Bonet B, Hauge-Gillenwater H, Zhu X D, Knopp R H
Universidad de San Pablo-CEU, Centro de CC Experimentales y Técnicas, Urbanización Montepríncipe, Madrid, Spain.
Proc Soc Exp Biol Med. 1998 Feb;217(2):203-11. doi: 10.3181/00379727-217-44224.
We have previously shown that LDL is oxidized by placental cells in primary tissue culture and that this process causes cytotoxicity proportional to LDL oxidation in the presence of sex steroid hormones. Here we describe further experiments linking LDL oxidation to placental cell cytotoxicity. Trophoblast and macrophages were isolated from healthy elective caesarean section placentas by enzymatic digestion and separated by centrifugation on a 40% Percoll gradient and maintained in primary culture for up to 5 days. LDL was oxidized by exposure to 5 microM CuCl2, cells were incubated in the absence of albumen to favor oxidation, and cytotoxicity was measured by 51Cr release from prelabelled cells and cell protein content. Native LDL was oxidized by both cell types with a 10%-50% increase in lipid peroxides and an approximately 4-fold increase in TBARS formation. Increasing concentrations of native LDL and oxidized-LDL increased 51Cr release and diminished protein content in cells incubated in HAM's F-10 medium. Addition of 5 microM Cu2+ augmented cytotoxicity of LDL in macrophage and trophoblast culture, but more in macrophages than trophoblast. Cytotoxicity was diminished by adding 0.001-0.1 mM EDTA to the system, diminishing 51Cr release from 91 +/- 0.5 to 40.8 +/- 1.0% in macrophages and 54.2 +/- 1.2 to 33.1 +/- 1.3% in trophoblast (P < 0.001 in both instances). Similarly, the absence of transition metal ion in culture (MEM medium) blocked an increase in 51Cr release compared to its presence (HAM's F-10 medium). An antioxidant, butylated hydroxytoluene, diminished 51Cr release and LDL electrophoretic mobility in HAM's F-10 medium in placental macrophage culture. LDL oxidation injures placental macrophages and trophoblast, the former more than the latter. The process is LDL- and transition metal ion-dependent and is inhibited by antioxidant. This model of LDL oxidation and placental cell damage in vitro provides a basis for studying mechanisms of placental dysfunction and senescence in human pregnancy.
我们之前已经表明，低密度脂蛋白（LDL）在原代组织培养中会被胎盘细胞氧化，并且在存在性类固醇激素的情况下，这一过程会导致与LDL氧化成正比的细胞毒性。在此，我们描述了将LDL氧化与胎盘细胞毒性联系起来的进一步实验。通过酶消化从健康择期剖宫产的胎盘中分离出滋养层细胞和巨噬细胞，并在40%的Percoll梯度上进行离心分离，然后在原代培养中维持长达5天。通过暴露于5微摩尔的氯化铜（CuCl2）来氧化LDL，在无白蛋白的情况下孵育细胞以促进氧化，并通过预标记细胞的51铬（51Cr）释放和细胞蛋白质含量来测量细胞毒性。两种细胞类型均能氧化天然LDL，脂质过氧化物增加10%-50%，硫代巴比妥酸反应物（TBARS）形成增加约4倍。在HAM's F-10培养基中孵育的细胞中，天然LDL和氧化型LDL浓度的增加会导致51Cr释放增加，蛋白质含量减少。添加5微摩尔的铜离子（Cu2+）会增强巨噬细胞和滋养层细胞培养中LDL的细胞毒性，但对巨噬细胞的增强作用比对滋养层细胞更强。向系统中添加0.001-0.1毫摩尔的乙二胺四乙酸（EDTA）可降低细胞毒性，使巨噬细胞中51Cr的释放从91±0.5%降至40.8±1.0%，滋养层细胞中从54.2±1.2%降至33.1±1.3%（两种情况均P<0.001）。同样，与存在过渡金属离子（HAM's F-10培养基）相比，培养物（MEM培养基）中不存在过渡金属离子可阻止51Cr释放的增加。抗氧化剂丁基羟基甲苯可降低胎盘巨噬细胞培养的HAM's F-10培养基中51Cr的释放和LDL的电泳迁移率。LDL氧化会损伤胎盘巨噬细胞和滋养层细胞，前者比后者更严重。该过程依赖于LDL和过渡金属离子，并受到抗氧化剂的抑制。这种体外LDL氧化和胎盘细胞损伤的模型为研究人类妊娠中胎盘功能障碍和衰老的机制提供了基础。
