CAJ | 학술논문

目的：构建BCSC-1基因慢病毒RNA干扰载体并感染人乳腺癌细胞MCF-7，实时荧光定量PCR （QPCR）检测干扰效果。方法设计并合成两条针对人BCSC-1基因的干扰序列（BCSC-1 shRNA1和BCSC-1 shRNA2）以及对照序列（NS），将其连接入载体PLKO．1-sp6-pgk-GFP中，得到两个干扰载体和一个阴性对照载体。通过菌落 PCR、双酶切和 DNA 序列测定等方法对其进行鉴定。将干扰载体与辅助包装质粒 Lipo-fectamine2000共转染HEK293T细胞包装慢病毒并收取病毒上清。所获慢病毒颗粒感染人乳腺癌细胞MCF-7， QPCR方法检测两种干扰载体对BCSC-1基因的抑制效率。结果菌落PCR结果显示目的片段插入PLKO．1-sp6-pgk-GFP载体，DNA测序证实插入序列无误。 QPCR结果显示两种干扰载体感染人乳腺癌细胞MCF-7后， BCSC-1 shRNA1组和BCSC-1 shRNA2组BCSC-1基因表达水平分别为0．34±0．046和0．63±0．043，较空白对照组和NS组均有明显下降（P＜0．05），且BCSC-1 shRNA1抑制效果优于BCSC-1 shRNA2（P＜0．05）。结论成功构建了两种BCSC-1基因慢病毒干扰载体，两载体均能下调人乳腺癌细胞MCF-7中BCSC-1基因表达水平。
목적：구건BCSC-1기인만병독RNA간우재체병감염인유선암세포MCF-7，실시형광정량PCR （QPCR）검측간우효과。방법설계병합성량조침대인BCSC-1기인적간우서렬（BCSC-1 shRNA1화BCSC-1 shRNA2）이급대조서렬（NS），장기련접입재체PLKO．1-sp6-pgk-GFP중，득도량개간우재체화일개음성대조재체。통과균락 PCR、쌍매절화 DNA 서렬측정등방법대기진행감정。장간우재체여보조포장질립 Lipo-fectamine2000공전염HEK293T세포포장만병독병수취병독상청。소획만병독과립감염인유선암세포MCF-7， QPCR방법검측량충간우재체대BCSC-1기인적억제효솔。결과균락PCR결과현시목적편단삽입PLKO．1-sp6-pgk-GFP재체，DNA측서증실삽입서렬무오。 QPCR결과현시량충간우재체감염인유선암세포MCF-7후， BCSC-1 shRNA1조화BCSC-1 shRNA2조BCSC-1기인표체수평분별위0．34±0．046화0．63±0．043，교공백대조조화NS조균유명현하강（P＜0．05），차BCSC-1 shRNA1억제효과우우BCSC-1 shRNA2（P＜0．05）。결론성공구건료량충BCSC-1기인만병독간우재체，량재체균능하조인유선암세포MCF-7중BCSC-1기인표체수평。
Objective To construct lentiviral vectors expressing shRNA for BCSC-1 gene and infect breast cancer cells MCF-7, real-time fluorescence quantitative PCR(QPCR)method was used to test the interference effect .Methods Two interfere sequences aiming to human BCSC-1 gene(BCSC-1 shRNA1 and BCSC-1 shRNA 2)and a control sequence(NS)were designed and synthesized ,inserting them in-to the shRNA expression vector PLKO.1-sp6-pgk-GFP.All the plasmids were proved correct by colony PCR ,restriction enzymes and DNA se-quencing.The positive plasmid was transfected into HEK293T cells with auxiliary plasmids via Lipofectamine2000 to produce lentivirus and supernatant containing viruses was collected .The viruses infecting human breast carcinoma cell line MCF-7.QPCR method were used to de-tect the suppression efficiency.Results Colony PCR results indicated all the designed interfere sequences were successfully inserted into PL -KO.1-sp6-pgk-GFP vector and DNA sequencing were correct .QPCR results indicated the BCSC-1 gene expression levels in MCF-7 cells were decreased significantly in two interference vectors groups than NS group (P<0.05 vs NS group).Moreover,the inhibition rate of BCSC-1 shRNA1 was higher than BCSC-1 shRNA1(P<0.05 BCSC-1 shRNA1 vs BCSC-1 shRNA2).Conclusion Two lentiviral vectors are suc-cessfully constructed.They can suppress the expression of BCSC-1 gene in MCF-7 cells.