Hnat Michael D, Meadows Juliana W, Brockman Diane E, Pitzer Brad, Lyall Fiona, Myatt Leslie
Department of Obstetrics and Gynecology, University of Cincinnati, College of Medicine, Cincinnati, OH, USA.
Am J Obstet Gynecol. 2005 Sep;193(3 Pt 1):836-40. doi: 10.1016/j.ajog.2005.01.059.
The purpose of this study was to compare immunohistochemical expression of heat shock protein-70 (hsp70), a marker for oxidative stress, and 4-hydroxy-2-nonenal adducts (HNE), a marker for lipid peroxidation, in placental villous tissue of normotensive, preeclampsia, and intrauterine growth restricted (IUGR) pregnancies.
Placentas were collected and flash frozen in liquid nitrogen after delivery from normotensive pregnancies (n=5), and pregnancies complicated by preeclampsia (n=5), IUGR (n=5), and preeclampsia plus IUGR (n=4). Cryosections were cut and immunostained with polyclonal anti-hsp70 and monoclonal anti-HNE antibodies using Vectastain Elite ABC kit. Normal rabbit serum or mouse IgG were used as negative controls. Three independent observers, blinded to identity of tissue, examined each slide to identify cellular localization and intensity of the immunostaining. Western blot analysis and scanning densitometry were used to quantify and compare the amount of hsp70 and HNE adducts present in tissue homogenates.
Positive immunostaining for both antibodies was observed in cytoplasm of syncytiotrophoblasts, extravillous trophoblasts, vascular smooth muscle, and endothelial cells for all groups. Expression of hsp70 and HNE adducts was reported as observers' mean stained intensity. Overall, kappa showed good agreement between observers. Immunostaining intensity was similar in all tissue types for each group with the exception that immunostaining was significantly more intense in the vascular endothelium of the preeclamptic group for HNE adducts (P=.02) and significantly less intense in the IUGR group for hsp70 (P=.013). Scanning densitometric analysis of the Western blots showed no significant difference in total hsp70 and HNE adducts expression in all 4 tissue groups.
Immunohistochemistry showed local changes for oxidative stress and lipid peroxidation in the vascular endothelium from placentas of preeclamptic and IUGR pregnancies. However, these changes were masked when studying tissue homogenates.
本研究旨在比较热休克蛋白-70（hsp70，一种氧化应激标志物）和4-羟基-2-壬烯醛加合物（HNE，一种脂质过氧化标志物）在血压正常、子痫前期及胎儿宫内生长受限（IUGR）妊娠的胎盘绒毛组织中的免疫组化表达情况。
收集血压正常妊娠（n = 5）、子痫前期妊娠（n = 5）、IUGR妊娠（n = 5）以及子痫前期合并IUGR妊娠（n = 4）分娩后的胎盘，并在液氮中速冻。制备冰冻切片，使用Vectastain Elite ABC试剂盒，用多克隆抗hsp70抗体和单克隆抗HNE抗体进行免疫染色。用正常兔血清或小鼠IgG作为阴性对照。三位独立观察者在不知组织身份的情况下检查每张切片，以确定免疫染色的细胞定位和强度。采用蛋白质印迹分析和扫描光密度测定法对组织匀浆中hsp70和HNE加合物的含量进行定量和比较。
所有组的合体滋养层细胞、绒毛外滋养层细胞、血管平滑肌和内皮细胞的细胞质中均观察到两种抗体的阳性免疫染色。hsp70和HNE加合物的表达以观察者的平均染色强度表示。总体而言，观察者之间kappa显示出良好的一致性。除子痫前期组HNE加合物在血管内皮中的免疫染色明显更强（P = 0.02），IUGR组hsp70的免疫染色明显更弱（P = 0.013）外，每组所有组织类型的免疫染色强度相似。蛋白质印迹的扫描光密度分析显示，所有4个组织组中hsp70和HNE加合物的总表达无显著差异。
免疫组化显示子痫前期和IUGR妊娠胎盘血管内皮中氧化应激和脂质过氧化的局部变化。然而，在研究组织匀浆时，这些变化被掩盖了。
