癌细胞相互作用的动态过程。那么,联体共生模型对肿瘤的生长是起到促进作用还是抑制作用。联体共生模型能否为肿瘤的治疗提供新的免疫治疗方向,两只共生小鼠中若其中一只为患肿瘤小鼠,另一只为健康小鼠,两者血液交互后,健康小鼠的细胞及体液因素能否影响到患肿瘤的小鼠,给予其免疫学支持,进而起到共同打击癌细胞的作用,若存在这一作用,那么是健康小鼠自身的细胞及体液因素对患肿瘤小鼠的影响还是患病小鼠自身的细胞及体液因素受到来自健康小鼠的激活后,产生接种疫苗的效果,自身分泌大量抗肿瘤因子从而抵抗或打击肿瘤细胞。而且,两只共生小鼠是如何做到移植耐受,嵌合体与耐受状态的关系。因此,本实验深入探讨异基因联体共生模型在肿瘤学方面的应用,寻找对肿瘤细胞具有杀伤及抑制作用的细胞及体液因素。本实验从以下几个方面进行了研究：1建立异基因联体共生模型,观察异基因联体共生模型小鼠的生理变化情况。2成功建立联体共生模型后,应用流式细胞学技术检测联体共生小鼠脾脏T细胞的情况,以及两只小鼠T细胞的相互影响,并对肿瘤的杀伤作用。3应用免疫组织化学技术,从形态学分析联体共生小鼠的肿瘤杀伤情况分析。本研究论文分为三个部分：第一部分联体共生模型小鼠的建立目的：建立异基因联体共生小鼠模型,观察在联体状态下荷瘤小鼠瘤体积变化,论证该模型的合理性方法：选取异基因联体共生小鼠,在两只小鼠肘关节远端4-5 mm处,沿着肱骨和肋骨侧面直到腰围线末端,连接皮肤和皮下组织,建立联体模型。观察联体共生模型的成模率,血液交互时间,荷瘤小鼠瘤体积变化。结果：成熟阶段的手术成功率率明显高于模拟阶段(P0.05),术后第三天建立血液交互,成模率为85.7%,且在手术后d2,d4,d6及d12,瘤体积较阳性对照组小,具有统计学差异(P0.05)。结论：成功建立异基因联体共生小鼠模型,小鼠可很好地适应联体共生状态,该模型在医学实验中可推广应用。第二部分联体共生模型对联体共生小鼠脾脏T细胞的影响目的：分析联体共生模型对B16肿瘤细胞的影响。方法：应用流式细胞技术,分别检测联体共生模型小鼠的脾细胞中CD4+T细胞,CD8+T细胞及IL-2, IL-4, IL-10,IFN-γ的表达。结果：血液交互产生后,GFP+C57小鼠的健康细胞经过连通的毛细血管网进入荷瘤小鼠血液循环内,刺激荷瘤小鼠CD4+,CD8+曾加,其中荷瘤小鼠CD4+的增加比阳性对照组高,具有统计学意义(P0.05)。GFP+在荷瘤小鼠体内检测到的含量较少,但是刺激荷瘤小鼠自身产生CD4+IL-4, CD4+IL-10(P0.05)。GFP+小鼠CD4+IL-2, CD4+IL-4, CD4+IL-10均高于阴性对照组(P0.05)。联体共生模型的两只小鼠,IFN-γ分泌均下降(P0.05)。联体GFP+小鼠CD4/CD8较阴性对照组CD4/CD8比值高,有统计学意义(P0.05)。结论：联体共生模型对肿瘤小鼠起到了免疫调节的作用,为今后肿瘤的免疫治疗提供依据。第三部分联体共生模型对肿瘤T细胞的影响目的：分析联体共生模型对B16肿瘤细胞的影响。方法：选用联体手术后d8,d14联体荷瘤小鼠的肿瘤组织,应用免疫组化的方法：观蔡CD3,CD4,CD8,CD31,IFN-γ,VEGF的变化情况。结果：(1)HE染色镜下观察肿瘤有不完整的包膜形成,肿瘤细胞呈巢状堆积,核大,深染,可见较多核分裂像,有多少不等的坏死灶,胞浆和胞核有棕黄色沉淀为阳性。(2)联体荷瘤组CD3, CD4, CD8, IFN-γ细胞阳性率较高,OD值分别为(0.32+0.63),(0.33+0.00),(0.31土0.91),(0.28+0.14),与阳性对照组比较,有统计学意义(P)0.05)。CD31和VEGF可见细胞阳性率表达较低,分别为(0.19+0.50),(0.19+0.21),与阳性对照组比较,无统计学意义(P0.05)。(3)阳性对照组CD31,VEGF细胞阳性率较高,OD值分别是(0.32+0.35),(0.29+0.35),但与联体荷瘤组比较,,无统计学意义(P0.05)。d8时CD3、CD4、CD8和]NF-r图中可见细胞阳性率表达较低,分别为0.22,0.17,0.15,0.16,与联体荷瘤组比较,无统计学意义(P0.05)。结论：联体共生模型可刺激联体荷瘤小鼠的CD3, CD4, CD8, IFN-γ的产生,起到免疫调节,肿瘤杀伤的作用,P0.05。联体荷瘤组的CD31和VEGF可见细胞阳性率表达较低,但与阳性对照组比较不具有统计学意义,P0.05。
[Abstract]:Parabiosis (parabiosis) is a kind of modeling method in medical experiments, this animal model can be well reflected by the two animal surgery CIS formation of blood, body fluid interaction, achieve the objective to observe the effect of CIS symbiosis, between the animal from the blood to the immunity at all levels of the body. Often used as a parabiosis animal model in animal mice, applied for more than 150 years in medical research, especially in the United States, Britain, Switzerland and Japan more research application of CIS symbiotic animal model, our country of the method is seldom used in.2013, Francesco S.L and other application parabiosis mouse model published a circulating factor GDF11 in cell magazine (Growth Differentiation Factor-11, GDF11) to reverse age-related myocardial hypertrophy caused a sensation, then the science magazine in 2014 published two articles to support this view, confirmed GDF11 Improvement of aging mice brain and skeletal muscle function, so this experiment model caused concern again. Tumor is a tumor immune escape from the immune surveillance and the growth of malignant cells which proposed by.R.DSchreiber editing theory that the immune system not only has the ability to eliminate tumor cells, also promote tumor growth the cancer cells in the body, the development is a dynamic process of the immune system and cancer cell interaction. Then, parabiosis on tumor growth model is to promote or inhibit the immune therapy. Parabiosis provides a new direction for the treatment of the tumor model can, if one of two mice in symbiosis just as the tumor mice, the other one is healthy mice, both blood interaction, cellular and humoral factors can affect the health of mice with tumors in mice, given the Immunological support, and to fight