胃内的分泌蛋白影响骨质代谢的相关机制研究
|
摘要
研究背景
随着社会的老龄化,骨质疏松症(osteoporosis,OP)的发生日渐增多,但OP的发病机理至今尚未被人们完全认识,能够导致OP的相关因素有很多,这包括:遗传因素、女性绝经、钙离子摄入或吸收的不足等等。近年,由于患消化道疾病而引发OP的现象逐渐引起了人们的重视。许多患胃肠道疾病的患者,尤其是经过全胃或部分胃切除的患者会发生明显的全身骨量减少的现象。进一步的实验及临床对照研究均证实消化道疾病与OP的发生关系密切。对其原因的认识最初认为是由于胃切除后胃酸减少而引起钙吸收不足,然而实际上并非如此简单。目前大量研究结果显示:胃粘膜所分泌的某种成份可以增加实验动物的骨量,而且其分泌受胃泌素(gastrin)的调控;经肽链端解酶酶解后这种促进成骨的作用消失。学者们由此推测出:胃粘膜中的分泌细胞可以分泌一种具有促进骨合成作用的激素肽,并将其命名为胃钙素(gastrocalcin),并把它认为是继甲状旁腺激素、雌激素、维生素D之后的第四种骨钙调解激素。
目前针对骨质疏松症的药物干预主要包括两方面:骨吸收抑制剂和骨和成促进剂。骨吸收抑制剂包括:雌激素、降钙素、双磷酸盐等。这些药物可以通过抑制破骨细胞的骨吸收功能减少骨量的损失,从而暂时防治骨质疏松症,但是由于骨的更新转化受抑制将导致骨质的老化、陈旧。因此治疗骨质疏松症不仅需要应用骨吸收抑制剂来防止骨量持续降低,而且还需同时使用刺激骨形成的药物以增加骨量,可采用联合用药。骨吸收抑制剂只能维持骨量和暂时骨形成增加,应用骨形成促进剂才会有效地提高骨量,这也是当前世界治疗骨质疏松症新药研究的主要方向。目前,骨形成刺激剂主要为甲状旁腺激素,已于2002年由美国FDA批准用于骨质疏松症治疗。但已有的PTH制剂价格昂贵,需注射给药,限制了其推广应用;而且有研究认为PTH的正性作用仅限于脊柱,其他部位可能均为负性效应,对骨折风险的影响尚待进一步研究。
总之,针对与胃粘膜中存在的这种促进骨合成活性物质的研究及应用有着广阔的研究、开发前景。然而到目前为止,这种从功能表现方面推测出的胃钙素到底是什么蛋白,仍旧未知。其性质不明则难以针对其进一步研究,难以阐明胃切除或病变诱发骨质疏松症的作用机制,也就无从采取基于此方面的各种具有针对性的干预措施。因此本课题为试图阐明胃钙素的性质,展开了一系列探索性研究。
研究目的:
1.观察胃粘膜提取物对成骨细胞生物学活性的作用。因为成骨细胞是机体骨质形成的执行者,以往的研究虽然已确定了胃粘膜提取物中含有促进机体骨量增加的因子,但是其对成骨细胞生物学活性的影响尚无研究,所以我们首先要明确胃粘膜提取物是否对成骨细胞的成骨活性有影响,有何影响。
2.对胃钙素候选蛋白进行筛选,从而确定胃钙素的性质。根据胃粘膜中促进骨形成因子可受胃泌素调控,即胃泌素可以促进这种因子释放的特点,我们采用了蛋白质组学技术,对受胃泌素干预前后,胃粘膜中所差异表达的蛋白进行筛选,试图从这些差异表达的蛋白中找到这种促进骨形成因子——即胃钙素。
研究意义:
1.观察胃粘膜提取物对成骨细胞生物学活性的作用将阐明其促进成骨的作用机制。
2.筛选出胃钙素并确定其性质,将有利于阐明胃切除或病变诱发骨代谢疾病的机制,有助于加深对骨代谢和OP的病理机制的认识;为防治OP提供新的干预靶点。
拟解决的科学问题
1.胃泌酸粘膜提取物是否直接通过成骨细胞来实现促进成骨的作用?
2.胃泌素干预后胃粘膜组织中所表达的差异蛋白有哪些?
3.胃泌酸粘膜提取物中这种促进骨合成因子——即胃钙素究竟是什么?
研究方法:
1.采用多次酶消化法,体外分离培养大鼠幼鼠的成骨细胞,利用细胞形态学观察、Giemsa染色、碱性磷酸酶染色、钙结节染色、荧光染色等方法对成骨细胞进行鉴定。
2.大鼠胃粘膜组织蛋白的制备。取大鼠胃囊,剥离下胃粘膜组织置于匀浆缓冲液中进行匀浆。离心、弃沉淀,上清液通过一系列空隙从大到小的微孔滤膜进行过滤。滤液离心、弃上清,沉淀溶于盐水中,超声冰上震荡,滤菌、测浓度后以生理盐水将其稀释为低(10~(-3)g/l)、中(10~(-2)g/l)、高(10~(-1)g/l)三种浓度,-80℃保存。
3.激光共聚焦显微镜观察。成骨细胞接种于激光共聚焦显微镜专用皿中,细胞贴壁后继续培养24小时后行细胞内钙离子的荧光标记。将培养皿置于激光共聚焦显微镜的载物台上,观察施加不同浓度胃粘膜提取物刺激前后细胞内荧光强度的变化,获得反映细胞内钙离子浓度动态变化的曲线。
4.成骨细胞的增殖观察。细胞接种于96孔培养板,每孔接种2000个细胞,过夜细胞贴壁后,每孔分别加入不同浓度的胃粘膜提取物或以生理盐水作为对照来干预细胞,按照CCK-8试剂盒的方法,采用紫外分光光度仪分别检测加入刺激因素前,刺激后1天、2天、3天的光密度值。
5.RT-PCR实验。成骨细胞接种于直径6cm的培养皿中,贴壁、融合更换培养液,以不同浓度的胃粘膜提取物或生理盐水对照来干预细胞,继续培养。TrizolReagent提取细胞总RNA后测浓度,定量后,将总RNA逆转录为cDNA,采用PCR技术分别扩增出collagen typeⅠ,osteocalcin和β-actin目的基因。PCR反应结束后,取反应液,琼脂糖凝胶电泳,紫外线下观察并用凝胶成像分析系统做PCR产物半定量分析。
6.Western-blot检测。细胞经不同浓度胃粘膜提取物干预后分别提取细胞的总蛋白、定量、电泳、转膜、封闭一抗杂交、洗膜、二抗杂交、再洗膜,ECL发光试剂盒发光,显影,定影。检测collagen typeⅠ,osteocalcin和β-actin蛋白的印迹图像并用光密度扫描仪半定量分析。
7.蛋白质组学实验。提取胃泌素干预前、后的胃粘膜组织的总蛋白,经除盐、定量等处理后,采用荧光差异双向电泳技术(2D-DIGE)分离,经质谱技术(MS)分析鉴定出经胃泌素干预的胃粘膜组织所差异表达的蛋白。
8.生物信息学技术分析:利用生物信息学技术,通过数据库检索,文献分析对所鉴定的差异蛋白进行分析,从中找到感兴趣的候选蛋,以进一步确定下一步的研究方向。
9.利用Western-blot技术对蛋白质组学所筛选出的蛋白进行验证。
10.利用免疫组化技术对所筛选出的蛋白在胃粘膜中的定位及分布情况进行观察。
结果:
1.形态学观察,碱性磷酸酶细胞化学染色和细胞结节染色表明:培养的细胞符合成骨细胞的形态特征,具有分泌碱性磷酸酶和形成矿化结节的生物学行为。
2.激光共聚焦显微镜观察胃粘膜提取物刺激前后细胞内钙离子浓度的变化:在每个视野中分为4个象限,在每个象限中随机选取5个细胞,共20个细胞进行观察,结果为:对照组细胞内的钙离子浓度无任何变化,低浓度实验组有15%的细胞(20个细胞中有3个),中浓度组有35%(20个细胞中有7个),高浓度组有95%(20个细胞中有19个)发生了钙离子浓度短暂、快速增高的变化。
3.成骨细胞增殖实验:不同浓度的胃粘膜提取物干预后第一、二、三天的细胞数量均比对照组增多。经统计学分析表明:3个实验组与对照组比较存在差异,而且差异具有显著性(p＜0.01)。
4.RT-PCR与western-blot实验:与对照组比较,三组不同浓度的实验组,均可提高collagen typeⅠ和osteocalcin的mRNA和蛋白的表达水平,经统计学分析其差异存在显著性(p＜0.05)。
5.蛋白质组学实验:经胃泌素干预后,胃粘膜组织中差异表达蛋白50个,其中增高表达21个,降低表达29个。经质谱分析后鉴定出了22个差异蛋白。
6.生物信息学分析:对上述22个差异蛋白进行了亚细胞定位,蛋白质之间相互作用预测,以及利用已有的文献对其功能、作用进行了分析。发现:蛋白质二硫异构酶A3(Protein disulfide-isomerase A3,PDIA3)经胃泌素干预后,在胃粘膜中高度表达;而且其在无机盐代谢过程中起着重要作用:其作为维生素D3在靶细胞膜上的受体蛋白,存在于肠粘膜细胞,成骨细胞、骨细胞、软骨细胞、成釉等细胞的膜上。PDIA3与维生素D3结合后可以快速促进钙磷在肠道内的吸收;诱导软骨细胞一系列的生化反应,包括:细胞膜通透性的改变,钙离子内流,磷脂的翻转,还有蛋白激酶C的激活,还具有促进软骨细胞分化、细胞外基质矿化的作用。另外,其不仅存在于细胞膜上,也在细胞外质中有表达。
7.经Western-blot检测发现经胃泌素干预后的大鼠胃粘膜中的PDIA3/1,25D3-MARRS蛋白表达量增加,这一结果与蛋白质组学的实验结果是一致的,从而从逆向实验角度对蛋白质组学实验结果进行了验证。
8.免疫组化对PDIA3/1,25D3-MARRS在胃粘膜中的定位及分布情况进行观测发现:在大鼠和人的胃粘膜下层中的胃腺管内遍布PDIA3/1,25D3-MARR蛋白的阳性表达。
结论:
1.胃粘膜提取物可呈剂量依赖关系引起成骨细胞内的钙离子迅速短暂地增加。
2.胃粘膜提取物能够地促进成骨细胞的增殖。
3.胃粘膜提取物可促进成骨细胞的成骨活性。
4.PDIA3/1,25D3-MARRS被认为是胃粘膜组织中促进骨合成作用的关键因素。
5.PDIA3/1,25D3-MARRS是可由胃粘膜内分泌的分泌蛋白。
本研究的创新之处:
1.以往的研究发现胃粘膜提取物具有促进实验动物骨量的作用,通过本研究项目不但验证了这一论点,而且阐明其成骨作用是通过直接促进成骨细胞的增殖和分化来完成的。
2.利用胃泌素干预后胃钙素表达增加的特点,采用蛋白质组学技术,包括:2D-DIGE,基质辅助激光解析飞行时间质谱(MALDI-TOF MS)技术对胃钙素的候选蛋白进行筛选。
3.提出PDIA3可能就是胃组织内促进骨合成的活性因子。目前,所谓的胃钙素只是根据其明确的功能推测出的肽类激素,因此其性质尚未明了,通过本研究提出PDIA3可能就是胃钙素,从而为进一步研究指明了方向。
4.对胃钙素的探索研究将有利于阐明胃切除或病变诱发骨质疏松症的机制,将创新性地提出继甲状旁腺激素、降钙素、雌激素之后的第四种骨钙调解激素,这是对传统钙离子代谢调节机制的重要补充。
5.对胃钙素的研究将为防治胃切除或病变诱发骨质疏松症甚至原发性骨质疏松症探索新的干预靶点。
Background
The morbility of Osteoporosis(OP)is increasing in the aging society.There are a lot of related effective factors result to OP,though its mechanism still unclear,which include hereditary factor,menopause,intake or absorption deficient about calcium or phosphorus,et al.There are certainly interesting focus on the OP induced by gastrectomy or gastropathy recently.It is well known that there is certain connection between the gastrectomy and osteoporosis.By now,more and more evidences show that the extracts of oxyntic mucosa(EOM)could increase the bone mess,Both gastrin-17 and the extract of oxyntic mucosa(EOM)induce transient hypocalcaemia and stimulate an uptake of Ca into bone in rats.Gastrin was without effect in rats that had undergone gastrectomy,whereas the EOM were equally effective in unoperated and gastrectomized rats.And this hypocalcemic effect was totally abolished by prior addition with leucine aminopeptidase.That suggests that the effective component of EOM is a small peptide.We propose the name gastrocalcin for this peptide hormone,which is produced in the oxyntic mucosa and released by gastrin.It accelerates calcium uptake into bone,thereby explaining the hypocalcaemia evoked by gastrin.To the three functionally interacting factors controlling Ca2+ concentration in blood(parathyroid hormone,calcitonin,and vitamin D),we wish to add a fourth---namely,gastrocalcin.
Medicine treatments for OP include anti-resorptive agents and bone-forming agents.Anti-resorptive agents,such as estrogens,calcitonin,bisphosphonates,can inhibit the bioactivity of osteoclasts and increase the bone mess relatively.Bone remodeling is an ongoing process in human bone biology that is necessary to repair micro-damage and renew skeletal integrity strength.Therefore,inhibition of bone resorption mechanism would induce the problem of bone aging.The idea medicine treatment of OP should contain both anti-resorptive agents and bone-forming agents, it is the major tend of OP management in the world.The main bone-forming agent is PTH which was approval to apply in clinic at 2002.But its cost is expensive and it would be administrated by intravenous injection only;these disadvantages limited its extensive utility.
