葛根素对骨质疏松模型大鼠细胞因子和骨代谢的影响

目的观察研究葛根素对绝经后骨质疏松大鼠细胞因子和骨代谢的影响,并探讨其作用机制。方法实验随机分为假手术组(Sham组)、模型组(OVX组)、雌二醇组(OVX+E2组)、葛根素组(OVX+PUE组),构建绝经后骨质疏松大鼠动物模型并给予不同药物干预。分别检测血清细胞因子指标和骨代谢指标,检测股骨BMD,观察骨组织形态结构变化。结果 OVX组血清TNF-α、TGF-β、IL-6和IL-1较Sham组均显著增高(P0.05),OVX+E2组和OVX+PUE组血清TNF-α、TGF-β、IL-6和IL-1较OVX组均显著降低(P0.05)。OVX组血清Ca、P较Sham组显著降低(P0.05),OVX+E2组和OVX+PUE组血清Ca、P较OVX组均显著升高(P 0.05)。OVX组血清ALP、BGP、PICP、TRAP-5b、ICIP、CTX水平较Sham组均显著升高(P 0.05),OVX+E2组和OVX+PUE组血清ALP、BGP、PICP、TRAP-5b、ICIP、CTX较OVX组均显著降低(P0.05)。OVX组较Sham组股骨BMD显著降低(P0.05),OVX+E2组和OVX+PUE组较OVX组股骨BMD均显著升高(P0.05)。OVX+PUE组骨皮质明显变厚,骨小梁数量增多增粗,排列较规则,髓腔明显缩小,骨细胞增多。结论葛根素通过调控绝经后骨质疏松大鼠炎性细胞因子分泌,调节骨代谢骨矿化、骨吸收和骨形成指标,改善骨组织形态学结构,发挥抗骨质疏松作用。     

密骨方对去势大鼠所致骨质疏松性骨折白细胞介素-1的影响

目的 观察密骨方对骨质疏松性骨折(OPF)白细胞介素-1(IL-1)的影响.方法 将90只雌性Wistar大鼠随机分为对照组(Sham),模型组(OVX)与密骨方治疗组(MGF),OVX组及MGF组行卵巢摘除术,同时MGF组和OVX组大鼠均致其右侧股骨上1/3处骨折.MGF组按100mg/(kg·d)的剂量灌胃给药,Sham组和OVX组给予同体积的生理盐水,每日1次,疗程分别为2、4、6周,采用免疫组织化学法观察骨折端IL-1的表达.结果 OVX组IL-1在骨折端的表达随时间的延长(2、4、6周)而显著升高(0.20660±0.03195、0.20790±0.03347、0.21120±0.02429,P＜0.05),而MGF组IL-1在骨折端的表达随时间的延长而显著降低(0.21380±0.02800、0.20870±0.03252、0.20480±0.03465,P＜0.05)；同时MGF组第6周IL-1在骨折端的表达显著低于OVX组(P＜0.01).结论 MGF能显著降低大鼠OPF端IL-1的合成分泌,从而抑制破骨细胞的活性,促进骨折愈合.     

铁蓄积小鼠去势前后骨吸收活性的差异

目的了解小鼠在去势前与去势后,铁对骨吸收影响的差异。方法建立高铁小鼠模型,将小鼠随机分为假手术组(SHAM组)、高铁去势组(F+OVX组)、去势组(OVX组)。铁剂干预两周后去势。在去势前(雌激素存在)与去势7周后(雌激素缺乏)检测小鼠体重,血清铁蛋白;反转录聚合酶链反应(RT-PCR)检测胫骨内抗酒石酸酸性磷酸酶(TRAP)、降钙素受体(CTR)、基质金属蛋白酶9(MMP9)、组织蛋白酶K(cathepsin K)、雌激素受体α(ERα)、雌激素受体β(ERβ)表达情况,股骨远端Micro-CT三维分析,提取各组小鼠股骨骨髓细胞进行破骨细胞培养,抗酒石酸酸性磷酸酶(TRAP)染色观察破骨细胞分化情况。结果各组小鼠体重无明显差异。F+OVX组铁蛋白含量明显升高(P<0.05)。Micro-CT分析表明:雌激素存在时,正常组与铁干预组骨密度及相关指标无明显差异(P>0.05);雌激素缺乏时,与OVX组比较,F+OVX组骨量下降更为严重(P<0.05)。半定量PCR结果显示:雌激素存在时,两组TRAP、CTR、MMP9、cathepsin K基因表达无统计学差异;雌激素缺乏时,F+OVX组各基因表达水平较OVX组显著升高(P<0.05)。去势后ERβ表达下降(P<0.05),ERα无明显差异。TRAP染色显示去势前各组小鼠破骨细胞数无明显差异(P>0.05);而去势后,F+OVX组破骨细胞数明显高于OVX组(P<0.05)。结论未去势时(有雌激素),FAC对骨吸收影响甚微;去势后(无雌激素),FAC能显著增强破骨活性。     

