【作者】 谢辉；

【导师】 廖二元；

【作者基本信息】 中南大学 ， 内科学， 2011， 博士

【摘要】 第一部分绝经后妇女雌激素受体-α36介导17β-雌二醇的骨保护作用目的雌激素受体-alpha36(ER-α36)是一种膜受体,不同于ER-α66、ER-α46和ER-β主要介导雌激素的核效应,ER-α36只能介导雌激素的膜效应,如促分裂原活化蛋白激酶/细胞外信号调节激酶(MAPK/ERK)信号通路的激活。本研究旨在探讨ER-α36是否与ER-α66一样,也参与调控骨代谢。方法免疫组织化学染色观察骨组织切片中ER-α36的表达。3H-胸腺嘧啶核苷(3H-TdR)掺入法检测细胞增殖。ELISA检测细胞凋亡。磷酸对硝基苯酚法测定碱性磷酸酶(ALP)活性。放射免疫分析法(RIA)测定骨钙素(OCN)含量。茜素红S染色评估成骨细胞基质的矿化程度。Western blot检测ER-α36、ER-α66、ERK1/2和p-ERK1/2蛋白表达水平。H2DCFDA荧光探针检测活性氧簇(ROS)水平。ER-α36 shRNA和真核表达载体调控成骨细胞和破骨细胞ER-α36表达,并采用ERK和ROS抑制剂观察E2调控成骨细胞与破骨细胞增殖、分化或凋亡的ER-α36受体及受体后信号途径。荧光实时定量PCR(QRT-PCR)检测骨组织OCN和抗酒石酸酸性磷酸酶(TRAP)基因表达量。将154例椎间盘突出、椎管狭窄或脊椎前移进行开窗术的患者分为绝经前组(60例)和绝经后组(94例,其中包括骨质疏松组30例,低骨量组31例,正常骨量组33例),用双能量X线吸收法(DXA)测定患者骨密度(BMD),取血后用ELISA检测血清骨形成标志物OCN和骨吸收标志物Ⅰ型胶原氨基末端肽(NTX)水平,收集脊椎松质骨标本并用QRT-PCR检测ER-α36、ER-α66、ER-α46和ER-p的表达水平。行动物试验,观察ER-α36选择性调节剂IC162对去卵巢大鼠骨代谢的影响结果(1)细胞培养试验结果：ER-α36介导绝经后低水平(10pM)E2诱导成骨细胞ERK短暂激活,从而促进成骨细胞增殖并抑制其分化和凋亡,而ER-α36介导10pM E2诱导破骨细胞ERK持续激活(依赖于ROS),进而促进破骨细胞凋亡；但10pM E2对ER-a36阴性成骨细胞/破骨细胞的增殖、分化和凋亡无作用。(2)骨组织培养试验结果：在高表达ER-α36的骨组织,10pM E2显著抑制骨形成细胞因子OCN和骨吸收细胞因子TRAP的表达；而在低表达ER-α36的骨组织,10pM E2对OCN和TRAP的表达无影响。(3)方差分析显示：绝经前组骨ER-α36表达水平显著低于绝经后各组；绝经后正常骨量组ER-α36表达水平较低骨量组和骨质疏松组高,低骨量组ER-α36表达水平较骨质疏松组高；绝经后各组骨ER-α66、ER-α46和ER-β表达水平均低于绝经前组,绝经后各组间ER-α66、ER-α46和ER-β表达水平差异无显著性。(4)相关分析显示：绝经后妇女骨ER-α36表达水平与BMD呈正相关,与血清OCN和NTX水平呈负相关；绝经前妇女骨ER-α36表达水平与BMD、OCN和NTX水平无相关性；绝经前和绝经后妇女骨ER-α66、ER-α46和ER-β表达水平与BMD、OCN和NTX水平均无相关性。(5)动物试验结果：IC162干预去卵巢大鼠可增加BMD,但对体重和子宫重量无影响。结论ER-α36在绝经后妇女骨代谢过程中发挥关键调控作用,高表达ER-α36的成骨细胞/破骨细胞对E2高度敏感,ER-α36介导绝经后低水平E2的骨保护作用。ER-a36的选择性调节剂IC162在去卵巢大鼠发挥骨保护作用,但对子宫无影响。第二部分假想蛋白LOC147710突变与常染色体隐性骨质硬化症的发病相关目的骨质硬化症是由于破骨细胞生成障碍或功能缺陷导致的一组以全身骨密度增高为主要特征的骨代谢疾病群,具有高度遗传异质性。迄今为止,已发现有11种基因的功能丢失性突变与人类骨质硬化症有关,其特点是单基因突变引起发病,且无重叠。本课题对一个常染色体隐性遗传的骨质硬化症家系(家系总成员11人,其中5名成员的基因型为纯合子患病基因型)进行了研究,以确定该家族性骨质硬化症的致病基因。方法双能X线吸收法(DXA)测定骨密度(BMD)。提取11名家系成员及180名正常对照者外周血DNA,用基因测序排除已知与骨质硬化症相关的11个基因的突变后,进行全基因组扫描、精细基因定位及单倍型分析确定致病基因的候选区,然后对该区基因外显子进行测序以确定该致病基因突变。Northern blot和Western blot检测LOC147710在人体各组织的表达量以及在破骨前体细胞CD14+外周血单核细胞(PBMCs)向破骨细胞分化过程中的表达模式。Western blot检测LOC147710蛋白的亚细胞定位。TRAP染色和甲苯胺蓝染色分别观察破骨细胞的形成和骨吸收活性。构建LOC147710真核表达载体,转染LOC147710-/- PBMCs,观察转染后是否能恢复其向破骨细胞分化的能力。结果全基因组扫描、精细基因定位及单倍型分析结果显示,染色体19q13.2-q13.3的D19S197至D19S545之间的8.36cM范围为致病基因候选区,连锁分析得最大LOD值Zmax=2.907(θ=0时)。基因测序发现,在LOC147710基因的2号外显子上存在一个纯合子无义突变(c.295C>T)。该无义突变使得终止密码子提前出现(p.R99X),导致其表达一种缺失93个氨基酸残基的截短蛋白。LOC147710基因表达于人类骨组织和小肠组织,而不表达于其它人类组织,提示该基因产物是一种与骨矿物质吸收和代谢密切相关的高特异性调节因子。本研究显示,LOC147710表达于原代人破骨细胞,而在原代人成骨细胞和破骨前体细胞PBMCs则不表达。在巨噬细胞集落刺激因子(M-CSF)和核因子κdB受体活化因子配体(RANKL)诱导PBMCs向破骨细胞分化的过程中,LOC147710的表达逐渐增强。LOC147710蛋白定位于破骨细胞的胞膜和胞质中,而在胞核中不表达。LOC147710-/- PBMCs向破骨细胞分化的能力极其微弱,而重新导入LOC147710基因后,可使其向破骨细胞分化的能力恢复至正常水平。结论LOC147710基因纯合子无义突变(c.295C>T)引起该基因的功能丢失,抑制破骨细胞分化能力,从而导致了一种特殊类型的常染色体隐性骨质硬化症,此型尚未报道第三部分网膜素-1在去卵巢小鼠和护骨素基因敲除小鼠发挥骨保护作用目的网膜素-1(Omentin-1)是近年来发现的一种主要由网膜脂肪组织分泌的细胞因子,其在血浆中含量丰富,达到ng/ml水平。本研究旨在探讨Omentin-1在体外和体内对骨代谢的影响。方法测定碱性磷酸酶(ALP)活性、骨钙素(OCN)和基质矿化以判断成骨细胞的分化程度。ELISA检测成骨细胞核因子κB受体活化因子配体(RANKL)和护骨素(OPG)蛋白分泌水平。在体外成骨细胞/破骨前体细胞共培养体系中,观察重组Omentin-1蛋白对破骨细胞生成与分化的影响。注射Omentin-1腺病毒表达载体到去卵巢(OVX)小鼠和OPG基因敲除(OPG-/-)小鼠体内,观察Omentin-1对这两种动物模型骨密度(BMD)、骨强度和骨代谢转换的影响。结果(1)Omentin-1直接抑制成骨细胞的分化,但并不直接抑制破骨细胞分化。在成骨细胞/破骨前体细胞体系中,Omentin-1刺激成骨细胞生成OPG,抑制成骨细胞合成RANKL,从而间接地抑制了破骨细胞的生成。(2)Omentin-1可以部分恢复OVX小鼠的BMD和骨强度,下调OVX小鼠血清骨形成标志物OCN和骨吸收标志物抗酒石酸酸性磷酸酶-5b(TRAP-5b)水平以及血清RANKL/OPG比率(3)Omentin-1可以部分恢复OPG-/-小鼠的BMD,下调OPG-/-小鼠血清RANKL、OCN和TRAP-5b水平。结论Omentin-1通过调控RANKL和OPG的表达抑制骨转换,从而减少因雌激素缺乏或OPG基因缺失所导致的骨丢失,因而,Omentin-1具有抗骨吸收作用。更多还原