Above all,there is another bone-forming agent in the oxyntic mucosa of rats, which is named gastrocalcin.Therefore the study about gastrocalcin has great prospective.However gastrocalcin was a hypothetic peptide result from its promoting bone-forming function,its nature still unclear by now.Since that,this study about the gastrocalcin is much more important for explore the mechanism of OP introduced by gastrectomy.
Objectives
1、Explore the effects of EOM on the bioactivity of osteoblast.
2、Screening the candidate protein of gastrocalcin and determining its nature.
Methods
1、Osteoblast isolation,culture,and identification:Osteoblasts were isolated from calvariae of 1-day-old Sprague-Dawley rats with the way of sequential enzymatic digestion,and than were identified by morphology,ALP staining, Alizarin red staining and fluorescence staining.
2、Preparation of EOM in rats:EOM was prepared as described previously. Briefly,oxyntic mucosa was collected from six male Sprague-Dawley rats.The stomach was opened,the antrum was removed,and the oxyntic gland area (fundus)rinsed in saline and placed on a cold silica gel plate surface.The mucosa free of mucus was scraped to separate from the muscle wall,then minced by Ultra-Turrax homogenizer and homogenized in buffer,using a Potter-Elvehjem glass homogenizer.The homogenate was centrifuged and the pellet was discarded.The supernatant was passed through cheesecloth. Filtration was followed by centrifugation.After the supernatant was discarded, the pellet was resuspended in the saline(with 1%proteinase inhibitor)by ultrasonication six times(six seconds each)on the ice.At last the solution was quantified according to the instruction of BCA protein assay kit and diluted to serial concentrations:10~(-1)g/l,10~(-2)g/l,10~(-3)g/l with saline respectively,then kept at-80℃.
3、Ca concentration in cytoplasm of osteoblasts:The cells at the third generation were digested and diluted to 1×10~7/L,then inoculated in four special 50mm culture dishes for laser scanning confocal microscopy,for 24 hours,followed by 5μM Fluo-3Am applied into each dish to label the intracytoplasm Ca~(2+). The dishes were placed on the sample holding plate of the confocal microscope and the change of[Ca~(2+)]i was investigated,under the normal(saline as control) or the influences of EOM in different concentrations.
4、Proliferation of osteoblast:Cells were inoculated into 96-wells plate at the density of 2×10~3 cells per well and divided into four groups and treated with low,mid,high concentrations of EOM and saline as control,respectively.24 hours after cell seeding,10μL EOM at different concentration or saline was added into each well.0,1,2 and 3 days after the treatment,cell proliferation was assessed according to the instruction of CCK-8 kit(BI Yuntian Co,China), respectively.In brief,at the end of each time point,10μL WST-8 was added to each well,and the plates were incubated for an additional 4 hours at 37℃to convert WST-8 into formazan.The absorbance of each plate was measured at 450 nm(absorbance)and 600 nm(background)with spectrophotometer.
5、Reverse transcriptase polymerase chain reaction(RT-PCR)test:The cells were inoculated in 6 cm culture dishes and designed into four groups:low,mid,high concentration EOM groups and saline control group.24 hours later,the new culture medium added in to culture system,together with 200μl EOM at different concentration or saline.After another 24 hour culture,total RNA was extracted from each group's osteoblasts using a TRIzol kit.The reverse transcription reaction was performed using 4μg of total RNA and oligo(dT) primer,and collagen typeⅠ,osteocalcin,P-actin(reference gene)gene were amplified using two oligonucleotide primers.The PCR products were separated electrophoretically in a 2%agarose DNA gel stained with ethidium bromide. Semi-quantitative analysis was performed using computing densitometer and Image Quant software.
6、Western-blot test:Cells were cultured in 6 cm culture dishes and treated with EOM.After 48 hours,the total proteins were extracted from each group. Proteins concentration were assessed by BCA protein assay kit,according to the manufacturer's instructions.Equal proteins(60μg)were separated electrophoretically in SDS-PAGE for collagen typeⅠ(138kDa)andβ-actin (43kDa)or Tricine-SDS-PAGE for osteocalcin(5.8kDa).The proteins were transferred to immobilon polyvinyldifluoride(PVDF)membranes.After blocked with 5%BSA for 1 h at room temperature,the membrane was incubated with rabbit anti-mouse antibodies against collagen typeⅠ,osteocalcin orβ-actin(for reference protein)(1:1000)for 1 h at room temperature,washed 6 times(5 minutes each)with TBS buffer containing 0.2%TW-20,followed by incubation with anti-rabbit peroxidase-conjugated secondary antibody(1:3000) for 1 h at room temperature and wash as above.The proteins recognized by antibodies were visualized by enhanced chemiluminescence using Kodak X-OMAT LS film.Quantitative data were obtained using computing densitometer and Image Quant software.
7、Proteomics experiment:Total protein was extracted from the gastric mucosa tissue of experiment and the control group respectively.And then total protein was treated for desalination and quantitation.The protein samples were separated with fluorescence two-dimensional differential in-gel electrophoresis (2D-DIGE),and the different proteins were identified with matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF MS).
8、Analysis by bioinformatics technology:The identified different proteins were estimated further by the technology of bioinformatics through data base and literature.
9、The result of 2D-DIGE was reconfirmed by western-blot test.
10、The location and the distribution of candidate protein defined by 2D-DIGE were explored by immunohistochemistry test.
Results
1、Osteoblast identification:According to the morphology observation and the staining of ALP,AZR and fluorescence,the isolated and cultured cells from rat cranial bone have the characters of osteoblasts.
