【作者】 陈月；

【导师】 邹大进；

【作者基本信息】 第二军医大学 ， 内分泌与代谢病， 2007， 博士

【摘要】 一、3T3-L1前脂肪细胞的培养、鉴定及标志物检测背景和目的:3T3-L1前脂肪细胞株,目前最常用的前脂肪细胞株之一,是一种从Swiss 3T3小鼠19天的胚胎中分离克隆获得的具有脂肪细胞分化潜力的细胞株,在融合成单层的情况下具有自发分化为成熟脂肪细胞的能力。该细胞株不但在体外经诱导后可分化为成熟的脂肪细胞,而且植入小鼠体内后也可分化并形成与正常脂肪组织无明显差别的脂肪团块。3T3-L1前脂肪细胞在形态学上类似于成纤维细胞,在经历了生长停滞和克隆性扩增后,在经典的激素鸡尾酒,即胰岛素(insulin, INS)、地塞米松(dexamethasone, DEX)和3-异丁基-1-甲基-黄嘌呤(3-isobutyl-1-methylxanthine, MIX)的刺激下,前脂肪细胞逐步转变为球形,细胞骨架以及细胞外基质成分和水平发生变化,脂质积聚,最终分化为成熟的脂肪细胞。由于3T3-L1前脂肪细胞可较好模拟脂肪细胞分化和活体脂肪组织的功能,因此目前已成为肥胖及体外脂肪细胞分化研究的一个常用工具。而如何在体外培养中,采用合适的培养条件、合理的培基配制及诱导剂的使用浓度对于脂肪细胞活力、生存状态及其分泌能力都有重要的影响。本部分实验通过对3T3-L1前脂肪细胞的形态、生长增殖、脂肪含量等细胞生物学特性的研究,诱导分化3T3-L1前脂肪细胞,摸索培养和诱导3T3-L1前脂肪细胞的最佳条件,为应用药物干预3T3-L1前脂肪细胞增殖分化以及后期研究3T3-L1前脂肪细胞分化过程中基因表达等奠定基础。方法:1、3T3-L1前脂肪细胞的复苏、体外培养、计数以及细胞冻存;2、3T3-L1前脂肪细胞的诱导分化;3、油红O染色法鉴定成熟脂肪细胞;4、采用逆转录聚合酶链反应(Reverse transcription-polymerase chain reaction, RT-PCR)检测3T3-L1前脂肪细胞的诱导分化过程中过氧化物酶体增殖物激活受体γ(Peroxisomal proliferation activated receptor-gamma , PPARγ2)基因表达变化。结果:1、采用含10%FBS、4mmol/l L-谷氨胺酰和100u/ml青、链霉素的高糖DMEM培养基成功进行了3T3-L1前体脂肪细胞的体外复苏、培养和传代;2、在体外培养的基础上,采用传统的鸡尾酒诱导剂(0.5 mmol/L MIX、0.25 umol/ L DEX和10ug/ ml INS)成功地完成了3T3-L1前脂肪细胞的诱导分化。3、油红O染色显示3T3-L1前脂肪细胞分化成熟过程中的脂滴形成情况,并证实3T3-L1脂肪细胞诱导分化成功。4、PPARγ2 mRNA水平在3T3-L1前脂肪细胞(加入诱导剂开始诱导分化,D0)中表达极低,随着诱导分化逐步成熟,其表达量逐步增加,至诱导分化D9~D10其表达量最高,提示脂肪细胞分化完全成熟。结论:本部分实验通过对3T3-L1前脂肪细胞的形态、生长增殖、脂肪含量等细胞生物学特性的研究,摸索出了培养和诱导3T3-L1前脂肪细胞的最佳培养条件和诱导剂浓度,油红O染色以及脂肪细胞中PPARγ2基因表达的检测均显示3T3-L1脂肪细胞诱导分化成熟,为应用药物干预3T3-L1前脂肪细胞增殖分化以及后期研究3T3-L1前脂肪细胞分化过程中基因表达等奠定基础。二、3T3-L1前脂肪细胞增殖和分化中PTP1B表达变化背景和目的:作为最早被纯化的蛋白酪氨酸磷酸酶之一,蛋白酪氨酸磷酸酶1B(protein tyrosine phosphatase 1B,PTP1B)一直被认为是胰岛素受体后信号转导的主要负性调控因子,近年来更是成为治疗肥胖、2型糖尿病等胰岛素抵抗相关疾病的新靶点。多项研究证实,无论是基因敲除PTP1B还是PTP1B反义核苷酸(antisense oligonucleotide, ASO)处理均可使实验小鼠肝脏和肌肉组织中胰岛素受体后胰岛素受体(insulin receptor, InsR)和胰岛素受体底物蛋白(insulin receptor substrate protein, IRS)的磷酸化增强,胰岛素信号传导增强,提高胰岛素敏感性。然而,在同样是胰岛素主要作用靶点的脂肪组织的研究中,关于PTP1B对脂肪组织胰岛素敏感性的影响则完全不同。在PTP1B基因敲除小鼠中,与肝脏及肌肉中胰岛素受体磷酸化增强、胰岛素敏感性提高相对应的是,脂肪组织中胰岛素受体的去磷酸化丝毫未受影响,无论是胰岛素受体磷酸化的最大水平还是达到该最大水平所需时间都无变化。