本文選題：聯體共生模型　切入點：血液交互　出處：《河北醫(yī)科大學》2015年博士論文　論文類型：學位論文

【摘要】：聯體共生(parabiosis)是醫(yī)學實驗中的一種造模方法,此動物模型可很好的體現兩只動物經聯體手術后形成血液、體液交互,達到共生目的,觀察聯體動物之間從血液到免疫等各個層面對機體的影響。聯體共生動物常以小鼠作為模型動物,在醫(yī)學實驗研究中應用超過了150年,其中以美國、英國、瑞士及日本應用聯體共生動物模型的研究較多,我國對這一實驗方法應用較少。2013年,Francesco S.L等應用聯體共生小鼠模型在cell雜志上發(fā)表了一篇循環(huán)因子GDF11 (Growth Differentiation Factor-11,GDF11)逆轉年齡相關性心肌肥厚的文章,引起轟動,隨后science雜志在2014年相繼發(fā)表兩篇文章佐證這一觀點,證實GDF11有改善衰老小鼠的大腦和骨骼肌的功能,使這一實驗造模方法再次引發(fā)關注。腫瘤是由逃避了機體的免疫監(jiān)視而生長繁殖的惡性細胞組成。R.DSchreiber提出的腫瘤免疫編輯理論認為,免疫系統不但具有排除腫瘤細胞的能力,也有促進腫瘤生長的作用。癌細胞在機體內發(fā)生、發(fā)展是一個免疫系統與癌細胞相互作用的動態(tài)過程。那么,聯體共生模型對腫瘤的生長是起到促進作用還是抑制作用。聯體共生模型能否為腫瘤的治療提供新的免疫治療方向,兩只共生小鼠中若其中一只為患腫瘤小鼠,另一只為健康小鼠,兩者血液交互后,健康小鼠的細胞及體液因素能否影響到患腫瘤的小鼠,給予其免疫學支持,進而起到共同打擊癌細胞的作用,若存在這一作用,那么是健康小鼠自身的細胞及體液因素對患腫瘤小鼠的影響還是患病小鼠自身的細胞及體液因素受到來自健康小鼠的激活后,產生接種疫苗的效果,自身分泌大量抗腫瘤因子從而抵抗或打擊腫瘤細胞。而且,兩只共生小鼠是如何做到移植耐受,嵌合體與耐受狀態(tài)的關系。因此,本實驗深入探討異基因聯體共生模型在腫瘤學方面的應用,尋找對腫瘤細胞具有殺傷及抑制作用的細胞及體液因素。本實驗從以下幾個方面進行了研究：1建立異基因聯體共生模型,觀察異基因聯體共生模型小鼠的生理變化情況。2成功建立聯體共生模型后,應用流式細胞學技術檢測聯體共生小鼠脾臟T細胞的情況,以及兩只小鼠T細胞的相互影響,并對腫瘤的殺傷作用。3應用免疫組織化學技術,從形態(tài)學分析聯體共生小鼠的腫瘤殺傷情況分析。本研究論文分為三個部分：第一部分聯體共生模型小鼠的建立目的：建立異基因聯體共生小鼠模型,觀察在聯體狀態(tài)下荷瘤小鼠瘤體積變化,論證該模型的合理性方法：選取異基因聯體共生小鼠,在兩只小鼠肘關節(jié)遠端4-5 mm處,沿著肱骨和肋骨側面直到腰圍線末端,連接皮膚和皮下組織,建立聯體模型。觀察聯體共生模型的成模率,血液交互時間,荷瘤小鼠瘤體積變化。結果：成熟階段的手術成功率率明顯高于模擬階段(P0.05),術后第三天建立血液交互,成模率為85.7%,且在手術后d2,d4,d6及d12,瘤體積較陽性對照組小,具有統計學差異(P0.05)。結論：成功建立異基因聯體共生小鼠模型,小鼠可很好地適應聯體共生狀態(tài),該模型在醫(yī)學實驗中可推廣應用。第二部分聯體共生模型對聯體共生小鼠脾臟T細胞的影響目的：分析聯體共生模型對B16腫瘤細胞的影響。方法：應用流式細胞技術,分別檢測聯體共生模型小鼠的脾細胞中CD4+T細胞,CD8+T細胞及IL-2, IL-4, IL-10,IFN-γ的表達。結果：血液交互產生后,GFP+C57小鼠的健康細胞經過連通的毛細血管網進入荷瘤小鼠血液循環(huán)內,刺激荷瘤小鼠CD4+,CD8+曾加,其中荷瘤小鼠CD4+的增加比陽性對照組高,具有統計學意義(P0.05)。GFP+在荷瘤小鼠體內檢測到的含量較少,但是刺激荷瘤小鼠自身產生CD4+IL-4, CD4+IL-10(P0.05)。GFP+小鼠CD4+IL-2, CD4+IL-4, CD4+IL-10均高于陰性對照組(P0.05)。聯體共生模型的兩只小鼠,IFN-γ分泌均下降(P0.05)。聯體GFP+小鼠CD4/CD8較陰性對照組CD4/CD8比值高,有統計學意義(P0.05)。結論：聯體共生模型對腫瘤小鼠起到了免疫調節(jié)的作用,為今后腫瘤的免疫治療提供依據。第三部分聯體共生模型對腫瘤T細胞的影響目的：分析聯體共生模型對B16腫瘤細胞的影響。方法：選用聯體手術后d8,d14聯體荷瘤小鼠的腫瘤組織,應用免疫組化的方法：觀蔡CD3,CD4,CD8,CD31,IFN-γ,VEGF的變化情況。結果：(1)HE染色鏡下觀察腫瘤有不完整的包膜形成,腫瘤細胞呈巢狀堆積,核大,深染,可見較多核分裂像,有多少不等的壞死灶,胞漿和胞核有棕黃色沉淀為陽性。(2)聯體荷瘤組CD3, CD4, CD8, IFN-γ細胞陽性率較高,OD值分別為(0.32+0.63),(0.33+0.00),(0.31土0.91),(0.28+0.14),與陽性對照組比較,有統計學意義(P)0.05)。CD31和VEGF可見細胞陽性率表達較低,分別為(0.19+0.50),(0.19+0.21),與陽性對照組比較,無統計學意義(P0.05)。(3)陽性對照組CD31,VEGF細胞陽性率較高,OD值分別是(0.32+0.35),(0.29+0.35),但與聯體荷瘤組比較,,無統計學意義(P0.05)。d8時CD3、CD4、CD8和]NF-r圖中可見細胞陽性率表達較低,分別為0.22,0.17,0.15,0.16,與聯體荷瘤組比較,無統計學意義(P0.05)。結論：聯體共生模型可刺激聯體荷瘤小鼠的CD3, CD4, CD8, IFN-γ的產生,起到免疫調節(jié),腫瘤殺傷的作用,P0.05。聯體荷瘤組的CD31和VEGF可見細胞陽性率表達較低,但與陽性對照組比較不具有統計學意義,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 緇勫寲鐨勬柟娉曪細瑙傝敗CD3,CD4,CD8,CD31,IFN-緯,VEGF鐨勫彉鍖栨儏鍐，

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