【Abstract】 Part one Estrogen Receptor-α36 Mediates a Bone-Sparing Effect of 17β-Estrodiol in Postmenopausal WomenObject Estrogen receptor-alpha36 (ER-α36) is a membrane-based protein. Unlike ER-α66、ER-α46 and ER-βwhich mediate nuclear effects of estrogens, ER-α36 only mediates membrane effects of estrodiol, such as activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway. This study was aimed to investigate whether ER-α36 is involved in the regulation of bone metabolism as done by ER-α66.Methods The expression of ER-α36 protein in bone sections was assessed by immunohistochemistry. Cell proliferation and apoptosis were analyzed by 3H-thymidine incorporation and ELISA, respectively. Alkaline phosphatase (ALP) activity was assayed by measurement of p-nitrophenol. Osteocalcin (OCN) was measured by radioimmunoassay (RIA). Matrix mineralization was examined with alizarin red S staining. Expression levels of ER-α36, ER-α66, ERK1/2 and p-ERK1/2 protein were detected by Western blot. Reactive oxygen species (ROS) level was measured by 2’,7’-dichlorodihydrofluorescein diacetate (H2-DCFDA) probe. To investigate the involvement of ER-α36 and post-receptor ERK and ROS signaling pathways in the regulation of proliferation, differentiation or apoptosis of osteoblasts and osteoclasts by E2, ER-α36 shRNA and eukaryon expression vector were used for regulating ER-α36 expression, and, inhibitors for ERK and ROS were also used. Expression levels of OCN and tartrate-resistant acid phosphatase (TRAP) mRNA were analyzed by quantitative real-time PCR (QRT-PCR). Furthermore, we selected 154 female operative cases (premenopausal:60; postmenopausal:33 normal,31 osteopenic, and 30 osteoporotic) who underwent surgery for intervertebral disk hernia, spinal stenosis or spondylolisthesis were selected in this study. BMD was measured using a dual-energy X-ray absorptiometry (DXA). Blood samples were collected for detection of serum levels of bone formation marker OCN and bone resorption marker cross-linked N-telopeptides of typeⅠcollagen (NTX). We also performed QRT-PCR for detection of the mRNA levels of ER-α36, ER-α66, ER-a46 and ER-βin collected vertebral cancellous bone explants in these patients. Furthermore, animal experiments were performed to observe the effects of the IC162, a selective ER-α36 modulator, on the bone metabolism in ovariectomized mice.Results (1) Cell culture experiments results:ER-α36 mediated mitogenic, antiapoptotic, and antiosteogenic effects of postmenopausal low level (10 pM) E2 in osteoblasts through transient activation of the ERK, whereas ER-α36 mediated proapoptotic effect of 10 pM E2 in osteoclasts through prolonged activation of the ERK with the involvement of ROS; 10 pM E2 had no effect on the proliferation, apoptosis or differentiation of ER-α36 negative osteoblasts and osteoclasts. (2) Bone tissue culture experiments results:10 pM E2 significantly inhibited the mRNA expression of osteoblast marker OCN and osteoclast marker TRAP in bone tissues that express high levels of endogenous ER-α36, while had no significant effect on the mRNA levels of OCN and TRAP in bone tissues that express extremely low levels of endogenous ER-α36. (3) The ANOVA showed that the mRNA