稳定表达小鼠甘丙肽的N-2a细胞株的建立及内源性过表达甘丙肽对N-2a细胞增殖和凋亡的影响

Establishment of An N-2a Cell Line Stably Expressing Mouse Galanin and the Effect of Over-expressed Galanin on the Proliferation and Apoptosis of N-2a Cell

目的建立稳定表达小鼠甘丙肽(GAL)的N-2a细胞株GAL-N-2a,探讨内源性过表达的甘丙肽对N-2a细胞增殖和凋亡的影响。方法脂质体法将重组载体PcDNA 3.1-PDGF-GAL转染N-2a细胞,通过G418筛选获得稳定过表达GAL的细胞克隆,利用PCR、反转录PCR和Western blot法检测细胞中GAL在N-2a细胞的过表达,通过MTT法检测N-2a细胞的增殖,借助流式细胞仪分别对过表达GAL的单克隆细胞进行细胞周期和细胞凋亡的检测。结果稳定转染N-2a细胞即GAL-N-2a实现GAL基因的高表达,MTT法绘制细胞生长曲线实验显示转染重组质粒PcDNA 3.1-PDGF-GAL的N-2a细胞增殖速度显著低于对照组(P<0.05),相比对照组未转染正常细胞,内源性过表达的GAL能使N-2a细胞周期阻滞在G0/G1期而且对N-2a细胞凋亡有明显的促进作用。结论真核表达载体PcDNA 3.1-PDGF-GAL在转染N-2a细胞中可高表达GAL蛋白,并对N-2a细胞的增殖和凋亡均有影响,为进一步研究GAL的生物学作用奠定了基础。

Objective To construct an N-2a cell line stably expressing PcDNA 3.1-platelet derived growth factor-galanin （GAL） and explore the effect of over-expressed GAL on proliferation and apoptosis of N-2a cell in vitro. Methods The vector containing the target gene was transfected into N-2a cells by liposome, and cell clones stably over-expressing GAL was obtained via G418 screening. GAL mRNA and protein levels were determined by reverse transcriotion-polymerase chain reaction （RT-PCR） and Western blot. The proliferation of N-2a cells was detected by MTY. The cell cycle and apoptosis were detected by flow cytometry. Results RT-PCT and Western blot indicated that GAL genes were highly expressed in the transfected N-2a cells （i. e. GAL-N-2a） . As shown by MTI＇, the proliferation of the N-2a cells transfected with PcDNA 3.1-PDGF-GAL was significantly slower than the control group （P 〈 0.05 ） . Compared with the non-transfected cells in the control group, the N- 2a cells with endogenously overexpressed GAL were arrested at the G0/G1 phases, and the over-expressed GAL protein significantly induced the N-2a cell apoptosis in a concentration-dependent fashion. Conclusion Eukaryotic ex- pression vector PcDNA 3.1-PDGF-GAL can encode the expression of GAL in N-2a cells. Aslo, it can inhibit cell proliferation and promote the cell apoptosis.