中华妇产科杂志
中華婦產科雜誌
중화부산과잡지
CHINESE JOUNAL OF OBSTETRICS AND GYNECOLOGY
2010年
4期
292-297
,共6页
康玉%孙庆文%余文博%程明军%张晓燕%武欣%陈春妹%郑煜芳%徐丛剑
康玉%孫慶文%餘文博%程明軍%張曉燕%武訢%陳春妹%鄭煜芳%徐叢劍
강옥%손경문%여문박%정명군%장효연%무흔%진춘매%정욱방%서총검
生殖器肿瘤,女(雌)性%DNA可移植因子%转染%转基因%单纯疱疹病毒属%胸苷激酶%发光蛋白质类
生殖器腫瘤,女(雌)性%DNA可移植因子%轉染%轉基因%單純皰疹病毒屬%胸苷激酶%髮光蛋白質類
생식기종류,녀(자)성%DNA가이식인자%전염%전기인%단순포진병독속%흉감격매%발광단백질류
Genital neoplasms,female%DNA transposable elements%Transfection%Transgenes%Simplexvirus%Thymidine kinase%Luminescent proteins
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.
目的 研究piggyBac(PB)轉座子介導轉移外源基因HSV-tk在多種婦科噁性腫瘤細胞中的錶達情況,探討其作為一種可整閤的非病毒載體在腫瘤基因治療中的應用價值.方法 通過PCR技術擴增HSV-tk基因的編碼區,定嚮剋隆至PB錶達載體PB[Act-RFP]DS,經酶切、測序鑒定為重組載體PB[Act-RFP,HSV-tk]DS(pPB/TK).聯閤3種小同的轉染試劑FuGENE HD、jetPEI及lipofectamine 2000,分彆將pPB/TK轉染入4種常見的婦科噁性腫瘤細胞株HeLa、JEG-3、SKOV3和HEC-1B.轉染後以mRFP1為報告基因,經熒光顯微鏡觀察和流式細胞儀分析轉染效率;逆轉錄(RT)-PCR技術檢測HSV-tk和mRFP1基因的錶達;四甲基偶氮唑藍實驗測定不同濃度的更昔洛韋(GCV)對轉染後細胞的殺傷作用.轉染後細胞經流式細胞儀分選,極限稀釋單剋隆培養建立穩定轉染株,併以反嚮PCR技術確定轉座子插入基因組的位點.結果 (1)成功構建重組載體pPB/TK.(2)在3種轉染試劑的輔助下,pPB/TK均可有效轉染入HeLa、HEC-1B、SKOV3和JEG-3細胞;不同轉染試劑輔助下pPB/TK在同種細胞中的轉染效率各不相同,在HeLa細胞中,FuGENE HD的轉染效率[(78.7±9.2)%]高于lipofectamine 2000[(54.1±11.4)%]和jetPEI][(46.5±7.4)%],分彆比較,差異均有統計學意義(P<0.05);同一轉染試劑輔助下pPB/TK在不同細胞中的轉染效率也不相同,以FuGENE HD為轉染試劑,pPB/TK在HeLa、JEG-3和SKOV3細胞中的轉染效率分彆為(78.7 ±9.2)%、(74.4±8.9)%和(83.2±9.7)%,高于HEC-1B細胞的(39.5±8.7)%,分彆比較,差異均有統計學意義(P<0.05).(3)RT-PCR結果顯示,4種細胞均有HSV-tk和mRFP1基因mRNA的錶達.(4)GCV對轉染有pPB/TK的HeLa、JEG-3、SKOV3和HEC-1B細胞的半數抑製濃度分彆為1.29、3.35、0.09和13.28 μg/ml.10μg/ml GCV對轉染有pPB/TK的SKOV3細胞抑製率高于單獨轉染pORF-HSVtk者,細胞抑製率分彆為(86±9)%和(52±12)%,兩者比較,差異有統計學意義(P<0.05).(5)穩定轉染株在轉染後3箇月仍可觀察到mRFP1錶達,併可榆測糾基因組中有TTAA位點的插入整閤.結論 PB轉座子可介導外源基因在多種婦科噁性腫瘤細胞中的長期穩定錶達,有望為腫瘤基因治療提供更為安全有效的轉基因技術平檯.
목적 연구piggyBac(PB)전좌자개도전이외원기인HSV-tk재다충부과악성종류세포중적표체정황,탐토기작위일충가정합적비병독재체재종류기인치료중적응용개치.방법 통과PCR기술확증HSV-tk기인적편마구,정향극륭지PB표체재체PB[Act-RFP]DS,경매절、측서감정위중조재체PB[Act-RFP,HSV-tk]DS(pPB/TK).연합3충소동적전염시제FuGENE HD、jetPEI급lipofectamine 2000,분별장pPB/TK전염입4충상견적부과악성종류세포주HeLa、JEG-3、SKOV3화HEC-1B.전염후이mRFP1위보고기인,경형광현미경관찰화류식세포의분석전염효솔;역전록(RT)-PCR기술검측HSV-tk화mRFP1기인적표체;사갑기우담서람실험측정불동농도적경석락위(GCV)대전염후세포적살상작용.전염후세포경류식세포의분선,겁한희석단극륭배양건립은정전염주,병이반향PCR기술학정전좌자삽입기인조적위점.결과 (1)성공구건중조재체pPB/TK.(2)재3충전염시제적보조하,pPB/TK균가유효전염입HeLa、HEC-1B、SKOV3화JEG-3세포;불동전염시제보조하pPB/TK재동충세포중적전염효솔각불상동,재HeLa세포중,FuGENE HD적전염효솔[(78.7±9.2)%]고우lipofectamine 2000[(54.1±11.4)%]화jetPEI][(46.5±7.4)%],분별비교,차이균유통계학의의(P<0.05);동일전염시제보조하pPB/TK재불동세포중적전염효솔야불상동,이FuGENE HD위전염시제,pPB/TK재HeLa、JEG-3화SKOV3세포중적전염효솔분별위(78.7 ±9.2)%、(74.4±8.9)%화(83.2±9.7)%,고우HEC-1B세포적(39.5±8.7)%,분별비교,차이균유통계학의의(P<0.05).(3)RT-PCR결과현시,4충세포균유HSV-tk화mRFP1기인mRNA적표체.(4)GCV대전염유pPB/TK적HeLa、JEG-3、SKOV3화HEC-1B세포적반수억제농도분별위1.29、3.35、0.09화13.28 μg/ml.10μg/ml GCV대전염유pPB/TK적SKOV3세포억제솔고우단독전염pORF-HSVtk자,세포억제솔분별위(86±9)%화(52±12)%,량자비교,차이유통계학의의(P<0.05).(5)은정전염주재전염후3개월잉가관찰도mRFP1표체,병가유측규기인조중유TTAA위점적삽입정합.결론 PB전좌자가개도외원기인재다충부과악성종류세포중적장기은정표체,유망위종류기인치료제공경위안전유효적전기인기술평태.
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.