另外,以PTP1B ASO处理ob/ob小鼠可见到小鼠体重减低,参与脂肪合成的多种基因如固醇调控元件结合蛋白-1(sterol regulatory element-binding protein, SREBP-1)表达减少,成脂作用减弱;而与胰岛素相关的蛋白激酶B(protein kinase B, PKB)磷酸化无改变,脂肪细胞中胰岛素介导的葡萄糖转运无影响。而一项最新的研究又提示在脂肪组织中特异性敲除PTP1B基因可见到小鼠的体重增加。那么,PTP1B在脂肪细胞分化成熟中的作用到底是什么呢?我们首次采用3T3-L1前脂肪细胞系,通过体外培养诱导分化,观察脂肪细胞分化成熟过程中PTP1B表达变化,为进一步揭示PTP1B在脂肪细胞分化和脂质积聚过程中的作用提供线索。方法:1、3T3-L1细胞的培养和诱导分化;2、油红O染色法鉴定成熟脂肪细胞;3、采用逆转录聚合酶链反应技术(Reverse transcription-polymerase chain reaction, RT-PCR)检测3T3-L1前脂肪细胞分化过程中PTP1B mRNA表达;4、采用实时荧光定量聚合酶链反应(Real-time fluorescence quantitative PCR, RT-FQ PCR )半定量检测3T3-L1前脂肪细胞分化过程中PTP1B mRNA表达;5、采用western blot方法检测3T3-L1前脂肪细胞分化过程中PTP1B蛋白水平表达。结果:1、在本实验中,我们成功培养了3T3-L1前体脂肪细胞并诱导其分化成熟。2、采用逆转录聚合酶链反应(RT-PCR)检测3T3-L1前脂肪细胞诱导分化过程中PTP1B基因表达显示,PTP1B mRNA表达在前脂肪细胞中(加入诱导剂,D0)最丰富,随着诱导分化的逐渐成熟,其表达也逐步减低,至诱导第10天(D10)脂肪细胞完全成熟时降至最低点。其中D1~3、D4~8、D9~10各阶段内表达未见明显差异,三组阶段之间可见PTP1B mRNA表达差异显著。3、实时荧光定量聚合酶链反应(RT-FQ PCR)半定量检测3T3-L1前脂肪细胞诱导分化过程中第1~10天的PTP1B基因表达显示,PTP1B mRNA表达在前脂肪细胞中最丰富,随着诱导分化的逐渐成熟,其表达也逐步减低,至D10脂肪细胞完全成熟时降至最低点。4、3T3-L1前脂肪细胞诱导分化过程中,PTP1B蛋白表达情况与其mRNA表达相平行,在前脂肪细胞中最丰富,随着诱导分化的逐渐成熟,蛋白水平逐步减低,至D9蛋白表达显著减少,D10脂肪细胞完全成熟时表达最少。其中D1~3、D4~8各阶段内表达未见明显差异,但D1~3、D4~8、D9和D10之间可见PTP1B蛋白表达差异显著。结论:本研究首次发现在3T3-L1前脂肪细胞分化成熟过程中,PTP1B mRNA及蛋白表达水平随着脂肪细胞分化成熟而逐步减低,至脂肪细胞完全成熟时达到最低点,提示PTP1B对于脂肪组织分化成熟的直接作用是抑制性的,PTP1B水平降低对于脂肪细胞分化成熟,脂质积聚则起到了“允许”甚至是“促进”作用。三、肿瘤坏死因子-α及罗格列酮对3T3-L1前脂肪细胞增殖和分化过程中PTP1B表达的影响背景和目的:已经证实,除了调节机体能量代谢平衡外,脂肪组织,作为一个重要的内分泌器官,分泌多种激素和细胞因子如瘦素、抵抗素、脂联素、肿瘤坏死因子等,参与了机体更广泛、更复杂、更重要的内分泌代谢调控。越来越多的研究表明,脂肪细胞分化异常,而并非仅仅的脂肪组织过度生成,才是发生2型糖尿病、高血压、冠心病等代谢性疾病的罪魁祸首。与糖尿病、冠心病等密切相关的中央型肥胖很可能是由于脂肪组织分化异常,无法产生足够数量的正常成熟脂肪细胞存储过多的能量,进而将生脂压力转嫁至肝脏、肌肉等器官,导致脂肪异位沉积,最终诱发一系列的代谢性疾患。蛋白酪氨酸磷酸酶1B,由于其可使胰岛素受体(InsR)及胰岛素受体底物蛋白(IRSs)等的酪氨酸脱磷酸化,一直被认为是胰岛素信号传导的负性调控因子。然而,PTP1B在机体各组织中的作用存在着明显的组织特异性。在肌肉、肝脏组织中已证实PTP1B水平降低,可使胰岛素受体和胰岛素受体底物蛋白磷酸化明显增强,反映胰岛素敏感性的指标如胰岛素诱导的机体葡萄糖处置力(insulin stimulated whole-body glucose disposal, ISGD)显著提升。而对于脂肪组织而言,PTP1B的作用目前仍不清楚,这些作用与机体胰岛素敏感性之间的关系,更是不得而知。