下颌功能性前伸后髁突部RANKL、MMP-2、MMP-9及VEGF水平变化的实验研究
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摘要
目的研究功能性矫正器引导大鼠下颌功能性前伸后髁突部核因子-κB受体活化因子配基(RANKL)、基质金属蛋白酶(MMP)-2、MMP-9、血管内皮生长因子(VEGF)的水平变化。方法选择健康SD大鼠20只,分为实验组和对照组,每组10只,对实验组大鼠进行下颌功能性前伸模型建造,正常对照组大鼠不做处理,自然生长。将两组大鼠于3、5、7 d时分别处死,选取髁突及周围3 mm组织作为实验标本,对两组大鼠3、5、7 d的组织标本进行检测:(1)酶联免疫吸附法检测RANKL的水平,测量牙间距离;(2)行HE染色光镜下观察组织学图像;(3)免疫组化染色光镜下观察MMP-2、MMP-9、VEGF蛋白的表达情况;(4)Western blot法检测MMP-2、MMP-9、VEGF蛋白相对表达量。结果实验组大鼠5、7 d时RANKL水平明显低于对照组(P<0.05,P<0.01),且实验组大鼠5、7 d时的牙间距离明显大于对照组(P均<0.01)。实验组大鼠5、7 d组织标本可见髁突软骨层结构紊乱、细胞排序散乱且发生软骨部分脱落,软骨厚度相比对照组大鼠普遍增加。免疫组化染色结果显示,实验组大鼠髁突部3、5 d时的MMP-2及3 d时的MMP-9、VEGF蛋白表达与对照组相比差异无统计学意义;7 d时的MMP-2和5、7 d时的MMP-9、VEGF蛋白表达明显低于对照组。Western blot结果显示,实验组大鼠髁突部3、5 d时MMP-2和3 d时MMP-9、VEGF表达水平与对照组相比差异无统计学意义(P均>0.05);5 d时MMP-2及5、7 d时MMP-9、VEGF表达水平显著低于对照组(P<0.05,P<0.01)。结论 RANKL、MMP-2、MMP-9和VEGF在下颌功能前伸后可能参与髁突部的骨重建和骨形成过程,对其水平的检测或可为髁突部的骨重建提供重要的参考价值。
Objective To study the changes of receptor activator of nuclear factor-κB ligand(RANKL),matrix metalloproteinase(MMP)-2,MMP-9 and vascular endothelial growth factor(VEGF) levels in condylar process after functional mandibular protrusion guided by functional orthodontic appliance in rats.Methods Twenty healthy SD rats were selected and were divided into experimental group and control group(n=10 each).The functional mandibular protrusion models were built in rats of experimental group.The rats in control group grew naturally without treatment.The rats in two groups were killed at 3-,5-,7-day of feeding,respectively.The tissues of condylar process and the tissues of 3 mm around it were served as experimental specimens.Selecting the tissue specimens of 3-,5-,7-day for detection:(1) RANKL was detected by enzyme-linked immunosorbent assay;interdental distance was measured;(2) histological image was observed under light microscope after HE staining;(3) expressions of MMP-2,MMP-9 and VEGF proteins were detected by immunohistochemical staining method and observed under light microscope;(4) relative expression levels of MMP-2,MMP-9 and VEGF proteins were detected by Western blot method.Results At 5-and 7-day,RANKL level in experimental group was significantly lower than that in control group(P<0.05,P<0.01),and the interdental distance in experimental group was significantly greater than that in control group(all P<0.01).Histological image showed that the structure of condylar cartilage layer was disordered,and the cell sorting was scattered with partial exfoliation of cartilage,and the cartilage thickness increased generally in the tissue specimen of experimental group at 5-and 7-day compared with control group.Immunohistochemical staining showed that there were no significant differences in the expression levels of MMP-2 protein at 3-and 5-day,MMP-9 and VEGF proteins at 3-day in condylar process between two groups,while the expression levels of MMP-2 protein at 7-day and MMP-9 and VEGF proteins at 5-and 7-day in condylar process in experimental group were significantly lower than those in control group.Western blot showed that there were no significant differences in the relative expression levels of MMP-2 protein at 3-and 5-day,MMP-9 and VEGF proteins in condylar process at 3-day between two groups(all P>0.05),while the expression levels of MMP-2 protein at 5-day,MMP-9 and VEGF proteins at 5-and 7-day in condylar process in experimental group were significantly lower than those in control group(P<0.05,P<0.01).Conclusion RANKL,MMP-2,MMP-9 and VEGF may be involved in the process of bone remodeling and bone formation of condylar process after mandibular functional protrusion,and detection of their level may provide important reference value for condylar bone reconstruction.
