【作者】 刘长庚；

【导师】 凌天牖；

【作者基本信息】 中南大学 ， 内科学， 2008， 博士

【摘要】 目的:观察大鼠正畸牙移动牙周组织改建中OPG(Osteoprotegerin,护骨素)、RANKL(receptor activator of nuclearfactor-κB ligand,细胞核因子-κB受体活化因子配体)的表达和分布以及灯盏花对其影响。方法:选用Wistar大鼠64只,随机分为A、B、C、D四组,每组均以右侧上颌第一磨牙近中腭侧根的牙周组织为实验观察部位。A组正畸加力合并局部注射灯盏花,B组单纯正畸加力,C组单纯注射灯盏花,D组为空白对照。A、B、C组又分设1、3、7、10、14d观察小组,每小组4只鼠。每小组鼠到期处死取材,分别进行HE染色法观察各组的组织学改变,原位杂交法检测OPG和RANKL阳性表达的强度和部位。结果:①实验观察部位的组织学改变符合正常正畸牙槽骨改建的变化;B组张力侧成骨细胞增多和新骨形成,压力侧破骨细胞增多和骨吸收;A组张力侧和压力侧除上述变化更明显外,新生血管增多。②OPG表达在加力第1、3、7d的张力侧和压力侧牙周组织中均逐渐增强,而在10、14d则逐渐减弱;OPG的表达部位主要在张力侧的成骨细胞、骨衬里细胞、成牙骨质细胞、成纤维细胞和压力侧的成牙骨质细胞、成纤维细胞,但在破骨细胞中不表达;合并灯盏花注射组的OPG表达部位不变,但阳性表达有所减弱。③RANKL表达在加力第1、3、7d的张力侧和压力侧牙周组织中均逐渐增强,而在10、14d则逐渐减弱;RANKL的表达部位在张力侧和压力侧的成骨细胞、破骨细胞、骨衬里细胞、成牙骨质细胞、成纤维细胞;合并灯盏花注射组的RANKL表达部位不变,阳性表达明显增强。结论:在大鼠正畸牙移动过程中,OPG和RANKL参与了牙周组织的改建;而灯盏花能抑制OPG的表达和增加RANKL的表达,增加RANKL/OPG的比值,从而加快正畸牙移动。目的:通过MTT法、[~3H]TdR掺入法和台盼蓝拒染法体外研究不同浓度灯盏花对成骨细胞和破骨前体细胞的增殖和分化的影响。方法:体外培养MG-63成骨样细胞和RAW264.7破骨前体细胞。破骨细胞诱导分化:RAW264.7细胞以1×10~5/孔接种于6孔板,分别用50ng/ml RANKL+25ng/ml M-CSF和不同浓度(0.001～1mg/m1)的灯盏花干预,第6天TRAP染色,显微镜下计数TRAP阳性多核破骨细胞。MTT法:MG63细胞或RAW264.7细胞以1×10~4个/孔接种于96孔板,培养24小时后用含不同浓度的灯盏花的无血清的DMEM培养液干预,继续培养36小时,每孔加入MTT,继续孵育4小时,每孔加入150μl的DMSO,测570nm波长的吸光值(OD值)。[~3H]TdR掺入法:MG63细胞或RAW264.7细胞以2×10~4个/孔密度培养于24孔板中,24小时后用0、0.001、0.01、0.1、1mg/ml灯盏花干预32小时后,每孔加入[~3H]TdR,测定细胞内标记的[~3H]TdR量。台盼蓝拒染法:MG63细胞或RAW264.7细胞以2.5×10~4/ml密度接种于24孔细胞培养板,实验组用1mg/ml灯盏花干预细胞1~6天。每天取细胞台盼蓝染色,计数活细胞,连续计数6天,绘制细胞生长曲线。结果:单独应用灯盏花不能使RAW264.7细胞形成TRAP阳性多核破骨细胞,但0.01-1mg/ml浓度灯盏花均能促进RANKL和M-CSF共同作用下诱导的RAW264.7细胞分化为成熟的破骨细胞。MTT法和[~3H]TdR掺入法检测显示灯盏花促进MG-63细胞和RAW264.7细胞增殖并呈现剂量依赖性,于1mg/ml剂量时,促增殖的作用最为显著。台盼蓝拒染法检测显示,MG63和RAW264.7细胞对照组和干预组在培养时间内生长呈现上升趋势,灯盏花干预组的细胞数增加更明显,且随着时间的延长,灯盏花的促增殖作用越明显。结论:灯盏花能促进成骨细胞和破骨前体细胞的增殖并呈现剂量和时间依赖性,同时能协同RANKL和M-CSF诱导破骨前体细胞分化为成熟的破骨细胞。目的:研究灯盏花在不同浓度不同时间下对体外培养的成骨细胞和破骨前体细胞中OPG/RANKL/RANK mRNA和蛋白表达的影响,以初步探讨灯盏花对骨改建的作用及其机理。方法:体外培养MG63细胞和RAW264.7细胞,取第三代细胞分为对照组和不同的实验组,分别应用不同浓度的灯盏花(0、0.001、0.01、0.1、1mg/ml干预不同时间(12、24、48h)后,提取总RNA和蛋白质,用半定量RT-PCR方法检测MG63细胞OPGmRNA和RANKL mRNA的表达以及RAW264.7细胞RANK mRNA的表达,用Western blot方法检测MG63细胞OPG蛋白和RANKL蛋白的表达以及RAW264.7细胞RANK蛋白的表达。结果:RT-PCR和Western blot结果显示,灯盏花随浓度(0、0.001、0.01、0.1、1mg/ml)增高,呈剂量依赖性抑制MG63细胞OPGmRNA和蛋白的表达,同时呈剂量依赖性促进MG63细胞RANKL mRNA和蛋白的表达。Western blot结果还显示灯盏花呈时间依赖性地促进MG63细胞RANKL蛋白的表达。1mg/ml灯盏花干预12h、24h、48h后,RANKL蛋白量均显著增高。RT-PCR和Western blot结果显示,灯盏花随浓度(0、0.001、0.01、0.1、1mg/ml)增高,呈剂量依赖性促进RAW264.7细胞RANKmRNA和蛋白的表达。Western blot结果还显示灯盏花呈时间依赖性地促进RAW264.7细胞RANK蛋白的表达。1mg/ml灯盏花干预12h、24h、48h后,RANK蛋白量均显著增高结论:灯盏花呈剂量和时间依赖性抑制成骨细胞OPG表达,促进成骨细胞RANKL的表达和破骨前体细胞RANK的表达,提示灯盏花可能有促进骨吸收的作用。目的:分别研究灯盏花、机械牵张力以及灯盏花与机械牵张力联合作用下对体外培养的成骨细胞和破骨前体细胞中OPG/RANKL/RANKmRNA和蛋白表达的影响,以进一步探讨灯盏花对骨改建的作用及其机理。方法:体外培养MG63细胞和RAW264.7细胞,采用SXG4201型四点弯曲细胞力学加载仪对细胞进行机械牵张力刺激。分别单用机械牵张力刺激(2000μstrain,0.2Hz)、单用灯盏花(1mg/ml)干预、以及机械牵张力(2000μstrain,0.2Hz)和灯盏花(1mg/ml)联合干预MG63细胞和RAW264.7细胞不同时间(3,6,12,24h)后,提取总RNA和蛋白质,用半定量RT-PCR方法检测MG63细胞OPGmRNA和RANKL mRNA的表达以及RAW264.7细胞RANKmRNA的表达,用Western blot方法检测MG63细胞OPG蛋白和RANKL蛋白的表达以及RAW264.7细胞RANK蛋白的表达。结果:在选定的3、6、12、24h四个时间段,随着机械牵张力对细胞刺激时间的增加,MG-63细胞OPG蛋白的表达逐渐增强,MG-63细胞RANKL蛋白和RAW264.7细胞RANK蛋白的表达没有显著改变。单用机械牵张力刺激MG63细胞后OPG蛋白表达上调,而RANKL蛋白表达无明显变化;单用灯盏花干预MG63细胞后OPG蛋白表达下调,而RANKL蛋白表达增加;两者联合干预MG-63细胞后,OPG蛋白表达量减少,而RANKL蛋白表达显著增加。单用机械牵张力刺激RAW264.7细胞后RANK蛋白表达无明显改变;单用灯盏花干预RAW264.7细胞后,RANK蛋白表达增加;两者联合干预RAW264.7细胞后,RANK蛋白表达量显著增加。结论:灯盏花能拮抗机械牵张力对成骨细胞OPG分泌的刺激作用,促进机械牵张力对成骨细胞RANKL分泌的刺激作用。灯盏花能使破骨前体细胞分化为成熟破骨细胞并分泌RANK,但机械牵张力却无明显作用。更多还原

