文章摘要
王殿忠,谢 敏,潘一明,朱海涛.小干扰RNA沉默NF-κB P65基因表达对胰腺癌细胞株PANC-1增殖与凋亡的影响[J].南京医科大学学报,2008,28(2):184~189
小干扰RNA沉默NF-κB P65基因表达对胰腺癌细胞株PANC-1增殖与凋亡的影响
Proliferative and apoptotic effects of siRNA-mediated NF-κB P65 gene silencing pancreatic carcinoma cell strain PANC-1
投稿时间:2007-06-16  
DOI:10.7655
中文关键词: RNA干扰  NF-κB  PANC-1细胞  细胞凋亡
英文关键词: RNA interference  NF-κB  PANC-1 cell  apoptosis
基金项目:南京市医学科技发展计划项目(YKK05104)
作者单位
王殿忠 南京医科大学鼓楼临床医学院普通外科,江苏 南京 210008 
谢 敏 南京医科大学鼓楼临床医学院普通外科,江苏 南京 210008 
潘一明 南京医科大学鼓楼临床医学院普通外科,江苏 南京 210008 
朱海涛 南京医科大学鼓楼临床医学院普通外科,江苏 南京 210008 
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中文摘要:
      目的:运用RNA干扰(RNA interference,RNAi)技术阻断胰腺癌细胞株PANC-1中NF-κB P65基因表达,并研究该基因沉默后对细胞增殖与凋亡的影响?方法:利用阳离子脂质体LipofectamineTM2000将化学合成的人NF-κB P65的小干扰RNA转染入胰腺癌细胞株PANC-1中?RT-PCR法测定胰腺癌细胞内NF-κB P65mRNA与细胞周期素D1(cyclinD1)mRNA的表达?ELISA法检测NF-κB亚单位P65的DNA结合活性的改变?MTT法测定细胞生长曲线观察细胞增殖的抑制情况?流式细胞仪测定细胞凋亡率及细胞周期的变化?结果:化学合成的人NF-κB P65 siRNA能有效地抑制PANC-1细胞中NF-κB P65与cyclinD1基因在mRNA水平上的表达(P < 0.01),同时ELISA结果显示,RelA siRNA组的p65亚单位与DNA结合活性明显低于阴性对照组和空白对照组(P < 0.05)?RelA siRNA组中细胞增殖减慢,凋亡率较对照组明显增加(P < 0.01),同时G1期细胞所占的比例较对照组增加了10%,S期和G2/M期细胞则分别减少了4%与6%?结论:体外实验初步证明NF-κB P65基因在胰腺癌细胞株增殖分化与凋亡方面扮演重要角色,通过沉默其表达可抑制胰腺癌细胞株PANC-1增殖并诱导其凋亡?
英文摘要:
      Objective:To observe the proliferative and apoptotic effects of RNAi-mediated NF-κB P65 gene on silencing pancreatic carcinoma cell strain PANC-1. Methods:Chemically synthesized small interference RNA(siRNA) directed against human NF-κB P65 was transfected into pancreatic carcinoma cell strain PANC-1 by using cationic liposome LipofectamineTM2000. The expression of NF-κB P65 and cyclinD1 gene was detected by using RT-PCR. The DNA binding activity of NF-κB was detected by the chemicon non-radioactive NF-κB P65 transcription factor assay kit. The effect of cell proliferation was studied by MTT. Flow cytometry was used to detect the apoptosis and cell cycle of transfected cells. Results:The results of RT-PCR indicated that chemically synthesized siRNA could knock down the transcription and expression of NF-κB P65 and cyclinD1 gene. The difference between the RelA siRNA group and control groups was significant(P < 0.01). After transfection,the DNA binding activity of NF-κB P65 in RelA siRNA group was much lower than that in the negative control group and blank control group(P < 0.05). The inhibitory action on proliferation of RelA siRNA group cells was confirmed by MTT and cell cycle test. The apoptotic rate of RelA siRNA group was significantly higher than control groups(P < 0.01) and the cell account during G1 phases increased 10%. During S and G2/M phases the cells account decreased about 4% and 6%. Conclusion:The study provided basis for researching the function of NF-κB P65 gene,indicating the expression of NF-κB down-regulating might inhibit proliferation and induce apoptosis in pancreatic carcinoma cell strain PANC-1.
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