【摘要】：目的通過建立小鼠炎癥性腸病乳腺癌肺轉(zhuǎn)移模型,觀察炎癥性腸病是否可加速乳腺癌的肺部轉(zhuǎn)移過程,探索其可能機制,以期為臨床上防治乳腺癌合并炎癥性腸病患者發(fā)生腫瘤遠處轉(zhuǎn)移提供新的干預策略。方法1依據(jù)實驗室前期實驗動物造模方法,以Balb/c雌鼠為實驗對象,建立2.5%葡聚糖硫酸鈉(dextran sulfate sodium,DSS)誘導的炎癥性腸病(inflammatory bowel disease,IBD)模型,并在此模型基礎上通過尾靜脈接種小鼠乳腺癌細胞4T1 100ul(1×106/ml)建立IBD乳腺癌肺轉(zhuǎn)移動物模型,動物實驗結果重復2次,每次實驗動物中每組動物至少10只(每組分別選取5只動物進行統(tǒng)計學分析)。2通過結腸組織病理切片評價IBD乳腺癌肺轉(zhuǎn)移模型中的結腸炎癥情況,通過肺組織病理切片觀察IBD乳腺癌肺轉(zhuǎn)移模型中的肺轉(zhuǎn)移情況。3應用流式細胞術、血細胞計數(shù)儀檢測小鼠外周血中中性粒細胞情況,應用免疫組織化學染色方法檢測小鼠肺組織中中性粒細胞浸潤情況。4應用脫氧核糖核酸酶(deoxyribonuclease,DNase-I)破壞中性粒細胞胞外捕獲網(wǎng)結構(neutrophil extracellular trap,NET),肺組織病理切片觀察小鼠肺轉(zhuǎn)移情況。5統(tǒng)計學方法:應用SAS9.1統(tǒng)計軟件包對數(shù)據(jù)進行統(tǒng)計分析,所有計量資料用(均數(shù)±標準差)來表示。兩組樣本之間差異的顯著性分析采用t檢驗,多組樣本間差異的顯著性分析采用One-way ANOVA單因素方差分析,多組之間兩兩比較(事后檢驗)采用Bonferroni法。在炎性細胞浸潤和組織破壞程度評估分析中,兩組樣本(多值有序變量的成組設計資料)之間采用Wilcoxon秩和檢驗。P0.05視為有統(tǒng)計學意義。結果1 2.5%DSS可誘導Balb/c雌鼠明顯的結腸炎癥反應,實驗過程中觀察可見經(jīng)DSS誘導的實驗小鼠發(fā)生明顯腹瀉、血便,解剖見結腸組織明顯充血,長度明顯縮短(P0.01),病理切片觀察結腸組織可見大量的炎性細胞浸潤及組織損傷。2在DSS誘導的IBD乳腺癌肺轉(zhuǎn)移模型中,肺組織病理切片可見經(jīng)DSS誘導的小鼠,與正常飲水組相比,其乳腺癌肺轉(zhuǎn)移明顯增加(P0.01)。3血細胞計數(shù)、流式細胞術結果顯示,DSS可誘導小鼠外周血中中性粒細胞的動員,其數(shù)量及比例均增加(P0.05),免疫組織化學染色結果顯示,轉(zhuǎn)移部位肺組織中浸潤的中性粒細胞也明顯增加(P0.01)。4應用DNase-I破壞中性粒細胞NET結構,可逆轉(zhuǎn)DSS誘導的IBD促進乳腺癌肺轉(zhuǎn)移增加(P0.05)。結論DSS誘導的小鼠IBD乳腺癌肺轉(zhuǎn)移模型可見明顯的乳腺癌肺轉(zhuǎn)移,其機制可能為DSS誘導小鼠外周血中中性粒細胞動員并浸潤至肺組織,動員的中性粒細胞可通過形成NET結構促進乳腺癌肺轉(zhuǎn)移,應用脫氧核糖核酸酶(DNase-I)破壞中性粒細胞NET結構,可逆轉(zhuǎn)DSS誘導的小鼠乳腺癌肺轉(zhuǎn)移增加。
[Abstract]:Objective to investigate whether inflammatory bowel disease can accelerate the process of lung metastasis of breast cancer and explore its possible mechanism by establishing a model of lung metastasis in mice with inflammatory bowel disease. In order to provide a new intervention strategy for the prevention and treatment of distant metastasis in patients with breast cancer complicated with inflammatory bowel disease. Methods 1 the model of inflammatory bowel disease (inflammatory bowel disease,IBD) induced by 2.5% glucosulfate sodium sulfate (dextran sulfate sodium,DSS) was established in Balb/c female rats according to the experimental animal model. On the basis of this model, mice breast cancer cell line 4T1 100ul (1 脳 10 ~ 6 / ml) was injected into the tail vein to establish the animal model of lung metastasis of IBD breast cancer. The results of the animal experiment were repeated twice. At least 10 animals in each group were selected for each experiment (5 animals in each group were selected for statistical analysis). 2 colonic inflammation in IBD breast cancer lung metastasis model was evaluated by histopathological sections of colon. The lung metastasis in the model of IBD breast cancer was observed by pathological sections of lung tissue. 