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过氧化物酶体增殖物激活受体γ对肥胖小鼠骨髓间充质干细胞分化的影响

The Relationship Between Pparγ and bone Marrow Mesenchymal Stem Cells in Obesity Mice

作者: 专业:老年医学 导师:丁国宪 年度:2010 学位:硕士  院校: 南京医科大学

Keywords

Peroxisome proliferator—activated receptorγ, Obesity, Bone marrow mesenchymal stem cells, Osteoporosis, Condition medium

        目的:在目前已有的几种胰岛素增敏剂中,噻唑烷二酮类(TZDs)可降低患糖尿病风险高达56%,远高于α-葡萄糖苷酶抑制剂和二甲双胍(仅分别为31%和32%)。因此,TZDs作为真正的胰岛素增敏剂,一直为国内外学者所推荐用于改善胰岛素抵抗。但是,随着TZDs在临床的广泛使用,它的副作用也越来越引人注目。它不仅导致水钠储留、体重增加、心血管事件明显增多,同时还发现,它能导致骨质疏松,使骨质疏松性骨折的发生明显增多。众所周知,噻唑烷二酮类属于过氧化物酶体增殖物激活受体(PPARγ)激动剂,而PPARγ在骨髓间充质干细胞(Mesenchymal Stem Cells, MSCs)向脂肪细胞和成骨细胞的定向分化潜能上起着“分子开关”样作用:促进PPARγ表达及增强其活性抑制MSC向成骨分化、增殖,增加其向脂肪细胞分化,而降低PPARγ表达和活性,相反则抑制MSCs向脂肪细胞分化、而促进向成骨细胞分化。另一方面,TZDs类药物改善胰岛素抵抗主要作用于脂肪组织,调节脂肪细胞分泌功能,而脂肪细胞分泌的脂肪因子如瘦素(Leptin)、脂联素(Adiponectin)以及炎症因子如白介素-6(IL-6)、肿瘤坏死因子α(TNF-α)等在骨代谢过程中发挥重要作用。因此本研究通过建立高脂饮食诱导的肥胖小鼠的模型,观察PPARγ激动剂吡格列酮对肥胖小鼠骨量变化的影响以及对MSCs分化的情况的影响,从体内外两方面探讨PPARγ与MSCs分化之间的关系及可能的作用机制,为进一步深入研究TZDs类药物的副作用提供初步研究基础。方法:1.将C57BL/6小鼠分为正常饮食对照组、高脂饮食对照组、吡格列酮给药组。对照组给予溶剂,给药组予吡格列酮(10mg.kg-1.d-1)灌胃4周。灌胃结束后检测各组小鼠的葡萄糖耐量,骨密度及骨组织形态学。2.取小鼠颅骨进行原代培养,加地塞米松(10-7M)维生素C(0.2mM)及β甘油磷酸钠(10mM)诱导成骨,检测碱性磷酸酶活性,定量PCR检测成骨分化指标如碱性磷酸酶、骨钙素、I型胶原等;取小鼠骨髓单核细胞,加入M-CSF(50ng/ml)、RANKL(30ng/ml)诱导形成破骨细胞,计数破骨细胞数目及破骨功能基因的变化;取小鼠股骨骨髓,分离、纯化、获得间充质干细胞,成骨诱导后计数成骨集落,并用定量PCR检测MSCs分化过程中成骨及成脂特异性基因的表达情况。3. 3T3-L1脂肪前体细胞生长接触至单层后,采用3-异丁基-1-甲基黄嘌呤(0.5mM)、地塞米松(10-6 M)及胰岛素(3.5ug/ml)的联合诱导方案诱导其分化,在分化过程中的第0-4天予PPARγ激动剂——吡格列酮进行刺激,收集分化8-10天的成熟脂肪细胞的上清液,制作成条件培养基,与新鲜培养基以1:1的比例刺激MSCs48h后,采用实时荧光定量PCR技术定量比较成脂成骨分化中关键因子变化。进一步用Western blot分析成脂成骨分化的首要调控因子PPARγ及Runx2。另外MSCs向成骨诱导后用茜素红法染色及氯化十六烷基吡啶萃取定量分析条件培养基对矿化的影响。结果:1.分别经过24周的正常饮食与高脂喂养,高脂饮食组的肥胖小鼠体重明显高于正常饮食对照组小鼠(P <0.01);肥胖小鼠的内脏脂肪的重量亦明显高于正常组(P <0.05);在口服葡萄糖耐量实验(OGTT)中,肥胖小鼠在15分钟的血糖水平显著升高(P <0.05)。肥胖小鼠经吡格列酮给药一月后,OGTT的结果显示血糖在空腹以及口服葡萄糖后15、30、60、120分钟都低于对照鼠,且在15分钟有显著性差异(P <0.05)。肥胖小鼠的对照组与吡格列酮给药组骨密度无明显差异。而给药组骨组织形态学中骨小梁面积百分数、骨小梁宽度、骨小梁数目均较对照组有所增加,骨小梁分离度下降,尤其是成骨细胞周长百分率较对照组有明显增加,且差异有显著性(P <0.01)。2.吡格列酮给药组与对照组肥胖小鼠相比,原代成骨细胞碱性磷酸酶活性及标志基因升高,破骨细胞数目及标志基因无差异;原代MSCs成骨分化基因Osterix、Dlx5明显升高,而成脂分化基因PPARγ明显下降(Dlx5、PPARγP <0.05; Osterix P <0.01),且MSCs向成骨分化的集落增多。3.体外实验中,用吡格列酮直接刺激MSCs后,成脂分化基因CEBPα、PPARγ较对照组表达量增加(P <0.05),成骨分化基因Osterix、Dlx5表达量下降(Osterix P <0.01; Dlx5 P <0.05);而我们进一步将成熟脂肪细胞培养液加入MSCs后,其成脂分化却下降,成骨分化反而升高。进一步研究还发现,用吡格列酮刺激脂肪细胞后,再将培养液刺激MSCs,成脂分化下降及成骨分化升高的现象更为明显。Western blot结果表明脂肪细胞上清能间接增加MSCs中成骨因子Runx2的表达同时降低成脂因子PPAR的表达。另外,脂肪细胞培养液刺激MSCs向成骨分化后的矿化程度明显高于对照组(P <0.05),而吡格列酮处理的条件培养基的促矿化作用更明显高于正常脂肪细胞条件培养基。结论:1. PPARγ激动剂吡格列酮在改善肥胖小鼠胰岛素抵抗的同时,不导致肥胖小鼠的骨质疏松,反而可以促使肥胖小鼠的骨髓MSCs向成骨细胞分化增多而导致骨形成增强,但是对破骨细胞的数目及功能无明显影响。这可能与PPARγ激动剂对肥胖小鼠的全身作用尤其是作用于脂肪细胞有关。2.脂肪细胞条件培养液作用于骨髓MSCs后可导致其向成骨分化增强,成脂分化减弱,吡格列酮刺激的脂肪细胞条件培养液的促成骨作用更为明显,这与吡格列酮直接刺激MSCs的作用完全相反。从这一结果可以推测吡格列酮可能通过作用于脂肪细胞调节其脂肪因子的分泌,然后脂肪因子间接作用于骨髓MSCs起到促进骨代谢的作用。
    Objective:Thiazolidinediones (TZDs) such as pioglitazone are peroxisome proliferator-activated receptor-γ(PPARγ) agonists and are therapeutic agents widely used in the treatment of type 2 diabetes. Several reports have demonstrated harmful effects of PPARγagonist not only on body weight gain、edma but also on the homeostasis of bone. As we all know, PPARγis the key regulator in adipocyte maturation and marrow mesenchymal stem cells (MSCs) adipogenesis. On the other hand, obesity has shown protective effects on bone metabolism. And it is well known that PPARγis a key player in the process of adipocyte differentiation. Activation of PPARγnot only