维生素D对基质GLA蛋白的影响

维生素D是人体健康所必要的元素,它参与体内钙平衡及骨代谢。基质GLA蛋白（matrix gla protein,MGP)是一种维生素K依赖性蛋白,是人体内血管和软骨钙化的抑制剂,对骨形成有重要的影响。了解维生素D对MGP表达的影响对探讨新的骨质疏松发病机理有着重要的意义。本文所述,维生素D与MGP这两种对骨形成有重要作用的影响因子之间存在着许多内在联系,维生素D除了能直接调节多种细胞MGP表达,也有可能通过调节钙磷代谢、雌二醇、维生素K2和BMP-2等多途径间接调节MGP表达,而且1,25( OH)2 D3有可能通过激活Wnt/β-catenin信号通路调节MGP表达而影响骨形成。这和以往我们所了解的维生素D在骨代谢中的作用机制有所不同。     

去卵巢大鼠骨髓基质细胞beta-catenin蛋白表达变化观察

目的观察卵巢切除大鼠骨髓基质细胞（BMSC）beta-catenin蛋白表达的变化。方法将18只健康3月龄未经产雌性SD大鼠随机平分为去卵巢和假手术两个组。术后14周,取左侧股骨,新鲜分离骨髓细胞,静置贴壁法体外培养9d后,采用间接免疫荧光方法检测BMSC中beta-catenin蛋白表达和定位的变化。取右侧股骨,在双能直线χ-射线骨密度仪上检测骨密度的变化。结果大鼠去卵巢14周后,股骨近端和远端的骨密度显著下降（P〈0.05）,而股骨干的骨密度没有变化（P〉0.05）。在体外培养的假手术组BMSC中,beta-catenin蛋白主要在胞核中表达。与假手术组相比,去卵巢组BMSC核中beta-catenin蛋白表达减少,而胞膜中表达增加。结论beta-catenin蛋白在卵巢切除大鼠BMSC核中表达的下降可能与绝经后骨质疏松症的发生有关。     

维生素K2对去卵巢大鼠股骨干骨折愈合影响的实验研究

目的探讨维生素K2能否促进卵巢切除骨质疏松(OVX)大鼠骨折愈合。方法将30只雌性大鼠随机分为3组(每组10只):假手术组(SHAM)、去卵巢组(OVX)、OVX+维生素K2组。大鼠卵巢切除术后3个月,在右侧股骨干制作单侧股骨干骨折。然后在通过维生素K2治疗8周后处死动物,行X线片检测;对骨折的股骨进行生物力学检测并观察血清骨钙素(BGLAP)、碱性磷酸酶(ALP)、抗酒石酸酸性磷酸酶(TRACP)和雌二醇水平的变化。结果与OVX组相比,OVX+维生素K2组观察到更多骨痂组织形成和更好的骨愈合,并且BGLAP、ALP和TRACP水平降低,但是血液雌二醇水平没有观察到显著增加。与OVX组相比,OVX+维生素K2组显示出骨强度、最大负荷和弹性显著增加。结论维生素K2具有作为绝经后骨质疏松症骨折愈合新型替代治疗剂的潜力。     

左归丸防治去卵巢大鼠骨质疏松症的实验研究

目的探讨左归丸对去卵巢所致骨质疏松大鼠的防治作用。方法 手术切除大鼠双侧卵巢建立绝经后骨质疏松大鼠模型,用左归丸(高、中、低剂量)进行灌胃,120 d后,取大鼠左后肢股骨远端1/3做病理切片,光镜下观察骨组织形态学变化,测定骨小梁面积百分比(%Tb.Ar)、骨小梁厚度(Tb.Th)、骨小梁分离度(Tb.Sp);采用双能X线骨密度仪测定右后肢离体股骨近端1/3骨密度(BMD);采用酶联免疫吸附法(ELISA)检测大鼠血清骨钙素(BGP),用比色法测定血清抗酒石酸酸性磷酸酶(TRAP)。结果切除大鼠双侧卵巢后,骨小梁明显紊乱,变细且较稀疏,缺少连接,断端增多,%Tb.Ar、Tb.Th显著降低,Tb.Sp明显升高,BMD下降,血清BGP及TRAP水平显著升高,统计学显示有显著性差异(P0.01);去卵巢大鼠灌服左归丸后,与模型空白组比较,左归丸各剂量组均能不同程度地改善骨小梁变化,增加BMD,降低血清BGP及TRAP,统计学显示有显著性差异(P0.01),各用药组之间比较无显著性差异(P0.05)。结论 左归丸对去卵巢所致绝经后骨质疏松大鼠有一定的防治作用。     

