摘要
目的构建包含有ACP1小干扰RNA序列的真核表达载体，并将构建的载体转染入前列腺癌T3B细胞中，检测ACP1在pGenesil-1／ACP1-shRNA转染的T3B中的表达，研究ACP1基因被下调后，前列腺癌T3B细胞的生物学特性变化。
方法针对人ACP1基因开放阅读框序列选择干扰目的片段，据干扰目的片段设计小发卡状RNA序列，人工合成设计好的片段，将合成片段与pGenesil-1质粒连接，构成克隆质粒。将重组的质粒转化入大肠杆菌DH5-α，进行扩增，经纯化、酶切、测序鉴定插入片段完整正确。用脂质体介导的基因转染技术将pGenesil-1／ACP1-shRNA质粒转染入T3B细胞，G418筛选得到稳定克隆，以RT-PCR方法在mRNA水平检测ACP1表达，以Western Blot方法检测蛋白水平的表达。将细胞分为PC-3组、T3B组、pGenesil-1转染组、pGenesil-1／ACP1-shRNA转染组，通过MTT法测生长曲线，流式细胞术测细胞周期和凋亡，粘附实验测粘附能力，划痕实验测迁移能力，Transwell法测侵袭能力。
结果成功构建了人ACP1-shRNA真核表达载体。筛选到成功转染，并能稳定表达的单克隆株，与对照组比较mRNA由0.45±0.03下降到0.09±0.02，蛋白相对表达由0.65±0.02下降到0.21±0.03，mRNA下调80％，蛋白下调76％，与对照组相比，下调ACP1后，前列腺癌细胞生长及细胞周期无明显变化，粘附能力明显下降，迁移能力和侵袭能力皆下降。
结论经shRNA对ACP1进行下调，可见ACP1在mRNA和蛋白水平均表达均得到有效抑制，ACP1下调后前列腺癌细胞的粘附、迁移、侵袭能力，对增殖无影响。
Objective To construct ACP1 short hairpin RNA eukaryotic expression vector,transfect reconstructed plasmid into prostatic carcinoma cell T3B, and to detect theexpression of ACP1 in T3B and PC-3. Study the biology characteristics changes ofprostatic carcinoma cells after down-regulation of ACP1.
Methods Selected the target gene fragment according the open reading frame ofACP1, and designed the short hairpin RNA which synthesized and inserted intopGenesil-1 vector to form cloning plasmid. The recombinant plasmid was identifiedby restriction enzyme digestion and sequencing analysis after E. coli transformation,amplification and purification. Plasmid pGenesil-1/ACP1-shRNA was transfected intoT3B cells with Lipoinfectamin~(TM) 2000 and then the stably transfected positive cloneswere screened with G418. The mRNA expression of ACP1 was detected by RT-PCRand protein by Western Blot. T3B group, pGenesil-1 group and pGenesil-1/ACP1-shRNA group, PC-3 group cells were detected on growth, cell circle by FCM,capacity of adhesion, migration and invasion.
Results Recombinant human ACP1-shRNA eukaryotic expression vector wasconstructed successfully The stably transfected positive clones was successfullyscreened which could express reconstructed ACP1-shRNA stably. The ACP1 mRNAof shRNA transfected group decreased from 0.45±0.03 to 0.09±0.02, compared withcontrol group; ACP1 protein expression dropped from 0.65±0.02 to 0.21±0.03.mRNA was down-regulated 80%, and protein 76%. Compared with pGenesil-1 group,cell growth rate and cell cycle of pGenesil-1/ACP1-shRNA group cells didn't change, the capacity of adhesion, migration and invasion decreased dramatically.
Conclusion The expression of ACP1 mRNA and protein was suppressed efficientlyby means of siRNA. Down-regulation of ACP1 could suppress the capacity ofadhesion, migration and invasion in prostatic carcinoma cells, and had no effect oncell proliferation.
引文
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