摘要
目的:分析增强型绿色荧光蛋白(EGFP)与蛋白激酶C-θ(PKCθ)的融合蛋白(PKCθ-EGFP)在HeLa细胞中的表达、亚细胞定位及其对细胞形态的影响。方法:用脂质转染胺(Lipofec-tAMINE 2000)为载体分别以pPKCθ-EGFP和pEGFP-N1转染HeLa细胞,流式细胞仪分析其表达率,激光共聚焦显微镜分析其亚细胞定位,同时分析二丁酸佛波醇酯(PDB)和小檗碱对其亚细胞定位和细胞形态的影响。结果:转染pEGFP-N1或pPKCθ-EGFP的HeLa细胞48 h后,表达绿色荧光的细胞百分率分别为(70.9±4.4)%和(45.8±2.3)%(P<0.05)。表达的PKCθ-EGFP融合蛋白均匀分布于细胞内,与EGFP蛋白在细胞内的分布相似。PDB刺激30 min后,表达PKCθ-EGFP的HeLa细胞的形态发生显著变化,即由原来的梭形变成圆形,荧光强度增强。但表达EGFP的HeLa细胞的形态及荧光分布均不受影响。小檗碱对PDB诱导的HeLa细胞的形态和EGFP-PKCθ融合蛋白的分布无明显影响。结论:表达PKCθ-EGFP融合蛋白的HeLa细胞在PDB作用下细胞形态发生显著变化。
Aim： To investigate the expression and subceilular localization of protein kinase Cθ（PKCθ） fiased with enhanced green fluorescent protein （EGFP） （PKCθ - EGFP） in HeLa ceils and to analyze its effect on the cell morphology. Methods： HeLa cells were transfected with expression vectors pPKCθ - EGFP and pEGFP- N1 using LipofectAMINE 2000, respectively. Their expression in the ceils was analyzed by flow cytometry and subceilular localization by confocal microscopy. The effect of phorbol 12, 13 - dibutyrate （PDB） and berberine on ceilmorphology was analyzed by confocal microscopy too. Results： The expression rates of green fluorescence in HeLa cells transfected with pEGFP- N1 and pPKCθ - EGFP plasmids were （70.9 ± 4.4） % and （45.8 ± 2.3） % respectively at 48 h after transfection （no statistics significance）. PKCθ- EGFP fusion protein was evenly distributed in the whole cells, which was similar to the distribution pattern of EGFP protein in HeLa cells. After 30 min stimulation of PDB, the shape of the HeLa cells expressing PKCθ - EGFP was dramatically changed. From spindle to small round, and the green fluorescence intensity increased and concentrated in the central of the cells. In contrast, the cells expressing EGFP had no significant change both in the green fluorescence distribution and in cell morphology after PDB stimulation. Besides, berberine exerted no obvious effect on the PDB - induced changes in HeLa cells expressing PKCθ - EGFP. Conclusion： The HeLa cells that expressed PKCθ - EGFP had significant morphological change under PDB stimulation.
出处
《暨南大学学报（自然科学与医学版）》
CAS
CSCD
北大核心
2005年第4期470-474,共5页
Journal of Jinan University(Natural Science & Medicine Edition)
基金
国家自然科学基金面上项目(No.30371651)
重点项目(No.30230350)资助
关键词
蛋白激酶CΘ
绿色荧光蛋白
融合蛋白
小檗碱
细胞转染
protein kinase Cθ
green fluorescent protein
fusion protein
berbefine
cell transfection
