【作者】 洪盾；

【导师】 李继承；

【作者基本信息】 浙江大学 ， 人体解剖学与组织胚胎学， 2011， 博士

【摘要】 研究背景：骨质疏松(Osteoporosis)是最常见的骨骼代谢性疾病,其特征包括正常骨密度的减少、骨质脆弱增加以及容易轻微外伤后骨折。糖皮质激素(Glucocorticoids, GCs)是一类包括天然或人工合成的类固醇激素,主要作用是抑制炎性反应和治疗自身免疫性疾病。在临床上,天然和人工合成的糖皮质激素,尤其是病人需要大量或长期激素治疗时,都可能导致患者骨量减少或骨质疏松。糖皮质激素性骨质疏松症(Glucocorticoids-induced Osteoporosis, GCOP)是20-45岁成人骨质疏松症以及医源性骨质疏松症(Iatrogenic osteoporosis)最常见的原因。虽然人工合成的糖皮质激素如强的松、地塞米松等有GCOP等严重的并发症,由于具有突出的免疫抑制和抗炎作用,所以广泛应用于治疗过敏、类风湿性关节炎,以及抑制器官移植排斥。尽管骨吸收是GCOP的重要原因,最近的研究也表明长期糖皮质激素应用的主要后果是成骨细胞功能障碍。糖皮质激素对成骨细胞分化和骨形成的抑制作用可能与多种机制有关。骨形成蛋白-2(Bone morphogenetic protein 2, BMP-2)具有骨诱导作用。糖皮质激素通过阻滞BMP-2的作用而抑制成骨细胞分化。Wnt信号通路也是骨形成的一个重要信号通路。研究表明,糖皮质激素也通过阻滞Wnt-b.catenin信号通路抑制成骨分化。此外,糖皮质激素也能使处于成骨分化的骨髓基质细胞转向脂肪细胞分化,并且诱导产生过氧化物酶增殖体激活受体Y(Peroxisome proliferator-activated receptorγ,PPARγ).然而除上述机制外,成骨分化中糖皮质激素信号通路目前不清楚。具有PDZ结合序列的转录共活化因子(Transcriptional coactivator with PDZ-binding motif,TAZ)是类似β-catenin的一种分子,通过共活化Runx2依赖的基因转录和抑制PPARγ依赖的基因转录而促使间充质干细胞(Mesenchymal stem cell,MSCs)成骨分化。地塞米松(Dexamethasone,DEX)是一种人工合成和临床广泛使用的糖皮质激素,目前地塞米松如何影响成骨分化仍不清楚。稳定同位素标记的氨基酸细胞培养(Stable-isotope labeling by amino acids in cell culture,SILAC)的通过代谢将同位素质靶导入蛋白质。两种不同生物状态的细胞在正常培养基(轻同位素)和稳定同位素标记(重同位素)培养基中生长,当细胞在培养基中增值6次以上时,稳定同位素的嵌入可达到近100%。通过液相色谱串联质谱法分析(LC-MS/MS)进行定量蛋白组学检测,肽质谱显示了两种培养基下的不同SILAC波峰,两种波峰之间的高度比代表了不同蛋白量之间准确比值,所以SIALC蛋白组学是一种精确的定量蛋白组学。材料与方法：我们从16只4周龄雌性SD大鼠中获得骨髓细胞原代培养,并通过表面抗原和多能分化能力鉴定MSCs.P1代MSCs重新以5×105/cm2的密度种植于六孔板。为诱导MSCs成骨分化,MSCs以各种不同的诱导剂诱导,并以钙盐沉积能力(茜素红染色)和ALP活性检测细胞的成骨分化能力,以RT-PCR. Real-time PCR.Western blot.免疫组化等检测MSCs诱导成骨分化时TAZ.ALP. BMP-2.Runx-2的mRNA和蛋白质表达情况。我们还将高浓度的地塞米松作用于成骨前体MC3T3-E1细胞,观察地塞米松对MC3T3-E1细胞的成骨和增值能力影响,并利用SILAC定量蛋白组学进行筛选蛋白进行分析。结果：我们的研究中原代培养贴壁细胞是CD54和CD90阳性而CD14和CD34阴性,而且这些细胞具有向成骨细胞、脂肪细胞、软骨细胞分化的多能分化能力,表明这些分离的细胞属于MSCs。我们的研究还发现,间充质干细胞10-8M地塞米松诱导14天后钙盐沉积能力明显增强,而抗坏血酸和β-甘油磷酸明显增强了地塞米松的成骨分化效果。Real-time PCR和免疫印迹结果支持10-8 M地塞米松处理间充质干细胞7天后TAZ mRNA和蛋白质水平均有提高,免疫组化也表明10-8M地塞米松处理后TAZ阳性的MSCs数量比例显著大于对照组。RU486是一种糖皮质受体拮抗剂，研究表明10-5M RU486拮抗了10-8 M DEX提高TAZ水平的能力。单纯抗坏血酸和β-甘油磷酸没有明显提高TAZ的表达水平。此外,我们的研究发现,10-8 M和10-9 M地塞米松显著提高了大鼠间充质干细胞ALP活性(P<0.01),而10-7 M地塞米松(P<0.05)和10-6 M地塞米松(P<0.01)显著抑制了间充质干细胞的ALP活性,而且10-7 M地塞米松处理间充质干细胞3至7天时显著降低了TAZ的表达水平,提示TAZ的表达和成骨分化活性相关。成骨前体MC3T3-E1细胞10-6M地塞米松治疗7天后,MTT和ALP酶活性检测表明地塞米松抑制了骨前体MC3T3-E1细胞的增值和成骨分化能力。SILAC定量蛋白组学检测发现10-6M地塞米松作用7天抑制了成骨前体细胞的成骨分化和细胞增值能力,上调了微管蛋白(TUBA1A、TUBB2B、TUBB5)、IQGAP1、S100蛋白(S100A11.S100A6、S100A4、S100A10)的表达水平,并下调了G3BP-1和Ras相关蛋白(Rab-1A. Rab-2A、Rab-7)的表达。结论：我们的研究表明地塞米松在诱导间充质干细胞成骨分化中调节了TAZ的表达,而这种作用与地塞米松的浓度有关。所以TAZ可能不仅是BMP-2诱导成骨分化中的重要通路,而且地塞米松诱导成骨中的信号通路有关。因此,TAZ作为一种β-catenin分子,可能是BMP-2信号通路和Wnt-β-catenin信号通路的重要交叉点。高浓度地塞米松抑制了成骨前体细胞的成骨分化和细胞增值能力。上调的微管蛋白、IQGAP1、S100蛋白以及下调的G3BP-1和Ras相关蛋白可能和地塞米松诱导的细胞成骨分化和增值功能抑制有关。本研究中的成骨分化中糖皮质激素信号通路的调节机制分析可能有助于将来GCOP治疗。更多还原

【Abstract】 Background:Osteoporosis is the most common metabolic disease characterized by loss of the normal density of bone, resulting in fragile bone and a higher risk of fractures in response to relatively minor injuries. Glucocorticoids (GCs) are naturally-produced steroid hormones, or synthetic compounds, that inhibit the process of inflammation or autoimmune disorders. Clinically, both of the natural or synthetic glucocorticoids can lead to the side effects of bone loss or osteoporosis while they are administrated especially of over dose and in long term. Patients under glucocorticoids treatment are one of the main groups of persons who suffer