去卵巢大鼠骨髓微环境中RUNX2\PPARγ基因及OPG\RANKL基因蛋白表达变化的实验研究

[目的]探讨在去卵巢大鼠骨质疏松模型骨髓微环境中成骨细胞、脂肪细胞和破骨细胞分化的相互关系及其机制.[方法]将3个月龄雌性SD大鼠16只随机分为2组:假手术组(SHAM)和去卵巢组(OVX),每组8只.采用双侧卵巢切除术复制骨质疏松大鼠模型.术后14周,应用双能X线吸收仪法(DXA)测第4腰椎和股骨骨密度(BMD).qRT-PCR法测量骨髓细胞RUNX2、PPARγ、OPG和RANKL mRNA表达量.石蜡组织切片HE染色测量第3腰椎和胫骨近端骨组织脂肪细胞数目.免疫组织化学染色测定胫骨近端骨组织OPG/RANKL蛋白表达量.[结果]与SHAM比较,OVX组腰椎和股骨BMD下降(P＜0.05).OVX组股骨骨髓细胞成骨分化转录因子RUNX2 mRNA水平比SHAM组增高(P＜0.05),成脂分化转录因子PPAR γ mRNA表达水平比SHAM组增高(P＜0.05).OVX组胫骨和第3腰椎脂肪细胞数目比SHMA组增多(P＜0.05).与SHAM组比较,OVX组股骨骨髓细胞RANKLmRNA和胫骨RANKL蛋白表达量增加(P＜0.05)且OPG/RANKL的比率都降低(P＜0.05),而两者OPG的mR-NA和蛋白表达量无统计学差异(P＞0.05).[结论]去卵巢大鼠骨量丢失可能是骨髓微环境中成骨细胞分化、脂肪细胞分化和破骨细胞分化紊乱导致.     

雌激素通过调节 EphB4／EphrinB2信号参与破骨细胞分化

目的研究雌激素通过调节EphB4/EphrinB2信号通路对破骨细胞分化的影响。方法 (1)采用RANKL诱导法培养卵巢摘除骨质疏松模型(OVX)组和假手术(Sham)组小鼠的破骨细胞,于第10天提取两组细胞的RNA和蛋白质样品,通过RT-PCR和Western blot检测细胞EphrinB2表达的变化;(2)采用雌激素及雌激素拮抗剂分别诱导培养RAW264.7破骨细胞,培养第8天提取RNA和蛋白质样品,利用RT-PCR和Western blot检测细胞EphrinB2表达的变化;(3)在OVX模型组小鼠的破骨细胞培养中添加EphrinB2配体EphB4-Fc片段,通过RT-PCR检测破骨细胞标志物的表达和抗酒石酸酸性磷酸酶(TRAP)染色并计数,观察破骨细胞分化能力的变化。结果卵巢摘除骨质疏松模型组小鼠EphrinB2的表达量低于假手术组(P0.01);雌激素拮抗剂组EphrinB2的表达量低于对照组(P0.01),而雌激素组EphrinB2的表达量高于对照组(P0.05);给予EphrinB2的配体EphB4-Fc片段后,OVX组小鼠的破骨细胞标志物表达量降低(P0.01,P0.001),TRAP染色阳性细胞数减少。结论雌激素可以通过调节破骨细胞EphrinB2的表达影响破骨细胞分化,采用EphB4-Fc片段处理后,OVX组小鼠增强的破骨细胞分化受到抑制。     

