摘要
目的 探讨佛波酯（phorbol 12-myristate 13-acetate, PMA）诱导THP-1细胞（人类单核/巨噬细胞）分化的最优条件，建立相关自噬模型，为基于细胞自噬模型的研究奠定实验基础。 方法 采用PMA诱导THP-1细胞分化成巨噬细胞；用表达融合蛋白pcDNA3.1-YFP-LC3质粒转染THP-1细胞，YFP为黄绿色荧光蛋白，以示踪自噬体的形成。采用倒置显微镜观察不同浓度（0、10、20、50、100、200 ng/ml）、不同时间（0、24、48、60 h）PMA分化细胞形态学变化；采用荧光显微镜示踪不同条件下LC3点聚集的增强；实时荧光定量聚合酶链式反应（reverse transcription-quantitative polymerase chain reaction ，RT-qPCR）检测自噬相关基因mRNA的表达。采用SPSS 17.0软件进行统计分析。mRNA相对含量2-ΔΔCt用“x±s”表示。经比较Ct值法分析基因表达差异。Earle’s balanced salts solution（EBSS，又称饥饿培养基），诱导细胞自噬相关基因mRNA表达量变化的比较采用t检验分析，以P0.05为差异有统计学意义。 结果 PMA浓度为100 ng/ml、诱导时间为24～48 h时，THP-1细胞分化成巨噬细胞的形态学状态较理想。饥饿培养基EBSS处理分化的THP-1细胞，能增加自噬体YFP-LC3点聚集，其自噬相关基因LC3、Atg5、Atg7、Beclin1的mRNA表达水平显著增加 （2-ΔΔCt均值分别为1.35±0.16、1.18±0.39、1.44±0.12、1.08±0.09，t值分别为4.00、2.90、5.16、3.57，P值均＜0.05）。 结论 PMA在浓度为100 ng/ml、24～48 h时诱导THP-1分化为巨噬细胞状态较理想；成功构建了THP-1源性细胞自噬模型。
Objective To optimized the conditions of PMA to stimulate THP-1 cell differentiation in order to establish a cell model of autophagy, which provides a scientific basis for the researches related to cell autophagy. Methods THP-1 cells differentiated into macrophages induced by PMA.Fusion protein pcDNA3.1-YFP-LC3 plasmid was transferred into THP-1 cell, and YFP（Yellow fluorescent protein） can trace the formation of autophagosome. Morphological changes of differentiated cells were photographed by inverted microscope respectively at diffe-rent PMA concentrations（0,10,20,50,100,200 ng/ml） and different times（0,24,48,60 h）. LC3 protein was traced by fluorescence microscopy in different conditions. The mRNA expression levels of autophagy-related genes were detected by RT-qPCR（reverse transcription-quantitative polymerase chain reaction ）. SPSS 17.0 was used to do statistical analyze, the relative amount 2-ΔΔCt of mRNA was showed as “x±s”, and the difference between gene expression was analyzed by Ct value. Paired t-test （P〈0.5） was used to analyse the expression of mRNA induced by EBSS（earle’s balanced salts solution）. Results When the PMA concentration was 100 ng/ml and the induction time was 24-48 h, the state of THP-1 derived cells reached best condition. After deal with EBSS medium, the formulation of YFP-LC3 autophagy in THP-1 derived cells was enhanced, and the expression levels of autophagy-rela-ted genes LC3、Atg5、Atg7、Beclin1 were increased at the same time, which indicated that autophagy cell model was successfully constructed （2-ΔΔCt value were 1.35±0.16,1.18±0.39,1.44±0.12,1.08±0.09,while t value were 4.00,2.90,5.16,3.57,P〈0.05）. Conclusion The optimization culture conditions were 100 ng/ml PMA and 24-48 h induced time. Based on this condition, an ideal autophagy model was established, which providing a solid scientific foundation for autophagy-related research.
出处
《中国防痨杂志》
CAS
2013年第12期997-1002,共6页
Chinese Journal of Antituberculosis
基金
广东省自然科学基金(WSTJJ20120319130623198210011211)
"十二五"国家科技重大专项(2012ZX10004903)
广东省"十二五"医学重点实验室
关键词
白血病
单核细胞
急性
细胞系
肿瘤
十四酰佛波乙酯
自噬
模型
生物学
Leukemia, monocytic, acute
Cell line, tumor
Tetradecanoylphorbol acetate
Autolahagy
Models, biological
