發(fā)布時間：2018-03-11 00:06

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

【摘要】：聯(lián)體共生(parabiosis)是醫(yī)學(xué)實驗中的一種造模方法,此動物模型可很好的體現(xiàn)兩只動物經(jīng)聯(lián)體手術(shù)后形成血液、體液交互,達到共生目的,觀察聯(lián)體動物之間從血液到免疫等各個層面對機體的影響。聯(lián)體共生動物常以小鼠作為模型動物,在醫(yī)學(xué)實驗研究中應(yīng)用超過了150年,其中以美國、英國、瑞士及日本應(yīng)用聯(lián)體共生動物模型的研究較多,我國對這一實驗方法應(yīng)用較少。2013年,Francesco S.L等應(yīng)用聯(lián)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(zhuǎn)年齡相關(guān)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(guān)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(tǒng)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(nèi)發(fā)生、發(fā)展是一個免疫系統(tǒng)與癌細胞相互作用的動態(tài)過程。那么,聯(lián)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(lián)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(xué)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(chǎn)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(guān)系。因此,本實驗深入探討異基因聯(lián)體共生模型在腫瘤學(xué)方面的應(yīng)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(lián)體共生模型,觀察異基因聯(lián)體共生模型小鼠的生理變化情況。2成功建立聯(lián)體共生模型后,應(yīng)用流式細胞學(xué)技術(shù)檢測聯(lián)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(yīng)用免疫組織化學(xué)技術(shù),從形態(tài)學(xué)分析聯(lián)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(lián)體共生模型小鼠的建立目的：建立異基因聯(lián)體共生小鼠模型,觀察在聯(lián)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(lián)體共生小鼠,在兩只小鼠肘關(guān)節(jié)遠端4-5 mm處,沿著肱骨和肋骨側(cè)面直到腰圍線末端,連接皮膚和皮下組織,建立聯(lián)體模型。觀察聯(lián)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(jié)果：成熟階段的手術(shù)成功率率明顯高于模擬階段(P0.05),術(shù)后第三天建立血液交互,成模率為85.7%,且在手術(shù)后d2,d4,d6及d12,瘤體積較陽性對照組小,具有統(tǒng)計學(xué)差異(P0.05)。結(jié)論：成功建立異基因聯(lián)體共生小鼠模型,小鼠可很好地適應(yīng)聯(lián)體共生狀態(tài),該模型在醫(yī)學(xué)實驗中可推廣應(yīng)用。第二部分聯(lián)體共生模型對聯(lián)體共生小鼠脾臟T細胞的影響目的：分析聯(lián)體共生模型對B16腫瘤細胞的影響。方法：應(yīng)用流式細胞技術(shù),分別檢測聯(lián)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(jié)果：血液交互產(chǎn)生后,GFP+C57小鼠的健康細胞經(jīng)過連通的毛細血管網(wǎng)進入荷瘤小鼠血液循環(huán)內(nèi),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(tǒng)計學(xué)意義(P0.05)。GFP+在荷瘤小鼠體內(nèi)檢測到的含量較少,但是刺激荷瘤小鼠自身產(chǎn)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(lián)體共生模型的兩只小鼠,IFN-γ分泌均下降(P0.05)。聯(lián)體GFP+小鼠CD4/CD8較陰性對照組CD4/CD8比值高,有統(tǒng)計學(xué)意義(P0.05)。結(jié)論：聯(lián)體共生模型對腫瘤小鼠起到了免疫調(diào)節(jié)的作用,為今后腫瘤的免疫治療提供依據(jù)。第三部分聯(lián)體共生模型對腫瘤T細胞的影響目的：分析聯(lián)體共生模型對B16腫瘤細胞的影響。方法：選用聯(lián)體手術(shù)后d8,d14聯(lián)體荷瘤小鼠的腫瘤組織,應(yīng)用免疫組化的方法：觀蔡CD3,CD4,CD8,CD31,IFN-γ,VEGF的變化情況。結(jié)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(lián)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(tǒng)計學(xué)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(tǒng)計學(xué)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(lián)體荷瘤組比較,,無統(tǒng)計學(xué)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(lián)體荷瘤組比較,無統(tǒng)計學(xué)意義(P0.05)。結(jié)論：聯(lián)體共生模型可刺激聯(lián)體荷瘤小鼠的CD3, CD4, CD8, IFN-γ的產(chǎn)生,起到免疫調(diào)節(jié),腫瘤殺傷的作用,P0.05。聯(lián)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(tǒng)計學(xué)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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