中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2014年
11期
2408-2410
,共3页
明佳%杜俊泽%王寅欢%姜军
明佳%杜俊澤%王寅歡%薑軍
명가%두준택%왕인환%강군
微小RNA-206%重组慢病毒载体%乳腺癌%转移
微小RNA-206%重組慢病毒載體%乳腺癌%轉移
미소RNA-206%중조만병독재체%유선암%전이
MicroRNA-206%Recombinant lenticiral vector%Breast cancer%Metastasis
目的 构建微小RNA-206(miR-206)的慢病毒载体转染MCF-7细胞,观察转染效率和细胞生物学特性的改变.方法 针对miR-206有效的靶序列设计合成寡链DNA,经过退火、连接、聚合酶链反应(PCR)筛选、测序鉴定、慢病毒包装、共转染等步骤获得慢病毒LV-hsa-miR-206,将其转染MCF-7细胞,实时定量逆转录聚合酶链反应(RT-qPCR)检测miR-206表达,细胞计数试剂盒(CCK-8)法检测细胞增殖,流式细胞仪检测细胞凋亡和细胞周期.结果 PCR扩增出插入片段,目的基因的测序结果与GeneBank序列一致.感染慢病毒后,MCF-7的miR-206表达由1.000增加至120.331±1.801;细胞增殖能力显著降低,在转染后第2天由1.420±0.052降低为1.275±0.147,第3天由1.762±0.089降低为1.387±0.093,第4天由2.045±0.235降低为1.269±0.103,凋亡细胞和G2期细胞比例显著增加.结论 成功构建LV-hsa-miR-206慢病毒载体,并在MCF-7细胞中成功表达,miR-206表达水平增加导致细胞的生物学特性发生明显变化.
目的 構建微小RNA-206(miR-206)的慢病毒載體轉染MCF-7細胞,觀察轉染效率和細胞生物學特性的改變.方法 針對miR-206有效的靶序列設計閤成寡鏈DNA,經過退火、連接、聚閤酶鏈反應(PCR)篩選、測序鑒定、慢病毒包裝、共轉染等步驟穫得慢病毒LV-hsa-miR-206,將其轉染MCF-7細胞,實時定量逆轉錄聚閤酶鏈反應(RT-qPCR)檢測miR-206錶達,細胞計數試劑盒(CCK-8)法檢測細胞增殖,流式細胞儀檢測細胞凋亡和細胞週期.結果 PCR擴增齣插入片段,目的基因的測序結果與GeneBank序列一緻.感染慢病毒後,MCF-7的miR-206錶達由1.000增加至120.331±1.801;細胞增殖能力顯著降低,在轉染後第2天由1.420±0.052降低為1.275±0.147,第3天由1.762±0.089降低為1.387±0.093,第4天由2.045±0.235降低為1.269±0.103,凋亡細胞和G2期細胞比例顯著增加.結論 成功構建LV-hsa-miR-206慢病毒載體,併在MCF-7細胞中成功錶達,miR-206錶達水平增加導緻細胞的生物學特性髮生明顯變化.
목적 구건미소RNA-206(miR-206)적만병독재체전염MCF-7세포,관찰전염효솔화세포생물학특성적개변.방법 침대miR-206유효적파서렬설계합성과련DNA,경과퇴화、련접、취합매련반응(PCR)사선、측서감정、만병독포장、공전염등보취획득만병독LV-hsa-miR-206,장기전염MCF-7세포,실시정량역전록취합매련반응(RT-qPCR)검측miR-206표체,세포계수시제합(CCK-8)법검측세포증식,류식세포의검측세포조망화세포주기.결과 PCR확증출삽입편단,목적기인적측서결과여GeneBank서렬일치.감염만병독후,MCF-7적miR-206표체유1.000증가지120.331±1.801;세포증식능력현저강저,재전염후제2천유1.420±0.052강저위1.275±0.147,제3천유1.762±0.089강저위1.387±0.093,제4천유2.045±0.235강저위1.269±0.103,조망세포화G2기세포비례현저증가.결론 성공구건LV-hsa-miR-206만병독재체,병재MCF-7세포중성공표체,miR-206표체수평증가도치세포적생물학특성발생명현변화.
Objective To construct a recombinant lenticiral vector of microRNA-206 (miR-206) gene.Methods Oligo-chain DNA complemented to miR-206 gene was designed and lentivirus-based vectors was constructed.Those colonies with positive polymerase chain reaction (PCR) were sequenced.All virus stocks were transfected by Lipofectamine 2000.The titer of virus was tested according to the expression level of green fluorescent protein.The expression of miR-206 in MCF-7 infected by the lentivirus was analyzed via real-time reverse transcriptase-polymerase chain reaction (RT-qPCR).The proliferation of MCF-7 was tested by cell counting kit-8 (CCK-8).The changes of apoptosis and cell cycle was studied by flow cytometry.Results The lentivirus vector of miR-206 (LV-hsa-miR-206) were successfully constructed.After MCF-7 was infected by LV-hsa-miR-206,the expression of miR-206 gene was significantly increased from 1.000 to 120.331 ± 1.801.The proliferation of MCF-7 was decreased from 1.420 ±0.052 to 1.275 ±0.147 (2nd day),from 1.762 ±0.089 to 1.387 ±0.093 (3rd day),from 2.045 ±0.235 to 1.269 ±0.103 (4th day).The cell number of apoptosis and in G2 phase was increased.Conclusion The LV-hsa-miR-206 is construted successfully and may be used to infect MCF-7 cells.After MCF-7 cells infected by this sort of vectors,the experession of miR-206 and biological ability is significantly changed.LV-hsa-miR-206 provides the basis for research on miR-206 in breast cancer.