【Abstract】 Objective:To study the expression and distribution of Osteoprotegerin（OPG）,receptor activator of nuclear factor-κB ligand （RANKL） in orthodontic rebuilding of the periodontal tissues,and the effection of erigeron breviscapus（EB） injection on it.Methods:64 Wistar mice were randomly divided into 4 groups（groups A,B,C and D）.Group A was given orthodontic appliance and injected with EB.Group B was only given orthodontic appliance. Group C was only injected EB injection.Group D was the control group. Groups A,B and C was observed 1,3,7,10,14d respectively,with 4 mice in each group.We selected the maxillary first molar as experimental tooth and observe the periodontal tissues of the mesi-glossia root.After killed on schedule,each mice of all groups was made into the sections of the periodontal tissues,the sections were studied histologically by HE stain and examined the expression and distribution of OPG and RANKL by hybridization in situ.Results:.①The histological results were in line with normal orthodontic remodeling of the alveolar bone.Osteoblastic new bone formation on the tension side of A and B groups,and osteoclastic bone resorption on the pressure side of A and B groups.From A groups,the another effection of EB on orthodontic remodeling of the periodontal tissues was neovascularization.②From 1 to 7 days,the expression of OPG was gradually enhanced in the periodontal tissues on the tension side and on the pressure side.OPG was seen in the cytoplasm of osteoblasts,bone stave cells,cementoblasts and fibrablasts on the tension side,also seen in the cytoplasm of cementoblasts and fibrablasts on the pressure side,but was not seen in the cytoplasm of osteoclasts.EB did not change the distribution of OPG,but inhibited the expression of OPG.③From 1 to 7 days,the expression of RANKL was gradually enhanced in the periodontal tissues on the tension side and on the pressure side. RANKL was seen in the cytoplasm of osteoblasts,osteoclasts,bone stave cells,cementoblasts and fibrablasts on the tension side and on the pressure side.EB did not change the distribution of RANKL,but increased apparently the expression of RANKL.Conclusions:OPG and RANKL can play an important role in orthodontic rebuilding of the periodontal tissues.EB can inhibit the expression of OPG and increase the expression of RANKL,thus accelerate tooth movement. Objective:To study the effection of erigeron breviscapus（EB） with different concentration on proliferation or differentiation of MG-63 osteoblast-like cells and RAW264.7 pre-osteoclast cells in vitro by methyl thiazolyl tetrazolium（MTT） approach,[³H]TdR incorporation assay and Trypan blue excluding test.Methods:MG-63 osteoblast-like cells and RAW264.7 pre-osteoclast cells were cultured in vitro.Induction and differentiation of osteoblast:RAW264.7 cells were planted in a 6-well culture plate and every well contained 1×10⁵ cells. They were cultured with RANKL（50ng/ml）,M-CSF（25ng/ml） and EB（0 mg/ml,0.001mg/ml,0.01mg/ml,0.1mg/ml,1mg/ml）,TRAP staning and counting multinucleated osteoclast.MTT approach:MG-63 osteoblast-like cells or RAW264.7 pre-osteoclast cells were planted in a 96-well culture plate and every well contained 1×10⁴ cells.After 24h,they were cultured with EB（0 mg/ml,0.001 mg/ml,0.01 mg/ml,0.1 mg/ml,1mg/ml）.After 36h,they were dyed by MTT method and OD（optical density） value of every well was detected by enzyme linked immunodetection meter.