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目的:建立肺腺癌干细胞荷瘤动物模型,体内验证鱼油组分共轭亚油酸(CLA)抑制肺腺癌增殖及转移的作用和分子机制。方法:采用新霉素和克隆挑选技术分别建立稳定表达绿色荧光蛋白(GFP)的A549和SPC-A-1细胞株,体外培养富集干细胞微球,建立肺癌干细胞原位荷瘤裸鼠模型,腹腔注射给予不同组分c9t11-CLA、t10c12-CLA和混合组,定期检测裸鼠体重和肿瘤质量,研究终点采用小动物活体荧光成像技术分别观察肿瘤的转移情况。采用Western blotting检测肿瘤组织COX-2、CXCR4的蛋白表达水平。结果:实验终点各组原发肿瘤重量,A549混合治疗组比各单药治疗组肿瘤质量显著降低(P<0.001),而各CLA单药治疗组比,A549 control组肿瘤质量显著降低(P<0.01),与SPC-A-1组结果相似。各组肿瘤转移情况:在动物活体荧光成像系统下,A549CSC和SPC-A-1 CSC空白对照组出现了全身多处转移;经c9,t11-CLA、t10,c12-CLA治疗显著减少转移位置和比率;而混合治疗组减少尤为明显。Western blotting检测OCT4、COX-2、CXCR4蛋白表达水平。在A549肺腺癌干细胞组,c9t11-CLA、t10c12-CLA均不同程度地抑制OCT4、COX-2、CXCR4蛋白,混合治疗组抑制作用最强。在SPC-A-1肺腺癌干细胞组结果类似。结论:c9,t11-CLA、t10,c12-CLA及其混合物能明显抑制肺腺癌干细胞荷瘤裸鼠原发肿瘤和转移灶的生长;CLA组分能明显抑制肿瘤组织OCT4、COX-2和CXCR4蛋白的表达。
OBJECTIVE: To establish an animal model of adenocarcinoma of lung adenocarcinoma and to verify the effect and mechanism of conjugated linoleic acid (CLA) on proliferation and metastasis of lung adenocarcinoma in vivo. METHODS: A549 and SPC-A-1 cell lines stably expressing green fluorescent protein (GFP) were established by neomycin and clonal selection techniques. Stem cell microspheres were enriched and cultured in vitro. Lung cancer stem cells in situ nude mice model was established, The c9t11-CLA, t10c12-CLA and mixed group were given intraperitoneally, and the body weight and tumor mass of nude mice were detected regularly. The end point of the study was to observe the metastasis of tumor with live animal fluorescence imaging. Western blotting was used to detect the protein expression of COX-2 and CXCR4 in tumor tissue. Results: At the end of the experiment, the weight of the primary tumor in each group was significantly lower than that in the A549 mixed treatment group (P <0.001), while the tumor mass in each CLA monotherapy group was significantly lower than that in the A549 control group (P < 0.01), similar to the results of SPC-A-1 group. Tumor metastasis in each group: In live animal fluorescence imaging system, A549CSC and SPC-A-1 CSC blank control group appeared systemic multiple metastases; c9, t11-CLA, t10, c12-CLA treatment significantly reduced the location and Ratio; and mixed treatment group decreased significantly. Western blotting was used to detect the expression of OCT4, COX-2 and CXCR4. In A549 lung adenocarcinoma stem cell group, c9t11-CLA and t10c12-CLA all inhibited OCT4, COX-2 and CXCR4 to some extent. The mixed group had the strongest inhibitory effect. The results were similar in the SPC-A-1 lung adenocarcinoma stem cell group. Conclusion: C9, t11-CLA, t10, c12-CLA and their mixture can significantly inhibit the growth of primary tumor and metastasis of lung adenocarcinoma stem cell-bearing nude mice. CLA can significantly inhibit the expression of OCT4, COX-2 and CXCR4 protein expression.