CAJ | 학술논문

目的：构建C57小鼠L型氨基酸转运载体1（ LAT1）的真核表达载体，转染Neuro-2a肿瘤细胞进行表达，探讨LAT1对Neuro-2a细胞增殖及凋亡的影响。方法采用RT-PCR扩增出LAT1全长目的基因，定向克隆pcDNA3.1表达载体，构建pcDNA3.1-LAT1重组表达质粒，通过脂质体法将阳性克隆转染Neuro-2a细胞，经G418筛选获得稳定表达株后，用RT-PCR和western blot 蛋白印迹法检测LAT1的表达情况。通过MTT法检测各组Neuro-2a细胞的增殖情况，并借助流式细胞仪检测LAT1对Neuro-2a细胞增殖和凋亡的影响。结果成功构建小鼠pcDNA3.1-LAT1真核表达载体，酶切和测序证实其序列正确，并成功转染Neuro-2a细胞建立稳定转染细胞系。MTT法显示转染重组质粒pcDNA3.1-LAT1的Neuro-2a细胞增殖速度显著高于对照组（P ＜0.05），且流式细胞术检测转染组对Neuro-2a细胞具有促进增殖抑制凋亡的作用。结论转染 pcDNA3.1-LAT1质粒的Neuro-2a细胞能成功表达LAT1蛋白，且LAT1对Neuro-2a细胞的增殖和凋亡均有显著影响，为进一步研究LAT1的生物学作用奠定了重要基础。
목적：구건C57소서L형안기산전운재체1（ LAT1）적진핵표체재체，전염Neuro-2a종류세포진행표체，탐토LAT1대Neuro-2a세포증식급조망적영향。방법채용RT-PCR확증출LAT1전장목적기인，정향극륭pcDNA3.1표체재체，구건pcDNA3.1-LAT1중조표체질립，통과지질체법장양성극륭전염Neuro-2a세포，경G418사선획득은정표체주후，용RT-PCR화western blot 단백인적법검측LAT1적표체정황。통과MTT법검측각조Neuro-2a세포적증식정황，병차조류식세포의검측LAT1대Neuro-2a세포증식화조망적영향。결과성공구건소서pcDNA3.1-LAT1진핵표체재체，매절화측서증실기서렬정학，병성공전염Neuro-2a세포건립은정전염세포계。MTT법현시전염중조질립pcDNA3.1-LAT1적Neuro-2a세포증식속도현저고우대조조（P ＜0.05），차류식세포술검측전염조대Neuro-2a세포구유촉진증식억제조망적작용。결론전염 pcDNA3.1-LAT1질립적Neuro-2a세포능성공표체LAT1단백，차LAT1대Neuro-2a세포적증식화조망균유현저영향，위진일보연구LAT1적생물학작용전정료중요기출。
Objective To construct the lamino acid transporter 1 eukaryotic expression vector of C 57 mouse and to express the gene inNeuro-2atumor cells,and explore the effect of LAT1on proliferation and apoptosis of Neuro-2a cell. Methods The full-length LAT1 cDNA was synthesized by RT-PCR and cloned into pcDNA3.1vector to construct recombinant plasmid.The constructed pcDNA3.1-LAT1vector was verified by Enzyme digestion and sequencing and then transfected intomurine Neuro-2acellsby liposome.The transfected cells were selected with G418 and stably expressed strain was constructed .The expression of LAT1 was detected by RT-PCR and western blot .Proliferation was analyzed by MTT , cell cycle and apoptosis were detected by flow cytometric analysis .Results The full-length LAT1 cDNA was amplified successfully and pcDNA3.1-LAT1eukaryoticvector was constructed successfully .Enzyme digestion and sequencing confirmed the sequence was correct .Neuro-2acells were transfected and Stably expressed strain was constructed successfully.MTT showed that the group of transfected restructuring plasmid could significantly affect Neuro -2a cell proliferation more than the control groups ( P <0.05 ) .From the flowcytometric analysis , LAT1 could promote cell proliferation and inhibit Neuro-2a cell apoptosis.Conclusion LAT1 can express successfully inNeuro-2acells which were transfected with recombinantpcDNA3.1-LAT1plasmid.LAT1 in Neuro-2a cells can promote cell proliferation and inhibit the cell apoptosis which provides a basis for the study of LAT 1.That lays the foundation for studying biological effects of LAT 1.