from osteoporosis. Glucocorticoids-induced Osteoporosis (GCOP) is the most common cause of adult osteoporosis in population aged from 20 to 45 and iatrogenic osteoporosis. Though the synthetic glucocorticoids such as prednisone and dexamethasone (DEX) have severe GCOP complication, they are still widely used in treatment of allergy, rheumatoid arthritis, and allograft rejection.Although bone resorption has been suggested to cause GIO, recent studies have revealed the main mechanism underlying long-term GCs-induced bone loss is the impairment of osteoblast function and bone formation. Glucocorticoids may inhibit osteoblasts through multiple mechanisms with the results of significant reduction in bone formation. Bone morphogenetic protein 2 (BMP-2) is proven to inducing bone formation and currently clinically used in orthopedic surgery. Glucocorticoid is revealed to inhibit osteoblast cell differentiation through the repression of bone morphogenetic protein 2, which leads to a decrease in bone formation. Wnt signaling pathway, a complex network of proteins most well known for their roles in embryogenesis and cancer, is also another key pathway for promoting osteoblastogenesis. In studies, glucocorticoids inhibit osteoblast cell differentiation by opposing Wnt-b-catenin signaling.Moreover, glucocorticoids are able to redirect differentiation of bone marrow stromal cells from the osteoblastic lineage to the adipocyte lineage. The underlying mechanisms involve the regulation of nuclear factors of the CAAT enhancer-binding protein family, which might also indirectly reduce osteoblast proliferation and decrease IGF-I transcription, and the induction of peroxisome proliferator-activated receptor y2 (PPARy2). However, the glucocorticoid signaling pathway during osteoblast differentiation remains unclear.Transcriptional coactivator with PDZ-binding motif (TAZ), aβ-catenin-like molecule, drives mesenchymal stem cells (MSCs) to differentiate into osteoblast lineage through co-activation of Runx2-dependent gene transcription and repression of peroxisome proliferator-activated receptor y (PPARy)-dependent gene transcription. Dexamethasone (DEX), a synthetic and widely used glucocorticoid, affects osteogenesis. However, the signaling pathway by which DEX affects osteoblastic differentiation remains obscure.Stable-isotope labeling by amino acids in cell culture (SILAC) enables metabolic incorporation of isotope mass tags into proteins [1]. The cells representing two biological conditions are grown in normal medium (light medium) and isotope amino acid labeled medium (heavy medium), respectively. After grown for six cell divisions in labeled medium to reach 100% of the incorporation of the isotope amino acid, the cells are processed for further treatment, followed by liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) analysis. Therefore the relative intensity ratio of the two pairs of "light" and "heavy "peaks in the peptide mass spectrum can reflect the protein quantitative expression levels under different conditions [1]. Materials and methods:The bone marrow cells from sixteen four-week-old virgin female Sprague-Dawley rats were harvested. The primary-MSCs were isolated and identified through detection of surface antigens and multi-differentiation ability. The passage 1 MSCs were