against cancer cells, if the existence of this role, so is the cellular and humoral factors on the influence of healthy mice tumor mice or diseased cells and humoral factors in mice by their activation from healthy mice after birth, vaccination effect, its secretion of anti tumor in order to combat resistance or factor of tumor cells. Moreover, two mice symbiosis is how to do transplantation tolerance, chimerism and tolerance relationship. Therefore, the application of the in-depth study of allogeneic parabiosis model in oncology, looking for the cellular and humoral factors killing and inhibitory effect on tumor cells. The experimental study of the from the following aspects: 1 establish allogeneic parabiosis model, physiological changes of.2 allogeneic parabiosis mouse model was successfully established with a total body Student model, symbiosis of mouse spleen T cells by flow cytometry to detect CIS technology, and the mutual influence of T cells of two mice, and the tumor killing effect of.3 immunohistochemical technique, morphological analysis from parabiosis mouse tumor killing case analysis. This thesis is divided into three parts: the establishment of objective the first part: the establishment of parabiosis model mice allogeneic parabiosis mouse model mice were observed, the tumor volume changes in body condition, the rationality of the model selection methods to demonstrate: allogeneic mice were born in CIS, two mice at 4-5 mm distal to the elbow, end of humerus and ribs along the side until the waist line, connecting the skin and subcutaneous tissue, the establishment of CIS model. Observe parabiosis model forming rate, blood interaction time and tumor volume changes in tumor bearing mice. Results: the mature stage The success rate of operation was significantly higher than that in the simulation stage (P0.05), blood interaction established third days after operation, incidence was 85.7%, and after D2, D4, D6 and D12, the tumor volume compared with the positive control group, with statistical difference (P0.05). Conclusion: the success of allogeneic paragentic conjoined mouse model mice, can well adapt to the CIS symbiotic state, the model can be applied in medical experiments. The second part parabiosis effect model of mouse spleen T cells of symbiotic system's objective: to analyze the effect of parabiosis model of B16 tumor cells. Methods: using flow cytometry, CD4+T cells were detected in parabiosis mouse model the spleen cells, CD8+T cells and IL-2, IL-4, IL-10, IFN- expression. Results: Blood interactions, GFP+C57 mice healthy cells through the communication network of capillaries into the blood circulation of tumor bearing mice, tumor stimulation Mouse CD4+, CD8+ added, which increased CD4+ of tumor bearing mice than the positive control group, with statistical significance (P0.05) less content of.GFP+ in mice was detected, but the stimulation of tumor bearing mice can produce CD4+IL-4, CD4+IL-10 (P0.05).GFP+ CD4+IL-2 CD4+IL-4, CD4+IL-10 mice, were higher than the negative control group (P0.05). Parabiosis model mice, IFN- secretion decreased (P0.05). The body GFP+ CD4/CD8 mice compared with negative control group high CD4/CD8 ratio, there was statistical significance (P0.05). Conclusion: parabiosis model immune regulation effect on tumor in mice to provide basis for immunotherapy in tumor third. Part parabiosis effect model of tumor T cells Objective: to analyze the effect of parabiosis model of B16 tumor cells. Methods: the CIS D8 after surgery, D14 combined tumor bearing mice tumor tissue, immunohistochemical 缁勫寲鐨勬柟娉曪細瑙傝敗CD3,CD4,CD8,CD31,IFN-纬,VEGF鐨勫彉鍖栨儏鍐，

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