为此我们采用体外培养3T3-L1前脂肪细胞,观察脂肪细胞诱导分化过程中PTP1B蛋白表达水平变化,再以罗格列酮和肿瘤坏死因子-α(tumor necrosis factor-α, TNF-α)分别干预脂肪细胞分化成熟过程并观察PTP1B蛋白表达情况,以期了解PTP1B在脂质合成和成脂过程的作用以及罗格列酮及肿瘤坏死因子-α分别影响脂肪细胞胰岛素敏感性的机制。方法:1、3T3-L1细胞的培养和诱导分化;2、油红O染色法鉴定成熟脂肪细胞;3、采用western blot方法检测不同诱导剂诱导分化三组3T3-L1前脂肪细胞分化过程中PTP1B蛋白水平表达。4、采用免疫沉淀和western blot方法检测三组3T3-L1前脂肪细胞分化成熟时胰岛素受体表达及胰岛素受体酪氨酸磷酸化水平。结果:1、在本实验中,我们成功建立了肿瘤坏死因子-α和罗格列酮干预3T3-L1前脂肪细胞诱导分化的适当工作浓度和条件。2、三组3T3-L1前脂肪细胞分化过程中,与正常诱导组相比,罗格列酮干预组脂肪细胞分化得到促进,肿瘤坏死因子-α干预组则脂肪分化成熟受到抑制。3、三组3T3-L1脂肪细胞分化成熟过程中,PTP1B蛋白表达趋势均表现为在前脂肪细胞中最为丰富,并随诱导分化成熟其表达逐步减少,至分化完全成熟时降至最低。但各组中PTP1B显著降低的拐点有所不同,正常诱导组与肿瘤坏死因子-α干预组在加入诱导剂诱导分化第9天(D9)出现,罗格列酮干预组则出现在第8天(D8)。4、比较三组脂肪细胞在诱导分化后期第7至第10天(D7~D10)PTP1B蛋白水平变化,进一步证实正常诱导组与肿瘤坏死因子-α干预组均在诱导分化第9天(D9)出现PTP1B蛋白表达水平显著降低,而罗格列酮干预组在第8天(D8)即出现PTP1B蛋白表达水平显著降低;PTP1B蛋白水平在肿瘤坏死因子-α干预组诱导分化第7天(D7)较同期正常诱导组显著增加,罗格列酮干预组的第8天(D8)及第10天(D10)则较正常诱导组同期显著减少,罗格列酮干预组的诱导分化后期第7至第10天(D7~D10)中PTP1B蛋白表达水平更是明显低于肿瘤坏死因子-α干预组诱导分化的同期水平。5、三组3T3-L1脂肪细胞分化成熟时胰岛素受体蛋白水平无差异,而胰岛素受体酪氨酸磷酸化水平在CR组最高,C组居中,CT组最低,提示PTP1B活性在CT组、C组和CR组依次降低。结论:本研究首次发现PTP1B蛋白表达水平与3T3-L1脂肪细胞分化成熟度呈负相关, PTP1B对于脂肪组织分化成熟的直接作用是抑制性的;肿瘤坏死因子-α及罗格列酮对于机体包括脂肪组织胰岛素敏感性的作用与其对脂肪细胞内PTP1B水平的影响有一定关系。更多还原

【Abstract】 Part I The Cultivation, Differentiation and Identification of 3T3-L1 Preadipocytes3T3-L1 preadipocyte cell line, one of the most used preadipocyte cell lines, was derived from immortalized 19 day-nonclonal Swiss 3T3 embryonic fibroblast cells of mesenchymal origin and is already committed to the adipocytic lineage. When treated with an empirically derived, prodifferentiation regimen that includes cAMP, insulin, and glucocorticoids, these cells undergo differentiation to mature fat cells over a 4- to 6-d period. Within 24–36 h of hormonal induction, cells re-enter the cell cycle, undergo one to two rounds of mitosis or clonal expansion, after which they permanently withdraw from the cell cycle, begin to accumulate lipid, and undergo terminal differentiation into mature adipocytes. As the good simulation of