维生素K3对去卵巢大鼠骨质疏松预防作用的实验研究

绝经后骨质疏松的防治以药物为主要手段．近年来一些实验初步表明，维生素K可通过促进骨矿化和抑制骨吸收等作用减少绝经后骨量丢失．且无明显副作用。本实验进一步探讨维生素K3对击卵巢大鼠骨质疏松的预防作用，并将其效果与尼尔雌醇(CEE8)进行比较。结果显示，维生素K3组椎体和股骨端骨密度显著高于()VX组(P＜0．001)．光镜下骨小粱较0VX组增宽、致密，透射电镜下维生素K2组骨细胞呈功能活跃状态．胶原纤维排列较()VX组致密，分布亦均匀。CEE3组各项结果优于维生素K3组，VTK3 CEE3组优于单一用药组。以上结果表明．维生素K。可在一定程度上预防去卵巢大鼠骨质疏松，效果逊于尼尔雌醇，二者联合用药效果优于单用药。     

维生素K2及其与性激素联合应用对去卵巢大鼠骨的影响

目的观察维生素K2（vitamin K2,VK2）及其与性激素（sex hormone,SH）联合应用对去卵巢大鼠骨质疏松模型预防的效果。方法30只SPF级10月龄雌性SD大鼠按体重随机分为4组,行卵巢切除（OVX,n=22）或假手术（Sham,n=8）,术后2周给以不同的药物干预：VK2组（OVX＋VK2,n=7）、VK2＋SH组（OVX＋VK2＋SH,n=7）;OVX组（n=8）和假手术组不做药物干预。14周后检测血骨特异性碱性磷酸酶（BALP）和尿脱氧吡啶啉（uDPYD）;测定骨密度（BMD）。牺牲后进行股骨三点弯曲实验和股骨远端骨组织形态计量学测定。结果①骨转换指标：OVX组较Sham组BALP和uDPYD显著升高,骨转换加快。与OVX组相比,VK2组uDPYD明显增加,而BALP没有统计学差异;VK2＋SH组BALP和uDPYD均下降。②BMD：标化体重,OVX组腰椎和股骨BMD明显低于Sham组,VK2组腰椎BMD显著高于OVX组,VK2＋SH组BMD没有变化。③股骨远端形态计量学：OVX组与Sham组相比,骨小梁厚度变薄,分离度增加,数量减少,但无统计学意义,骨形成和骨吸收参数均升高,其中骨形成率（BFR/Bs）、类骨质周长百分数（%O.Pm）与Sham组有统计学差异。与OVX组相比,用药组对骨量和骨小梁结构无改善,均有降低骨形成参数的趋势,从数值上看,VK2＋SH组降低的程度〉VK2组。VK2＋SH组骨矿化延迟时间（MLT）、BFR/Bs下降最为显著。④股骨三点弯曲实验结果：VK2＋SH组最大载荷较其他各组显著降低,最大应变和弹性模量各组之间无统计学差异。结论单用VK2可以增加去卵巢大鼠腰椎BMD,但作用机制尚不清楚。VK2与SH合用未发现对骨健康有利。     

去卵巢对大鼠骨密度的影响

目的：探讨去卵巢对大鼠骨密度（BMD）的影响。方法；20只3．5月龄SD雌性大鼠分别除双侧卵巢（OVX）或假性去卵巢（Sham)，术后14周处死，应用QDR－4500A型扇形束双能X射线吸收法（DXA）测量大鼠全身、离体股骨、胫骨、腰维及兴趣区的BMD。结果：①术后6周OVX组全身BMD显低Sham组（P＝0．048），术后14周两组无显性差异；②术后14周OVX组离体股骨BMD显低于Sham组（P＜0．01），股骨远侧干骺端平均降低11．6％（P＜0．001）；③术后14周右侧离体胫骨BMD两组间差异无显性，但OVX组胫骨的端干骺端BMD显低于Sham组（P＜0．001）；④术后14周OVX组腰椎（L4－L6）的BMD显低于Sham组（P＝0．014），第六腰椎降低明显，平均降低8．1％（P＝0．005）。结论：去卵巢所致骨丢失以松质骨含量丰富的兴趣区明显。     