[³H]TdR incorporation assay:MG-63 osteoblast-like cells or RAW264.7 pre-osteoclast cells were planted in a 24-well culture plate and every well contained 2×10⁴ cells.After 24h,they were cultured with EB（0 mg/ml,0.001mg/ml,0.01mg/ml,0.1mg/ml,1mg/ml）.After 32h,they were incorporated by[³H]TdR,and were detected[³H]TdR.Trypan blue excluding test:MG-63 osteoblast-like cells or RAW264.7 pre-osteoclast cells were planted in a 24-well culture plate. Experimental group were cultured with EB,they were dyed by Trypan blue,and counted cells from 1to 6 day.Then the curve of growth was drawed.Results:EB could not induce directly RAW264.7 pre-osteoclast cells into multinucleated osteoclast of TRAP positive staining,but EB（0.01-1mg/ml） could promote RANKL and M-CSF to induce RAW264.7 into multinucleated osteoclast.The result of MTT and[³H]TdR incorporation assay indicated that EB could promote dose-dependently the proliferation of MG-63 osteoblast-like cells and RAW264.7 pre-osteoclast cells in vitro.Trypan blue excluding test:The curve of growth of MG63 osteoblast-like cells and RAW264.7 pre-osteoclast cells rised gradually in the control groups and the experiment groups,and the experiment groups were apparent than the control groups.Conclusions:With some range of its concertration,EB could promote the proliferation of osteoblast in vitro in dose and time dependent.EB could promote RANKL and M-CSF to induce pre-osteoclast cells into multinucleated osteoclast. Objective:To study the effection of erigeron breviscapus（EB） with different concentrations and different intervention time on the expression of OPG/RANKL/RANK in MG-63 osteoblast-like cells and RAW264.7 pre-osteoclast cells in vitro,thus to propose the role that EB can play in bone rebuilding.Methods:MG-63 osteoblast-like cells and RAW264.7 pre-osteoclast cells were cultured in vitro.The 3^th passage of cells were divided into the control group and the different experiment group.The different EB,including 0、0.001、0.01、0.1、1.0 mg/mL concentration,were join respectively into the different groups.Total RNA and protein were respectively isolated from cells after treatment with different concentration of EB for 12,24,and 48 hours.Expressions of OPGmRNA and RANKLmRNA in MG-63 osteoblast-like cells and RANKmRNA in RAW264.7 pre-osteoclast cells were detected by semiquantitative RT-PCR..Expressions of OPG protein and RANKL protein in MG-63 osteoblast-like cells and RANK protein in RAW264.7 pre-osteoclast cells were detected by Western blot.Results:RT-PCR and Western blot indicated that EB could inhibit the expression of OPGmRNA and protein in MG-63 osteoblast-like cells in dose dependent,and EB could promote the expression of RANKLmRNA and protein in MG-63 osteoblast-like cells in dose dependent.Western blot also indicated that EB could promote the expression of RANKL protein in MG-63 osteoblast-like cells in time dependent,and RANKL protein was increased remarkbly