弱激光照射对受张应力刺激的人牙周膜细胞成骨分化相关蛋白表达的影响
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摘要
目的通过建立人牙周膜细胞(hPDLCs)体外张应力加载模型,探讨弱激光照射(LLLI)对受张应力刺激的hPDLCs成骨分化相关蛋白表达的影响。方法取健康青少年(12~16岁)因正畸治疗拔除的双尖牙根中1/3牙周膜组织,进行hPDLCs原代分离培养。取第4代细胞,随机分为空白组(A组)、张应力加载组(B组)和张应力加载并LLLI组(C组),接种于6孔板中。采用免疫印迹法检测加力前及加力后2、6、12、24h时各组细胞成骨分化相关蛋白碱性磷酸酶(ALP)和核心结合因子(Runx2)的表达。结果 C组细胞加力2h时ALP即出现高表达,6h时Runx2出现高表达,而B组细胞两指标则随时间延长表达量逐渐增高,至24h时达到峰值。加力后不同时间ALP、Runx2蛋白表达量C组>B组>A组,各组之间差异有统计学意义(F=7.29~71.19,P<0.05)。结论LLLI能够加快并增强受周期性张应力刺激的hPDLCs成骨分化相关蛋白的表达。
Objective To establish an in vitro tensile stress loading model of human periodontal ligament cells(hPDLCs),and to investigate the effect of low-level laser irradiation(LLLI)on the expression of proteins involved in osteogenic differentiation of hPDLCs stimulated by tensile stress. Methods One third of periodontal tissue in bicuspid root was collected from healthy adolescents(aged 12-16years)who underwent orthodontic treatment,and primary hPDLCs were isolated and cultured.The fourth-generation cells were divided into three groups:blank group(group A),tensile stress loading group(group B),and LLLI+tensile stress loading group(group C)and inoculated in 6-well plates.Western blotting was used to measure the expression of alkaline phosphatase(ALP)and Runx2 involved in osteogenic differentiation before loading and at 2,6,12,and 24 hours after loading. Results Group C had high expression of ALP at 2hours and high expression of Runx2 at 6hours,while group B had gradual increases in the expression of ALP and Runx2 over the time of loading and had peak expression at 24 hours.At different time points after tensile stress loading,group C had the highest expression of ALP and Runx2,followed by group B and group A(F=7.29-71.19,P<0.05). Conclusion LLLI can accelerate and enhance the expression of ALP and Runx2 involved in osteogenic differentiation of hPDLCs stimulated by cyclic tensile stress.
Objective To establish an in vitro tensile stress loading model of human periodontal ligament cells(hPDLCs),and to investigate the effect of low-level laser irradiation(LLLI)on the expression of proteins involved in osteogenic differentiation of hPDLCs stimulated by tensile stress. Methods One third of periodontal tissue in bicuspid root was collected from healthy adolescents(aged 12-16years)who underwent orthodontic treatment,and primary hPDLCs were isolated and cultured.The fourth-generation cells were divided into three groups:blank group(group A),tensile stress loading group(group B),and LLLI+tensile stress loading group(group C)and inoculated in 6-well plates.Western blotting was used to measure the expression of alkaline phosphatase(ALP)and Runx2 involved in osteogenic differentiation before loading and at 2,6,12,and 24 hours after loading. Results Group C had high expression of ALP at 2hours and high expression of Runx2 at 6hours,while group B had gradual increases in the expression of ALP and Runx2 over the time of loading and had peak expression at 24 hours.At different time points after tensile stress loading,group C had the highest expression of ALP and Runx2,followed by group B and group A(F=7.29-71.19,P<0.05). Conclusion LLLI can accelerate and enhance the expression of ALP and Runx2 involved in osteogenic differentiation of hPDLCs stimulated by cyclic tensile stress.
引文
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[12]HAXSEN V,SCHIKORA D,SOMMER U,et al.Relevance of laser irradiance threshold in the induction of alkaline phosphatase in human osteoblast cultures[J].Lasers Med Sci,2008,23(4):381-384.
[13]孙新华,公柏娟,叶耐永,等.弱激光照射对实验性牙齿移动骨改建影响作用的组织化学研究[J].激光杂志,2003,24(1):74-75.
[14]陈莉花,陈文静,顾敏,等.大鼠牙移动过程中牙周膜中Runx2的表达[J].口腔医学,2010,30(5):286-288.
[15]PARK S J,NA K.The transfection efficiency of photosensitizer-induced gene delivery to human MSCs and internalization rates of EGFP and Runx2genes[J].Biomaterials,2012,33(27):6485-6494.
[2]SU J K,CHOU M Y,PARK Y G.Effect of low-level laser on the rate of tooth movement[J].Seminars in Orthodontics,2015,21(3):210-218.
[3]苑子艺,刘珺,伊吉翠,等.低能量激光照射对大鼠正畸牙牵张侧牙周膜细胞中p-ERK1/2表达的影响[J].青岛大学医学院学报,2016,52(3):315-318.
[4]汤楚华,施生根,牛忠英,等.酶消化组织块法原代培养人牙周膜成纤维细胞的初步研究[J].中华医学杂志,2004,84(8):656-658.
[5]彭庭莉,杨军,周继祥,等.不同张应力对体外培养的人牙周膜成纤维细胞形态和纤维型肌动蛋白改变的影响[J].第三军医大学学报,2005,27(9):898-900.
[6]WU J Y,CHEN C H,YEH L Y,et al.Low-power laser irradiation promotes the proliferation and osteogenic differentiation of human periodontal ligament cells via cyclic adenosine monophosphate[J].Int J Oral Sci,2013,5(2):85-91.
[7]DOSHI-MEHTA G,BHAD-PATIL W A.Efficacy of low-intensity laser therapy in reducing treatment time and orthodontic pain:a clinical investigation[J].Am J Orthod Dentofacial Orthop,2012,141(3):289-297.
[8]XIAOTING L,YIN T,YANGXI C.Interventions for pain during fixed orthodontic appliance therapy.A systematic review[J].Angle Orthod,2010,80(5):925-932.
[9]SHIMIZU N,MAYAHARA K,KIYOSAKI T,et al.Lowintensity laser irradiation stimulates bone nodule formation via insulin-like growth factor-Ⅰexpression in rat calvarial cells[J].Lasers Surg Med,2007,39(6):551-559.
[10]STEIN A,BENAYAHU D,MALTZ L,et al.Low-level laser irradiation promotes proliferation and differentiation of human osteoblasts in vitro[J].Photomed Laser Surg,2005,23(2):161-166.
[11]李秋实,陈英新,周延民.低能量激光治疗对成骨细胞的生物效应研究进展[J].中国激光医学杂志,2011,20(5):328-332.
[12]HAXSEN V,SCHIKORA D,SOMMER U,et al.Relevance of laser irradiance threshold in the induction of alkaline phosphatase in human osteoblast cultures[J].Lasers Med Sci,2008,23(4):381-384.
[13]孙新华,公柏娟,叶耐永,等.弱激光照射对实验性牙齿移动骨改建影响作用的组织化学研究[J].激光杂志,2003,24(1):74-75.
[14]陈莉花,陈文静,顾敏,等.大鼠牙移动过程中牙周膜中Runx2的表达[J].口腔医学,2010,30(5):286-288.
[15]PARK S J,NA K.The transfection efficiency of photosensitizer-induced gene delivery to human MSCs and internalization rates of EGFP and Runx2genes[J].Biomaterials,2012,33(27):6485-6494.