染铅大鼠胎盘一氧化氮、基质金属蛋白酶-9表达与胎盘组织超微结构的相关性

The relationship between the expression of NO,MMP-9 and placenta cytoarchitecture in lead exposed rat

目的观察NO和基质金属蛋白酶-9(MMP-9)在孕期铅暴露大鼠胎盘组织中的变化特征,及其与胎盘组织超微结构的相关性。方法108只大鼠分为4组(A、B、C组和对照组),对照组孕期饮服蒸馏水,A、B和C组分别于孕期不同阶段饮服0.025%醋酸铅溶液。原子吸收光谱法测定血铅水平。硝酸还原酶法测定胎盘组织NO含量。免疫组化法测定胎盘滋养层细胞MMP-9表达阳性率。电镜观察胎盘组织超微结构。结果①A和B组孕鼠胎盘组织NO含量均高于对照组,差异有显著统计学意义(P<0.01);②MMP-9评分4组间总体差异有显著统计学意义(H=39.226,P<0.01),以B组的MMP-9阳性表达率最高,C组最低;③各组孕鼠胎盘组织超微结构示:对照组合体滋养层细胞微绒毛致密,胞质内细胞器丰富,内质网致密,有较多的线粒体,细胞间连接和基底膜清晰完整;A组胎盘微血管内皮细胞肿胀,基底膜增厚,微绒毛突起;B组滋养层巨细胞增生,空泡化细胞岛增多;C组滋养层细胞坏死,微绒毛稀疏,线粒体数目减少和粗面内质网池扩张。结论铅暴露孕鼠胎盘组织中NO含量和MMP-9表达阳性率与胎盘组织超微结构的病理改变密切相关。

Objective To investigate the effect of lead exposure during various gestational periods on the expression of rat placental nitric oxide （NO） and matrix metalloproteinase-9 （ MMP-9 ）, and their relationship with the placenta cytoarchitecture. Methods 108 Wistar rats were randomly divided into four groups and fed with respective drinking solutions with or without 0.025% lead acetate during various gestational periods. Controls were given distilled water without lead; Group A was given distilled water containing 0. 025% lead acetate throughout days 1 to 10 of gestation. Both group B and group C were fed with the same lead acetate water during days 11 to 20 or 1 to 20 of gestation, respectively. Blood lead levels were determined by atomic absorption spectrophotometry. The levels of NO were determined by nitrate reductase test. The expression of MMP-9 in placenta was determined by immunohistochemistry. Five microscopic fields （ x 400） in each placenta were randomly selected. The total cells positive for MMP-9 were counted by same observer for the entire study and verified by a second observer blinded to the study. Five random fields per placenta were scored. The immunoreactive intensity was determined according to immunoreactive cell scores and the immunoreactive chromatosis scores. The immunoreactive cells were scored according to the nmnber of positive cells in each field ：0 （ ~〈5% ）, 1 （ -25% ） ,2 （ -50% ） , 3 （ -75% ） and 4 （ 〉 75% ）. The immunoreactive chromatosis scores were classified into three grades： hadro-chromatosis was scored 3, superficial- chromatosis was scored 1 and that between 1 and 3 was scored 2. The immunoreactive intensity was classified into four grades ： 0 （ - , negative）, - 2 （ ＋ , weakly positive）, - 4 （ ＋ ＋ , moderately positive） and 〉 5 （ ＋ ＋ ＋, strongly positive）. Placenta cytoarchitecture was examined by an unmasked observer with a transmission electron microscope （TEM, Model JEM-120OEX, Jeol Jem, Tokyo）. Pieces of placenta were fixed with 2.5% glutaraldehyde and I% osmium tetroxide, dehydrated in ethanol of gradient concentrations, and permeated using EPON812 embedding solution. Uhrathin sections were prepared and stained with uranyl acetate and lead citrate. Results @ The NO levels of the lead-exposed rat placenta were significantly higher compared to controls （P 〈 0.01 ）. @ The expressions of MMP-9 were detected as yellow- brown pigment localizing in the langhans cells and zoarium trophoblastic ceils of placental villus, mesenchymal fibroblasts, vascular endothelial cells and contractile fiber cells of vessel wall. The expression of MMP-9 in lead-exposed rats was significantly higher than that in controls（ H = 39. 226, P 〈0.01 ）. The expressions of NF-B between any two groups showed significantly differences. The highest level of expression of MMP-9 was observed in group B, while group C showed the lowest level. @ In group C, swollen placental microangium endotheliocytes, basal membrane thickening and microvilli evagination were observed under electronic microscopy. In group B, trophoblast giant corpuscles and vacuolization cells were increased in number. In group C, rat placental trophoblast showed cell necrosis, rough endoplasmic reticula appeared distended, mitochondria decreased in number and microvilli quantity decreased. Conclusions The expressions of rat placental NO and MMP-9 were associated with placenta cytoarchitectural changes.