维生素K2与绝经后骨质疏松症

绝经后骨质疏松症（postmenapausal osteoporosis，PMO）是以雌激素水平下降致骨代谢高转换，从而使破骨细胞（osteoclast，OC）及骨小梁丢失增多，骨折风险增高为主要特征的常见骨科疾患，居代谢性骨病第1位，目前尚无安全有效的根治方法。为此，国内外学者不断探索，近年来试图采用维生素K（Vitamin K2，VK2）防治PMO，并取得相当进展，现综述如下。     

密骨颗粒对去卵巢大鼠TGF-β1表达的影响

目的 观察密骨颗粒对去卵巢大鼠TGF-β1表达的影响，探讨其对绝经后骨质疏松症的预防机制。方法 运用切除大鼠双侧卵巢方法建立骨质疏松症模型，应用中药密骨颗粒防治12周，采用双能X线骨密度测定法、Elisa、免疫组化SABC法、反转录聚合酶链方法，分别观察其对实验鼠离体骨密度、血清TGF-β1活性及其在骨组织中的蛋白和mRNA表达的影响。结果 中药密骨颗粒可以显著升高骨密度，提高血清TGF-β1活性，上调其在骨组织中的mRNA和蛋白表达水平，其蛋白广泛表达于胫骨干骺端。结论 中药密骨颗粒可通过调节TGF-βI活性及其mRNA表达水平而对去卵巢大鼠骨质疏松有预防或延缓发生的作用。     

雌激素预防性给药对去卵巢大鼠骨和脏器的影响

目的采用双侧卵巢切除术建立绝经后骨质疏松(postmenopausal osteoporosis,PMOP)大鼠模型,探讨雌激素预防性给药对绝经后大鼠骨和脏器的影响。方法将SD大鼠分为假手术(sham-operated,Sham)组、去卵巢(ovariectomized,OVX)组、雌二醇组(β-estradiol-treated OVX,OVX/E2)组。术后第10 d开始皮下注射给药并称量大鼠体重,术后61 d处死大鼠,取脏器和骨,称量脏器重量,计算脏器指数。制备组织切片,进行HE染色。结果组织形态学观察表明,OVX组大鼠的股骨和胫骨均出现骨小梁断裂、间距变大、结构紊乱等骨质疏松症状,而OVX/E2组并未出现明显的发病症状。相较于Sham组,OVX组大鼠子宫内膜固有层中的子宫腺数目增多,腺腔增大,子宫黏膜上皮明显增厚,而OVX/E2组的大鼠子宫形态结构并未发生明显病变。大鼠体重和脏器指数分析表明,摘除卵巢不仅会引起大鼠术后早期的体重增加,还会导致大鼠肝、肺、肾和脾的脏器指数增加,而雌激素预防性给药能一定程度上缓解去卵巢手术引发的脏器指数的异常变化。结论适时进行一定剂量的雌激素给药能够较好地预防绝经后骨质疏松症的发生,为绝经后骨质疏松症的预防和治疗提供参考。     

MGP基因的重组及在大肠杆菌中的表达

目的重组骨质疏松候选基因基质Gla蛋白（MGP）基因使其蛋白在大肠杆菌中高效表达。方法利用反转录聚合酶链反应（RT—PCR）从正常人肺组织总RNA中扩增出MGP基因cDNA序列，与克隆pGEM—Teasy载体相连，测序为完整的编码序列后与表达载体pTrcHisB构建重组体，转化入大肠杆菌Top10后用IPTG诱导，Western bloting证实蛋白表达。结果克隆至pGEM-Teasy载体及pTreHisB载体中的MGP基因cDNA序列与基因库完全一致。转入大肠杆菌后经IPTG诱导有蛋白的表达，Western bloting证实诱导后2、3、4h蛋白的表达量显著增加。结论成功重组的人MGP基因，重组体在大肠杆菌内能成功高效地表达。IPTG诱导后蛋白的表达为时间依赖性。     