reseeded in the density of 5x105/cm2 in six-well-plates. To induce osteoblast differentiation, the MSCs were cultured in different differentiation mediums. The calcium deposition ability (AR-S staining) and ALP activity were detected to evaluate the osteoblast differentiation ability of the MSCs. In addition, we carried out RT-PCR, Real-time PCR, Western blot, and immunochemistry to measure the mRNA and protein levels of TAZ, ALP, BMP-2, and Runx-2 during the osteoblast differentiation.Results:In this study, we found that isolated adhere cells in our study were CD54 positive and CD90 positive, but CD14 negative and CD34 negative. In addition, the MSCs possess the abilities of differentiating into osteoblast, adipocyte, and chondrocyte. Osteoprogenitor MC3T3-E1 cells were treated with or without 10-6 M DEX for 7 days and the differentiation ability, proliferation, the cells were measured. The protein level changes were analyzed using SILAC and liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS). Therefore, the surface antigen identification and mutilineage differentiation ability supported that these isolated cells were MSCs. Present study showed that 10-8 M DEX increased calcium formation in MSCs at day 14 and the effect was synergized by AA andβ-GP. Real-time PCR and immunobloting analysis showed that TAZ mRNA level and protein expression were enhanced in MSCs treated with 10-8 M DEX at day 7. In addition, the number of TAZ-positive MSCs after 10-8 M DEX treatment significantly increased compared to control using immunochemistry assay. The stimulative effect of 10-8 M DEX on TAZ was blocked by 10-5M RU486, an antagonist of glucocorticoid receptor and MSCs were treated with AA orβ-GP did not show increased TAZ level. In the present study,DEX at 10-8 M and 10-9 M significantly elevated ALP activity (P< 0.01) whereas 10-7 M DEX (P<0.05) and 10-6M DEX (P< 0.01) inhibited ALP activity in rat MSCs. We also demonstrated that higher concentration of DEX (10-7M) decreased TAZ mRNA level and ALP activity from day 3 to day 7, which indicates that TAZ level is closely related with osteoblast differentiation ability. In this study,10-6 M DEX inhibited both osteoblast differentiation and proliferation but induced apoptosis in osteoprogenitor MC3T3-E1 cells on day 7. We found that 10-6 M DEX increased the levels of tubulins (TUBA1A, TUBB2B, and TUBB5), IQGAP1, and S100 proteins (S100A11, S100A6, S100A4, and S100A10), while inhibited the protein levels of G3BP-1 and Ras related proteins (Rab-1A, Rab-2A and Rab-7) in MC3T3-E1 cells.Conclusions:These findings suggests that TAZ is not only involved in the signal pathway of BMP-2 induced osteoblastic differentiation, but also involved in the signaling pathway of DEX-induced osteoblastic differentiation, supporting the notion that TAZ is a convergence point of two signaling pathways, BMP-2 signaling pathway and Wnt-β-catenin signaling pathway.10-6 M DEX inhibited the osteoblast differentiation and proliferation in MC3T3-E1 cells on day 7. The upregulated levels of tubulin, IQGAP1, and S100 proteins and the downregulated protein levels of G3BP-1 and Ras related proteins (Rab-1A, Rab-2A and Rab-7) may be critical in the mechanism of the DEX-induced results. Such protein expression changes may be of pathological significance in coping with GCOP.更多还原