2、Intracytoplasm Ca2+of osteoblast assay:Under laser scanning confocal microscope,four quadrants total to 20 cells of each visual field were chosen randomly for intracytoplasm[Ca~(2+)]i recording,before or after administration of different stimulate factors.None cell changed in control group,but 15%(three out of twenty cells)in 10~(-3)g/L EOM group,35%(seven out of twenty cells)in 10~(-2)g/Lgroup and 95%(nineteen out of twenty cells)in 10~(-1)g/Lgroup had demonstrated a kind of response that the[Ca~(2+)]i in those cells showed increasing rapidly and temporally just after different concentration of EOM was administered。
3、Osteoblast proliferation assay:The number of cells in three experimental groups increased as compared with the control group,This difference is significant after being analysised by statistics(p＜0.05).
4、RT-PCR and Western-blot test:Contrast to the control group,the mRNA expression of collagen typeⅠand osteocalcin increased in three experimental groups This difference is significant after being analysised by statistics (p＜0.05).
5、Proteomics experiment:There are 50 different proteins expressed in experimental groups compared as control groups,among those different proteins,21 proteins increased expression and 29 decreased expression.22 different proteins were identified by MS.Analysis by bioinformatics technology: The 22 different proteins were located detailly into subcellular structure by WOLF PSORT data base.The interactivity of 22different proteins were predicted by STRING data base.Among the 22 different proteins,our interesting focus on the PDIA3 protein,which is highly expressed after the administration of gastrin,and plays an important role in the mechanism of mineral salt.In intestinal epithelial cells and perfused duodena,the PDIA3 also known as PDIA3/1,25D3-MARRS receptor mediates the rapid(seconds to minutes)stimulation of phosphate and calcium.And it is notable that osteoblasts,osteoclasts,and odontoblasts have been reported to contain PDIA3/1,25D3-MARRS.
6、According to the result of western-blot test,the expression of PDIA3/1,25D3-MARRS in rat stomach mucosa was increasing after the administration of gastrin.This result is coincident with 2D-DIGE.That is the confirmation for 2D-DIGE.
7、According to the result of immunohistochemistry,the expression of PDIA3/ 1,25D3-MARRS is positive in the rat mucosa,and the location of expression is in the glandular tube.
Conclusions
1、We have demonstrated that EOM elevated intracellular[Ca~(2+)]i,after the administration of EOM with doses relationship.
2、EOM enhanced osteoblast cells' proliferation significantly.
3、EOM increased the expression of collagen type I and osteocalcin in primary cultured osteoblasts,which means it can promote the bioactivity of osteoblasts.
4、PDIA3 was regarded as the key factor of promoting bone-forming derives from gastric mucosa.
5、PDIA3/1,25D3-MARRS can be regard as the endocrine protein screted from stomach mucosa.
随着社会的老龄化,骨质疏松症(osteoporosis,OP)的发生日渐增多,但OP的发病机理至今尚未被人们完全认识,能够导致OP的相关因素有很多,这包括:遗传因素、女性绝经、钙离子摄入或吸收的不足等等。近年,由于患消化道疾病而引发OP的现象逐渐引起了人们的重视。许多患胃肠道疾病的患者,尤其是经过全胃或部分胃切除的患者会发生明显的全身骨量减少的现象。进一步的实验及临床对照研究均证实消化道疾病与OP的发生关系密切。对其原因的认识最初认为是由于胃切除后胃酸减少而引起钙吸收不足,然而实际上并非如此简单。目前大量研究结果显示:胃粘膜所分泌的某种成份可以增加实验动物的骨量,而且其分泌受胃泌素(gastrin)的调控;经肽链端解酶酶解后这种促进成骨的作用消失。学者们由此推测出:胃粘膜中的分泌细胞可以分泌一种具有促进骨合成作用的激素肽,并将其命名为胃钙素(gastrocalcin),并把它认为是继甲状旁腺激素、雌激素、维生素D之后的第四种骨钙调解激素。
目前针对骨质疏松症的药物干预主要包括两方面:骨吸收抑制剂和骨和成促进剂。骨吸收抑制剂包括:雌激素、降钙素、双磷酸盐等。这些药物可以通过抑制破骨细胞的骨吸收功能减少骨量的损失,从而暂时防治骨质疏松症,但是由于骨的更新转化受抑制将导致骨质的老化、陈旧。因此治疗骨质疏松症不仅需要应用骨吸收抑制剂来防止骨量持续降低,而且还需同时使用刺激骨形成的药物以增加骨量,可采用联合用药。骨吸收抑制剂只能维持骨量和暂时骨形成增加,应用骨形成促进剂才会有效地提高骨量,这也是当前世界治疗骨质疏松症新药研究的主要方向。目前,骨形成刺激剂主要为甲状旁腺激素,已于2002年由美国FDA批准用于骨质疏松症治疗。但已有的PTH制剂价格昂贵,需注射给药,限制了其推广应用;而且有研究认为PTH的正性作用仅限于脊柱,其他部位可能均为负性效应,对骨折风险的影响尚待进一步研究。
总之,针对与胃粘膜中存在的这种促进骨合成活性物质的研究及应用有着广阔的研究、开发前景。然而到目前为止,这种从功能表现方面推测出的胃钙素到底是什么蛋白,仍旧未知。其性质不明则难以针对其进一步研究,难以阐明胃切除或病变诱发骨质疏松症的作用机制,也就无从采取基于此方面的各种具有针对性的干预措施。因此本课题为试图阐明胃钙素的性质,展开了一系列探索性研究。
研究目的:
1.观察胃粘膜提取物对成骨细胞生物学活性的作用。因为成骨细胞是机体骨质形成的执行者,以往的研究虽然已确定了胃粘膜提取物中含有促进机体骨量增加的因子,但是其对成骨细胞生物学活性的影响尚无研究,所以我们首先要明确胃粘膜提取物是否对成骨细胞的成骨活性有影响,有何影响。
2.对胃钙素候选蛋白进行筛选,从而确定胃钙素的性质。根据胃粘膜中促进骨形成因子可受胃泌素调控,即胃泌素可以促进这种因子释放的特点,我们采用了蛋白质组学技术,对受胃泌素干预前后,胃粘膜中所差异表达的蛋白进行筛选,试图从这些差异表达的蛋白中找到这种促进骨形成因子——即胃钙素。
研究意义:
1.观察胃粘膜提取物对成骨细胞生物学活性的作用将阐明其促进成骨的作用机制。
2.筛选出胃钙素并确定其性质,将有利于阐明胃切除或病变诱发骨代谢疾病的机制,有助于加深对骨代谢和OP的病理机制的认识;为防治OP提供新的干预靶点。
拟解决的科学问题
1.胃泌酸粘膜提取物是否直接通过成骨细胞来实现促进成骨的作用?
2.胃泌素干预后胃粘膜组织中所表达的差异蛋白有哪些?
3.胃泌酸粘膜提取物中这种促进骨合成因子——即胃钙素究竟是什么?
研究方法:
1.采用多次酶消化法,体外分离培养大鼠幼鼠的成骨细胞,利用细胞形态学观察、Giemsa染色、碱性磷酸酶染色、钙结节染色、荧光染色等方法对成骨细胞进行鉴定。
2.大鼠胃粘膜组织蛋白的制备。取大鼠胃囊,剥离下胃粘膜组织置于匀浆缓冲液中进行匀浆。离心、弃沉淀,上清液通过一系列空隙从大到小的微孔滤膜进行过滤。滤液离心、弃上清,沉淀溶于盐水中,超声冰上震荡,滤菌、测浓度后以生理盐水将其稀释为低(10~(-3)g/l)、中(10~(-2)g/l)、高(10~(-1)g/l)三种浓度,-80℃保存。
3.激光共聚焦显微镜观察。成骨细胞接种于激光共聚焦显微镜专用皿中,细胞贴壁后继续培养24小时后行细胞内钙离子的荧光标记。将培养皿置于激光共聚焦显微镜的载物台上,观察施加不同浓度胃粘膜提取物刺激前后细胞内荧光强度的变化,获得反映细胞内钙离子浓度动态变化的曲线。
4.成骨细胞的增殖观察。细胞接种于96孔培养板,每孔接种2000个细胞,过夜细胞贴壁后,每孔分别加入不同浓度的胃粘膜提取物或以生理盐水作为对照来干预细胞,按照CCK-8试剂盒的方法,采用紫外分光光度仪分别检测加入刺激因素前,刺激后1天、2天、3天的光密度值。
5.RT-PCR实验。成骨细胞接种于直径6cm的培养皿中,贴壁、融合更换培养液,以不同浓度的胃粘膜提取物或生理盐水对照来干预细胞,继续培养。TrizolReagent提取细胞总RNA后测浓度,定量后,将总RNA逆转录为cDNA,采用PCR技术分别扩增出collagen typeⅠ,osteocalcin和β-actin目的基因。PCR反应结束后,取反应液,琼脂糖凝胶电泳,紫外线下观察并用凝胶成像分析系统做PCR产物半定量分析。
6.Western-blot检测。细胞经不同浓度胃粘膜提取物干预后分别提取细胞的总蛋白、定量、电泳、转膜、封闭一抗杂交、洗膜、二抗杂交、再洗膜,ECL发光试剂盒发光,显影,定影。检测collagen typeⅠ,osteocalcin和β-actin蛋白的印迹图像并用光密度扫描仪半定量分析。
7.蛋白质组学实验。提取胃泌素干预前、后的胃粘膜组织的总蛋白,经除盐、定量等处理后,采用荧光差异双向电泳技术(2D-DIGE)分离,经质谱技术(MS)分析鉴定出经胃泌素干预的胃粘膜组织所差异表达的蛋白。
8.生物信息学技术分析:利用生物信息学技术,通过数据库检索,文献分析对所鉴定的差异蛋白进行分析,从中找到感兴趣的候选蛋,以进一步确定下一步的研究方向。
9.利用Western-blot技术对蛋白质组学所筛选出的蛋白进行验证。
10.利用免疫组化技术对所筛选出的蛋白在胃粘膜中的定位及分布情况进行观察。
结果:
1.形态学观察,碱性磷酸酶细胞化学染色和细胞结节染色表明:培养的细胞符合成骨细胞的形态特征,具有分泌碱性磷酸酶和形成矿化结节的生物学行为。
2.激光共聚焦显微镜观察胃粘膜提取物刺激前后细胞内钙离子浓度的变化:在每个视野中分为4个象限,在每个象限中随机选取5个细胞,共20个细胞进行观察,结果为:对照组细胞内的钙离子浓度无任何变化,低浓度实验组有15%的细胞(20个细胞中有3个),中浓度组有35%(20个细胞中有7个),高浓度组有95%(20个细胞中有19个)发生了钙离子浓度短暂、快速增高的变化。
3.成骨细胞增殖实验:不同浓度的胃粘膜提取物干预后第一、二、三天的细胞数量均比对照组增多。经统计学分析表明:3个实验组与对照组比较存在差异,而且差异具有显著性(p＜0.01)。
4.RT-PCR与western-blot实验:与对照组比较,三组不同浓度的实验组,均可提高collagen typeⅠ和osteocalcin的mRNA和蛋白的表达水平,经统计学分析其差异存在显著性(p＜0.05)。
5.蛋白质组学实验:经胃泌素干预后,胃粘膜组织中差异表达蛋白50个,其中增高表达21个,降低表达29个。经质谱分析后鉴定出了22个差异蛋白。
6.生物信息学分析:对上述22个差异蛋白进行了亚细胞定位,蛋白质之间相互作用预测,以及利用已有的文献对其功能、作用进行了分析。发现:蛋白质二硫异构酶A3(Protein disulfide-isomerase A3,PDIA3)经胃泌素干预后,在胃粘膜中高度表达;而且其在无机盐代谢过程中起着重要作用:其作为维生素D3在靶细胞膜上的受体蛋白,存在于肠粘膜细胞,成骨细胞、骨细胞、软骨细胞、成釉等细胞的膜上。PDIA3与维生素D3结合后可以快速促进钙磷在肠道内的吸收;诱导软骨细胞一系列的生化反应,包括:细胞膜通透性的改变,钙离子内流,磷脂的翻转,还有蛋白激酶C的激活,还具有促进软骨细胞分化、细胞外基质矿化的作用。另外,其不仅存在于细胞膜上,也在细胞外质中有表达。
7.经Western-blot检测发现经胃泌素干预后的大鼠胃粘膜中的PDIA3/1,25D3-MARRS蛋白表达量增加,这一结果与蛋白质组学的实验结果是一致的,从而从逆向实验角度对蛋白质组学实验结果进行了验证。
8.免疫组化对PDIA3/1,25D3-MARRS在胃粘膜中的定位及分布情况进行观测发现:在大鼠和人的胃粘膜下层中的胃腺管内遍布PDIA3/1,25D3-MARR蛋白的阳性表达。
结论:
1.胃粘膜提取物可呈剂量依赖关系引起成骨细胞内的钙离子迅速短暂地增加。
2.胃粘膜提取物能够地促进成骨细胞的增殖。