by EB intervention after 12,24,48h.RT-PCR and Western blot indicated that EB could promote the expression of RANKmRNA and protein in RAW264.7 pre-osteoclast cells in dose dependent from 0、0.001、0.01、0.1 to 1.0 mg/mL concentration.Western blot also indicated that EB could promote the expression of RANK protein in RAW264.7 pre-osteoclast cells in time dependent,and RANK protein was increased remarkbly by EB intervention after 12,24,48h.Conclusions:EB could inhibit the secretion of OPG in osteoblasts,and could promote the secretion of RANKL in osteoblasts and the secretion of RANK in pre-osteoclast,thus indicated EB could promote bone resorption. Objective:To study the effection of erigeron breviscapus（EB） and（or） mechanical force on the expression of OPG/RANKL/RANK mRNA and protein in MG-63 osteoblast-like cells and RAW264.7 pre-osteoclast cells in vitro,thus to propose the role that EB can play in bone rebuilding.Methods:MG-63 osteoblast-like cells and RAW264.7 pre-osteoclast cells were cultured in vitro,and were stimulated by a mechanical instrument called SXG4201 model.The 3th passage of cells were divided into the control group and the different experiment group.The different groups were intervented respectively by EB（1mg/ml）,mechanical force（2000μstrain,0.2Hz）, EB（1mg/ml） combined with mechanical force（2000μstrain, 0.2Hz）.Total RNA and protein were respectively isolated from cells after 3,6,12 and 24 hours.Expressions of OPGmRNA and RANKLmRNA in MG-63 osteoblast-like cells and RANKmRNA in RAW264.7 pre-osteoclast cells were detected by semiquantitative RT-PCR.Expressions of OPG protein and RANKL protein in MG-63 osteoblast-like cells and RANK protein in RAW264.7 pre-osteoclast cells were detected by Western blot.Results:From 3,6,12 to 24 hours after intervented by mechanical force,the expression of OPG protein in MG-63 osteoblast-like cells was increased in time dependent,but the expression of RANKL protein in MG-63 osteoblast-like cells and the expression of RANK protein in RAW264.7 pre-osteoclast cells did not be changed apparently.Mechanical force（2000μstrain,0.2Hz） increased the expression of OPG protein in MG-63 osteoblast-like cells,but could not change the expression of RANKL protein in MG-63 osteoblast-like cells.EB（1 mg/ml） decreased the expression of OPG protein in MG-63 osteoblast-like cells,and increased the expression of RANKL protein in MG-63 osteoblast-like cells.EB（1mg/ml） combined with mechanical force （2000μstrain,0.2Hz） decreased the expression of OPG protein in MG-63 osteoblast-like cells,and increased apparently the expression of RANKL protein in MG-63 osteoblast-like cells.Mechanical force（2000μstrain,0.2Hz） could not change the expression of RANK protein in RAW264.7 pre-osteoclast cells.EB（1mg/ml） increased the expression of RANK protein in RAW264.7 pre-osteoclast cells.EB（1mg/ml） combined with mechanical force （2000μstrain,0.2Hz） increased apparently the expression of RANK protein in RAW264.7 pre-osteoclast cells.Conclusions:EB could inhibit the secretion of OPG in osteoblasts by mechanical force,and could promote the secretion of RANKL in osteoblasts and could also promote the secretion of RANK in pre-osteoclast by EB combined with mechanical force.更多还原