雌激素缺乏上调TNF-α促发卵巢切除大鼠骨细胞程序性坏死

目的探讨雌激素缺乏对去卵巢骨质疏松大鼠骨细胞程序性坏死的影响及其发生机制。方法 60只雌性SD大鼠随机分为Control组(12只)和OVX组(48只),分别行假手术或OVX术;再将48只OVX大鼠分为OVX+vehicle、OVX+Nec-1、OVX+Z-VAD和OVX+E2组,每组12只。Micro-CT扫描评价胫骨近端骨量改变;免疫组化法测定骨细胞TNF-α、RIP1、RIP3的表达情况,并计算各组阳性细胞百分率;用透射电镜鉴定细胞形态学变化;Western blot检测程序性坏死下游信号通路蛋白MLKL、Drp1的表达情况。结果 OVX+vehicle组大鼠胫骨近端出现明显的骨量丢失,即OVX+vehicle组Tb.Th、BV/TV、Tb.N小于Control组(P0.01);而OVX+vehicle组Tb.Sp大于Control组(P0.01)。TNF-α、RIP1、RIP3蛋白在骨细胞胞浆中均有表达;在OVX+vehicle组,TNF-α、RIP1、RIP3阳性骨细胞百分率较Control组显著增多(P0.01),在OVX+vehicle组可见大量坏死样结构的骨细胞。应用Nec-1治疗后可有效抑制RIP1蛋白表达,而Z-VAD对RIP1、RIP3蛋白表达无影响,在OVX+Nec-1组未见到坏死样结构的骨细胞。在OVX+vehicle组,Western blot结果显示MLKL、Drp1蛋白表达较Control组显著增多(P0.01),应用Nec-1治疗后可有效抑制MLKL、Drp1蛋白表达,而Z-VAD对MLKL、Drp1蛋白表达无影响。结论雌激素缺乏可能是通过上调TNF-α表达而促发骨细胞发生程序性坏死的,MLKL、Drp1可能是骨细胞程序性坏死的下游关键信号分子。     

骨碎补通过骨髓间充质干细胞调节RANKL/OPG抑制破骨细胞的实验研究

目的　研究骨碎补对绝经后骨质疏松大鼠骨髓间充质干细胞(bone marrow stromal cells,BMSCs)RANKL/OPG及破骨细胞分化成熟的影响并探查其可能的作用机制。方法 实验大鼠去双侧卵巢造模,分为实验组（OVXDF,造模+骨碎补水煎液灌胃）、模型组（OVX,造模+0.9%生理盐水灌胃）、假手术组（SHAM,假手术+0.9%生理盐水灌胃）,造模成功后提取BMSCs,将BMSCs和骨髓单核细胞共培养于Transwell小室的上室和下室,分为实验组+破骨细胞（OVXDF+OC）、模型组+破骨细胞（OVX+OC）、假手术组+破骨细胞（SHAM+OC）。下室加入破骨细胞诱导剂,倒置相差显微镜观察破骨细胞的分化成熟情况并计数,酶联免疫吸附剂测定（ELISA）检测下室培养液中骨保护素(osteoprotegerin,OPG)、RANKL的含量,并计算RANKL/OPG,实时荧光定量PCR和蛋白质印迹法(Western blot)检测BMSCs中Wnt10b、β-catenin、RANKL、OPG mRAN及蛋白表达并计算RANKL/OPG。结果 在共培养系统中,与去卵巢灌胃大鼠BMSCs共培养的破骨细胞（OVXDF+OC）数量较单纯模型组+破骨细胞（OVX+OC）明显减少（P＜0.05）。下室培养液OPG含量及共培养BMSCs中Wnt10b、β-catenin、OPG mRAN及蛋白表达模型组+破骨细胞（OVX+OC）最低,RANKL及RANKL/OPG最高,经骨碎补灌胃后（OVXDF+OC）培养液中OPG含量及BMSCs细胞中Wnt10b、β-catenin、OPG mRAN及蛋白表达明显升高,培养液及BMSCs细胞中RANKL及RANKL/OPG明显降低（P＜0.05）。结论 骨碎补可调节BMSCs细胞OPG、RANKL的表达,激活OPG/RANKL/RANK信号通路抑制破骨细胞的分化和成熟,此作用可能与BMSCs的Wnt/β-catenin信号通路的激活有关。     

鲑鱼降钙素对去卵巢大鼠骨髓细胞OPG、RANKL基因和蛋白表达的影响

[目的]观察鲑鱼降钙素(sCT)对去卵巢大鼠骨密度(BMD)、血清Ⅰ型胶原交联羧基末端肽(ICTP)变化的影响,以及骨髓细胞骨保护素(OPG)和核因子κB受体活化因子配体(RANKL)的基因表达和两者在胫骨骨骺端蛋白含量的变化.[方法]取3个月龄雌性SD大鼠24只,随机平均分3组:假手术组(Sham)、鲑鱼降钙素处理组(sCT)、安慰剂组(OVX).采用双侧卵巢切除术复制骨质疏松大鼠模型.术后2周CT组予鲑鱼降钙素皮下注射12周,应用双能X线吸收仪法(DXA)测BMD,ELISA法测量血清ICTP浓度,qRT-PCR法定量骨髓细胞OPG和RANKL的mRNA表达量,免疫组织化学染色法测定胫骨干骺端OPG和RANKL蛋白表达量.[结果]与OVX组比较,sCT组的腰椎BMD上升显著(P＜0.05),但股骨BMD改变不明显(P＞0.05);血清ICTP含量显著降低(P＜0.05);骨髓细胞RANKL的mRNA表达量变化不大(P＞0.05),但OPG的mRNA表达量升高(P＜0.05),OPG/RANKL的比率升高(P＜0.05);胫骨干骺端也呈现出RANKL蛋白改变不明显(P＞0.05),而OPG的蛋白分泌增加(P＜0.05),从而OPG/RANKL的比率高于OVX组(P＜0.05)的现象.[结论]降钙素可以预防腰椎BMD的丢失,降低血清ICTP水平,在体内可能主要通过上调OPG的mRNA表达和蛋白分泌,影响OPG/RANKL/RANK系统,影响破骨细胞功能,抑制骨吸收,进而达到预防绝经后骨质疏松的目的.     