3.胃粘膜提取物可促进成骨细胞的成骨活性。
4.PDIA3/1,25D3-MARRS被认为是胃粘膜组织中促进骨合成作用的关键因素。
5.PDIA3/1,25D3-MARRS是可由胃粘膜内分泌的分泌蛋白。
本研究的创新之处:
1.以往的研究发现胃粘膜提取物具有促进实验动物骨量的作用,通过本研究项目不但验证了这一论点,而且阐明其成骨作用是通过直接促进成骨细胞的增殖和分化来完成的。
2.利用胃泌素干预后胃钙素表达增加的特点,采用蛋白质组学技术,包括:2D-DIGE,基质辅助激光解析飞行时间质谱(MALDI-TOF MS)技术对胃钙素的候选蛋白进行筛选。
3.提出PDIA3可能就是胃组织内促进骨合成的活性因子。目前,所谓的胃钙素只是根据其明确的功能推测出的肽类激素,因此其性质尚未明了,通过本研究提出PDIA3可能就是胃钙素,从而为进一步研究指明了方向。
4.对胃钙素的探索研究将有利于阐明胃切除或病变诱发骨质疏松症的机制,将创新性地提出继甲状旁腺激素、降钙素、雌激素之后的第四种骨钙调解激素,这是对传统钙离子代谢调节机制的重要补充。
5.对胃钙素的研究将为防治胃切除或病变诱发骨质疏松症甚至原发性骨质疏松症探索新的干预靶点。
Background
The morbility of Osteoporosis(OP)is increasing in the aging society.There are a lot of related effective factors result to OP,though its mechanism still unclear,which include hereditary factor,menopause,intake or absorption deficient about calcium or phosphorus,et al.There are certainly interesting focus on the OP induced by gastrectomy or gastropathy recently.It is well known that there is certain connection between the gastrectomy and osteoporosis.By now,more and more evidences show that the extracts of oxyntic mucosa(EOM)could increase the bone mess,Both gastrin-17 and the extract of oxyntic mucosa(EOM)induce transient hypocalcaemia and stimulate an uptake of Ca into bone in rats.Gastrin was without effect in rats that had undergone gastrectomy,whereas the EOM were equally effective in unoperated and gastrectomized rats.And this hypocalcemic effect was totally abolished by prior addition with leucine aminopeptidase.That suggests that the effective component of EOM is a small peptide.We propose the name gastrocalcin for this peptide hormone,which is produced in the oxyntic mucosa and released by gastrin.It accelerates calcium uptake into bone,thereby explaining the hypocalcaemia evoked by gastrin.To the three functionally interacting factors controlling Ca2+ concentration in blood(parathyroid hormone,calcitonin,and vitamin D),we wish to add a fourth---namely,gastrocalcin.
Medicine treatments for OP include anti-resorptive agents and bone-forming agents.Anti-resorptive agents,such as estrogens,calcitonin,bisphosphonates,can inhibit the bioactivity of osteoclasts and increase the bone mess relatively.Bone remodeling is an ongoing process in human bone biology that is necessary to repair micro-damage and renew skeletal integrity strength.Therefore,inhibition of bone resorption mechanism would induce the problem of bone aging.The idea medicine treatment of OP should contain both anti-resorptive agents and bone-forming agents, it is the major tend of OP management in the world.The main bone-forming agent is PTH which was approval to apply in clinic at 2002.But its cost is expensive and it would be administrated by intravenous injection only;these disadvantages limited its extensive utility.
Above all,there is another bone-forming agent in the oxyntic mucosa of rats, which is named gastrocalcin.Therefore the study about gastrocalcin has great prospective.However gastrocalcin was a hypothetic peptide result from its promoting bone-forming function,its nature still unclear by now.Since that,this study about the gastrocalcin is much more important for explore the mechanism of OP introduced by gastrectomy.
Objectives
1、Explore the effects of EOM on the bioactivity of osteoblast.
2、Screening the candidate protein of gastrocalcin and determining its nature.
Methods
1、Osteoblast isolation,culture,and identification:Osteoblasts were isolated from calvariae of 1-day-old Sprague-Dawley rats with the way of sequential enzymatic digestion,and than were identified by morphology,ALP staining, Alizarin red staining and fluorescence staining.
2、Preparation of EOM in rats:EOM was prepared as described previously. Briefly,oxyntic mucosa was collected from six male Sprague-Dawley rats.The stomach was opened,the antrum was removed,and the oxyntic gland area (fundus)rinsed in saline and placed on a cold silica gel plate surface.The mucosa free of mucus was scraped to separate from the muscle wall,then minced by Ultra-Turrax homogenizer and homogenized in buffer,using a Potter-Elvehjem glass homogenizer.The homogenate was centrifuged and the pellet was discarded.The supernatant was passed through cheesecloth. Filtration was followed by centrifugation.After the supernatant was discarded, the pellet was resuspended in the saline(with 1%proteinase inhibitor)by ultrasonication six times(six seconds each)on the ice.At last the solution was quantified according to the instruction of BCA protein assay kit and diluted to serial concentrations:10~(-1)g/l,10~(-2)g/l,10~(-3)g/l with saline respectively,then kept at-80℃.
3、Ca concentration in cytoplasm of osteoblasts:The cells at the third generation were digested and diluted to 1×10~7/L,then inoculated in four special 50mm culture dishes for laser scanning confocal microscopy,for 24 hours,followed by 5μM Fluo-3Am applied into each dish to label the intracytoplasm Ca~(2+). The dishes were placed on the sample holding plate of the confocal microscope and the change of[Ca~(2+)]i was investigated,under the normal(saline as control) or the influences of EOM in different concentrations.
4、Proliferation of osteoblast:Cells were inoculated into 96-wells plate at the density of 2×10~3 cells per well and divided into four groups and treated with low,mid,high concentrations of EOM and saline as control,respectively.24 hours after cell seeding,10μL EOM at different concentration or saline was added into each well.0,1,2 and 3 days after the treatment,cell proliferation was assessed according to the instruction of CCK-8 kit(BI Yuntian Co,China), respectively.In brief,at the end of each time point,10μL WST-8 was added to each well,and the plates were incubated for an additional 4 hours at 37℃to convert WST-8 into formazan.The absorbance of each plate was measured at 450 nm(absorbance)and 600 nm(background)with spectrophotometer.
5、Reverse transcriptase polymerase chain reaction(RT-PCR)test:The cells were inoculated in 6 cm culture dishes and designed into four groups:low,mid,high concentration EOM groups and saline control group.24 hours later,the new culture medium added in to culture system,together with 200μl EOM at different concentration or saline.After another 24 hour culture,total RNA was extracted from each group's osteoblasts using a TRIzol kit.The reverse transcription reaction was performed using 4μg of total RNA and oligo(dT) primer,and collagen typeⅠ,osteocalcin,P-actin(reference gene)gene were amplified using two oligonucleotide primers.The PCR products were separated electrophoretically in a 2%agarose DNA gel stained with ethidium bromide. Semi-quantitative analysis was performed using computing densitometer and Image Quant software.
