摘要
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／Fe304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．M1Tr法检测胃癌细胞BGC823的增殖能力．以上实验均以正常肝细胞L02为对照。结果重组质粒pGL3-hTERT-tk构建成功，其长度为1100bp。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。MqT检测结果示．转染pGL3．hTERT-tk质粒4d后．BGC823细胞增殖受抑制．其A，，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。
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. MT＇F assay was used to evaluate the effect of HSV-tk on the proliferation of BGC823 cells. Normal hepatic cells L02 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-basie-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 transfectedby 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.
出处
《中华胃肠外科杂志》
CAS
2012年第7期727-731,共5页
Chinese Journal of Gastrointestinal Surgery
