乌司他丁对骨水泥颗粒诱导MC3T3-E1鼠前成骨细胞凋亡的干预作用

背景：前期研究发现，乌司他丁对RANKL诱导RAW264.7细胞活化为成熟破骨细胞具有一定的抑制作用，并可降低基质金属蛋白酶9的表达，但其对聚甲基丙烯酸甲酯骨水泥促成骨细胞凋亡效应是否有干预作用尚不清楚。 目的：探讨乌司他丁对骨水泥颗粒诱导MC3T3-E1鼠前成骨细胞凋亡及Ⅰ型胶原、骨钙素、基质金属蛋白酶2 mRNA表达的影响。 方法：取第六七代生长状态良好的MC3T3-E1鼠前成骨细胞，分4组培养，骨水泥组加入1 g/L的聚甲基丙烯酸甲酯骨水泥悬液；高、低剂量乌司他丁组在加入1g/L 的聚甲基丙烯酸甲酯骨水泥悬液后，再分别加入5000，500 U/mL的乌司他丁；设置单独培养的细胞为空白对照。MTT法检测细胞增殖活性，茜素红染色检测细胞矿化程度，流式细胞仪检测细胞凋亡率，半定量RT-PCR检测细胞中Ⅰ型胶原、骨钙素、基质金属蛋白酶2 mRNA的表达。 结果与结论：与空白对照组比较，骨水泥抑制了MC3T3-E1细胞增殖活性（P＜0.05），促进了细胞凋亡（P＜0.05），在加入不同浓度乌司他丁后，细胞增殖活性逐渐增强（P＜0.05），凋亡率降低（P＜0.05），且呈剂量时间依赖关系；骨水泥降低了细胞中Ⅰ型胶原、骨钙素表达（P ＜0.05），升高了基质金属蛋白酶2表达（P ＜0.05），在加入5000 U/mL乌司他丁后，细胞基质金属蛋白酶2表达降低，Ⅰ型胶原、骨钙素表达升高（P＜0.05）。骨水泥组无矿化结节，其他组均有矿化结节形成。结果表明乌司他丁对骨水泥诱导MC3T3-E1鼠前成骨细胞凋亡具有一定的抑制作用，可促进成骨细胞中Ⅰ型胶原、骨钙素的生成，并降低基质金属蛋白酶2的表达水平。

Ulinastatin intervention for polymethyl methacrylate-induced MC3T3-E1 mouse preosteoblast apoptosis

BACKGROUND：Previous studies have indicated that ulinastatin can inhibit RANKL-induced osteoclastogenesis on RAW264.7 cells and also lower matrix metal oproteinase-9 expression and activity. However, it remains be unclear whether ulinastatin has the intervention effect on polymethyl methacrylate （PMMA）-induced MC3T3-E1 mouse preosteoblast apoptosis or not. OBJECTIVE：To explore the intervention role of ulinastatin on the PMMA-induced MC3T3-E1 mouse preosteoblast apoptosis and its effects on type I col agen, osteocalcin, matrix metal oproteinase-2 mRNA expression. METHODS：MC3T3-E1 mouse preosteoblasts at passages 6 and 7 were divided into four groups：blank group＆amp;nbsp;（only cultured MC3T3-E1 mouse preosteoblast）, PMMA-induced group （MC3T3-E1 mouse preosteoblast＋1 g/L PMMA bone cement suspension）, low dose ulinastatin group （MC3T3-E1 mouse preosteoblast＋1 g/L PMMA bone cement suspension＋500 U/mL ulinastatin） and high dose ulinastatin group （MC3T3-E1 mouse preosteoblast＋1 g/L PMMA bone cement suspension＋5 000 U/mL ulinastatin）. MTT method was adopted to detect the proliferation activity of proliferative activity of MC3T3-E1 mouse preosteoblast;alizarin red staining method was used to observe mineralization nodules of MC3T3-E1 mouse preosteoblast among different groups;the change of apoptosis rate for MC3T3-E1 cells was detected by flow cytometry analysis;semi-quantitative RT-PCR was taken to analyze type I col agen, osteocalcin, matrix metal oproteinase-2 mRNA expression level in MC3T3-E1 mouse preosteoblasts among different groups. RESULTS AND CONCLUSION：Compared with the blank group, PMMA significantly inhibited the proliferation activity of MC3T3-E1 mouse preosteoblast （P〈0.05）, and however significantly promoted cells apoptosis （P〈0.05）. After addition of different concentrations of ulinastatin （500, 5 000 U/mL）, the proliferation activity of MC3T3-E1 mouse preosteoblasts significantly raised （P〈0.05）, and cells apoptosis rate significantly decr