转座子piggyBac介导的HSV-tk基因在妇科恶性肿瘤细胞中长期稳定表达的研究

Long-term and stable expression of transgene mediated by piggyBac transposon in gynecological malignant tumor cells

目的 研究piggyBac（PB）转座子介导转移外源基因HSV-tk在多种妇科恶性肿瘤细胞中的表达情况,探讨其作为一种可整合的非病毒载体在肿瘤基因治疗中的应用价值.方法 通过PCR技术扩增HSV-tk基因的编码区,定向克隆至PB表达载体PB[Act-RFP]DS,经酶切、测序鉴定为重组载体PB[Act-RFP,HSV-tk]DS（pPB/TK）.联合3种小同的转染试剂FuGENE HD、jetPEI及lipofectamine 2000,分别将pPB/TK转染入4种常见的妇科恶性肿瘤细胞株HeLa、JEG-3、SKOV3和HEC-1B.转染后以mRFP1为报告基因,经荧光显微镜观察和流式细胞仪分析转染效率;逆转录（RT）-PCR技术检测HSV-tk和mRFP1基因的表达;四甲基偶氮唑蓝实验测定不同浓度的更昔洛韦（GCV）对转染后细胞的杀伤作用.转染后细胞经流式细胞仪分选,极限稀释单克隆培养建立稳定转染株,并以反向PCR技术确定转座子插入基因组的位点.结果 （1）成功构建重组载体pPB/TK.（2）在3种转染试剂的辅助下,pPB/TK均可有效转染入HeLa、HEC-1B、SKOV3和JEG-3细胞;不同转染试剂辅助下pPB/TK在同种细胞中的转染效率各不相同,在HeLa细胞中,FuGENE HD的转染效率[（78.7±9.2）%]高于lipofectamine 2000[（54.1±11.4）%]和jetPEI][（46.5±7.4）%],分别比较,差异均有统计学意义（P〈0.05）;同一转染试剂辅助下pPB/TK在不同细胞中的转染效率也不相同,以FuGENE HD为转染试剂,pPB/TK在HeLa、JEG-3和SKOV3细胞中的转染效率分别为（78.7 ±9.2）%、（74.4±8.9）%和（83.2±9.7）%,高于HEC-1B细胞的（39.5±8.7）%,分别比较,差异均有统计学意义（P〈0.05）.（3）RT-PCR结果显示,4种细胞均有HSV-tk和mRFP1基因mRNA的表达.（4）GCV对转染有pPB/TK的HeLa、JEG-3、SKOV3和HEC-1B细胞的半数抑制浓度分别为1.29、3.35、0.09和13.28 μg/ml.10μg/ml GCV对转染有pPB/TK的SKOV3细胞抑制率高于单独转染pORF-HSVtk者,细胞抑制率分别为（86±9）%和（52±12）%,两者比较,差异有统计学意义（P〈0.05）.（5）稳定转染株在转染后3个月仍可观察到mRFP1表达,并可榆测纠基因组中有TTAA位点的插入整合.结论 PB转座子可介导外源基因在多种妇科恶性肿瘤细胞中的长期稳定表达,有望为肿瘤基因治疗提供更为安全有效的转基因技术平台.

Objective To investigate the expression of exogenous gene transferred by piggyBac （PB） transposon in various gynecological malignant cell lines and reveal its potential application of gene therapy in gynecological cancer.Methods Amplified herpes simplex virus thymidine kinase （HSV-tk） gene coding region by PCR and integrated it into PB expression vector, PB[Act-RFP]DS, for reconstructing PB[Act-RFP, HSV-tk]DS （pPB/TK）.By using different transfection reagents： FuGENE HD, jetPEI, lipofectamine 2000, pPB/TK together with helper plasmid Act-PBase were cotransfected into four mostly common gynecological malignant tumor cell lines HeLa, JEG-3, SKOV3 and HEC-1B.The mRFP1 report gene expressions was observed and detected by fluorescence microscope and flow cytometry to analyze transfection efficiency.The expressions of HSV-tk and mRFP1 gene were detected by reverse transcription PCR （RT-PCR）.The cytotoxic effect of various concentration of pro-drug ganciclovir （GCV） for transfected cells was detected by methyl thiazole tetrazolium assay.The transfected cells were positive sorted by flow cytometry and limiting diluted to obtain the stable transfected cell line.The insertion sites of foreign gene tranferred by PB transposon in genome were analyzed by inverse PCR.Results （1） Double digests analysis and sequences test demonstrated that pPB/TK vector was reconstructed successfully.（2） Using three different transfective reagents, PB trausposon transferred HSV-tk gene and mRFP1 gene into HeLa, HEC-1 B, SKOV3 and JEG-3 cell efficiently, and the transfection efficiency of pPB/TK for the same cell was different by using different transfective reagents; in Hela cell, the transfection efficiency of FuGENE HD [（78.7 ± 9.2） %]was higher than that of lipofectamine 2000 [（54.1 ± 11.4） %]and jetPEI [（46.5 ± 7.4） %, all P 〈 0.05] ; using the same transfective reagent, the transfection efficiency of pPB/TK was also different on various cell lines, using FuGENE HD, the transfeetion efficiency of pPB/TK on HeLa, JEG-3 and SKOV3 cell was （78.7 ± 9.2） %, （74.4 ± 8.9） % and （83.2 ± 9.7） % respectively, which all were higher than that on HEC-1B [（39.5 ± 8.7） %, P 〈 0.05] .（3） RT-PCR showed that there were the mRNA expression of HSV-tk and mRFP1 in all cell lines.（4） 50% inhibitory concentration of GCV for transfected cells, HeLa, JEG-3, SKOV3 and HEC-1B, was 1.29, 3.35, 0.09 and 13.28 μg/ml respectively.Inhibitory effect of GCV （10 μg/ml） on SKOV3 transfected with pPB/TK was （86 ± 9） %, which was superior to that transfected with pORF-HSVtk alone [（52 ± 12）%, P 〈 0.05] .（5） The insertion sites of PB transposon in the target cells genome were located at TTAA sites, mRFP1 expression still could be detected in three months after transfected.Conclusions PB transposon could transfer exogenous gene into various gynecological malignant cells, which could integrated into genome and obtain a long-term and stable expression.It is expected that PB transposon may supply a more efficient and safer transgene technology platform for gene therapy in gynecological cancer.