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作 者:肖卫东[1] 刘智文[2] 文武[1] 王福飞[1] 廖国良[1] 李勇[1]
机构地区:[1]南昌大学第一附属医院普通外科 [2]江西省儿童医院新生儿外科,南昌330006
出 处:《南昌大学学报(医学版)》2014年第1期7-9,F0003,共4页Journal of Nanchang University:Medical Sciences
基 金:江西省自然科学基金(2010GZY0316);江西省教育厅科学技术研究项目(GJJ10320)
摘 要:目的构建miR-148a/b真核表达质粒,转染胰腺癌AsPC-1细胞,并观察其miR-148a/b的表达水平。方法根据miRBase提供的序列合成miR-148a/b前体,PCR扩增后形成双链,插入线性化真核表达质粒pcDNA3.1,构建pcDNA3.1/miR-148a/b,进行酶切和测序鉴定。将胰腺癌AsPC-1细胞分4组进行质粒转染:转染pcDNA3.1/miR-148a(miR-148a组)、转染pcDNA3.1/miR-148b(miR-148b组)、转染pcDNA3.1(空质粒对照组)和仅加脂质体(空白对照组)。转染48 h后,采用定量PCR法检测各组细胞中miR-148a/b的表达量。结果 pcDNA3.1/miR-148a/b的酶切和测序与miRBase提供的序列完全一致。转染48 h后,miR-148a组AsPC-1细胞中的miR-148a表达量明显高于空质粒对照组和空白对照组(6.76±0.35比1.00±0.54、1.02±0.40,均P<0.05),miR-148b组AsPC-1细胞中的miR-148b表达量明显高于空质粒对照组和空白对照组(3.28±0.08比1.02±0.35、1.01±0.28,均P<0.05)。结论成功构建了miR-148a/b真核表达质粒pcDNA3.1/miR-148a/b,该质粒能显著上调胰腺癌AsPC-1细胞的miR-148a/b表达水平。Objective To construct miR-148a/b eukaryotic expression vector,and to explore its expression in pancreatic cancer AsPC-1 ceils. Methods The priiR-148a/b was synthesized according to the miRBase and double-stranded oligonucleotides were generated by PCR. The miR- 148a/b were cloned into pcDNA3.1 vector and identified by enzyme digestion and sequence analysis. AsPC-1 cells were divided into four groups: pcDNA3. 1/miR-148a transfection group(miR- 148a group),pcDNA3.1/miR-148b transfection group(miR-148b group),pcDNA3.1 transfection group(empty vector control group), lipofectamine transfection group (blank control group). The expression of miR-148a/b was detected by real-time quantitative PCR at 48 hours after transfection. Results The pcDNA3.1/miR-148a/b identified by enzyme digestion and sequence analysis was completely consistent with the sequence from miRBase database. After of transfection 48 hours,the AsPC-1 cells expression of miR-148a in miR-148a group(6.76±0.35) was significantly higher than that in empty vector control group (1.00 ± 0.54) and blank control group (1.02±0.40) (P〈0.05). In addition, the significantly higher than in empty expression of miR-148b in miR-148b group(3.28±0.08) was vector control group(1.02±0.35) and blank control group(1. 01±0.28)(P〈0.05). Conclusion The miR-148a/b eukaryotic expression vector has been successfully constructed. The pcDNA3. 1/miR-148a/b can significantly increase the expression of miR-148a/b in pancreatic cancer AsPC-1 ceils.
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