论文部分内容阅读
目的探讨胃泌素对胃癌细胞SGC7901 Reg Ⅰ(Reg Ⅰ)基因转录因子的效应。方法应用巢式PCR技术从胃癌细胞SGC7901基因组DNA扩增Reg Ⅰ基因启动子1414bp片段,将该片段插入pMD19-T载体,序列分析鉴定。应用随机引物法以地高辛分别标记1414bp及其HindⅢ酶切800bp和614bp片段,经灵敏度检测后,作为探针。应用Genomatix MatInspector在线分析软件分析Reg Ⅰ基因启动子1414bp片段的转录因子结合位点。分别以10-7mol/L和10-8mol/L胃泌素G-17处理胃癌细胞SGC7901 48h,提取核蛋白。应用DNA-蛋白质印迹法(Southernblotting),分别以地高辛标记的1414bp、800bp和614bp片段为探针检测胃泌素对胃癌细胞SGC7901 Reg Ⅰ基因转录因子的效应。结果 1414bp探针可检测到20条蛋白主带。胃泌素孵育后,带型没有变化,但是一些条带的灰度值改变,带9、12、13、14、15和16的灰度值明显降低(P<0.05);不同浓度胃泌素处理组之间上述6个条带的灰度值差异不明显(P>0.05)。614bp探针可检测到灰度值变化的6条主带中的带9、12和13,胃泌素处理后,此3条主带的灰度值明显降低(P<0.05)。800bp探针可检测到灰度值变化的6条主带中的带9、12和14,胃泌素处理后,仅带14的灰度值明显降低(P<0.05)。614bp和800bp探针均未检出带15和带16。结论胃癌细胞SGC7901 Reg Ⅰ基因表达由多个转录因子协同调控。降低几个转录因子的结合活性可能是胃泌素上调胃癌细胞SGC7901Reg Ⅰ基因表达的途径之一。
Objective To investigate the effect of gastrin on Reg Ⅰ gene transcription in gastric cancer cell line SGC7901. Methods A 1414bp fragment of promoter of Reg Ⅰ gene was amplified from gastric cancer cell SGC7901 by nested PCR. The fragment was inserted into pMD19-T vector and sequenced. Random primers were used to label 1414bp and 800np and 614bp fragments respectively with digoxigenin. After sensitivity test, the probe was used as a probe. The transcription factor binding sites of the 1414 bp fragment of Reg Ⅰ gene promoter were analyzed by Genomatix MatInspector online analysis software. The gastric cancer cell line SGC7901 was treated with 10-7mol / L and 10-8mol / L gastrin G-17 for 48h respectively, and the nuclear protein was extracted. The effects of gastrin on the transcription factor of Reg Ⅰ gene of gastric cancer SGC7901 cells were detected by Southern blotting with digoxigenin-labeled 1414bp, 800bp and 614bp fragments respectively. Results The 1414bp probe could detect 20 major protein bands. After gastrin incubation, there was no change in banding pattern, but the gray value of some bands changed, the gray values of bands 9, 12, 13, 14, 15 and 16 decreased significantly (P <0.05) There was no significant difference in gray value between the above six bands (P> 0.05). The 614bp probe could detect bands 9, 12 and 13 of the 6 main bands with gray value changes. After gastrin treatment, the gray values of the three main bands were significantly decreased (P <0.05). 800bp probes detected bands of 9, 12, and 14 in the 6 main bands with gray value changes. Only the gray value of 14 with gastrin treatment decreased significantly (P <0.05). Neither band 15 nor band 16 was detected for the 614bp and 800bp probes. Conclusion The expression of Reg Ⅰ gene in gastric cancer cell SGC7901 is coordinated by multiple transcription factors. Reducing the binding activity of several transcription factors may be one of the mechanisms by which gastrin upregulates the expression of Reg Ⅰ gene in gastric cancer cell line SGC7901.