人肺癌A549细胞原位移植瘤模型建立以及高胆固醇饮食对其发展的影响

发布时间：2018-11-25 12:28

【摘要】：背景:肺癌发病率居高不下,而病因尚未明确,所以肺癌动物模型的构建对于其病因学研究显得尤其重要。同时肺癌病死率高,发现时多为晚期,应加强一级预防;而目前高胆固醇饮食与肺癌的流行病学研究结果并不一致,也缺乏相关的机制探讨,高胆固醇饮食和肺癌发生之间的关系仍亟待研究。目的:建立稳健的肺癌原位移植瘤模型,以及初步探讨高胆固醇饮食对其发展的影响。方法:选取4-6周龄BABL/c雄性裸鼠,经气管植入5*10^6个A549肺癌细胞(含0.01M EDTA)建立肺癌原位移植模型,并根据需要改进手术操作条件。移植6周后收取肺组织,HE染色验证肿瘤形态学特征以及蛋白免疫印记检测移植瘤增殖相关蛋白(STAT3、AKT、m TOR)的表达等。选取4-6周龄BABL/c雄性裸鼠随机分为两组,后分别喂养普通饮食和高胆固醇饮食。饮食适应3周后,进行手术移植和空白对照手术。之后继续喂养6周,收集小鼠肺组织等进行以下检测:计数肿瘤数目;使用游标卡尺测量肿瘤大小;对收集的血清脂质四项进行检测;对裸鼠组织增殖相关蛋白进行检测:免疫组化检测PCNA的表达;同时对脂质筏中STAT3、AKT、PI3K蛋白表达水平进行检测。结果:手术最适宜麻醉为75mg/kg体重腹腔注射。A549细胞移植瘤具有明显腺癌特征,且分化程度较低。肿瘤负荷肺组织增殖相关通路蛋白STAT3,AKT,m TOR的磷酸化水平即p-STAT3/STAT3,p-AKT/AKT,p-m TOR/m TOR比值均高于正常肺组织,差异具有统计学意义(p值分别为0.041,0.011,0.038)。与普通饮食组相比,高胆固醇饮食组裸鼠血清总胆固醇(TC)含量显著升高,证明高胆固醇血症裸鼠模型构建成功。在本移植模型中高胆固醇饮食组裸鼠肺部成瘤(4/11)较正常饮食组(8/11)低,且PCNA免疫组化结果显示高胆固醇饮食组PCNA阳性率低于正常饮食。进一步探讨可能机制发现,在脂质筏中STAT3、PI3k、AKT蛋白表达量在高胆固醇组明显下调(p=0.027,0.047,0.041)。结论:经过改进手术条件建立的经气管原位移植A549细胞实验方法可以构建具有典型特征的肺腺癌原位移植瘤模型。高胆固醇饮食可能对该原位移植瘤的生长具有保护作用,其机制可能与脂质筏中锚定蛋白STAT/PI3K/AKT的抑制有关。
[Abstract]:Background: the incidence of lung cancer remains high, but the etiology is not clear, so the construction of lung cancer animal model is particularly important for its etiology. At the same time, the mortality of lung cancer is high, most of them are late, so we should strengthen the primary prevention. However, the results of epidemiological studies on high cholesterol diet and lung cancer are inconsistent and lack of relevant mechanism. The relationship between high cholesterol diet and lung cancer still needs to be studied. Objective: to establish a stable orthotopic tumor model of lung cancer and to explore the effect of high cholesterol diet on its development. Methods: BABL/c male nude mice aged 4-6 weeks were implanted with 5 10 ^ 6 A549 lung cancer cells (including 0.01M EDTA) to establish a lung cancer orthotopic transplantation model, and the operation conditions were improved according to the need. Lung tissues were collected 6 weeks after transplantation. The morphological features of tumor were examined by HE staining and the expression of proliferation-associated protein (STAT3,AKT,m TOR) was detected by protein imprinting. BABL/c male nude mice aged 4 to 6 weeks were randomly divided into two groups, and fed with common diet and high cholesterol diet respectively. After 3 weeks of dietary adaptation, surgical transplantation and blank control surgery were performed. After 6 weeks of feeding, the following tests were performed: counting the number of tumor, measuring tumor size with Vernier caliper, detecting the four items of serum lipid. The expression of proliferation-associated protein (PCNA) in nude mice was detected by immunohistochemistry, and the expression of STAT3,AKT,PI3K protein in lipid raft was detected by immunohistochemistry. Results: the most suitable anesthesia for operation was intraperitoneal injection of 75mg/kg. The phosphorylation level of proliferation-related pathway protein (STAT3,AKT,m TOR) in lung tissue under tumor loading was significantly higher than that in normal lung tissue, i.e., the ratio of p-STAT3 / STAT3, p-AKT / AKTP-m TOR/m TOR. The difference was statistically significant (p = 0.041, 0.011, 0.038, respectively). Compared with the normal diet group, the serum total cholesterol (TC) content in the hypercholesterolemic diet group was significantly higher than that in the normal diet group, which proved that the hypercholesterolemia nude mice model was successfully constructed. The lung tumorigenesis of nude mice in high cholesterol diet group (4 / 11) was lower than that in normal diet group (8 / 11). PCNA immunohistochemical results showed that the positive rate of PCNA in high cholesterol diet group was lower than that in normal diet group. Further study on the possible mechanism showed that the expression of STAT3,PI3k,AKT protein in lipid rafts was significantly down-regulated in high cholesterol group (p0. 027 ~ 0. 047 ~ 0. 041). Conclusion: the experimental method of transtracheal orthotopic transplantation of A549 cells can be used to construct the model of lung adenocarcinoma orthotopic transplantation. High cholesterol diet may have a protective effect on the growth of orthotopic transplanted tumor, and its mechanism may be related to the inhibition of STAT/PI3K/AKT in lipid raft.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R734.2

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