CAJ | 학술논문

目的建立猪主动脉内皮细胞株,为以猪为供体的异种移植研究提供必要的细胞水平研究平台.方法分离健康幼猪主动脉内皮细胞并用携带链霉素抗性和猿猴病毒40大T抗原(SV40LT)基因的慢病毒转染细胞.检测SV40LT基因和α-1,3-半乳糖转移酶(α-1,3-GT)基因和d-1,3-半乳糖抗原表达.最后测定2类细胞与人血清发生反应的能力.结果聚合酶链反应(PCR)结果显示,SV40LT基因在转染后细胞内高表达(P＜0.05).原代细胞倍增时间约为21 h,而细胞株倍增时间约18h.2类细胞均具有吞噬低密度脂蛋白(LDL)和表达血管性假性血友病因子(vWF)因子特性(P＞0.05).α-1,3-GT基因和α-1,3-半乳糖抗原在2类细胞内表达均无差异[荧光强度分别为(498.2±3.5)和(500.6±3.2),P＞0.05].与人血清共培养时,随血清浓度升高,2类内皮细胞的增殖能力下降、细胞凋亡率增加,并均可结合人IgM抗体、C3和C5b-9 (P ＞0.05).结论猪动脉内皮细胞株具有与原代内皮细胞相同的形态结构和表型特征,并具有和人血清发生免疫反应的能力.
목적 건립저주동맥내피세포주,위이저위공체적이충이식연구제공필요적세포수평연구평태.방법 분리건강유저주동맥내피세포병용휴대련매소항성화원후병독40대T항원(SV40LT)기인적만병독전염세포.검측SV40LT기인화α-1,3-반유당전이매(α-1,3-GT)기인화d-1,3-반유당항원표체.최후측정2류세포여인혈청발생반응적능력.결과 취합매련반응(PCR)결과현시,SV40LT기인재전염후세포내고표체(P＜0.05).원대세포배증시간약위21 h,이세포주배증시간약18h.2류세포균구유탄서저밀도지단백(LDL)화표체혈관성가성혈우병인자(vWF)인자특성(P＞0.05).α-1,3-GT기인화α-1,3-반유당항원재2류세포내표체균무차이[형광강도분별위(498.2±3.5)화(500.6±3.2),P＞0.05].여인혈청공배양시,수혈청농도승고,2류내피세포적증식능력하강、세포조망솔증가,병균가결합인IgM항체、C3화C5b-9 (P ＞0.05).결론 저동맥내피세포주구유여원대내피세포상동적형태결구화표형특정,병구유화인혈청발생면역반응적능력.
Objective To establish pig aortic endothelial cell lines for providing necessary cell model for xenotransplantation research.Methods The cell lines were established following the transfection of primary endothelial cells isolated from the aortas of the immature pig with lentivirus carrying genes for neomycin resistance and Simian Virus 40 Large T (SV40LT) antigen.The SV40LT mRNA,α-1,3-galactosyltransferase (α-1,3-Gal) mRNA and α-1,3-galactose were detected.At last,the response between the cells and human serum was tested.Results The expression of the SV40LT mRNA in cell lines were very high (P ＜0.05).Doubling time of the primary cells and the cell lines was about 21 h and 18 h,respectively.The cells also displayed endothelial cell characteristics since they endocytosed acetylated low density lipoproteins and steadily continuously expressed the yon Willebrand factor (P ＞ 0.05).The high expression of α-1,3-Gal mRNA and α-1,3-galactose [the fluorescence intensity was (498.2 ± 3.5) and (500.6 ± 3.2),respectively] had no difference in the two types of cells (P ＞ 0.05).When co-incubation with human serum,human natural antibodies bound to the two types of cells.Conclusion These results suggest that pig endothelial cell lines retained their original morphological structure and phenotypic characterization,and also had ability to respond to human serum.