6、Western-blot test:Cells were cultured in 6 cm culture dishes and treated with EOM.After 48 hours,the total proteins were extracted from each group. Proteins concentration were assessed by BCA protein assay kit,according to the manufacturer's instructions.Equal proteins(60μg)were separated electrophoretically in SDS-PAGE for collagen typeⅠ(138kDa)andβ-actin (43kDa)or Tricine-SDS-PAGE for osteocalcin(5.8kDa).The proteins were transferred to immobilon polyvinyldifluoride(PVDF)membranes.After blocked with 5%BSA for 1 h at room temperature,the membrane was incubated with rabbit anti-mouse antibodies against collagen typeⅠ,osteocalcin orβ-actin(for reference protein)(1:1000)for 1 h at room temperature,washed 6 times(5 minutes each)with TBS buffer containing 0.2%TW-20,followed by incubation with anti-rabbit peroxidase-conjugated secondary antibody(1:3000) for 1 h at room temperature and wash as above.The proteins recognized by antibodies were visualized by enhanced chemiluminescence using Kodak X-OMAT LS film.Quantitative data were obtained using computing densitometer and Image Quant software.
7、Proteomics experiment:Total protein was extracted from the gastric mucosa tissue of experiment and the control group respectively.And then total protein was treated for desalination and quantitation.The protein samples were separated with fluorescence two-dimensional differential in-gel electrophoresis (2D-DIGE),and the different proteins were identified with matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF MS).
8、Analysis by bioinformatics technology:The identified different proteins were estimated further by the technology of bioinformatics through data base and literature.
9、The result of 2D-DIGE was reconfirmed by western-blot test.
10、The location and the distribution of candidate protein defined by 2D-DIGE were explored by immunohistochemistry test.
Results
1、Osteoblast identification:According to the morphology observation and the staining of ALP,AZR and fluorescence,the isolated and cultured cells from rat cranial bone have the characters of osteoblasts.
2、Intracytoplasm Ca2+of osteoblast assay:Under laser scanning confocal microscope,four quadrants total to 20 cells of each visual field were chosen randomly for intracytoplasm[Ca~(2+)]i recording,before or after administration of different stimulate factors.None cell changed in control group,but 15%(three out of twenty cells)in 10~(-3)g/L EOM group,35%(seven out of twenty cells)in 10~(-2)g/Lgroup and 95%(nineteen out of twenty cells)in 10~(-1)g/Lgroup had demonstrated a kind of response that the[Ca~(2+)]i in those cells showed increasing rapidly and temporally just after different concentration of EOM was administered。
3、Osteoblast proliferation assay:The number of cells in three experimental groups increased as compared with the control group,This difference is significant after being analysised by statistics(p＜0.05).
4、RT-PCR and Western-blot test:Contrast to the control group,the mRNA expression of collagen typeⅠand osteocalcin increased in three experimental groups This difference is significant after being analysised by statistics (p＜0.05).
5、Proteomics experiment:There are 50 different proteins expressed in experimental groups compared as control groups,among those different proteins,21 proteins increased expression and 29 decreased expression.22 different proteins were identified by MS.Analysis by bioinformatics technology: The 22 different proteins were located detailly into subcellular structure by WOLF PSORT data base.The interactivity of 22different proteins were predicted by STRING data base.Among the 22 different proteins,our interesting focus on the PDIA3 protein,which is highly expressed after the administration of gastrin,and plays an important role in the mechanism of mineral salt.In intestinal epithelial cells and perfused duodena,the PDIA3 also known as PDIA3/1,25D3-MARRS receptor mediates the rapid(seconds to minutes)stimulation of phosphate and calcium.And it is notable that osteoblasts,osteoclasts,and odontoblasts have been reported to contain PDIA3/1,25D3-MARRS.
6、According to the result of western-blot test,the expression of PDIA3/1,25D3-MARRS in rat stomach mucosa was increasing after the administration of gastrin.This result is coincident with 2D-DIGE.That is the confirmation for 2D-DIGE.
7、According to the result of immunohistochemistry,the expression of PDIA3/ 1,25D3-MARRS is positive in the rat mucosa,and the location of expression is in the glandular tube.
Conclusions
1、We have demonstrated that EOM elevated intracellular[Ca~(2+)]i,after the administration of EOM with doses relationship.
2、EOM enhanced osteoblast cells' proliferation significantly.
3、EOM increased the expression of collagen type I and osteocalcin in primary cultured osteoblasts,which means it can promote the bioactivity of osteoblasts.
4、PDIA3 was regarded as the key factor of promoting bone-forming derives from gastric mucosa.
5、PDIA3/1,25D3-MARRS can be regard as the endocrine protein screted from stomach mucosa.
引文
[1]Bussabarger RA F S,Ivy AC.The experimental production of severe homogeneous osteoporosis by gastrectomy in puppies[J].Am J Physiol,1938:137-148.
[2]Mellstro¨m D J C,Johnell O,Lindstedt G,Lundberg P-A,Obrant K,et al.Osteoporosis,metabolic aberrations,and increased risk for vertebral fractures after partial gastrectomy[J].Calcif Tissue Int,1993,53.
[3]Zittel TT Z B,Maier GW,Kaiser GW,Zwirner M,Liebich H,et al.High prevalence of bone disorders after gastrectomy.[J].Am J Surg,1997,174:431-438.
[4]Surve V V,Andersson N,Lehto-Axtelius D,et al.Comparison of osteopenia after gastrectomy,ovariectomy and prednisolone treatment in the young female rat[J].Acta Orthop Scand,2001,72:525-532.
[5]Hakanson R P P,Axelson J,Johnell O,Sundler F.Evidence that gastrin enhances Ca uptake into bone through release of gastric hormone[J].Regul Pept,1990,28:107-118.
[6]Adachi Y S E,Matsumata T,Iso Y,Yoh R,Kitano S.Bone mineral density in patients taking H2-receptor antagonist[J].Calcif Tissue Int,1998,62:283-285.
[7]Tovey F I,Hall M L,Ell P J,et al.Postgastrectomy osteoporosis[J].Br J Surg,1991,78:1335-1337.
[8]Klinge B,Lehto-Axtelius D,Akerman M,et al.Structure of calvaria after gastrectomy.An experimental study in the rat[J].Scand J Gastroenterol,1995,30:952-957.
[9]Davies M H S,Selby PL,Berry JL,Mawer EB..Increased catabolism of 25-hydroxyvitamin D in patients with partial gastrectomy and elevated 1,25-dihydroxyvitamin D levels,Implications for metabolic bone disease[J].Clin Endocrinol Metab,1997,82:209-212.
[10]Maier GW K M,Zittel TT,Becker HD.Calcium regulation and bone mass loss after total gastrectomy in pig[J].Ann Surg,1997,225:181-192.
[11]Krishnamra N,Limlomwongse L.Acute and long-term effects of gastrin on muscle and bone calcium uptake in rats[J].J Nutr Sci Vitaminol(Tokyo),1995,41:687-697.
[12]Limlomwongse L K,N.The mechanism of action and target organ of gastrin-induced hypocalcemia[J].Proc Soc ExpBiol Med,1981,168:72-76.
[13]Schulak J A K,E.L.The importance of the stomach in gastrin-induced hypocalcemia in the rat[J].Endocrinology 1975,96:1217-1220.
[14]Persson P,Grunditz,T.,Axelson,J.,Sundler,F,Hakanson,R.Chole cystokinins but not gastrin-17 release calcitonin from thyroid C-cells in the rat[J].RegulPept,1988,21:45-56.
[15]Larsson B G L A,Norlen P,et al.Extracts of ECL-cell granules / vesicles and of isolated ECL cells from rat oxyntic mucosa evoke a Ca second messenger response in osteoblastic cells[J].Regul Pept,2001,97:153-161.
[16]Persson P,Hakanson R,Axelson J,et al.Gastrin releases a blood calcium-lowering peptide from the acid-producing part of the rat stomach[J].Proc Natl Acad Sci U S A,1989,86:2834-2838.
[17]Larsson B N P,Lindstrom E,et al.Effects of ECL cell extracts and granule/vesicle-enriched fractions from rat oxyntic mucosa on cAMP and IP3 in rat osteoblast-like cells[J].Regul Pept,2002,106:13-18.
[18]Niklas Andersson S M r S,Rolf Ha°kanson,Claes Ohlsson.A gene expression fingerprint of mouse stomach ECL cells[J].Biochemical and Biophysical Research Communications,2005,332:404-410.
[19]Schipani E,Provot S.PTHrP,PTH,and the PTH/PTHrP receptor in endochondral bone development[J].Birth Defects Res C Embryo Today,2003,69:352-362.
[20]Horwitz M J,Tedesco M B,Gundberg C,et al.Short-term,high-dose parathyroid hormone-related protein as a skeletal anabolic agent for the treatment of postmenopausal osteoporosis[J].J Clin Endocrinol Metab,2003,88:569-575.
[21]Bisello A,Horwitz M J,Stewart A F.Parathyroid hormone-related protein:an essential physiological regulator of adult bone mass[J].Endocrinology,2004,145:3551-3553.
[22]Friis-Hansen L,Schjerling C K,de la Cour C D,et al.Characteristics of gastrin controlled ECL cell specific gene expression[J].Regul Pept,2007,140:153-161.
[23]Larsson B,Gritli-Linde A,Norlen P,et al.Extracts of ECL-cell granules/vesicles and of isolated ECL cells from rat oxyntic mucosa evoke a Ca2+ second messenger response in osteoblastic cells[J].Regul Pept,2001,97:153-161.
