CAJ | 학술논문

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。
배경：전기연구발현，오사타정대RANKL유도RAW264.7세포활화위성숙파골세포구유일정적억제작용，병가강저기질금속단백매9적표체，단기대취갑기병희산갑지골수니촉성골세포조망효응시부유간예작용상불청초。 　　목적：탐토오사타정대골수니과립유도MC3T3-E1서전성골세포조망급Ⅰ형효원、골개소、기질금속단백매2 mRNA표체적영향。 　　방법：취제륙칠대생장상태량호적MC3T3-E1서전성골세포，분4조배양，골수니조가입1 g/L적취갑기병희산갑지골수니현액；고、저제량오사타정조재가입1g/L 적취갑기병희산갑지골수니현액후，재분별가입5000，500 U/mL적오사타정；설치단독배양적세포위공백대조。MTT법검측세포증식활성，천소홍염색검측세포광화정도，류식세포의검측세포조망솔，반정량RT-PCR검측세포중Ⅰ형효원、골개소、기질금속단백매2 mRNA적표체。 　　결과여결론：여공백대조조비교，골수니억제료MC3T3-E1세포증식활성(P<0.05)，촉진료세포조망(P<0.05)，재가입불동농도오사타정후，세포증식활성축점증강(P<0.05)，조망솔강저(P<0.05)，차정제량시간의뢰관계；골수니강저료세포중Ⅰ형효원、골개소표체(P <0.05)，승고료기질금속단백매2표체(P <0.05)，재가입5000 U/mL오사타정후，세포기질금속단백매2표체강저，Ⅰ형효원、골개소표체승고(P<0.05)。골수니조무광화결절，기타조균유광화결절형성。결과표명오사타정대골수니유도MC3T3-E1서전성골세포조망구유일정적억제작용，가촉진성골세포중Ⅰ형효원、골개소적생성，병강저기질금속단백매2적표체수평。
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 (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 decreased (P<0.05), showing the dose and time-dependent relation. Type I col agen and osteocalcin mRNA expression levels both significantly decreased after co-culture in PMMA group compared with the blank group (P<0.05), matrix metal oproteinase-2 mRNA expression level, however, significantly increased (P<0.05). After intervention with 5000 U/mL ulinastatin, type I col agen and osteocalcin mRNA expression levels both significantly increased, while matrix metal oproteinase-2 mRNA expression level significantly decreased (P<0.05). PMMA group showed no obvious mineralization nodules. Yet, mineralization nodules were formed in the blank group, high and low dose ulinastatin groups. These results indicate that ulinastatin could have the inhibitory effect on the PMMA-induced MC3T3-E1 mouse preosteoblast apoptosis, and it could promote type I col agen and osteocalcin mRNA expression and yet suppress matrix metal oproteinase-2 mRNA expression.