Objective To study the changes of receptor activator of nuclear factor-κB ligand(RANKL),matrix metalloproteinase(MMP)-2,MMP-9 and vascular endothelial growth factor(VEGF) levels in condylar process after functional mandibular protrusion guided by functional orthodontic appliance in rats.Methods Twenty healthy SD rats were selected and were divided into experimental group and control group(n=10 each).The functional mandibular protrusion models were built in rats of experimental group.The rats in control group grew naturally without treatment.The rats in two groups were killed at 3-,5-,7-day of feeding,respectively.The tissues of condylar process and the tissues of 3 mm around it were served as experimental specimens.Selecting the tissue specimens of 3-,5-,7-day for detection:(1) RANKL was detected by enzyme-linked immunosorbent assay;interdental distance was measured;(2) histological image was observed under light microscope after HE staining;(3) expressions of MMP-2,MMP-9 and VEGF proteins were detected by immunohistochemical staining method and observed under light microscope;(4) relative expression levels of MMP-2,MMP-9 and VEGF proteins were detected by Western blot method.Results At 5-and 7-day,RANKL level in experimental group was significantly lower than that in control group(P<0.05,P<0.01),and the interdental distance in experimental group was significantly greater than that in control group(all P<0.01).Histological image showed that the structure of condylar cartilage layer was disordered,and the cell sorting was scattered with partial exfoliation of cartilage,and the cartilage thickness increased generally in the tissue specimen of experimental group at 5-and 7-day compared with control group.Immunohistochemical staining showed that there were no significant differences in the expression levels of MMP-2 protein at 3-and 5-day,MMP-9 and VEGF proteins at 3-day in condylar process between two groups,while the expression levels of MMP-2 protein at 7-day and MMP-9 and VEGF proteins at 5-and 7-day in condylar process in experimental group were significantly lower than those in control group.Western blot showed that there were no significant differences in the relative expression levels of MMP-2 protein at 3-and 5-day,MMP-9 and VEGF proteins in condylar process at 3-day between two groups(all P>0.05),while the expression levels of MMP-2 protein at 5-day,MMP-9 and VEGF proteins at 5-and 7-day in condylar process in experimental group were significantly lower than those in control group(P<0.05,P<0.01).Conclusion RANKL,MMP-2,MMP-9 and VEGF may be involved in the process of bone remodeling and bone formation of condylar process after mandibular functional protrusion,and detection of their level may provide important reference value for condylar bone reconstruction.
引文
[1] 何琴,张佐,王春玲,等.下颌前移式矫治器治疗OSAHS患者引起髁突表面应力变化的三维有限元研究[J].宁夏医科大学学报,2016,38(1):43-46,50,封2.
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[3] 秦波,润丹,徐卫华,等.大鼠下颌持续前导后髁突软骨骨形态发生蛋白-2的表达[J].贵州医科大学学报,2016,41(5):570-572.
[4] 蔺栋鹏,田海锁,赵天一,等.下颌髁突软骨与股骨头软骨体外生长发育的比较研究[J].实用口腔医学杂志,2015,31(3):369-373.