[24]Sch(a|¨)gger H.Tricine-SDS-PAGE[J].nature protocols,2006,l:16-22.
[25]Zayzafoon M.Calcium/calmodulin signaling controls osteoblast growth and differentiation[J].J Cell Biochem,2006,97:56-70.
[26]Zhao B,Nemere I.l,25(OH)2D3-mediated phosphate uptake in isolated chick intestinal cells:effect of 24,25(OH)2D3,signal transduction activators,and age[J].J Cell Biochem,2002,86:497-508.
[27]Farach-Carson M C,Nemere I.Membrane receptors for vitamin D steroid hormones:potential new drug targets[J].Curr Drug Targets,2003,4:67-76.
[28]Rohe B,Safford S E,Nemere I,et al.Identification and characterization of l,25D3-membrane-associated rapid response,steroid(l,25D3-MARRS)-binding protein in rat IEC-6 cells[J].Steroids,2005,70:458-463.
[29]Larsson B,Nemere I.Effect of growth and maturation on membrane-initiated actions of 1,25-dihydroxyvitamin D(3).I.Calcium transport,receptor kinetics,and signal transduction in intestine of male chickens[J].Endocrinology,2003,144:1726-1735.
[30]Swain L D,Schwartz Z,Caulfield K,et al.Nongenomic regulation of chondrocyte membrane fluidity by l,25-(OH)2D3 and 24,25-(OH)2D3 is dependent on cell maturation[J].Bone,1993,14:609-617.
[31]Schwartz Z,Langston G G,Swain L D,et al.Inhibition of l,25-(OH)2D3- and 24,25-(OH)2D3-dependent stimulation of alkaline phosphatase activity by A23187 suggests a role for calcium in the mechanism of vitamin D regulation of chondrocyte cultures[J].J Bone Miner Res,1991,6:709-718.
[32]Sylvia V L,Schwartz Z,Schuman L,et al.Maturation-dependent regulation of protein kinase C activity by vitamin D3 metabolites in chondrocyte cultures[J].J Cell Physiol,1993,157:271-278.
[33]Berdal A,Mesbah M,Papagerakis P,et al.Putative membrane receptor for l,25(OH)2 vitamin D3 in human mineralized tissues during prenatal development[J].Connect Tissue Res,2003,44 Suppl 1:136-140.
[1]Bussabarger RA,F.S.,Ivy AC,The experimental production of severe homogeneous osteoporosis by gastrectomy in puppies.Am J Physiol,1938.121:p.137-148.
[2]Mellstro¨m D,J.C.,Johnell O,Lindstedt G,Lundberg P-A,Obrant K,et al.,Osteoporosis,metabolic aberrations,and increased risk for vertebral fractures after partial gastrectomy.Calcif Tissue Int,1993.53(370-377).
[3]Zittel TT,Z.B.,Maier GW,Kaiser GW,Zwirner M,Liebich H,et al,High prevalence of bone disorders after gastrectomy.Am J Surg,1997.174:p.431-438.
[4]Persson P,G.-P.R.,Chen D,Axelson J,Nylander A-G,Johnell O,H(a|¨)kanson R,Gastrectomy causes bone loss in the rat:is lack of gastric acid responsible? Scand J Gastroenterol,1993.28:p.301-306.
[5]Adachi Y,S.E.,Matsumata T,Iso Y,Yoh R,Kitano S,Bone mineral density in patients taking H2-receptor antagonist.Calcif Tissue Int,1998.62:p.283-285.
[6]Tovey FI,H.M.,Ell PJ,Hobsley M.,Postgastrectomy osteoporosis.Br J Surg,1991.78:p.1335-1347.
[7]Klinge B,L.-A.D.,A kerman M,Hakanson R,Structure of calvaria after gastrectomy:An experimental study in the rat.Scand J Gastroenterol,1995.30:p.952-957.
[8]Davies M,H.S.,Selby PL,Berry JL,Mawer EB,.Increased catabolism of 25-hydroxyvitamin D in patients with partial gastrectomy and elevated 1,25-dihydroxyvitamin D levelsjmplications for metabolic bone disease.Clin Endocrinol Metab,1997.82:p.209-212.
[9]Maier GW,K.M.,Zittel TT,Becker HD,Calcium regulation and bone mass loss after total gastrectomy in pig.Ann Surg,1997.225:p.181-192.
[10]Gregory RA,e.a.,The construction and properties of two gastrins extracted from hog antral mucosa.Gut.,1964.5:p.103-114.
[11]Persson P,H.R.,Axelson J,Sundler R,Gastrin releases a blood calcium-lowering peptide from the acid-producing part of the rat stomach.Proc Natl Acad Sci USA,1989.86:p.2834-2838.
[12]Hakanson R,P.P.,Axelson J,Johnell O,Sundler F,Evidence that gastrin enhances Ca uptake into bone through release of gastric hormone.Regul Pept,1990.28:p.107-118.
[13]H(a|¨)kanson R,P.P.,Axelson J,Elevated serum gastrin after food intake or acid blockade evokes hypocalcemia..Regul Pept,1990.28:p.131-136.
[14]Krishnamra,N.and L.Limlomwongse,Acute and long-term effects of gastrin on muscle and bone calcium uptake in rats.J Nutr Sci Vitaminol(Tokyo),1995.41(6):p.687-97.
[15]Limlomwongse,L.K.,N,The mechanism of action and target organ of gastrin-induced hypocalcemia.Proc.Soc.Exp.Biol.Med,1981.168:p.72-76.
[16]Schulak,J.A.K.,E.L,The importance of the stomach in gastrin-induced hypocalcemia in the rat.Endocrinology 1975.96:p.1217-1220.
[17]Persson,P.,Grunditz,T.,Axelson,J.,Sundler,F,Hakanson,R,Chole cystokinins but not gastrin-17 release calcitonin from thyroid C-cells in the rat.Regul.Pept,1988. 21:p.45-56.
[18]Larsson B,G.L.A.,Norlen P,et al,Extracts of ECL-cell granules / vesicles and of isolated ECL cells from rat oxyntic mucosa evoke a Ca second messenger response in osteoblastic cells.Regul Pept,2001.97:p.153-161.
[19]Larsson B,N.P.,Lindstrom E,et al,Effects of ECL cell extracts and granule/vesicle-enriched fractions from rat oxyntic mucosa on cAMP and IP3 in rat osteoblast-like cells.Regul Pept,2002.106:p.13-18.
[20]Niklas Andersson,S.M.r.S.,Rolf Ha°kanson,Claes Ohlsson,A gene expression fingerprint of mouse stomach ECL cells.Biochemical and Biophysical Research Communications,2005.332:p.404-410.
[21]Schipani,E.and S.Provot,PTHrP,PTH,and the PTH/PTHrP receptor in endochondral bone development.Birth Defects Res C Embryo Today,2003.69(4):p.352-62.
[22]Horwitz,M.J.,et al.,Short-term,high-dose parathyroid hormone-related protein as a skeletal anabolic agent for the treatment of postmenopausal osteoporosis.J Clin Endocrinol Metab,2003.88(2):p.569-75.
[23]Bisello,A.,M.J.Horwitz,and A.F.Stewart,Parathyroid hormone-related protein:an essential physiological regulator of adult bone mass.Endocrinology,2004.145(8):p.3551-3.
[1]Abbott TA,M.L.,Manfredonia D,et al,Efficient patient identification strategies for women with osteoporosis.J Clin Densitom,1999,.2(3):p.223-230.
[2]冯燕茹,老年性骨质疏松症诊断的进展.中国骨伤杂志,2001.14(11):P.682-686.
[3]伍贤平,廖二元,破骨细胞及其调节机制的某些进展.中华内分泌代谢杂志,2002.18(2):P.157-160.
[4]Gangji V,Hauzeur JP,S.A.,et al Abnormalties in the replicative capacity of osteoblastic cells in the proximal femur of fatients with osteonecrosis of the femoral head J Rhermaatol,2003.30(2):p.348-351.
[5]Charles P,M.L.,Risteli L,et al,Assessment of bone remodeling using biochemical indicators of type I collagen synthesis and degredation:relation to calcium kinetics.(J).Bone Miner,1994.24:p.81～94.
[6]Fledelins C,R.B.,Overgaard K,et al,The diagnostic validity of urinary free pyridinolines to identify women at risk of osteoporosis.(J).Calcif Tissue Int,1994.54:p.381～384.
[7]Gertz BJ,S.P,Hanson DA,et al,Monitoring bone resorption in early postmenopausal women by an immunoassay for crosslinked collagen peptides in wine..(J).J Bone Min Res,1994.9:p.135～141.
[8]Hoshino H,T.M.,kushida K,et al,Urinary encretion of type I collagen degradation products in healthy women and osteoporotic patients with ventebral and hip fractures.(J).Calcif Tissue Int,1998.62:p.36～39.
[9]Cosman F,N.J.,Wilkinson C,et al,Bone density change and biochemical indices of skeletal turnover.(J).Calcif Tissue Int,1996.58:p.236～243.
[10]J.,F.,Verapamil induces PTH resistance but increases duodenal calcium absorption in rats.Am F Phsiol 1988.255:p.702-707.
[11]Kronenberg HM,L.B.,Kovacs CS,et al,Functional analysis of the PTH/ PTHrP network of ligands and receptors.[J].Recent Prog Horm Res,1998.53:p.283-301.
[12]McSheehy PM,C.T.,Osteoblastic cells mediate osteoclastic resposiveness to parathyroid hormone Endocrinology 1986.118(2):p.824--828.
[13]Dietrich JW,C.E.,Maina DM,et al Hormonal control of bone collagen synthesis in vitro:effects of parathyroid hormone and calcitonin.Endocrinology,1976.98(4):p.943-949.
