中华肝胆外科杂志
中華肝膽外科雜誌
중화간담외과잡지
CHINESE JOURNAL OF HEPATOBILIARY SURGERY
2014年
2期
137-141
,共5页
潘耀振%孙诚谊%詹磊%张浩%田舍%张宏%姜楠
潘耀振%孫誠誼%詹磊%張浩%田捨%張宏%薑楠
반요진%손성의%첨뢰%장호%전사%장굉%강남
胰腺癌%MUC1启动子%双荧光素酶活性检测
胰腺癌%MUC1啟動子%雙熒光素酶活性檢測
이선암%MUC1계동자%쌍형광소매활성검측
Pancreatic cancer%MUC1 Promoter%Dual luciferase assays
目的 克隆MUC1黏蛋白启动子,研究MUC1启动子在人类胰腺癌细胞株Panc-1细胞和人类宫颈癌细胞株Hela细胞中的转录活性.方法 采用巢式PCR扩增MUC1启动子片段并酶切连接至含有EGFP报告基因的pEGFP-N1载体中构建质粒pEGFP-MUC1-N1,采用基因重组方法将MUC1启动子片段及EGFP报告基因构建于pShuttle质粒上形成pShuttle-MUC1-EGFP质粒.通过脂质体共转染人胰腺癌细胞株Panc-1和人宫颈癌细胞株Hela.使用荧光素酶检测系统(Luciferase assay system)测定细胞的荧光素酶活性,通过荧光显微镜和流式细胞仪检测MUC1启动子在Panc-1细胞中的特异转录活性.结果 成功克隆出MUC1启动子,双酶切、PCR检测和DNA测序证实pEGFPMUC1-N1、pShuttle-MUC1-EGFP载体构建成功.重组报告载体荧光素酶活性显著升高(t=18.975,P =0.001).经pEGFPMUC1-N1、pShuttleMUC 1-EGFP质粒转染后MUC1启动子在Panc-1细胞中的转录活性为阳性对照CMV启动子活性的69.6%及63.6%,明显高于Hela细胞中的4.2%及3.7%,在胰腺癌细胞中具有较高特异性,且在胰腺癌细胞中的活性明显高于阴性对照pGL3-Basic的0.093%.结论 MUC1启动子在淋巴瘤细胞中具有较高的特异性,可以作为胰腺癌细胞基因转染的肿瘤特异性启动子使用.此研究为进一步运用MUC1启动子在基因水平靶向治疗胰腺癌奠定了基础.
目的 剋隆MUC1黏蛋白啟動子,研究MUC1啟動子在人類胰腺癌細胞株Panc-1細胞和人類宮頸癌細胞株Hela細胞中的轉錄活性.方法 採用巢式PCR擴增MUC1啟動子片段併酶切連接至含有EGFP報告基因的pEGFP-N1載體中構建質粒pEGFP-MUC1-N1,採用基因重組方法將MUC1啟動子片段及EGFP報告基因構建于pShuttle質粒上形成pShuttle-MUC1-EGFP質粒.通過脂質體共轉染人胰腺癌細胞株Panc-1和人宮頸癌細胞株Hela.使用熒光素酶檢測繫統(Luciferase assay system)測定細胞的熒光素酶活性,通過熒光顯微鏡和流式細胞儀檢測MUC1啟動子在Panc-1細胞中的特異轉錄活性.結果 成功剋隆齣MUC1啟動子,雙酶切、PCR檢測和DNA測序證實pEGFPMUC1-N1、pShuttle-MUC1-EGFP載體構建成功.重組報告載體熒光素酶活性顯著升高(t=18.975,P =0.001).經pEGFPMUC1-N1、pShuttleMUC 1-EGFP質粒轉染後MUC1啟動子在Panc-1細胞中的轉錄活性為暘性對照CMV啟動子活性的69.6%及63.6%,明顯高于Hela細胞中的4.2%及3.7%,在胰腺癌細胞中具有較高特異性,且在胰腺癌細胞中的活性明顯高于陰性對照pGL3-Basic的0.093%.結論 MUC1啟動子在淋巴瘤細胞中具有較高的特異性,可以作為胰腺癌細胞基因轉染的腫瘤特異性啟動子使用.此研究為進一步運用MUC1啟動子在基因水平靶嚮治療胰腺癌奠定瞭基礎.
목적 극륭MUC1점단백계동자,연구MUC1계동자재인류이선암세포주Panc-1세포화인류궁경암세포주Hela세포중적전록활성.방법 채용소식PCR확증MUC1계동자편단병매절련접지함유EGFP보고기인적pEGFP-N1재체중구건질립pEGFP-MUC1-N1,채용기인중조방법장MUC1계동자편단급EGFP보고기인구건우pShuttle질립상형성pShuttle-MUC1-EGFP질립.통과지질체공전염인이선암세포주Panc-1화인궁경암세포주Hela.사용형광소매검측계통(Luciferase assay system)측정세포적형광소매활성,통과형광현미경화류식세포의검측MUC1계동자재Panc-1세포중적특이전록활성.결과 성공극륭출MUC1계동자,쌍매절、PCR검측화DNA측서증실pEGFPMUC1-N1、pShuttle-MUC1-EGFP재체구건성공.중조보고재체형광소매활성현저승고(t=18.975,P =0.001).경pEGFPMUC1-N1、pShuttleMUC 1-EGFP질립전염후MUC1계동자재Panc-1세포중적전록활성위양성대조CMV계동자활성적69.6%급63.6%,명현고우Hela세포중적4.2%급3.7%,재이선암세포중구유교고특이성,차재이선암세포중적활성명현고우음성대조pGL3-Basic적0.093%.결론 MUC1계동자재림파류세포중구유교고적특이성,가이작위이선암세포기인전염적종류특이성계동자사용.차연구위진일보운용MUC1계동자재기인수평파향치료이선암전정료기출.
Objective To evaluate the MUC1 promoter's role in driving gene expression in pancreatic cancer and its therapeutic significance.Methods Two plasmids were made.The plasmid pEGFP-MUC1N1 contained MUC1 promoter fragment connected to the pEGFP-N1 vector with the EGFG reporter gene.The pShuttle-MUC1-EGFP plasmid contained MUC1 promoter fragment and EGFP reporter gene connected to pShuttle plasmid.Lipofectamine 2000 was used to transfect the two plasmids into cells of MUC1-positive human pancreatic cell line Panc-1 and MUC1-negative human cervical carcinoma Hela.Fluorescence microscopy and flow cytometry compared the specificity and activity of the MUC1 promoter and CMV promoter.Results Reporter gene EGFP-positive cells 48 hours after transfection with pEGFP-MUC1-N1 and pShuttleMUC1-EGFP plasmid were 69.6% and 63.6% respectively,in Panc-1 cells,and 4.2% and 3.7% respectively,in Hela cells.Conclusions MUC1 promoter can drive reporter gene activity in MUC1-positive tumor cells targeting functional expression.There is potentially a use of targeted therapy in pancreatic cancer at the genetic level.