人肺癌A549細胞原位移植瘤模型建立以及高膽固醇飲食對其發(fā)展的影響
發(fā)布時間：2018-11-25 12:28
【摘要】：背景:肺癌發(fā)病率居高不下,而病因尚未明確,所以肺癌動物模型的構建對于其病因學研究顯得尤其重要。同時肺癌病死率高,發(fā)現(xiàn)時多為晚期,應加強一級預防;而目前高膽固醇飲食與肺癌的流行病學研究結果并不一致,也缺乏相關的機制探討,高膽固醇飲食和肺癌發(fā)生之間的關系仍亟待研究。目的:建立穩(wěn)健的肺癌原位移植瘤模型,以及初步探討高膽固醇飲食對其發(fā)展的影響。方法:選取4-6周齡BABL/c雄性裸鼠,經氣管植入5*10^6個A549肺癌細胞(含0.01M EDTA)建立肺癌原位移植模型,并根據(jù)需要改進手術操作條件。移植6周后收取肺組織,HE染色驗證腫瘤形態(tài)學特征以及蛋白免疫印記檢測移植瘤增殖相關蛋白(STAT3、AKT、m TOR)的表達等。選取4-6周齡BABL/c雄性裸鼠隨機分為兩組,后分別喂養(yǎng)普通飲食和高膽固醇飲食。飲食適應3周后,進行手術移植和空白對照手術。之后繼續(xù)喂養(yǎng)6周,收集小鼠肺組織等進行以下檢測:計數(shù)腫瘤數(shù)目;使用游標卡尺測量腫瘤大小;對收集的血清脂質四項進行檢測;對裸鼠組織增殖相關蛋白進行檢測:免疫組化檢測PCNA的表達;同時對脂質筏中STAT3、AKT、PI3K蛋白表達水平進行檢測。結果:手術最適宜麻醉為75mg/kg體重腹腔注射。A549細胞移植瘤具有明顯腺癌特征,且分化程度較低。腫瘤負荷肺組織增殖相關通路蛋白STAT3,AKT,m TOR的磷酸化水平即p-STAT3/STAT3,p-AKT/AKT,p-m TOR/m TOR比值均高于正常肺組織,差異具有統(tǒng)計學意義(p值分別為0.041,0.011,0.038)。與普通飲食組相比,高膽固醇飲食組裸鼠血清總膽固醇(TC)含量顯著升高,證明高膽固醇血癥裸鼠模型構建成功。在本移植模型中高膽固醇飲食組裸鼠肺部成瘤(4/11)較正常飲食組(8/11)低,且PCNA免疫組化結果顯示高膽固醇飲食組PCNA陽性率低于正常飲食。進一步探討可能機制發(fā)現(xiàn),在脂質筏中STAT3、PI3k、AKT蛋白表達量在高膽固醇組明顯下調(p=0.027,0.047,0.041)。結論:經過改進手術條件建立的經氣管原位移植A549細胞實驗方法可以構建具有典型特征的肺腺癌原位移植瘤模型。高膽固醇飲食可能對該原位移植瘤的生長具有保護作用,其機制可能與脂質筏中錨定蛋白STAT/PI3K/AKT的抑制有關。
[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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R734.2
本文編號：2356105
[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.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R734.2
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