levels of ER-α36 in the premenopausal group were significantly lower than in the three postmenopausal groups; the mRNA levels of ER-α36 in the normal postmenopausal group were significantly higher than in the other two postmenopausal groups (osteoporotic or osteopenic); the mRNA levels of ER-α36 in the osteopenic group were significantly higher than in the osteoporotic group; there was a decrease of ER-α46, ER-α66 and ER-βlevels in all postmenopausal groups compared with the premenopausal group; there was no significant difference in mRNA levels of ER-α46, ER-α66 and ER-βexpression among all postmenopausal groups. (4) The correlation analysis showed that in postmenopausal women, a significant positive correlation was observed between the levels of ER-α36 mRNA in bone and BMD, while a significantly negative relationship was observed between the levels of ER-α36 mRNA in bone and serum OCN and NTX; no significant correlations were observed between the levels of ER-α36 mRNA in bone and BMD and serum OCN and NTX in premenopausal women; no significant correlations were found between the levels of ER-α46, ER-Ⅵ66 and ER-βmRNA in bone with BMD and serum OCN and NTX in both pre- and post-menopausal women. (5) Animal experiment results:In ovariectomized mice, IC162 increased BMD but had no effect on the body weight and uterus weight.Conclusions ER-α36, but not ER-α66, ER-a46 or ER-β, is a critical regulator of bone metabolism in postmenopausal women—osteoblasts and osteoclasts expressing high levels of ER-α36 are hypersensitive to E2, ER-α36 mediates a bone-sparing effect of the low level of E2 in postmenopausal women. IC162, a selective ER-α36 modulator, exerts bone-sparing effect in ovariectomized mice. Part twoIdentification of a hypothetical protein LOC147710 mutation associated with autosomal recessive osteopetrosisObject Osteopetrosis is a highly heterogeneous group of inherited skeleton disorder characterized by a generalized increase in bone mineral density due to a failure of osteoclast differentiation or function. To date, loss-of-function mutations in 11 genes have been identified as being associated with human osteopetrosis. All of the heritage osteopetrosis patients were caused by single gene mutation. In this study, the homozygous genotype of 5 members of a Chinese family (including 11 members) affected by an autosomal recessive osteopetrosis was investigated with the aim of identifying novel gene mutations associated with osteopetrosis in this family.Methods BMD was measured using a dual-energy X-ray absorptiometry (DXA). Total genomic DNA was isolated from peripheral blood of all 11 family members and 180 normal controls. Genome-wide scan and fine mapping study and haplotype analysis were performed to identify the critical region. Exons of genes located in the critical interval were sequenced to identify gene mutation. Northern blot and Western blot were performed to reveal the the expression pattern of LOC147710 in human tissues and in the osteoclast precursor CD14+ peripheral blood mononuclear cells (PBMCs) which undergoing osteoclastic differentiation. Western blot was also used for detection of the subcellular localization of LOC147710 protein. TRAP staining and Toluidine blue staining were used for observing osteoclast formation and activity, respectively. LOC147710 