骨质疏松症大鼠肾组织中Smmf1 mRNA和蛋白表达的实验研究

目的通过研究摘除卵巢致骨质疏松症大鼠肾组织中Smurf1的mRNA和蛋白的表达,揭示摘除卵巢致骨质疏松症的病理机制,并研究补肾中药防治疗效及其作用机制。方法摘除雌性大鼠双侧卵巢的方法复制骨质疏松症模型,采用具有补肾壮骨益精作用的纳米钙补肾中药（高、低剂量）对实验大鼠治疗12w,以骨疏康颗粒剂、盖天力片作为阳性对照组,正常大鼠、假手术组作为标准对照组,模型大鼠作为空白对照组,并与补脾方剂（补中益气汤）作疗效的比较。用RT-PCR法、Western印迹法检测骨质疏松症大鼠肾组织中Smurf1的mRNA和蛋白表达。结果RT-PCR及Western方法检测,正常大鼠肾组织中存在Smurf1的mRNA和蛋白表达;与正常组、假手术组比较,模型组Smurf1 mRNA和蛋白的表达水平在肾组织中明显降低。用药12w后,与模型组比较,补肾高、低剂量组、补脾组、盖天力组、骨疏康组均可明显上调Smurf1 mRNA和蛋白在肾组织中的表达水平。其中补肾高剂量组上调幅度大于其他各用药组。结论①正常大鼠肾组织中Smurf1可以在基因、蛋白水平表达;②骨质疏松症的发生,可能与肾组织中Smurf1的mRNA和蛋白表达的变化有关;③纳米钙补肾中药通过调控肾组织中Smurf1的mRNA和蛋白表达,对于骨质疏松症有一定的防治作用。     

纳米钙补肾中药对骨质疏松症大鼠肠黏膜中 1，25(OH)2D3受体的mRNA和蛋白表达影响

目的研究骨质疏松症大鼠肠黏膜中维生素1,25(OH)2D3受体的mRNA和蛋白表达,揭示骨质疏松症的发病机制;同时,阐述纳米钙补肾中药的调节作用机制。方法切除大鼠双侧卵巢的方法复制骨质疏松症模型,采用纳米钙组及具有益肾填精、补钙壮骨作用的纳米钙补肾中药(大、中、小)剂量对实验大鼠治疗12周,以骨疏康颗粒剂、雌二醇(E2)及钙尔奇D600作为阳性对照组,正常大鼠和模型空白组为空白对照组;用PCR法、Western印迹法检测骨质疏松症大鼠肠黏膜中维生素1,25(OH)2D3受体的mRNA和蛋白表达。结果RT-PCR及Western方法检测,正常大鼠肠黏膜中含有维生素1,25(OH)2D3受体的mRNA和蛋白表达;模型空白组大鼠肠黏膜组织中的mRNA和蛋白表达水平有明显的下降。纳米钙、纳米钙补肾中药各剂量组、钙尔奇D600组、骨疏康和雌二醇E2组用药后12周,与模型空白组比较,可不同程度上调肠黏膜组织中的mRNA和蛋白表达水平。但其中各治疗用药组的效果有所不同,一般表现为纳米钙补肾中药剂量组作用较好。结论①正常大鼠肠黏膜组织可以在基因、蛋白水平表达维生素1,25(OH)2D3受体,而且在肠钙吸收中可能起到重要作用。②大鼠骨质疏松症的发生,与肠黏膜组织中维生素1,25(OH)2D3受体的mRNA和蛋白表达的变化有关,从而影响肠钙的吸收。③纳米钙补肾中药通过调控大鼠肠黏膜维生素1,25(OH)2D3受体的mRNA和蛋白表达,促进肠钙的吸收,对于骨质疏松症有一定的防治作用。     