强骨饮对去卵巢大鼠腰椎骨形态计量学的影响

目的：观察自拟强骨饮对骨质疏松症的骨形态计量学的影响,并探讨其机制。方法：选健康雌性SD大鼠60只,体重230～280g。随机分为3组,强骨饮组（治疗组）、密钙息组（对照组）和空白组,每组20只。分别采取切除双侧卵巢方法进行骨质疏松造模,10周造模成功后,开始给药,治疗组用自拟强骨饮灌胃,每日1次,每次0.001ml/kg;对照组用密钙息皮下注射,每日1次,每次0.72U/kg。空白组,不做处理。在给药后45、90、135及180d每组各取5只大鼠进行检测,先测量体重,然后空气栓塞处死,分离腰椎,获得腰椎样本,经切片等处理后,显微镜下作骨形态计量学检测（包括骨体积分数、骨小梁厚度、骨小梁间距）。结果：给药后135d治疗组骨小梁厚度与对照组相比差异有统计学意义。给药后180d治疗组骨小梁厚度、骨小梁间距、骨体积分数与对照组相比均具有统计学意义。治疗组与空白组相比骨小梁厚度、骨小梁间距、骨体积分数差异均具有统计学意义。结论：自拟强骨饮可以明显改善骨形态计量学指标,可能是通过刺激成骨细胞生长,抑制破骨细胞活性,并抑制高骨转换趋势来实现的。     

Effect of vitamin K2 on three-dimensional trabecular microarchitecture in ovariectomized rats.

Menatetrenone, a vitamin K2 with four isoprene units, has been reported to improve osteoporotic bone loss. The purpose of this investigation was to clarify the effect of menatetrenone on the three-dimensional (3D) trabecular microarchitecture in ovariectomized (OVX) rats by using microcomputed tomography (MCT). Forty-two 13-week-old female rats were used and divided into four groups: the OVX (OVX + MK-4) group treated with menatetrenone, the (OVX untreated) group, the sham-operated (Sham + MK-4) group treated with menatetrenone, and the sham-operated group not treated with menatetrenone (Sham untreated) group. OVX rats were fed a calcium-deficient diet. Menatetrenone treatment was begun just after the ovariectomy, and the mean menatetrenone oral intake over the 8-week period was adjusted to 30 mg/kg BW per day. The proximal metaphyseal region of the right tibia was evaluated by dual X-ray absorptiometry (DXA) and MCT. A parametric analysis of the reconstructed trabecular volume was carried out using bone volume fractions, the fractal dimension calculated by the 3D box-counting method, and the connectivity density as determined by topological analysis. Menatetrenone significantly increased the trabecular bone volume, fractal dimension, and connectivity in the OVX + MK-4 group compared with the OVX-untreated group (p < 0.01). Our results suggest that an 8-week administration of menatetrenone protects against the loss of trabecular bone volume and its connectivity when treatment is begun just after the ovariectomy. Despite this apparent protection, it remains unknown whether it is possible to reestablish trabecular connectivity if therapeutic intervention occurs after the trabecular connectivity has been lost.     

Effect of vitamin K2 (menaquinone-7) in fermented soybean (natto) on bone loss in ovariectomized rats

The effect of dietary vitamin K₂ (menaquinone-7) on bone loss in ovariectomized (OVX) rats was investigated. OVX rats were freely given experimental diets containing menaquinone-4 (MK-4; 12 mg/100 g diet) or menaquinone-7 (MK-7; 18.1 mg/100 g diet) for 24 days; MK-4 and MK-7 were equal in molar concentrations. This feeding caused a remarkable increase of MK-4 and MK-7 concentrations in the serum and femur of OVX rats. OVX-induced decrease in the femoral dry weight and femoral calcium content was prevented by the feeding of dietary MK-4 or NK-7. In separate experiments, OVX rats were freely given experimental diets containing the fermented soybean (natto; including 9.4 μg MK-7/100 g diet) without or with added MK-7 (37.6 μg/100 g diet) for 77 days. Feeding produced a significant elevation of MK-4 and MK-7 concentrations in the serum of OVX rats. In this case, a significant increase in the femoral MK-4 content was observed but MK-7 was not detected in the femoral tissues. OVX-induced decreases in the femoral dry weight and femoral calcium content were significantly prevented by the feeding of diets containing natto with MK-7 added (37.6 μg/100 g diets). This study demonstrates that the intake of dietary MK-7 has a preventive effect on bone loss caused by OVX. This effect may be partly caused by MK-4, which is formed by degradation of MK-7. Received: July 23, 1998 / Accepted: Sept. 28, 1998     