BEMF对去卵巢骨质疏松大鼠骨组织bFGF表达的影响及意义

目的 观察BEMF对去卵巢骨质疏松大鼠骨组织bFGF蛋白表达的影响,探讨BEMF治疗骨质疏松的机理.方法 6月龄雌性未孕Wistar大鼠40只,按体质量随机分为模型组（OVX）、假手术组（Sham）、仿生脉冲电磁场治疗组（BEMF＋OVX,EM）、雌激素治疗组（Estrogen＋OVX,E）.OVX、EM、E组行双侧卵巢切除术,Sham组行假手术.术后第9 W开始治疗：E组行苯甲酸雌二醇肌肉注射,0.5mg/kg,1次/2 W.EM组大鼠暴露于仿生脉冲电磁场治疗,1 h/1次/d,OVX、Sham组不予以任何处理,作为对照组.治疗10 W后处死各组实验动物,测量腰椎骨密度、椎体的最大载荷、扫描电镜观察椎体骨结构的变化.采用免疫组化检测椎体骨组织中bFGF蛋白表达情况并以图像分析进行组间比较.结果 治疗10 W后EM组大鼠骨密度、椎体的最大载荷较OVX组显著性增高（P＜0.05）,骨结构明显改善.bFGF的表达主要位于成骨细胞、骨细胞,EM组椎体骨组织中bFGF表达显著性高于OVX组（P＜0.01）.结论 提高骨组织中bFGF的表达是BEMF治疗骨质疏松的重要机理之一.     

骨碎补经骨髓间充质干细胞调节OPG/RANKL/RANK通路抑制破骨细胞的实验研究

目的研究骨碎补作用绝经后骨质疏松大鼠骨髓间充质干细胞(bone marrow stromal cells,BMSCs)调节OPG/RANKL/RANK通路及对破骨细胞分化成熟的影响并探讨其可能的作用机制。方法实验大鼠去双侧卵巢造模,分为实验组(OVXDF,造模+骨碎补水煎液灌胃)、模型组(OVX,造模+0.9%生理盐水灌胃)、假手术组(SHAM,假手术+0.9%生理盐水灌胃),造模成功后提取BMSCs,将BMSCs和骨髓单核细胞共培养于Transwell小室的上室和下室,分为实验组+破骨细胞(OVXDF+OC)、模型组+破骨细胞(OVX+OC)、假手术组+破骨细胞(SHAM+OC)。下室加入破骨细胞诱导剂,倒置相差显微镜观察破骨细胞的分化成熟情况并计数,酶联免疫吸附剂测定(ELISA)检测下室培养液中骨保护素(osteoprotegerin,OPG)、RANKL的含量,实时荧光定量PCR和蛋白质印迹法(Western blot)检测BMSCs中Wnt10b、β-catenin、RANKL、OPG mRAN及蛋白表达并计算RANKL/OPG。结果在共培养系统中,与去卵巢灌胃大鼠BMSCs共培养的破骨细胞(OVXDF+OC)数量较单纯模型组+破骨细胞(OVX+OC)明显减少(P0.05)。下室培养液OPG含量及共培养BMSCs中Wnt10b、β-catenin、OPG mRAN及蛋白表达模型组+破骨细胞(OVX+OC)最低,RANKL及RANKL/OPG最高,经骨碎补灌胃后(OVXDF+OC)培养液中OPG含量及BMSCs细胞中Wnt10b、β-catenin、OPG mRAN及蛋白表达明显升高,培养液及BMSCs细胞中RANKL及RANKL/OPG明显降低(P0.05)。结论骨碎补可调节BMSCs细胞OPG、RANKL的表达,激活OPG/RANKL/RANK信号通路抑制破骨细胞的分化和成熟,此作用可能与BMSCs的Wnt/β-catenin信号通路的激活有关。     