川芎嗪对膝骨性关节炎大鼠软骨BMP-2、Smad1及BMP-2mRNA、Smad1mRNA表达的影响

目的观察川芎嗪治疗膝骨关节炎(knee osteoarthritis,KOA)大鼠模型关节软骨中骨形态发生蛋白-2(bone morphogenetic protein-2,BMP-2)、Smad1表达的变化,并探讨川芎嗪治疗KOA的作用机制。方法制作大鼠膝关节炎模型,将建模成功的大鼠纳入模型对照组,常规饲养,川芎嗪高剂量组、川芎嗪低剂量组、阳性对照组行灌,正常组和模型组行等量生理盐水治疗6 w,实验结束后,各组大鼠分别行HE切片软骨Mankin评分、Western Blot检测软骨组织BMP-2、Smad1及实时荧光定量PCR检查BMP-2mRNA、Smad1mRNA的变化。结果各组软骨Mankin评分中,川芎嗪各剂量组和阳性对照组均较空白组高(P0.05),均较模型组低(P0.05),且川芎嗪高剂量组阳性对照组川芎嗪低剂量组(P0.05)。正常组、川芎嗪各剂量组及阳性对照组中软骨BMP-2、Smad1及BMP-2mRNA、Smad1mRNA相对表达量较模型组均高于模型组,差异有统计学意义(P0.05);与阳性对照组相比,川芎嗪高剂量组BMP-2、Smad1及BMP-2mRNA、Smad1mRNA表达量明显高于阳性对照组(P0.05),而川芎嗪低剂量组其表达则均低于阳性对照组。结论川芎嗪可能通过上调早期KOA大鼠BMP-2mRNA及Smad1mRNA基因的表达,从而促进BMP-2及Smad1蛋白的表达,这可能是在某种程度上减轻关节软骨退变、修复软骨损伤作用的机制之一。     

Ang-1在去卵巢骨质疏松症大鼠骨组织中表达变化以及中医不同治法对其影响的实验研究

目的观察去卵巢骨质疏松症模型大鼠的骨组织中血管生成素1(Ang-1)mRNA和蛋白含量变化,及中药不同方剂对其影响,探讨中药组方治疗骨质疏松的可行性。方法骨质疏松症模型的建立采用切除雌性大鼠双侧卵巢的方法。运用补肾填精中药复方、活血化瘀中药复方、补肾活血中药复方对模型大鼠灌胃12周,用骨疏康作为阳性药物对照组,还有正常组和模型空白组。RT-PCR检测各组骨组织Ang-1mRNA相对表达量;ELISA法检测各组骨组织Ang-1蛋白含量。结果去卵巢骨质疏松症模型大鼠的骨组织中Ang-1mRNA相对表达量与正常组比较,显著降低(P0.01);与模型空白组比较,各个用药组Ang-1mRNA相对表达量显著升高(P0.01);但各个用药组间比较无统计学意义。去卵巢骨质疏松症模型大鼠骨组织中Ang-1蛋白含量与正常组比较,显著降低(P0.01);与模型空白组比较,各个用药组Ang-1含量显著升高(P0.01);各个用药组间比较,补肾活血组Ang-1含量升高最为显著,与补肾填精组和活血化瘀组比较有显著性差异(P0.01)。结论补肾活血中药复方可提高Ang-1mRNA相对表达量及其蛋白含量,且优于单纯的补肾填精和活血化瘀中药复方,起到防止骨质疏松症的作用。     

益肾填精中药对骨质疏松症模型大鼠的骨组织中Smad1/5的表达影响

目的 通过观察去卵巢骨质疏松症模型大鼠股骨中Smad1/5在细胞中的表达,探索肾虚骨质疏松症的发病机理.方法 实验采用切除大鼠双侧卵巢的方法复制骨质疏松症模型,同时应用益肾填精作用的纳米钙补肾中药低、高剂量组,对模型大鼠给药干预12周,用骨疏康颗粒剂、盖天力作为阳性对照组,以及正常对照组、假手术对照组和模型空白组,并与补脾中药补中益气汤作比较;应用双能X线骨密度仪检测离体股骨的骨密度;HE染色光镜下观察股骨形态;免疫组化SABC法检测股骨中的Smad1/5蛋白活性表达.结果 与正常组比较,模空组大鼠股骨头的骨密度显著降低,P<0.01;与模空组比较,用药12周之后各用药组均可提高模型大鼠股骨的骨密度值;HE染色光镜下观察各组大鼠右侧股骨头病理切片,模空组骨髓腔增大,骨小梁断裂,各用药组可见骨髓腔较小,形态结构较完整;与正常组相比较,模空组Smad1/5阳性表达显著下降,P<0.01;用药12周后,与模空组相比较,各用药组均可上调其表达情况,P<0.01.结论 骨组织中Smad1/5阳性表达降低可能是骨质疏松症发生的机制之一,纳米钙补肾填精中药可能通过改善和调控股骨中Smad1/5的表达,而起到防治肾虚骨质疏松症的作用.     

