Establishment Of Orthotopic A549 Cell Transplantation Lung Cancer Model And The Effect Of High Cholesterol Diet On Lung Cancer Develepment

Background: Although the incidence of lung cancer was still increasing,the etiology is unclear.Construction of animal model of lung cancer is important for its etiology research.At the same time,the mortality of lung cancer is still on the top among cancers,and it is hard to be diagnosed until very late stages.So,primary prevention should be strengthened.The results of the epidemiological study of high cholesterol diet on lung cancer is equivocal,and due to lack of mechanism research.It is urgent to investigate the relationships between high cholesterol diet and lung cancer risk.Objective:Establish a moderate model of lung cancer by intra-tracheal orthotopic transplantation.Explore the effect of high cholesterol diet on the development of transplanted lung cancer.Methods:Four-six weeks old BABL/c male nude mice was intra-tracheal orthotopic transplanted with 5*10^6 A549 lung cancer cell(0.01 M EDTA)to establish lung cancer model.And the operation conditions was improved as needed.Collected the tissues after 6 weeks of transplantation.H & E staining was adopted to verify tumor morphology characteristics,and Western Blot was used to test the expression of proliferation related protein(STAT3,AKT,m TOR).4-6 weeks old BABL/c male nude mice were randomly divided into two groups and fed with normal diet(ND)and high cholesterol diet(HCD)respectively.Following three weeks of diet adapting,mice were carried out the transplantation or sham operation.Then continued diet intervention for six weeks,all mice were sacrificed and the lung tissues were removed.The lung tissues were collected to indentify: the number of tumors;the tumor size(mearsured by vernier caliper);serum lipids profile;expression of proliferation related proteins,such as PCNA(detected by immouhistochemistry),STAT3,AKT,PI3K(tested by western blot).Results: The appropriate anesthesia for the operation is 75mg/kg body weight of pentobarbital sodium by intraperitoneal injection.The xenografts of A549 was with obvious lung adenocarcinoma structure and poor differentiation degree.The lung tissues beared tumors showed upregulations of STAT3,AKT and m TOR protein with p-STAT3/STAT3,p-AKT/AKT,p-m TOR/m TOR were higher than those in normal lungs(p=0.041,0.011,0.038 respectively).Compared with the ND-fed mice,the serum TC in the HCD-fed mice was significantly increased,which proved that the model of hypercholesterolemia was successful established.The lung tumor formation(4/11)in the HCD group was lower than that in the ND group(8/11),and the immunohistochemistry results of PCNA showed that the positive rates of PCNA in the HCD group was lower than that in the ND group.To further explore the possible mechanism,we found that,in lipid rafts,the orthtopic transplanted HCD-fed mice showed lower expression STAT3,PI3 K,AKT than those in the HCD-fed cancer group(p=0.027,0.047,0.041).Conclusion: The method of orthotopic intra-tracheal transplantation of A549 cell can be used to establish a stable lung adenocarcinoma model.The high cholesterol diet may have a protective effect on the growth of transplanted tumor,which may be related to the downregulate of STAT3/PI3K/AKT in lipid rafts.