[14]14.Martin TJ,M.J.,Williams ED.,Parathyroid hormone2related protein:hormone and cytokine.[J].Endocrinol,1997.154(suppl):p.s23-s37.
[15]AE,S.,PTHrP(l - 36) as a skeletal anabolic agent for the treatment of osteoporosis.[J].Bone,1996.19(4):p.303-306.
[16]Everhart-Caye M,I.S.,Guinness-Henry J,et al.Parathyroid hormone(PTH)2related protein(1 - 36) is equipotent to PTH(1 - 34) in humans.[J].J Clin Endocrinol Metab ? 1996.81(1):p.199-208.
[17]Liu BY,W.J.,Leu JS.,Effects of continous calcitonin treatment on osteoblasts derived from cocultures of mouse marrow stromal and spleen cells.J Formos Med Assoc 2000.99(2):p.140-150.
[18]Samura A,w.S.,suda S,et al.Calcitonin receptor regulation and responsiveness to calcitonin in human osteoclast-like cells prepared in vitro using receptor activator of nuclear foctor-kappa B linland and macrophage colony-stimulating factor Endocrinology,2000.141(10):p.3774-3782.
[19]Schuit SC,d.J.F,Stolk L,et al,Estrogen receptor alpha gene polymorphisms are associated with estradiol levels in postmenopausal women.Eur J Endocrinol,,2005.153(2):p.327-334.
[20]SA,D.,Recent important clinical trials of drugs in osteoporosis.Expert Opin Pharmacother,2004.5(7):p.1635-1638.
[21]Mone Z,B.S.,Li S,et al,Understanding osteoclast formation and function:Implications for future therapies for osteoporosis Curr Opin Orthop,2003.14(5):p.341-350.
[22]Schubert M,B.C.,Minnemann T,et al,Osteoporosis in male hypogonadism:responses to androgen substit ution differ among men wit h primary and secondary hypogonadism.Horm Res,2003.60(1):p.21-28.
[23]C,L.,Adrenal and gonadal androgen secretion in normal females.Clin Endocrinol Metab,1986.15:p.213-228.
[24]Gurnell EM,C.V.K.,Dehydroepiandrosterone replacement t herapy.Eur J Endocrinol,2001.145(2):p.103-106.
[25]Labrie F,B.A.,Cusan L,et al,Candas B Marked decline in serum concent rations of adrenal C19 sex steroid precursors and conjugated androgen metabolites during aging.Endocrinology,1997.82:p.2396-2402.
[26]Tok EC,E.D.,Oz U,et al,The effect of circulating androgens on bone mineral density in postmenopausal women.Maturitas,2004.48(3):p.235-242.
[2]Mellstro¨m D J C,Johnell O,Lindstedt G,Lundberg P-A,Obrant K,et al.Osteoporosis,metabolic aberrations,and increased risk for vertebral fractures after partial gastrectomy[J].Calcif Tissue Int,1993,53.
[3]Zittel TT Z B,Maier GW,Kaiser GW,Zwirner M,Liebich H,et al.High prevalence of bone disorders after gastrectomy.[J].Am J Surg,1997,174:431-438.
[4]Surve V V,Andersson N,Lehto-Axtelius D,et al.Comparison of osteopenia after gastrectomy,ovariectomy and prednisolone treatment in the young female rat[J].Acta Orthop Scand,2001,72:525-532.
[5]Hakanson R P P,Axelson J,Johnell O,Sundler F.Evidence that gastrin enhances Ca uptake into bone through release of gastric hormone[J].Regul Pept,1990,28:107-118.
[6]Adachi Y S E,Matsumata T,Iso Y,Yoh R,Kitano S.Bone mineral density in patients taking H2-receptor antagonist[J].Calcif Tissue Int,1998,62:283-285.
[7]Tovey F I,Hall M L,Ell P J,et al.Postgastrectomy osteoporosis[J].Br J Surg,1991,78:1335-1337.
[8]Klinge B,Lehto-Axtelius D,Akerman M,et al.Structure of calvaria after gastrectomy.An experimental study in the rat[J].Scand J Gastroenterol,1995,30:952-957.
[9]Davies M H S,Selby PL,Berry JL,Mawer EB..Increased catabolism of 25-hydroxyvitamin D in patients with partial gastrectomy and elevated 1,25-dihydroxyvitamin D levels,Implications for metabolic bone disease[J].Clin Endocrinol Metab,1997,82:209-212.
[10]Maier GW K M,Zittel TT,Becker HD.Calcium regulation and bone mass loss after total gastrectomy in pig[J].Ann Surg,1997,225:181-192.
[11]Krishnamra N,Limlomwongse L.Acute and long-term effects of gastrin on muscle and bone calcium uptake in rats[J].J Nutr Sci Vitaminol(Tokyo),1995,41:687-697.
[12]Limlomwongse L K,N.The mechanism of action and target organ of gastrin-induced hypocalcemia[J].Proc Soc ExpBiol Med,1981,168:72-76.
[13]Schulak J A K,E.L.The importance of the stomach in gastrin-induced hypocalcemia in the rat[J].Endocrinology 1975,96:1217-1220.
[14]Persson P,Grunditz,T.,Axelson,J.,Sundler,F,Hakanson,R.Chole cystokinins but not gastrin-17 release calcitonin from thyroid C-cells in the rat[J].RegulPept,1988,21:45-56.
[15]Larsson B G L A,Norlen P,et al.Extracts of ECL-cell granules / vesicles and of isolated ECL cells from rat oxyntic mucosa evoke a Ca second messenger response in osteoblastic cells[J].Regul Pept,2001,97:153-161.
[16]Persson P,Hakanson R,Axelson J,et al.Gastrin releases a blood calcium-lowering peptide from the acid-producing part of the rat stomach[J].Proc Natl Acad Sci U S A,1989,86:2834-2838.
[17]Larsson B N P,Lindstrom E,et al.Effects of ECL cell extracts and granule/vesicle-enriched fractions from rat oxyntic mucosa on cAMP and IP3 in rat osteoblast-like cells[J].Regul Pept,2002,106:13-18.
[18]Niklas Andersson S M r S,Rolf Ha°kanson,Claes Ohlsson.A gene expression fingerprint of mouse stomach ECL cells[J].Biochemical and Biophysical Research Communications,2005,332:404-410.
[19]Schipani E,Provot S.PTHrP,PTH,and the PTH/PTHrP receptor in endochondral bone development[J].Birth Defects Res C Embryo Today,2003,69:352-362.
[20]Horwitz M J,Tedesco M B,Gundberg C,et al.Short-term,high-dose parathyroid hormone-related protein as a skeletal anabolic agent for the treatment of postmenopausal osteoporosis[J].J Clin Endocrinol Metab,2003,88:569-575.
[21]Bisello A,Horwitz M J,Stewart A F.Parathyroid hormone-related protein:an essential physiological regulator of adult bone mass[J].Endocrinology,2004,145:3551-3553.
[22]Friis-Hansen L,Schjerling C K,de la Cour C D,et al.Characteristics of gastrin controlled ECL cell specific gene expression[J].Regul Pept,2007,140:153-161.
[23]Larsson B,Gritli-Linde A,Norlen P,et al.Extracts of ECL-cell granules/vesicles and of isolated ECL cells from rat oxyntic mucosa evoke a Ca2+ second messenger response in osteoblastic cells[J].Regul Pept,2001,97:153-161.
[24]Sch(a|¨)gger H.Tricine-SDS-PAGE[J].nature protocols,2006,l:16-22.
[25]Zayzafoon M.Calcium/calmodulin signaling controls osteoblast growth and differentiation[J].J Cell Biochem,2006,97:56-70.
[26]Zhao B,Nemere I.l,25(OH)2D3-mediated phosphate uptake in isolated chick intestinal cells:effect of 24,25(OH)2D3,signal transduction activators,and age[J].J Cell Biochem,2002,86:497-508.
[27]Farach-Carson M C,Nemere I.Membrane receptors for vitamin D steroid hormones:potential new drug targets[J].Curr Drug Targets,2003,4:67-76.
[28]Rohe B,Safford S E,Nemere I,et al.Identification and characterization of l,25D3-membrane-associated rapid response,steroid(l,25D3-MARRS)-binding protein in rat IEC-6 cells[J].Steroids,2005,70:458-463.
[29]Larsson B,Nemere I.Effect of growth and maturation on membrane-initiated actions of 1,25-dihydroxyvitamin D(3).I.Calcium transport,receptor kinetics,and signal transduction in intestine of male chickens[J].Endocrinology,2003,144:1726-1735.
[30]Swain L D,Schwartz Z,Caulfield K,et al.Nongenomic regulation of chondrocyte membrane fluidity by l,25-(OH)2D3 and 24,25-(OH)2D3 is dependent on cell maturation[J].Bone,1993,14:609-617.
[31]Schwartz Z,Langston G G,Swain L D,et al.Inhibition of l,25-(OH)2D3- and 24,25-(OH)2D3-dependent stimulation of alkaline phosphatase activity by A23187 suggests a role for calcium in the mechanism of vitamin D regulation of chondrocyte cultures[J].J Bone Miner Res,1991,6:709-718.
[32]Sylvia V L,Schwartz Z,Schuman L,et al.Maturation-dependent regulation of protein kinase C activity by vitamin D3 metabolites in chondrocyte cultures[J].J Cell Physiol,1993,157:271-278.
[33]Berdal A,Mesbah M,Papagerakis P,et al.Putative membrane receptor for l,25(OH)2 vitamin D3 in human mineralized tissues during prenatal development[J].Connect Tissue Res,2003,44 Suppl 1:136-140.
[1]Bussabarger RA,F.S.,Ivy AC,The experimental production of severe homogeneous osteoporosis by gastrectomy in puppies.Am J Physiol,1938.121:p.137-148.