补肾益髓中药对糖皮质激素性骨质疏松症大鼠骨组织Runx2的mRNA及蛋白表达的影响

目的 观察糖皮质激素性骨质疏松症(GIOP)大鼠模型骨组织Runt相关基因2(Runx2)mRNA及蛋白表达,探讨GIOP的发病机制以及补肾益髓中药的疗效及其调控作用.方法 采用后肢肌注地塞米松(2.5 mg/kg,每周2次,连续9w)的方法复制GIOP大鼠模型,按体重分层随机分为正常组、模型空白组、补肾益髓中药组、补中益气颗粒组、血府逐瘀胶囊组、骨疏康颗粒阳性对照组.灌胃给药9w.应用XR-26型双能X线骨密度仪测定股骨骨密度,以评价动物模型的成立及药物疗效.实时定量RT-PCR(Real-Time Quantitative RT-PCR)检测骨组织Runx2 mRNA表达,Western blot检测骨组织Runx2蛋白表达.结果 (1)股骨骨密度:与正常组比较,模型空白组明显降低(P<0.01);与模型空白组比较,补肾益髓中药组明显升高(P<0.01),骨疏康颗粒阳性对照组明显升高(P<0.05),补中益气颗粒组和血府逐瘀胶囊组虽有升高趋势,但无统计学意义(P>0.05).(2)骨组织Runx2 mRNA及蛋白表达:与正常组比较,模型空白组明显降低(P<0.01);与模型空白组比较,补肾益髓中药组明显上调(P<0.01),骨疏康颗粒阳性对照组也有明显上调(P<0.05),补中益气颗粒组和血府逐瘀胶囊组虽有所升高,但无统计学意义(P>0.05).结论 (1)肌注地塞米松可以成功复制GIOP大鼠模型,(2)骨组织Runx2 mRNA及蛋白表达水平降低可能是GIOP的发病机制之一,(3)应用补肾益髓中药具有明显防治效果,疗效优于健脾中药和活血中药,其作用机理与上调骨组织Runx2 mRNA及蛋白表达密切相关.     

补肾复方对去卵巢大鼠MEK1、ERK2基因表达的调控 作用

目的研究补肾中药复方(鹿茸、牡蛎、淫羊藿)对去卵巢骨质疏松症大鼠的影响,探讨肾虚骨质疏松症的病理机制,为中医药防治骨质疏松症提供实验依据。方法采用摘除雌性大鼠双侧卵巢的方法复制骨质疏松症模型,以正常组作为标准对照,模型组作为空白对照,盖天力组作为阳性对照,补肾中药复方低、中、高剂量组作为实验组。各用药组灌胃给药12周。采用腹主动脉取血法采集大鼠血清测定血骨钙素(BGP)和抗酒石酸酸性磷酸酶(TRACP),并用DEXA骨密度仪检测左侧股骨骨密度;右侧股骨匀浆提取骨组织中的Total RNA,RT-Real Time PCR,检测MEK1及ERK2的mRNA表达水平。结果模型组BGP水平显著低于正常组(P0.01),TRAP水平较正常组显著升高(P0.01);正常组和补肾中药复方组的股骨骨密度均高于模型组(P0.05);ERK2 mRNA相对表达量明显高于模型组(P0.01),MEK1mRNA相对表达量明显低于模型组(P0.01)。结论补肾中药复方具有防治去卵巢大鼠骨质疏松的作用,其机制与调控MEK1、ERK2基因表达有关。     

补肾、活血复方对骨质疏松症模型大鼠 VEGF 表达的影响

目的比较补肾、活血复方对骨质疏松症模型大鼠VEGF表达的影响,探讨补肾、活血复方治疗骨质疏松症的分子机制。方法以去卵巢骨质疏松症模型大鼠为实验研究对象,不同实验药物灌胃12周后,双能X线法测定骨密度,ELISA法测定骨、肾及血清中VEGF含量,定量PCR法测定骨、肾中VEGF mRNA相对表达量。结果骨质疏松症模型大鼠骨密度、VEGF蛋白及mRNA表达量显著降低,运用补肾、活血中药复方干预后,各组大鼠骨密度、VEGF蛋白含量及mRNA表达量显著升高。结论肾虚血瘀骨代谢失常与VEGF表达降低有关,补肾、活血复方通过调节VEGF表达水平,起到治疗骨质疏松症的作用。     