stimulates preadipocytes to differentiate into mature adipocytes, but also exhibits improved adipokine, inflammatory profiles, including the increased secretion of adiponectin, the decreased production of lepin, and the anti-inflammatory effects by lowering the expression of TNF-α, IL-6 in white adipocyte tissues. We hypothesize that TZDs would be beneficial to bone through stimulating adipocytes indirectly. The purpose of this study was to investigate the effect of pioglitazone on bone utilizing diet induce obese mice model, and in vitro, pioglitazone was used for the investigation of the direct effect of osteogenesis/adipogenesis on mouse primary MSCs, as well as the indirect effect caused by 3T3-L1 adipocyte conditioned medium stimulated with pioglitazone.Methods:1. C57BL/6 mice were randomly divided into normal control group, obesity control group and pioglitazone-treatment obesity group, and they were given normal chow or high-fat diet respectively for 24 weeks continuously. And they were intragastric administrated with placebo or pioglitazone(10mg.kg-1.d-1)for four weeks. Then BMD, static bone histomorphometry and oral glucose tolerance test (OGTT) were performed.2. Murine osteoblasts were generated from crinial bone and were induced. The activity of ALP was detected, the mRNA expression levels of ALP and COLIa1 were analyzed by real-time quantitative PCR; M-CSF-dependent bone marrow macrophages (M-BMM)was cultured with 30ng/mL of RANKL and 50ng/mL of M-CSF, TRAP-positive mononuclear and multinucleated cells were formed. The number of osteoclast was calculated and marker genes were detected by real-time quantitative PCR; MSCs were obtained from the bone marrow in the mice femurs. For the CFU-osteo assay, MSCs were grown in the basal culture medium for 6 days. The cells were then grown in an osteogenesis-inducing culture medium that comprised basal culture medium supplemented with 10-7 M dexamethasone, 0.2mM ascorbic acidphosphate, and 10 mMβ-glycerophosphate, After 21 days, the cells were fixed with 10% formalin, stained with 0.1% alizarin red, and the number of positive colonies was counted under a light microscope.3. 3T3-L1 preadipocytes were incubated in the adipogenic cocktail (10-6M Dex, 0.5mM isobutylmethylxanthine, 5ug/ml insulin) from 0d to 4d. 3T3-L1 preadipocytes were treated with or without pioglitazone during differentiation, and the obtained conditioned medium (CM) from mature adipocytes was added to mouse MSCs. Several osteogenic and adipogenic genes were determined by real-time quantitative PCR in mouse MSCs after direct treatment with pioglitazone or indirect treatment with pioglitazone stimulated CM (PCM) for 48 h. Western blotting was further performed to analyze the representative protein expression levels including PPARγand Runx2. The induced mineralization of mouse MSCs was also tested with the Alizarin Red-based assay.Results:1. As expected, body weight and visceral adipose tissue (VAT) weight were significantly higher in HFD mice which had been maintained with HFD for 24 weeks versus control (p < 0.01). To assess the effect of the HFD in adipose tissues on glucose homeostasis, oral glucose tolerance test (OGTT) was performed. The level of sugar of blood in the obese mice was significantly elevated at 15 m (p < 0.05). After obese mice were intragastric administrated with pioglitazone for one month, they exhibited sustained hypoglycemia at