CAJ | 학술논문

目的：探寻小鼠牙乳头来源MDPC?23细胞自发形成的体外破牙细胞的培养方法。方法 MDPC?23细胞常规培养6 d，利用抗酒石酸酸性磷酸酶（TRAP）染色法与RANKL诱导RAW 264.7细胞形成的破骨细胞相比较；金相显微镜及扫描电镜（SEM）观察牙本质片上细胞形态及吸收陷窝形成情况；免疫荧光染色观察丝状肌动蛋白（F?actin）细胞骨架结构；免疫印迹、免疫荧光和（或） ELISA法检测破牙细胞形成相关蛋白RANKL、RANK、TRAF6以及破牙细胞标志蛋白TRAP、组织蛋白酶K（Cathepsin K）的表达情况。Cathepsin K和TRAP蛋白表达的灰度值比较采用两独立样本的t检验进行统计；0、2、4、6 d四个时间点RANKL分泌水平的比较采用方差分析进行统计分析。结果MDPC?23细胞常规培养6 d可自发形成少量TRAP染色阳性的多核巨细胞，其形态有别于RAW 264.7细胞形成的破骨细胞；自发形成的多核巨细胞仅能在牙本质片上形成少量较浅的吸收陷窝；免疫荧光结果显示，自发形成的类破牙细胞具有破牙细胞特征性丝状肌动蛋白环结构；免疫印迹、免疫荧光和（或）ELISA结果表明，MDPC?23细胞体外常规培养下表达RANK、TRAF6蛋白并自分泌RANKL，第0、2、4、6天RANKL分泌水平总体差异有统计学意义（F=5.373，P=0.026），第2天RANKL分泌水平较第0天增加（P=0.007）；第6天TRAP及Cathepsin K蛋白表达上调（tTRAP=5.033，PTRAP=0.0024；tCathepsin K=12.95，PCathepsin K=0.0002）。结论 MDPC?23细胞体外常规培养下可自发形成少量吸收能力较弱的TRAP染色阳性的多核巨细胞，具有特征性肌动蛋白环结构，同时能上调破牙细胞特征性蛋白TRAP和Cathepsin K表达，自分泌RANKL并表达RANK、TRAF6蛋白，是与成熟破牙细胞形态、结构及蛋白表达相似的类破牙细胞。
목적：탐심소서아유두래원MDPC?23세포자발형성적체외파아세포적배양방법。방법 MDPC?23세포상규배양6 d，이용항주석산산성린산매（TRAP）염색법여RANKL유도RAW 264.7세포형성적파골세포상비교；금상현미경급소묘전경（SEM）관찰아본질편상세포형태급흡수함와형성정황；면역형광염색관찰사상기동단백（F?actin）세포골가결구；면역인적、면역형광화（혹） ELISA법검측파아세포형성상관단백RANKL、RANK、TRAF6이급파아세포표지단백TRAP、조직단백매K（Cathepsin K）적표체정황。Cathepsin K화TRAP단백표체적회도치비교채용량독립양본적t검험진행통계；0、2、4、6 d사개시간점RANKL분비수평적비교채용방차분석진행통계분석。결과MDPC?23세포상규배양6 d가자발형성소량TRAP염색양성적다핵거세포，기형태유별우RAW 264.7세포형성적파골세포；자발형성적다핵거세포부능재아본질편상형성소량교천적흡수함와；면역형광결과현시，자발형성적류파아세포구유파아세포특정성사상기동단백배결구；면역인적、면역형광화（혹）ELISA결과표명，MDPC?23세포체외상규배양하표체RANK、TRAF6단백병자분비RANKL，제0、2、4、6천RANKL분비수평총체차이유통계학의의（F=5.373，P=0.026），제2천RANKL분비수평교제0천증가（P=0.007）；제6천TRAP급Cathepsin K단백표체상조（tTRAP=5.033，PTRAP=0.0024；tCathepsin K=12.95，PCathepsin K=0.0002）。결론 MDPC?23세포체외상규배양하가자발형성소량흡수능력교약적TRAP염색양성적다핵거세포，구유특정성기동단백배결구，동시능상조파아세포특정성단백TRAP화Cathepsin K표체，자분비RANKL병표체RANK、TRAF6단백，시여성숙파아세포형태、결구급단백표체상사적류파아세포。
Objective To identify whether the mouse papilla?derived MDPC?23 cells could spontaneously differentiate into odontoclast?like cells and function as odontoclasts. Methods MDPC?23 cells and RAW 264.7 cells stimulated with RANKL were cultured for 6 and 4 days respectively,then the cells were stained for TRAP. The TRAP?positive multinuclear cells induced by MDPC?23 cells were observed and compared with osteoclasts by RAW 264.7 cells. The resorption pits and morphology of MDPC?23 cells on the dentin discs were further determined by metallurgical microscope and scanning electron microscope(SEM). F?actin rings stained with phalloidin were photographed using a confocal microscope. Western blot analysis or immunostaining was used to investigate the protein expression level of odontoclast?specific markers TRAP and Cathepsin K as well as the key protein in odontoclastgenesis—RANKL, RANK and TRAF6. The secretion of RANKL was measured by ELISA in the supernatant harvested from MDPC?23 cells. Two independent samples t test was used to statistically analyze the Cathepsin K and TRAP expression. Statistical analysis of the secretion of RANKL on day 0,2,4,6 was performed using one?way ANOVA. Results MDPC?23 cells could spontaneously differentiate into a small amount of TRAP?positive multinuclear cells on day 6,which were morphologically different from osteoclasts induced in RAW 264.7 by RANKL. The resorption pits by these multinuclear cells on the dentin discs were quite shallow. Immunostaining results showed the ringlike structure of F?actin in odontoclast?like cells. Western blot,immunofluorescent staining and ELISA revealed that MDPC?23 cells express RANK and TRAF6 protein and secrete RANKL. The differnce among the level of secreted RANKL from 0 to 6 days was statistically significant(F=5.373,P=0.026). In addition,RANKL release levels were upregulated on day 2 when compared with day 0(P=0.007). Protein expression of TRAP and Cathepsin K were upregulated in MDPC?23 cells on day 6(tTRAP=5.033,PTRAP=0.0024;tCathepsin K=12.95,PCathepsin K=0.0002). Conclusions Papilla?derived MDPC?23 cells could spontaneously differentiate into odontoclast?like cells,but with weak resorption capacity. RANKL, RANK and TRAF6 may play an important role in the regulation of odontoclastgenesis.