雌激素缺乏脊柱神经肽P物质变化的实验研究

目的阐明雌激素缺乏时椎体、椎间盘及小关节等部位ＳＰ免疫反应阳性神经纤维的变化规律。方法选用健康２月龄雌性ＳＤ大鼠９６只,随机分为卵巢切除（Ｏｖａｒｉｅｃｔｏｍｙ,ＯＶＸ）和假手术（Ｓｈａｍ－ｏｐｅｒａｔｅｄ,Ｓｈａｍ－Ｏ）两组。ＯＶＸ组行双侧卵巢切除术,Ｓｈａｍ－Ｏ组行假手术。分别于术后１５天、１、２、３、６、９月时每组各处死８只动物,切取Ｌ３～５脊柱小关节、棘上韧带、椎体和椎间盘。免疫组织化学Ｓ－Ｐ法观察各部位ＳＰ免疫反应阳性神经纤维分布。结果①ＯＶＸ后骨小梁及小梁膜上ＳＰ免疫反应阳性神经纤维增多,增粗。②软骨组织内ＳＰ反应阳性纤维增多;２月时椎间盘ＳＰ阳性反应物增加,分布至纤维环中带,３月时观察到纤维环内带,６月在纤维环髓核中观察到ＳＰ免疫反应阳性神经纤维。结论雌激素缺乏时大鼠腰椎椎体、椎间盘、小关节ＳＰ阳性反应神经纤维增多,与骨质疏松性腰背疼痛有密切关系。     

Activin increases bone mass and mechanical strength of lumbar vertebrae in aged ovariectomized rats

Activin is a member of the transforming growth factor-beta superfamily and is thought to be involved in the regulation of bone formation due to its presence in bone tissue and its osteogenic activity both in vitro and in vivo. We recently found that systemic administration of activin increased both tibial bone mass and mechanical strength in young growing rats. The present study investigated the effects of activin in aged ovariectomized (ovx) rats. Twelve-month-old Fischer rats were ovariectomized and maintained for 10 months. Recombinant human activin A (activin) or human parathyroid hormone 1-34 (PTH) was administered intramuscularly three times a week for 12 weeks. Activin (1 and 5 microg/kg) markedly increased lumbar vertebral bone mineral content and bone mineral density. Activin also increased the mechanical strength of the vertebral body, which was highly correlated to the bone mineral density of the vertebral body. The maximal response in bone mass and strength was observed at 1 microg/kg of activin, which was approximately equal to that induced by PTH at 40 microg/kg. Peripheral quantitative computed tomography revealed that activin enlarged the cross-sectional size of the vertebrae without changing the foramen area, indicating its effects on cortical shells. Histomorphometric analysis of cancellous bone of vertebral body in similar experiment showed that activin (3 microg/kg) increased bone volume and the mineralizing surface, although its effects were less than PTH. The present results indicate that low doses of activin are effective against vertebral bone loss in aged ovx rats.     

Effect of vitamin K2 on lumbar vertebral bone: histomorphometric analyses in experimental osteoporotic rats

The in-vivo effect of vitamin K₂ on bone metabolism was investigated by histochemical and morphometric methods, using an animal model of osteoporosis. Eighteen female Wistar rats were divided into three groups. Rats in group A had sham ovariectomies, group B were ovariectomized, and group C were ovariectomized and received vitamin K₂, at 10 mg/kg per day, injected subcutanously. The lumbar vertebral bones were evaluated 8 weeks after the operation by a modified tetrachrome method after decalcification. Mineralized bone areas, osteoid, and deficectively mineralized bone areas in group B were markedly decreased compared with findings in group A, but these features in group C were not severely decreased. There was no significant difference in total bone areas and total bone volumes among the three groups. Accordingly, it appeared that vitamin K₂ had an effect in reducing mineralized bone loss after the ovariectomy. In conclusion, vitamin K₂ is thought to be beneficial for the properties of bone microarchitecture in the condition of osteoporosis. Received: September 14, 2000 / Accepted: June 11, 2001