fasting blood glucose levels and at 15 m, 30 m, 60 m, 120 m after the glucose treatment by intragastric administration. Especially, the level of sugar of blood was significantly diminished at 15 m (p < 0.05). There was no significance in the bone mineral density of proximal femur by dual energy X-ray absorptiometry (DEXA) between obesity control and pioglitazone-treatment mice. More extensive histomorphometric studies were performed in both groups. Static histomorphometric parameters of femurs were fixed in sections and stained with toluidine blue and Masson-Goldner Trichrome, both of which showed more bone trabecula in pioglitazone-treatment mice than in control mice. Ob.S.Pm (%) was significantly higher in pioglitazone-treatment mice (p = 0.002). In addition, no significant differences were detected in any of the parameters related to bone absorption rate such as osteoclast number and osteoclast perimeter.2. Tartrate-resistant acid phosphatase (TRAP) staining revealed no significant changes in the number of osteoclasts, and specific gene including TRAP, Integrinαand CathepsinK expressed by osteoclasts showed no differences between two group mice. The expression of ALP in pioglitazone-treatment mice increased when compared with the control, and so do the mRNA levels of specific gene including BMP2, COLIa1 expressed by osteoblasts. When the differentiation potential of stromal cells was estimated by plating out CFUs in osteogenic conditions, there was a significantly increased CFU-osteo numbers (stained by alizarin red) in the pioglitazone-treatment mice. The expression level of Dlx5、Osterix were significantly higher, while of PPARγwas significantly lower in MSCs from pioglitazone-treatment mice compared to control(Dlx5、PPARγp <0.05;Osterix p <0.01)3. Opposite to the results from direct treatment, after stimulation with PCM obtained from 3T3-L1 adipocytes, RNA levels of PPARγand C/EBPαwere down-regulated significantly, while Runx2、Dlx5 and Osterix levels were up-regulated significantly. The genic alteration patterns in CM and PCM stimulated MSCs were similar, but the effects from PCM treated groups were much more obvious compared with the CM treated groups. Moreover, PCM treated MSCs presented a significantly higher mineralization( p <0.01), as well as a higher protein expression level of Runx2 and a lower expression level of PPARγcompared with the CM treated MSCs.Conclusion:1. PPARγagonist pioglitazone not only enhanced insulin sensibility in obesity mice, but also increases the expression of osteogenic specific differentiation genes, and suppresses the adipogenic expression of genetic markers. Thus, in conclusion, the effects of PPARγagonist on bone metabolism in vivo may be bilateral and the key point is to keep balance between the two contrary effects.2. It is highlighted that TZDs might play a protective role in bone formation by promoting osteogenic differentiation of MSCs through indirectly stimulating adipocytes. So targeting TZDs to fat tissues would be an innovative approach to improve insulin resistance and reduce osteoporosis simultaneously.
        

过氧化物酶体增殖物激活受体γ对肥胖小鼠骨髓间充质干细胞分化的影响

中文摘要4-8
英文摘要8-11
前言13-16
方法16-27
    一、材料16-18
    二、动物实验方法18-20
    三、细胞实验方法20-27
结果27-38
    一、动物实验结果27-34
    二、体外实验结果34-38
讨论38-42
参考文献42-49
综述 噻唑烷二酮类与骨代谢的研究现状与进展49-59
    参考文献55-59
附录一 在读期间发表的论著59-60
附录二 英文缩略词60-61
致谢61
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