Cleverley D Z, Geller H M, Lenard J
Department of Physiology and Biophysics, UMDNJ-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
Exp Cell Res. 1997 Jun 15;233(2):288-96. doi: 10.1006/excr.1997.3573.
The patented cell line from the cabbage looper Trichoplusia ni (High Five from Invitrogen) was found to grow readily under cholesterol-free (CF) culture conditions. Cellular cholesterol became undetectable by CF passage 4, while growth rate and overall cell morphology remained unaffected for at least 59 CF passages. The Golgi apparatus in CF cells was significantly smaller than in control cells, and the CF cells also concentrated a ceramide-based fluorescent Golgi marker to a greater extent, but endoplasmic reticulum morphology appeared unaffected. Two proteins were expressed in High Five cells from recombinant baculoviruses under CF and control conditions: the vesicular stomatitis virus (VSV) fusion glycoprotein G and the influenza virus ion channel M2. Both proteins were expressed in comparable amounts in CF and control cells. Both were properly assembled and transported to the plasma membrane in CF cells, indicating the presence of functional Golgi. Wild-type G protein expression resulted in extensive syncytia formation in both CF and control cells, showing that cholesterol is not required for VSV fusion. However, a mutant G protein lacking six transmembrane domain residues was inactive in both CF and control cells. Influenza M2 protein was functional in control cells, as indicated by its amantadine-inhibitable cytotoxicity, but cytotoxicity was absent in CF cells expressing this protein, indicating a cholesterol-dependence for the cytotoxic action of this protein. CF and control cells were both infectible with VSV. However, infected cell centers were modestly decreased (ca. 3.5-fold) in CF cells. CF cells offer a convenient and novel approach to the study of specific cholesterol functions.
研究发现，来自粉纹夜蛾（Trichoplusia ni）的专利细胞系（Invitrogen公司的High Five细胞系）在无胆固醇（CF）培养条件下易于生长。到CF传代4次时，细胞内胆固醇已无法检测到，而生长速率和整体细胞形态在至少59次CF传代过程中未受影响。CF细胞中的高尔基体明显小于对照细胞，并且CF细胞还能更显著地浓缩一种基于神经酰胺的荧光高尔基体标记物，但内质网形态似乎未受影响。在CF和对照条件下，两种蛋白质通过重组杆状病毒在High Five细胞中表达：水泡性口炎病毒（VSV）融合糖蛋白G和流感病毒离子通道M2。这两种蛋白质在CF细胞和对照细胞中的表达量相当。它们在CF细胞中均能正确组装并转运至质膜，表明存在功能性高尔基体。野生型G蛋白的表达在CF细胞和对照细胞中均导致广泛的多核细胞形成，表明VSV融合不需要胆固醇。然而，一种缺少六个跨膜结构域残基的突变型G蛋白在CF细胞和对照细胞中均无活性。流感M2蛋白在对照细胞中具有功能，其金刚烷胺可抑制的细胞毒性表明了这一点，但在表达该蛋白的CF细胞中不存在细胞毒性，表明该蛋白的细胞毒性作用依赖胆固醇。CF细胞和对照细胞均能被VSV感染。然而，CF细胞中感染细胞中心数量略有减少（约3.5倍）。CF细胞为研究特定胆固醇功能提供了一种便捷且新颖的方法。
