生殖细胞膜脂筏中精原糖脂质的分离纯化及初步鉴定  

Purification and Preliminary Identification of Seminolipid in the Lipid-raft of Germ Cell Membrane

作　　者：张彦龙[1] 

机构地区：[1]东北林业大学野生动物资源学院动物医学系,哈尔滨150040

出　　处：《中国医科大学学报》2011年第1期38-41,共4页Journal of China Medical University

摘　　要：目的哺乳动物的硫酸化糖脂质由负电荷硫酸离子和两亲性的鞘糖脂组成,硫酸盐半乳糖神经酰胺的硫酸糖脂质和硫酸盐半乳糖烷基甘油酯的精原糖脂质是存在与哺乳动物的两种主要糖脂质,为验证它是否以脂筏形式存在于生殖细胞膜上,提纯生殖细胞的精原糖脂质及其脂筏。方法应用DEAE-Sephadex A-25和硅胶交替层析法,提纯牛的睾丸中精原糖脂质;在裂解缓冲液下应用匀浆机裂解生殖细胞,在5%～40%的蔗糖密度梯度离心下,提取生殖细胞中的脂筏;在氯仿/甲醇/水=10/20/1溶剂下分离脂筏的脂类和蛋白质,经薄层层析(TLC)和蛋白电泳(SDS-PAGE)检测精原糖脂质存在方式。结果经离子交换层析和硅胶层析,获得高纯度的精原糖脂质;经蔗糖梯度离心,在离心管中可见的脂筏两条带被回收,经薄层层析(TLC)检验,精原糖脂质存在于生殖细胞膜脂筏中。结论由此推测精原糖脂质在生殖细胞细胞膜内,作为一个为受体信号传导和运输的功能性平台,并作为脂筏的组成部分而发挥关键作用。Objective The sulfatides of mammals are composed of anionic and amphiphilic glycosphingolipids （GSLs）.Sulfated galactosylceramide （galactosylsulfatide,SM4s;Sulfatide） and sulfated galactosylalkylacylglycerol （seminolipid,SM4g） are two major sulfoglycolipids in mammals.To certify the seminolipid in the germ cell membrane as lipid raft,we purified the seminolipid in the germ cells.Methods Seminolipid was purified the bovine testicles by silica gel chromatography and DEAE-Sephadex A-25.The germ cells were separated with a refiner in lysate buffer and the lipid rafts were purified by gradient centrifugation of the 5-40% sucrose density.Finally,protein and lipid of the lipid rafts were separated by the Chloroform / methanol / water = 0/20/1 solvent and then subjected to thin-layer chromatography （TLC） and SDS-PAGE to confirm the existence of the seminolipid.Results We obtained highly pure seminolipid by the ion-exchange column chromatography and silica gel chromatography.Two bands of lipid rafts were eluted from the centrifuge tube by the sucrose gradient centrifugation.It was found that the seminolipid exists in the lipid rafts of germ cell membrane by the test of thin-layer chromatography.Conclusion Seminolipid exists in the germ cell membrane and plays an important role in receptor signal tranduction as the constituent of the lipid raft.

关 键 词：精原糖脂质 生殖细胞 层析 蔗糖梯度 脂筏 

分 类 号：R322.8[医药卫生—人体解剖和组织胚胎学]