中华胃肠外科杂志
中華胃腸外科雜誌
중화위장외과잡지
CHINESE JOURNAL OF GASTROINTESTINAL SURGERY
2012年
7期
727-731
,共5页
阿不都外力·吾守尔%李玲%张邦昌
阿不都外力·吾守爾%李玲%張邦昌
아불도외력·오수이%리령%장방창
纳米磁流体%HTERT增强子%胸苷激酶基因%胃肿瘤%基因治疗
納米磁流體%HTERT增彊子%胸苷激酶基因%胃腫瘤%基因治療
납미자류체%HTERT증강자%흉감격매기인%위종류%기인치료
Nano-magnetic fluids%hTERT enhancer%tk gene%Gastric cancer%BGC823 cells
目的 研究hTERT启动子调控下单纯疱疹病毒胸苷激酶(HSV-tk)重组质粒的构建、在人胃癌细胞BGC823中的表达及对胃癌细胞BGC823增殖的影响.方法 构建重组质粒pGL3 -hTERT-tk和相应的荧光报告质粒pGL3-hTERT-tk-Luc+；经纳米磁流体PEG-PEI/Fe3O4转染胃癌细胞BGC823,荧光显微镜观察细胞形态变化和转染效率,免疫组织化学(免疫组化)方法检测目的基因在胃癌细胞BGC823中的表达,MTT法检测胃癌细胞BGC823的增殖能力,以上实验均以正常肝细胞LO2为对照.结果 重组质粒pGL3-hTERT-tk构建成功,其长度为1100 bp.荧光素酶标记的阳性、阴性对照及治疗报告质粒pGL3-hTERT-TK- Luc+均能有效转染高表达端粒酶活性的胃癌细胞BGC823,转染效率为(28.1±2.3)％.免疫组化结果显示,重组质粒组胃癌细胞BGC823的细胞质中可见大量HSV -tk基因编码的表达.MTT检测结果示,转染pGL3-hTERT-tk质粒4d后,BGC823细胞增殖受抑制,其A570值为0.254±0.011,低于L02细胞的0.322±0.013;亦低于pGL3-basic-tk转染的BGC823细胞(0.357±0.014)(均P＜0.05).结论 纳米磁流体能将重组质粒pGL3-hTERT-tk转染BGC823细胞并获得表达,PEG-PEI/Fe3O4纳米磁流体-tk可显著抑制BGC823细胞增殖,有望成为胃癌基因治疗的新型生物制剂之一.
目的 研究hTERT啟動子調控下單純皰疹病毒胸苷激酶(HSV-tk)重組質粒的構建、在人胃癌細胞BGC823中的錶達及對胃癌細胞BGC823增殖的影響.方法 構建重組質粒pGL3 -hTERT-tk和相應的熒光報告質粒pGL3-hTERT-tk-Luc+；經納米磁流體PEG-PEI/Fe3O4轉染胃癌細胞BGC823,熒光顯微鏡觀察細胞形態變化和轉染效率,免疫組織化學(免疫組化)方法檢測目的基因在胃癌細胞BGC823中的錶達,MTT法檢測胃癌細胞BGC823的增殖能力,以上實驗均以正常肝細胞LO2為對照.結果 重組質粒pGL3-hTERT-tk構建成功,其長度為1100 bp.熒光素酶標記的暘性、陰性對照及治療報告質粒pGL3-hTERT-TK- Luc+均能有效轉染高錶達耑粒酶活性的胃癌細胞BGC823,轉染效率為(28.1±2.3)％.免疫組化結果顯示,重組質粒組胃癌細胞BGC823的細胞質中可見大量HSV -tk基因編碼的錶達.MTT檢測結果示,轉染pGL3-hTERT-tk質粒4d後,BGC823細胞增殖受抑製,其A570值為0.254±0.011,低于L02細胞的0.322±0.013;亦低于pGL3-basic-tk轉染的BGC823細胞(0.357±0.014)(均P＜0.05).結論 納米磁流體能將重組質粒pGL3-hTERT-tk轉染BGC823細胞併穫得錶達,PEG-PEI/Fe3O4納米磁流體-tk可顯著抑製BGC823細胞增殖,有望成為胃癌基因治療的新型生物製劑之一.
목적 연구hTERT계동자조공하단순포진병독흉감격매(HSV-tk)중조질립적구건、재인위암세포BGC823중적표체급대위암세포BGC823증식적영향.방법 구건중조질립pGL3 -hTERT-tk화상응적형광보고질립pGL3-hTERT-tk-Luc+；경납미자류체PEG-PEI/Fe3O4전염위암세포BGC823,형광현미경관찰세포형태변화화전염효솔,면역조직화학(면역조화)방법검측목적기인재위암세포BGC823중적표체,MTT법검측위암세포BGC823적증식능력,이상실험균이정상간세포LO2위대조.결과 중조질립pGL3-hTERT-tk구건성공,기장도위1100 bp.형광소매표기적양성、음성대조급치료보고질립pGL3-hTERT-TK- Luc+균능유효전염고표체단립매활성적위암세포BGC823,전염효솔위(28.1±2.3)％.면역조화결과현시,중조질립조위암세포BGC823적세포질중가견대량HSV -tk기인편마적표체.MTT검측결과시,전염pGL3-hTERT-tk질립4d후,BGC823세포증식수억제,기A570치위0.254±0.011,저우L02세포적0.322±0.013;역저우pGL3-basic-tk전염적BGC823세포(0.357±0.014)(균P＜0.05).결론 납미자류체능장중조질립pGL3-hTERT-tk전염BGC823세포병획득표체,PEG-PEI/Fe3O4납미자류체-tk가현저억제BGC823세포증식,유망성위위암기인치료적신형생물제제지일.
Objective To construct an hTERT promoter-controlled recombinant plasmid HSV-tk,and to investigate its expression in human gastric cancer cells BGC823 and its effect on proliferation of gastric cancer cells BGC823 in vitro.Methods Recombinant plasmid pGL3-hTERT-tk and the corresponding reporter plasmid pGL3-hTERT-tk-Luc+ were constructed by gene engineering.The recombinant plasmids were then transfected into gastric cancer cells BGC823 via PEG-PEI/Fe3O4 magnetic nanoparticles.Fluorescence microscope was used to observe the changes of cell morphology and the transfection efficiency. The expression of the target gene in gastric cancer cells BGC823 was detected by immunohistochemistry.MTT assay was used to evaluate the effect of HSV-tk on the proliferation of BGC823 cells.Normal hepatic cells LO2 were used as controls in all the experiments.Results Recombinant plasmid pGL3-hTERT-tk was successfully constructed,and the length was 1100 bp.pGL3-control-tk-Luc+,pGL3-basic-tk-Luc+ and pGL3-hTERT-tk-Luc+ all could effectively transfect BGC823 cells with high telomerase activity,with the transfection rate being (28.1±2.3)％.Immunohistochemistry study showed significant expression of HSV-tk gene in the cytoplasm of BGC823 cells.MTT showed that 4 days after transfection of pGL3-hTERT-tk,the proliferation of BGC823 was inhibited,and the A570 value was (0.254±0.011),significantly lower than that of L02 cells (0.322-±0.013) and that of BGC823 transfected by pGL3-basic-tk (0.357±0.014) (all P＜0.05).Conclusions Magnetic nano-particles can transfect pGL3-hTERT-tk into BGC823 cells and the expression is achieved.PEG-PEI/Fe3O4 magnetic nanoparticles can significantly inhibit the proliferation of BGC823 and may become a potential biological agent for gene therapy of gastric cancer.