3The neutrophils in peripheral blood of mice were detected by flow cytometry and blood cell counter. Immunohistochemical staining was used to detect neutrophil infiltration in the lung tissue of mice. (4) the extracellular capture net structure (neutrophil extracellular trap,NET) of neutrophils was destroyed by deoxyribonuclease (deoxyribonuclease,DNase-I). The pathological sections of lung tissue were used to observe the lung metastasis in mice. 5 Statistical methods: the data were statistically analyzed by SAS9.1 software package, and all the data were expressed by the mean 鹵standard deviation (mean 鹵standard deviation). T-test was used to analyze the significance of the difference between the two groups, One-way ANOVA single factor analysis of variance was used to analyze the significance of the difference between multiple samples, and Bonferroni method was used to compare the differences between the two groups (post-test). In the evaluation analysis of inflammatory cell infiltration and tissue destruction, Wilcoxon rank sum test was used between the two groups of samples (group design data of multi-valued ordered variables). P0.05 was regarded as statistically significant. Results 1 2.5%DSS could induce obvious colonic inflammation in Balb/c female rats. In the course of the experiment, the mice induced by DSS had obvious diarrhea, blood stool, and the colon tissue was obviously hyperemia in anatomy, and in the course of the experiment, there were obvious diarrhea and blood stool in the experimental mice induced by DSS. The length was significantly shortened (P0.01), and a large number of inflammatory cell infiltration and tissue injury were observed in colonic tissue by pathological section. 2 in the lung metastasis model of IBD breast cancer induced by DSS, the mice induced by DSS could be seen in the pathological section of lung tissue. Compared with the normal drinking water group, the lung metastasis of breast cancer was significantly increased (P0.01). 3Blood cell count and flow cytometry showed that DSS could induce the mobilization of neutrophils in peripheral blood of mice, and the number and proportion of neutrophils were increased (P0.05). Immunohistochemical staining showed that the infiltration of neutrophils in the metastatic lung tissue was also significantly increased (P0.01). (4) the NET structure of neutrophils was destroyed by DNase-I. Reversible DSS-induced IBD enhanced lung metastasis in breast cancer (P0.05). Conclusion the lung metastasis of breast cancer in IBD mice induced by DSS may be related to the mobilization of neutrophils in peripheral blood and infiltration of neutrophils into lung tissue induced by DSS, and the mechanism may be that DSS can induce the mobilization of neutrophils in peripheral blood of mice. The mobilized neutrophils could promote the lung metastasis of breast cancer by forming NET structure and destroy the NET structure of neutrophils by using deoxyribonuclease (DNase-I) to reverse the increase of DSS-induced lung metastasis of breast cancer in mice.
【學位授予單位】：華北理工大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R574;R737.9

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