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目的 :应用Southwestern技术研究 8 Br cAMP和槲皮素 (quercetin)对Eca 10 9细胞DNA结合蛋白与p16基因启动子区的结合调控mRNA表达的影响。方法 :将贴壁培养的Eca 10 9细胞随机分成 3组 ,Br组 :加 8 Br cAMP至终浓度为 2× 10 5mol/L ;Q组 :加槲皮素至终浓度为 4 3μmol/L ;C组 :不加任何药物 ,只加入新培养液培养。同时培养 4 8h ,应用质粒提取及限制性酶切回收目的DNA片段 ,采用生物素随机引物标记探针 ;应用DNA斑点印迹技术检测探针的灵敏度后以Southwestern印迹技术检测与p16基因启动子区特异性结合的DNA结合蛋白 (DBP) ;以原位杂交检测p16基因mRNA的表达情况。结果 :实验组条带强于对照组 ,Br组强于Q组 ;核基质DNA结合蛋白主带位于Mr6 6 0 0 0、5 80 0 0和 2 0 0 0 0 ;核外DNA结合蛋白主带位于Mr4 0 0 0 0、2 80 0 0和 2 0 0 0 0 ;2者中 2 0 0 0 0是共同成分。原位杂交技术显示p16基因mRNA蓝紫色信号颗粒位于胞质 ,杂交信号以实验组较强。结论 :8 Br cAMP和quercetin尤其是前者作用于人食管癌Eca 10 9细胞 ,诱导产生与p16基因启动子结合较强的DBP ,启动p16基因表达
OBJECTIVE: To study the effect of 8 Br cAMP and quercetin on the binding of DNA binding protein to p16 gene promoter in Eca 10 9 cells by Southwestern technique. Methods: The adherent cultured Eca109 cells were randomly divided into three groups: Br group: 8 Br cAMP to a final concentration of 2 × 10 5 mol / L; Q group: quercetin to a final concentration of 4 3 μmol / L; Group C: without any drug, only adding new culture medium. At the same time, the target DNA fragment was recovered by plasmid extraction and restriction enzyme digestion, and the probe was labeled with biotin random primers. The sensitivity of the probe was detected by using dot blot technique and the specificity of p16 gene promoter region was detected by Southwestern blotting Sex-binding DNA binding protein (DBP); p16 gene mRNA expression was detected by in situ hybridization. Results: The stripe of the experimental group was stronger than that of the control group, while the Br group was stronger than that of the Q group. The main stroma of nuclear stromal DNA binding protein was located on Mr660, 58080 and 20000; Located in Mr4 0 0 0 0, 2 80 0 0 and 2 0 0 0 0; 2 among 2 0 0 0 0 is a common component. In situ hybridization showed that p16 gene blue-violet signal was located in the cytoplasm, and the hybridization signal was stronger in experimental group. CONCLUSION: 8 Br cAMP and quercetin, especially the former, act on human esophageal cancer Eca 10 9 cells and induce the production of DBP with strong binding to the promoter of p16 gene, and initiate the expression of p16 gene