[5] 施美霞,高婧,许艳华,等.下颌髁突软骨生长改建基因调控的研究进展[J].医学综述,2018,24(6):1052-1056,1061.
[6] Chrcanovic BR.Surgical versus non-surgical treatment of mandibular condylar fractures:a meta-analysis[J].Int J Oral Maxillofac Surg,2015,44(2):158-179.
[7] 梁鑫,张波,刘苹,等.FGFR2功能增强对小鼠下颌骨髁突发育影响[J].遗传,2015,37(6):561-567.
[8] 张亚梅,雷勇华,邹双双,等.功能性矫治器前伸下颌对颞下颌关节影响的研究进展[J].中华口腔正畸学杂志,2016,23(2):94-97.
[9] Gredes T,Mack H,Spassov A,et al.Changes in condylar cartilage after anterior mandibular displacement in juvenile pigs[J].Arch Oral Biol,2012,57(6):594-598.
[10] 汪浩然,左艳萍,闫宝勇,等.牙本质基质蛋白-1参与下颌功能前伸过程中髁突软骨增殖的实验研究[J].中国实用口腔科杂志,2017,10(9):554-557.
[11] 张钧,江莉婷,章翊钟,等.下颌骨功能性偏斜对发育期大鼠髁突软骨胶原改建的影响[J].口腔颌面外科杂志,2015,25(1):12-18.
[12] 辛江波,袁渭,兴辰,等.两种颌后切口入路治疗下颌骨髁突骨折的比较研究[J].中国美容医学,2015,24(13):35-37.
[13] 杨帅,李雪,高洁,等.下颌持续前导对成年大鼠髁突软骨骨形态发生蛋白表达及超微结构的影响[J].华西口腔医学杂志,2016,34(6):632-638.
[14] Xiong J,Piemontese M,Onal M,et al.Osteocytes,not Osteoblasts or Lining Cells,are the Main Source of the RANKL Required for Osteoclast Formation in Remodeling Bone[J].PLoS One,2015,10(9):e0138189.
[15] 黎彦龙,何明,陈秉雄,等.OPG-RANKL-RANK信号系统是调节破骨细胞及骨质疏松症的重要途径[J].中国组织工程研究,2015,19(24):3894-3898.
[16] 潘克清,张朋梅,邓婧,等.OPG/RANK/RANKL在牙周炎联合血管钙化大鼠牙髓组织中的表达及意义[J].上海口腔医学,2016,25(4):391-395.
[17] 付颖,董庆文,王稚英,等.富血小板纤维蛋白对下颌骨牵引成骨区RANKL表达的影响[J].中国临床解剖学杂志,2016,34(3):322-325,330.
[18] 姚金丹,韩光红,胡敏,等.IL-17对破骨细胞中MMP-9表达水平的影响及其意义[J].吉林大学学报(医学版),2016,42(3):462-466,后插1.
[19] 张帆,余国玺.不同状态人牙髓组织中基质金属蛋白酶的表达水平及临床意义[J].解放军医药杂志,2017,29(7):50-53.
[20] 刘红,卢军,张真,等.MMP-2和TIMP-2参与大鼠实验性正畸牙齿移动及牙根吸收机制的研究[J].牙体牙髓牙周病学杂志,2017,27(7):382-386.
[21] 张向凤,邓锋,张翼,等.功能性下颌偏斜青春期模型大鼠髁突软骨中血管内皮细胞生长因子的表达[J].中国组织工程研究,2015,19(20):3141-3146.
[22] 侯丽雯,焦婷,谢明,等.血管内皮生长因子与牙发生发育的关系[J].国际口腔医学杂志,2016,43(5):605-608.
[2] Berner T,Essig H,Schumann P,et al.Closed versus open treatment of mandibular condylar process fractures:a meta-analysis of retrospective and prospective studies[J].J Craniomaxillofac Surg,2015,43(8):1404-1408.
[3] 秦波,润丹,徐卫华,等.大鼠下颌持续前导后髁突软骨骨形态发生蛋白-2的表达[J].贵州医科大学学报,2016,41(5):570-572.