跖骨感染骨外露的显微外科治疗

[目的]回顾总结跖骨骨感染骨外露的显微外科治疗方法。[方法]自1995年～2005年采用显微外科技术治疗214例跖骨骨外露骨感染患者。[结果]全部病例获得随访1～10年，平均随访3年，14例游离植皮术后皮肤成活良好，199例术后皮瓣全部成活，1例腓肠神经营养血管皮瓣移位修复术出现远端部分皮肤坏死，后经换药处理后，伤口自然愈合。皮瓣移植术后质地良好，无溃疡复发。患足均可负重走路。[结论]应用显微外科技术治疗跖骨骨感染骨外露可获得较好的疗效。     

重组合异种骨植骨修复骨囊肿所致骨缺损

2001年10月～2003年9月，笔者共收治28例骨囊肿患者，均采用病灶刮除，瘤腔灭活和重组合异种骨植骨治疗，获得满意疗效，体会如下。     

Genetic regulation of bone mass: from bone density to bone strength

Osteoporosis is a common disease characterized in adults by diminished bone density. Bone is an organ that evolves and grows throughout life, and establishing optimal bone density in childhood and adolescence serves to buffer bone loss later in life. Bone density, a measurable entity, is the clinical substitute for bone strength, or the ability to defend against fracture. Chronic diseases may adversely affect optimal peak bone density. Bone density is under genetic control, as revealed by three lines of investigations. These include (1) the finding of quantitative trait loci for bone density, (2) the finding that specific mutations in genes that are important in the development of osteoblast or osteoclast lineages alter bone density, and (3) the linkeage of known polymorphisms for genes involved in mineral homeostasis to bone density and/or fracture. Future therapeutics for improving peak bone density or delaying bone loss later in life may take advantage of the genetic nature of bone density development.This work was presented in part at the IPNA Seventh Symposium on Growth and Development in Children with Chronic Kidney Disease: The Molecular Basis of Skeletal Growth, 1–3 April 2004, Heidelberg, Germany     

Aneurysmal bone cyst of the frontal bone

Aneurysmal bone cyst rarely affects the skull. We report two cases of aneurysmal bone cyst of the frontal bone. One of the cases is associated with pregnancy. The association of pregnancy with aneurysmal bone cyst and enlargement of the aneurysmal bone cyst during the pregnancy have been discussed.     

Development of bone canaliculi during bone repair

We recently found that silver impregnation staining with protargol (silver protein), that is, a modified Bodian method, is useful for histologically identifying the details of bone canaliculi structure, using thin sections of decalcified bone tissues. With this staining method, we conducted the present study to assess the development of bone canaliculi during the process of intramembranous ossification using a fracture-like stimulation model of the rat femur. After making a drill-hole in the cortex of the rat femur, decalcified thin sections were obtained after 3, 5, 7, and 14 days by the standard paraffin-embedding procedure. Silver staining for bone canaliculi was performed using our previously reported technique. The results showed that woven bone covered the fracture surface of the cortex after 5 days, then immature lamellar bone attached to the woven bone after 7 days, and finally the lamellar bone matured and became thick with appositional growth after 14 days. The osteocytes in the woven bone appeared at an early stage of bone repair and developed a few canaliculi that were short and irregularly distributed in the osteoid matrix, while the osteocytes in the lamellar bone at a late stage formed many bone canaliculi that were long and regularly distributed in mature bone matrix. Therefore, we concluded that woven bone osteocytes may be necessary for induction of the lamellar bone osteocytes followed by active appositional growth of the lamellar bone at the early stage of bone repair, and also that both bone tissues could be clearly distinguished from one another based on the pattern of development of bone canaliculi by the osteocytes, as seen with the use of our sensitive staining method.     

Aneurysmal bone cysts of the occipital bone

Two cases of aneurysmal bone cysts are reported. Each patient presented with a palpable mass in the occipital region and signs of compression of structures in the posterior fossa. One of the cases is unique, in that the aneurysmal bone cyst was associated with an epidural hematoma in the posterior fossa. The pertinent literature is reviewed.     

Assessment of bone density and bone loss

Osteoporosis International - Dual-energy X-ray absorptiometry has become the standard for the evaluation of osteoporosis. It is useful both for identifying those people who are going to be at risk...     

Aneurysmal bone cyst of the occipital bone

A 13 years boy presented with a painless hard and fixed swelling in occipital region for the last three months. Plain X-ray, CT scan and MRI showed an expansile multi loculated cystic lesion in occipital bone. Histopathological examination revealed it to be an aneurysmal bone cyst. Treatment of choice is surgery. However, radiotherapy may be helpful in incompletely excised lesions.