CAJ | 학술논문

目的观察阻断MBD1表达后对胰腺癌细胞生长增殖的影响,探讨MBD1在胰腺癌发生发展中的作用.方法采用RNA干扰技术,构建MBD1-siRNA重组质粒,脂质体介导转染人胰腺癌细胞株BxPC-3,RT-PCR和Western印迹检测转染前后MBD1 mRNA与蛋白的表达变化,MTT法检测转染前后BxPC-3细胞的生长曲线,将细胞接种于裸鼠,观察转染前后胰腺癌细胞体内移植瘤的生长情况.结果转染siRNA-MBD1质粒后胰腺癌细胞株BxPC-3 MBD1的mRNA与蛋白表达水平明显降低.MTT法检测细胞的生长曲线,发现转染组较转染空质粒组和未转染组生长明显缓慢(P<0.01);体内实验显示,将转染前后的细胞接种于裸鼠,转染组移植瘤生长速度明显较其他两组减慢(P<0.01),RT-PCR检测移植瘤MBD1mRNA表达的变化,转染组中未能测到明显的MBD1表达.而转染组中CDH1、Rb等抑癌基因mRNA的表达明显高于转染空质粒组和未转染组.结论通过RNA干扰技术,可在转录和翻译水平降低MBD1在胰腺癌细胞株BxPC-3中的表达,并能抑制肿瘤细胞的生长,但其作用机制尚不清楚,MBD1介导的转录抑制作用可能在胰腺癌的发生发展过程中起到重要的作用.
목적 관찰조단MBD1표체후대이선암세포생장증식적영향,탐토MBD1재이선암발생발전중적작용.방법 채용RNA간우기술,구건MBD1-siRNA중조질립,지질체개도전염인이선암세포주BxPC-3,RT-PCR화Western인적검측전염전후MBD1 mRNA여단백적표체변화,MTT법검측전염전후BxPC-3세포적생장곡선,장세포접충우라서,관찰전염전후이선암세포체내이식류적생장정황.결과 전염siRNA-MBD1질립후이선암세포주BxPC-3 MBD1적mRNA여단백표체수평명현강저.MTT법검측세포적생장곡선,발현전염조교전염공질립조화미전염조생장명현완만(P<0.01);체내실험현시,장전염전후적세포접충우라서,전염조이식류생장속도명현교기타량조감만(P<0.01),RT-PCR검측이식류MBD1mRNA표체적변화,전염조중미능측도명현적MBD1표체.이전염조중CDH1、Rb등억암기인mRNA적표체명현고우전염공질립조화미전염조.결론 통과RNA간우기술,가재전록화번역수평강저MBD1재이선암세포주BxPC-3중적표체,병능억제종류세포적생장,단기작용궤제상불청초,MBD1개도적전록억제작용가능재이선암적발생발전과정중기도중요적작용.
Objective To observe the effect of MBD1 siRNA on the growth of pancreatic cancer cell line BxPC-3, and explore the role of MBD1 in the carcinogenesis of the pancreatic carcinoma. Methods One siRNA sequence targeting MBD1 was designed by software and cloned into the expres-sion plasmid pGCsi-U6/Neo/GFP. DNA sequencing was used to confirm that the recombinant plasmid was constructed correctly. The constructed plasmid and the control plasmid were stably transfected in-to human pancreatic cancer cell line BxPC-3. RT-PCR and Western blot were used to detect the MBD1 mRNA and protein expression after RNA interference. The cell growth curve was measured by MTT assay. The three kind of cells (BxPC-3, BxPC-3/vector, BxPC-3/MBD1-siRNA) were injected subcu-taneously to nucle mice, which were observed for 8 weeks for tumor formation respectively. Results RT-PCR and Western blot showed that the MBD1 mRNA and protein expression were significantly lower in the BxPC-3/MBD1-siRNA group than in BxPC-3 and BxPC-3/veetor group (P<0. 01). In vivo and vitro, the growth of BxPC-3 cell was suppressed after transfected with MBD1 siRNA. The MBD1 mRNA in tumor of BxPC-3/MBD1-siRNA group could not De detected by RT-PCR. The mRNA levels of some tumor suppressor genes including CDH1 and RB were up-regulated significantly after RNA interference targeting MBD1. Conclusion The stable MBD1-siRNA recombinant plasmid can decrease MBD1 expression in human pancreatic cancer cell BxPC-3 and inhibit the growth of the cell notably in vivo and vitro, the mechanism is still unclear. MBD1 mediating transcriptional repres-sion may play an important part in the development of pancreatic cancer.