eukaryon expression vector was constructed and transfected to LOC147710-/- PBMCs for investigating whether the osteoclastic differentiation defect of LOC147710-/- PBMCs could be restored by re-introduction of LOC147710.Results A single critical region of homozygosity on chromosome 19q13.2-ql3.3 between D19S197 and D19S545 spanning 8.36 cM with maximum LOD score of 2.907 atθ=0 was identified. Sequencing of the genes in this region revealed a homozygous nonsense mutation (c.295C>T) in exon 2 of the LOC147710 gene. This mutation introduced a premature stop codon, resulting in p.R99X in the translated protein. LOC147710 mRNA is primarily detected in human bone and small intestine, but not in other tissues. In vitro, LOC147710 mRNA was expressed in human primary osteoclasts, but not in huamn osteoblasts and PBMCs. LOC147710 mRNA and protein expression in PBMCs increased progressively during the osteoclastic differentiation process induced by macrophage colony-stimulating factor (M-CSF) and receptor activator for nuclear factorκB ligand (RANKL). LOC147710-/- PBMCs from the osteopetrosis subject exhibited poor ability to differentiate into osteoclasts upon exposure to M-CSF and RANKL. Furthermore, reintroduction of LOC147710 restored the ability of LOC147710-/ PBMCs to differentiate into functional osteoclasts in the presence of M-CSF and RANKL.Conclusions This study showed that a loss-of-function homozygous mutation in LOC147710 gene caused a novel type of autosomal recessive osteopetrosis via down-regulating osteoclast differentiation. Part threeOmentin-1 exerts bone-sparing effect in ovariectomized mice and osteoprotegerin gene knockout miceObject Omentin-1 is a recently identified visceral adipose tissue-derived cytokine and is highly abundant in plasma. This study was undertaken to investigate the effect of omentin-1 on bone metabolism.Methods Osteoblast differentiation was assessed by measuring ALP activity, osteocalcin production and matrix mineralization. ELISA was used to detect receptor activator for nuclear factorκB ligand (RANKL) and osteoprotegerin (OPG) protein expression in osteoblasts. The effect of recombinant omentin-1 on osteoclasts formation was examined in the co-culture systems of osteoblasts and osteoclast precursors. The effects of omentin-1 on bone mass, bone strength and bone turnover were examined in the ovariectomized (OVX) mice or OPG gene knockout (OPG-/-) mice by injection of omentin-1 adenovirus.Results (1) In vitro, omentin-1 directely inhibited osteoblast differentiation, but had no direct effect on osteoclast differentiation; however, it indirectly reduced osteoclast formation in the co-culture systems of osteoblasts and osteoclast precursors through stimulating OPG and inhibiting RANKL production in osteoblasts. (2) In vivo, omentin-1 partially restored bone mineral density (BMD) and bone strength in OVX mice, accompanied by decreased levels of plasma osteocalcin (OCN) and tartrate-resistant acid phosphatase-5b (TRAP-5b) and a lower serum RANKL/OPG ratio. (3) Omentin-1 partially restored BMD in OPG-mice, accompanied by decreased levels of serum RANKL, OCN and TRAP-5b.Conclusions The present study suggests that omentin-1 ameliorates bone loss induced by estrogen-deficiency or OPG gene knockout via regulating RANKL and OPG expression.更多还原