补肾益髓中药复方对去卵巢骨质疏松症大鼠骨组织Runx2 mRNA及蛋白表达的影响

目的观察去卵巢骨质疏松症大鼠骨组织Runx2 mRNA及蛋白表达,探讨绝经后骨质疏松症(Postmenopausal Osteoporosis,PMOP)的发病机制以及补肾益髓中药复方的疗效机理。方法去卵巢复制骨质疏松症大鼠模型,实验设正常组、模型组、假手术组、补肾益髓中药复方组、钙尔奇D组、骨疏康组。术后7d开始灌胃给药12周。应用XR-36型双能X线骨密度仪测定股骨骨密度,应用OLYMPUS BX51显微镜观察股骨头石蜡切片HE染色显微形态结构,实时定量RT-PCR及Western Blot检测骨组织Runx2 mRNA及蛋白表达。结果 (1)与正常组、假手术组比较,模型组股骨骨密度明显降低(P0.001、P0.05),骨组织Runx2 mRNA及蛋白表达明显降低(P0.001)。(2)与模型组比较,补肾益髓中药复方组、骨疏康组股骨骨密度明显升高(P0.05),补肾益髓中药复方组(P0.001)、钙尔奇D组(P0.05)、骨疏康组(P0.01)骨组织Runx2 mRNA及蛋白表达明显上调。(3)与钙尔奇D组、骨疏康组比较,补肾益髓中药复方组骨组织Runx2 mRNA(P0.01、P0.05)及蛋白(P0.001)表达均明显升高。(4)股骨头石蜡切片HE染色显微形态结构:与正常组、假手术组比较,模型组骨小梁形态结构完整性差,有些部位破坏、断裂,余留骨重建空间较多;各给药组骨小梁形态结构均改善,结构较紧密,余留骨重建空间减少,其中以补肾益髓中药复方组改善最明显。结论骨组织Runx2 mRNA及蛋白表达降低可能是PMOP的发病机制之一;补肾益髓中药复方可能通过上调骨组织Runx2 mRNA及蛋白表达有效防治PMOP,其作用优于钙尔奇D、骨疏康。     

Hedgehog信号通路与“肾虚血瘀”骨代谢失常的实验研究

目的观察去卵巢骨质疏松症模型大鼠的骨组织和肾组织中Hedgehog信号通路细胞表面受体Patched-1(PTCH1)和Gli3mRNA和蛋白含量变化及补肾填精、活血化瘀中药复方对其影响,探讨中药组方治疗绝经后骨质疏松症的可行性以及绝经后骨质疏松症的发病机制与Hedgehog信号通路的关系。方法采用切除雌性大鼠双侧卵巢的方法建立绝经后骨质疏松症模型,分别给予补肾填精中药复方、活血化瘀中药复方、骨疏康对模型大鼠灌胃12周。然后将大鼠分为补肾填精组、活血化瘀组、阳性对照组、正常组和模型空白组,其中骨疏康作为阳性药物为阳性对照组。运用RT-PCR检测各组大鼠骨组织和肾组织PTCH1和Gli3mRNA相对表达量;通过ELISA法检测各组大鼠骨组织和肾组织PTCH1和Gli3蛋白含量。结果各组大鼠股骨和肾组织中PTCH1mRNA及蛋白表达方面:与正常组比较,模型组显著升高(P0.01);与模型组比较,补肾填精组、活血化瘀组、阳性对照组显著降低(P0.01);各个用药组间比较,补肾填精组下调优于活血化瘀组,具有显著性差异(P0.01)。各组大鼠股骨和肾组织中Gli3mRNA及蛋白表达方面:与正常组比较,模型组显著升高(P0.01);与模型组比较,补肾填精组、活血化瘀组、阳性对照组显著下降(P0.01);各个用药组间比较,补肾填精组下调优于活血化瘀组,具有显著性差异(P0.01)。结论 PTCH1mRNA及蛋白含量和Gli3mRNA及蛋白含量在去卵巢骨质疏松症模型大鼠骨组织和肾组织中显著升高,提示PTCH1和Gli3可能参与绝经后骨质疏松症骨代谢失衡的发生和进展。补肾填精中药复方和活血化瘀中药复方可下调PTCH1和Gli3mRNA相对表达量及其蛋白含量,这可能是起到防治绝经后骨质疏松症的作用机制之一,并且补肾填精法优于活血化瘀法。     