[4] 蔺栋鹏,田海锁,赵天一,等.下颌髁突软骨与股骨头软骨体外生长发育的比较研究[J].实用口腔医学杂志,2015,31(3):369-373.
[5] 施美霞,高婧,许艳华,等.下颌髁突软骨生长改建基因调控的研究进展[J].医学综述,2018,24(6):1052-1056,1061.
[6] Chrcanovic BR.Surgical versus non-surgical treatment of mandibular condylar fractures:a meta-analysis[J].Int J Oral Maxillofac Surg,2015,44(2):158-179.
[7] 梁鑫,张波,刘苹,等.FGFR2功能增强对小鼠下颌骨髁突发育影响[J].遗传,2015,37(6):561-567.
[8] 张亚梅,雷勇华,邹双双,等.功能性矫治器前伸下颌对颞下颌关节影响的研究进展[J].中华口腔正畸学杂志,2016,23(2):94-97.
[9] Gredes T,Mack H,Spassov A,et al.Changes in condylar cartilage after anterior mandibular displacement in juvenile pigs[J].Arch Oral Biol,2012,57(6):594-598.
[10] 汪浩然,左艳萍,闫宝勇,等.牙本质基质蛋白-1参与下颌功能前伸过程中髁突软骨增殖的实验研究[J].中国实用口腔科杂志,2017,10(9):554-557.
[11] 张钧,江莉婷,章翊钟,等.下颌骨功能性偏斜对发育期大鼠髁突软骨胶原改建的影响[J].口腔颌面外科杂志,2015,25(1):12-18.
[12] 辛江波,袁渭,兴辰,等.两种颌后切口入路治疗下颌骨髁突骨折的比较研究[J].中国美容医学,2015,24(13):35-37.
[13] 杨帅,李雪,高洁,等.下颌持续前导对成年大鼠髁突软骨骨形态发生蛋白表达及超微结构的影响[J].华西口腔医学杂志,2016,34(6):632-638.
[14] Xiong J,Piemontese M,Onal M,et al.Osteocytes,not Osteoblasts or Lining Cells,are the Main Source of the RANKL Required for Osteoclast Formation in Remodeling Bone[J].PLoS One,2015,10(9):e0138189.
[15] 黎彦龙,何明,陈秉雄,等.OPG-RANKL-RANK信号系统是调节破骨细胞及骨质疏松症的重要途径[J].中国组织工程研究,2015,19(24):3894-3898.
[16] 潘克清,张朋梅,邓婧,等.OPG/RANK/RANKL在牙周炎联合血管钙化大鼠牙髓组织中的表达及意义[J].上海口腔医学,2016,25(4):391-395.
[17] 付颖,董庆文,王稚英,等.富血小板纤维蛋白对下颌骨牵引成骨区RANKL表达的影响[J].中国临床解剖学杂志,2016,34(3):322-325,330.
[18] 姚金丹,韩光红,胡敏,等.IL-17对破骨细胞中MMP-9表达水平的影响及其意义[J].吉林大学学报(医学版),2016,42(3):462-466,后插1.
[19] 张帆,余国玺.不同状态人牙髓组织中基质金属蛋白酶的表达水平及临床意义[J].解放军医药杂志,2017,29(7):50-53.
[20] 刘红,卢军,张真,等.MMP-2和TIMP-2参与大鼠实验性正畸牙齿移动及牙根吸收机制的研究[J].牙体牙髓牙周病学杂志,2017,27(7):382-386.
[21] 张向凤,邓锋,张翼,等.功能性下颌偏斜青春期模型大鼠髁突软骨中血管内皮细胞生长因子的表达[J].中国组织工程研究,2015,19(20):3141-3146.
[22] 侯丽雯,焦婷,谢明,等.血管内皮生长因子与牙发生发育的关系[J].国际口腔医学杂志,2016,43(5):605-608.