[2]Mellstro¨m D,J.C.,Johnell O,Lindstedt G,Lundberg P-A,Obrant K,et al.,Osteoporosis,metabolic aberrations,and increased risk for vertebral fractures after partial gastrectomy.Calcif Tissue Int,1993.53(370-377).
[3]Zittel TT,Z.B.,Maier GW,Kaiser GW,Zwirner M,Liebich H,et al,High prevalence of bone disorders after gastrectomy.Am J Surg,1997.174:p.431-438.
[4]Persson P,G.-P.R.,Chen D,Axelson J,Nylander A-G,Johnell O,H(a|¨)kanson R,Gastrectomy causes bone loss in the rat:is lack of gastric acid responsible? Scand J Gastroenterol,1993.28:p.301-306.
[5]Adachi Y,S.E.,Matsumata T,Iso Y,Yoh R,Kitano S,Bone mineral density in patients taking H2-receptor antagonist.Calcif Tissue Int,1998.62:p.283-285.
[6]Tovey FI,H.M.,Ell PJ,Hobsley M.,Postgastrectomy osteoporosis.Br J Surg,1991.78:p.1335-1347.
[7]Klinge B,L.-A.D.,A kerman M,Hakanson R,Structure of calvaria after gastrectomy:An experimental study in the rat.Scand J Gastroenterol,1995.30:p.952-957.
[8]Davies M,H.S.,Selby PL,Berry JL,Mawer EB,.Increased catabolism of 25-hydroxyvitamin D in patients with partial gastrectomy and elevated 1,25-dihydroxyvitamin D levelsjmplications for metabolic bone disease.Clin Endocrinol Metab,1997.82:p.209-212.
[9]Maier GW,K.M.,Zittel TT,Becker HD,Calcium regulation and bone mass loss after total gastrectomy in pig.Ann Surg,1997.225:p.181-192.
[10]Gregory RA,e.a.,The construction and properties of two gastrins extracted from hog antral mucosa.Gut.,1964.5:p.103-114.
[11]Persson P,H.R.,Axelson J,Sundler R,Gastrin releases a blood calcium-lowering peptide from the acid-producing part of the rat stomach.Proc Natl Acad Sci USA,1989.86:p.2834-2838.
[12]Hakanson R,P.P.,Axelson J,Johnell O,Sundler F,Evidence that gastrin enhances Ca uptake into bone through release of gastric hormone.Regul Pept,1990.28:p.107-118.
[13]H(a|¨)kanson R,P.P.,Axelson J,Elevated serum gastrin after food intake or acid blockade evokes hypocalcemia..Regul Pept,1990.28:p.131-136.
[14]Krishnamra,N.and L.Limlomwongse,Acute and long-term effects of gastrin on muscle and bone calcium uptake in rats.J Nutr Sci Vitaminol(Tokyo),1995.41(6):p.687-97.
[15]Limlomwongse,L.K.,N,The mechanism of action and target organ of gastrin-induced hypocalcemia.Proc.Soc.Exp.Biol.Med,1981.168:p.72-76.
[16]Schulak,J.A.K.,E.L,The importance of the stomach in gastrin-induced hypocalcemia in the rat.Endocrinology 1975.96:p.1217-1220.
[17]Persson,P.,Grunditz,T.,Axelson,J.,Sundler,F,Hakanson,R,Chole cystokinins but not gastrin-17 release calcitonin from thyroid C-cells in the rat.Regul.Pept,1988. 21:p.45-56.
[18]Larsson B,G.L.A.,Norlen P,et al,Extracts of ECL-cell granules / vesicles and of isolated ECL cells from rat oxyntic mucosa evoke a Ca second messenger response in osteoblastic cells.Regul Pept,2001.97:p.153-161.
[19]Larsson B,N.P.,Lindstrom E,et al,Effects of ECL cell extracts and granule/vesicle-enriched fractions from rat oxyntic mucosa on cAMP and IP3 in rat osteoblast-like cells.Regul Pept,2002.106:p.13-18.
[20]Niklas Andersson,S.M.r.S.,Rolf Ha°kanson,Claes Ohlsson,A gene expression fingerprint of mouse stomach ECL cells.Biochemical and Biophysical Research Communications,2005.332:p.404-410.
[21]Schipani,E.and S.Provot,PTHrP,PTH,and the PTH/PTHrP receptor in endochondral bone development.Birth Defects Res C Embryo Today,2003.69(4):p.352-62.
[22]Horwitz,M.J.,et al.,Short-term,high-dose parathyroid hormone-related protein as a skeletal anabolic agent for the treatment of postmenopausal osteoporosis.J Clin Endocrinol Metab,2003.88(2):p.569-75.
[23]Bisello,A.,M.J.Horwitz,and A.F.Stewart,Parathyroid hormone-related protein:an essential physiological regulator of adult bone mass.Endocrinology,2004.145(8):p.3551-3.
[1]Abbott TA,M.L.,Manfredonia D,et al,Efficient patient identification strategies for women with osteoporosis.J Clin Densitom,1999,.2(3):p.223-230.
[2]冯燕茹,老年性骨质疏松症诊断的进展.中国骨伤杂志,2001.14(11):P.682-686.
[3]伍贤平,廖二元,破骨细胞及其调节机制的某些进展.中华内分泌代谢杂志,2002.18(2):P.157-160.
[4]Gangji V,Hauzeur JP,S.A.,et al Abnormalties in the replicative capacity of osteoblastic cells in the proximal femur of fatients with osteonecrosis of the femoral head J Rhermaatol,2003.30(2):p.348-351.
[5]Charles P,M.L.,Risteli L,et al,Assessment of bone remodeling using biochemical indicators of type I collagen synthesis and degredation:relation to calcium kinetics.(J).Bone Miner,1994.24:p.81～94.
[6]Fledelins C,R.B.,Overgaard K,et al,The diagnostic validity of urinary free pyridinolines to identify women at risk of osteoporosis.(J).Calcif Tissue Int,1994.54:p.381～384.
[7]Gertz BJ,S.P,Hanson DA,et al,Monitoring bone resorption in early postmenopausal women by an immunoassay for crosslinked collagen peptides in wine..(J).J Bone Min Res,1994.9:p.135～141.
[8]Hoshino H,T.M.,kushida K,et al,Urinary encretion of type I collagen degradation products in healthy women and osteoporotic patients with ventebral and hip fractures.(J).Calcif Tissue Int,1998.62:p.36～39.
[9]Cosman F,N.J.,Wilkinson C,et al,Bone density change and biochemical indices of skeletal turnover.(J).Calcif Tissue Int,1996.58:p.236～243.
[10]J.,F.,Verapamil induces PTH resistance but increases duodenal calcium absorption in rats.Am F Phsiol 1988.255:p.702-707.
[11]Kronenberg HM,L.B.,Kovacs CS,et al,Functional analysis of the PTH/ PTHrP network of ligands and receptors.[J].Recent Prog Horm Res,1998.53:p.283-301.
[12]McSheehy PM,C.T.,Osteoblastic cells mediate osteoclastic resposiveness to parathyroid hormone Endocrinology 1986.118(2):p.824--828.
[13]Dietrich JW,C.E.,Maina DM,et al Hormonal control of bone collagen synthesis in vitro:effects of parathyroid hormone and calcitonin.Endocrinology,1976.98(4):p.943-949.
[14]14.Martin TJ,M.J.,Williams ED.,Parathyroid hormone2related protein:hormone and cytokine.[J].Endocrinol,1997.154(suppl):p.s23-s37.
[15]AE,S.,PTHrP(l - 36) as a skeletal anabolic agent for the treatment of osteoporosis.[J].Bone,1996.19(4):p.303-306.
[16]Everhart-Caye M,I.S.,Guinness-Henry J,et al.Parathyroid hormone(PTH)2related protein(1 - 36) is equipotent to PTH(1 - 34) in humans.[J].J Clin Endocrinol Metab ? 1996.81(1):p.199-208.
[17]Liu BY,W.J.,Leu JS.,Effects of continous calcitonin treatment on osteoblasts derived from cocultures of mouse marrow stromal and spleen cells.J Formos Med Assoc 2000.99(2):p.140-150.
[18]Samura A,w.S.,suda S,et al.Calcitonin receptor regulation and responsiveness to calcitonin in human osteoclast-like cells prepared in vitro using receptor activator of nuclear foctor-kappa B linland and macrophage colony-stimulating factor Endocrinology,2000.141(10):p.3774-3782.
[19]Schuit SC,d.J.F,Stolk L,et al,Estrogen receptor alpha gene polymorphisms are associated with estradiol levels in postmenopausal women.Eur J Endocrinol,,2005.153(2):p.327-334.
[20]SA,D.,Recent important clinical trials of drugs in osteoporosis.Expert Opin Pharmacother,2004.5(7):p.1635-1638.
[21]Mone Z,B.S.,Li S,et al,Understanding osteoclast formation and function:Implications for future therapies for osteoporosis Curr Opin Orthop,2003.14(5):p.341-350.
[22]Schubert M,B.C.,Minnemann T,et al,Osteoporosis in male hypogonadism:responses to androgen substit ution differ among men wit h primary and secondary hypogonadism.Horm Res,2003.60(1):p.21-28.
[23]C,L.,Adrenal and gonadal androgen secretion in normal females.Clin Endocrinol Metab,1986.15:p.213-228.
[24]Gurnell EM,C.V.K.,Dehydroepiandrosterone replacement t herapy.Eur J Endocrinol,2001.145(2):p.103-106.
[25]Labrie F,B.A.,Cusan L,et al,Candas B Marked decline in serum concent rations of adrenal C19 sex steroid precursors and conjugated androgen metabolites during aging.Endocrinology,1997.82:p.2396-2402.
[26]Tok EC,E.D.,Oz U,et al,The effect of circulating androgens on bone mineral density in postmenopausal women.Maturitas,2004.48(3):p.235-242.