牙体牙弓颌骨阻力中心及其临床意义

牙体、牙弓及颌骨的阻力中心在正畸矫治力系统中具有重要的意义,也是正畸学领域争论较多的一个问题。Dermaut等研究表明,当力作用于物体阻力中心时,物体将发生平动,否则将发生平动和转动的复合运动。目前,国内外多数学者认为牙体、牙弓及颌骨存在阻力中心,但其位置存在争议。本文就牙体、牙弓及颌骨的阻力中心及其临床意义作一综述。     

Hazards and complications of endoscopic transurethral ureterolithotripsy and lithoextraction and means of prevention

Complications related to ureterolithotomy and ultrasonic ureterolithotripsy performed under the control of visual endoscope were analyzed in 86 ureterolithiasis patients, methods of their prevention discussed. All the aforementioned complications were distributed into three groups: inapplicability of surgery due to anatomic and functional defects of lower and upper urinary tracts, intraoperative, and postoperative complications. The commonest ones were ureteral abruption and perforation, acute pyelonephritis, temporary vesicoureteral reflux. Their control measures were considered as relative methods of treatment: immediate surgical intervention in case of ureteral abruption, renal catheterization in patients with insignificant ureteral perforation or acute pyelonephritis. Adequate ureteroscopy, careful consideration of pro- and contraindications, catheterization of renal pelvis and urinary bladder performed within 2-3 days after the surgery and adequate antibacterial therapy are the most decisive steps in the control of aforementioned complications.     

Characterisation and differentiation of chondroblastomas and chondromyxoidfibromas - presence and expression of collagen types I and II

AIM: Chondroblastomas and chondromyxoidfiibromas are rare benign skeletal neoplasms with reported overlapping histology. Aim of this study was to analyse the biochemical composition of the matrix of these tumour entities in order to further characterise the cellular phenotypes of these neoplasms using typical cell biological marker genes. METHODS: The matrix compositions of chondroblastomas and chondromyxoidfibromas were analyzed by HE-histology, histochemistry, and immunolocalization techniques. Cellular gene expression patterns were detected by mRNA in situ hybridization. RESULTS: Chondroblastomas are rich in collagen type I and show foci of an osteoid-like matrix, whereas collagen type II as a typical marker of chondrocytic differentiation was not detected in any of the specimens. Chondromyxoidfiibromas had foci of chondroid appearance with chondroblastic cellular differentiation characterised by collagen type II expression. CONCLUSION: These results characterise chondroblastomas and chondromyxoidfiibromas as skeletal neoplasms that have a different biology and which can be distinguished by matrix protein expression products: collagen type II, the typical marker of chondroblast differentiation, could only be detected in chondromyxoidfibromas, but not in chondroblastomas. Thus, both neoplasms are clearly different on the cell biological level.     

Histochemical and contractile properties of striated muscles of urethra and levator ani of dogs and sheep

AIMS: To understand their possible importance in long- and short-term control of continence, some properties of the striated muscles of the urethra and pelvic floor (levator ani) of dogs and sheep were investigated, especially fiber types and contractile characteristics. MATERIALS AND METHODS: Striated muscles of urethra and levator ani of 29 male and 6 female dogs and 11 male and 6 female sheep were removed and cut into strips. Some strips were frozen and stained for ATPase at pH 9.4 and 4.3 for fiber typing; others were set up in an organ bath to study contractile responses to nerve stimulation. RESULTS: All muscles contained both type I (slow) and type II fibers, ranging from 97% type II in female greyhound urethra to 60% in female sheep levator ani. For each muscle, there were fewer type II muscles in sheep than in dog. The diameters of the urethral fibers were about 60% of the levator ani in dogs and 34% in sheep. Contraction of the urethral muscle was faster than for levator ani and declined to about 80% of the peak, 500 msec after the beginning of stimulation at 20 Hz. The levator ani contraction rose to a steady level as long as stimulation continued. CONCLUSIONS: Both the levator ani and urethral striated muscles contain slow and fast fiber types. The levator ani muscles are capable of sustained contraction with rapid onset which will produce long-term closure of the urethra. The circular urethral muscle contraction was faster but less well maintained.