增强型绿色荧光蛋白标记的Hela细胞株的建立

Construction of enhanced green fluorescent (EGFP) tagged Hela cell subline

目的构建增强型绿色荧光蛋白(EGFP)标记的Hela细胞系。方法PCR和DNA测序鉴定pEGFP-C1真核表达质粒,采用Qiagen tip 500进行pEGFP-C1真核表达质粒DNA的提取和纯化;cellfectin转染Hela细胞,G418筛选,有限稀释法单克隆培养,荧光显微镜进行荧光检测EGFP的表达,采用激光共聚焦显微镜观测器检测荧光特性。结果PCR和DNA测序证实pEGFP质粒结构正确;在倒置荧光显微镜下,转染24h后,部分Hela细胞可见绿色荧光,转染72h时,大约80%Hela细胞内均可见绿色荧光。加入含G418的选择性培养基进行选择性培养,选择荧光较强的Hela细胞经有限稀释,持续筛选及克隆化培养,获得稳定表达绿色荧光的Hela细胞克隆,扩大培养并传至10代以上,将此细胞株命名为Hela-EGFP。激光共聚焦显微镜观察发现:在蓝光(-395nm)激发时,Hela-EGFP细胞绿色荧光激发波长为395nm,最大发射峰为509nm。结论Hela-EGFP细胞株具备稳定表达EGFP的能力,为实时可视化进行宫颈癌侵袭转移机制的研究奠定了基础。

Objective To construct a novel enhanced green fluorescent protein （EGFP） tagged Hela cell subline. Methods EGFP gene fragment was amplified from pEGFP-C1 vector and confirmed by PCR and gene sequencing, pEGFP-C1 vector DNA was isolated and purified by Qiagen tip 500 for transfection into Hela cells. After Hela ceils were transfected with pEGFP-C1 by cellfectin reagent, single cell clones were isolated by the selective medium containing geneticin （G418） and limiting dilution in 96-well flat-bottomed culture plates. We replenished the selective media every 3--4 days, and observed the percentage of surviving cells. The fluorescent signal of Hela cells was detected using the convert fluorescent microscope every 24 hours. The character of EGFP was detected with laser confocal microscopy. Results The pEGFP-C1 vector was sequenced and amplified respectively to confirm that EGFP gene was in the correct orientation for expression and contained an ATG and a stop codon. After Hela ceils were transfected with the indicated expression pEGFP-C1 vectors 24 hours, the green fluorescence could be found from Hela ceils. The intensity of green fluorescence and the number of Hela cells expression green fluorescence increased steadily until 80% Hela cells, after 72 hours. From then on, Hela cells were cultured with selective media containing G418 while untransfected Hela cells were killed. This process took up to 4 weeks. EGFP-labeled Hela cells displayed green fluorescence under fluorescent microscopy and showed high-level stable expression of EGFP. The excitation peak of EGFP was at 395 nm （blue）, and its emission peak was at 509 nm （green） for laser confocal microscopy. Conclusion The high-level stable EGFP-expressing Hela cell line （HeLa- EGFP） provided a simple and reliable tool for studying human cervical cancer in vivo. It may be valuable for further study on molecular mechanisms of cervical cancer metastasis.