Schreiber B M, Jones H V, Franzblau C
Department of Biochemistry, Boston University School of Medicine, MA 02118.
J Lipid Res. 1994 Jul;35(7):1177-86.
The expression of apolipoprotein E in cultured neonatal rabbit aortic smooth muscle cells was examined. Northern blot analysis determined that there was a single RNA transcript of approximately 1.2 kb. Moreover, a polyclonal antibody against rabbit apolipoprotein E was prepared in a goat and used in immunoprecipitations to demonstrate that the cultured cells secreted apolipoprotein E into the media. A double band typical of apolipoprotein E migrated at apparent molecular masses of 37 and 39 kDa. Analysis of steady-state levels of apolipoprotein E mRNA demonstrated that expression increased as cell seeding density increased. When examined as a function of time in culture, there were two peaks of expression evident 1 day and 8 days after seeding. The addition of beta VLDL (beta-very low density lipoprotein) to smooth muscle cells increased both [3H]thymidine incorporation into DNA as well as cell number and these increases were accompanied by a decrease in the levels of apolipoprotein E mRNA in cells treated with the lipoprotein for 1 and 7 days. After incubation of the cultures with beta VLDL for 1 week, the cells were radiolabeled with [35S]methionine and the media was subjected to immunoprecipitation with anti-apolipoprotein E. The data revealed that the amount of apolipoprotein E secreted into the media decreased in the presence of beta VLDL. In summary, these results show that apolipoprotein E expression in aortic smooth muscle cells is regulated by cell density, time in culture, cell proliferative state, and beta VLDL addition. These observations may have relevance to the conditions that are known to accompany the development of the atherosclerotic lesion.
检测了载脂蛋白E在培养的新生兔主动脉平滑肌细胞中的表达。Northern印迹分析确定存在一条约1.2 kb的单一RNA转录本。此外，用山羊制备了抗兔载脂蛋白E的多克隆抗体，并用于免疫沉淀，以证明培养的细胞将载脂蛋白E分泌到培养基中。载脂蛋白E典型的双链在表观分子量为37 kDa和39 kDa处迁移。对载脂蛋白E mRNA的稳态水平分析表明，其表达随着细胞接种密度的增加而增加。当作为培养时间的函数进行检测时，接种后1天和8天出现两个明显的表达峰值。向平滑肌细胞中添加β-VLDL（β-极低密度脂蛋白）可增加[3H]胸苷掺入DNA以及细胞数量，并且在用脂蛋白处理1天和7天的细胞中，这些增加伴随着载脂蛋白E mRNA水平的降低。用β-VLDL孵育培养物1周后，用[35S]甲硫氨酸对细胞进行放射性标记，并用抗载脂蛋白E对培养基进行免疫沉淀。数据显示，在β-VLDL存在的情况下，分泌到培养基中的载脂蛋白E量减少。总之，这些结果表明，主动脉平滑肌细胞中载脂蛋白E的表达受细胞密度、培养时间、细胞增殖状态和β-VLDL添加的调节。这些观察结果可能与已知伴随动脉粥样硬化病变发展的情况相关。