adipocyte differentiation and function of live adipocyte, 3T3-L1 preadipocyte cell line has been thought as one of the best characterized and widely used invitro models to study adipocyte differentiation. The cultural condition, optimal medium and inducer also make great sense to the livingness and potency of differentiated adipocyte. In this part of our study, with the observation of the resuscitation, growth, confluence and differentiation of 3T3-L1 preadipocytes, we make great efforts to get the optimal condition(DMEM with high glucose level and supplemented with 10%FBS, 100u/ml penicillin and streptomycin ) and inducing concerntration of differentiation(0.5 mmol/L MIX, 0.25 umol/ L DEX and 10ug/ ml INS), and we used oil red O staining to identify the fully differentiated adipocytes, all of which make good basis for the after work of our study. Part II The Expression of Protein Tyrosine Phosphatase 1B during The Differentiation of 3T3-L1 PreadipocyteAs one of the first purified protein tyrosine phophatases, PTP1B has been thought as the negative regulator of insulin signal transducting. The researches about the inhibition of PTP1B have been new hotspots for the treatment of metabolic diseases such as obesity and type 2 diabetes.It has already been confirmed that whether knockout PTP1B-/- mice or PTP1B antisense oligonucleotide(ASO) pretreated mice showed enhanced phosphorylation of insulin receptor(InsR) and insulin receptor substrate(IRS) in liver and skeletal muscles, with enhanced insulin signal transducting and improvement of insulin sensitivity. But in adipose tissue, these studies gave us so different results. There is no change in the phosphorylation of InsR and IRS-1 in adipose tissue, and both the highest level of phosphorylation in InsR and the time needed to get that highest level being unaltered. PTP1B ASO can decrease the weight of ob/ob mice and diminish the expression of many genes involved in adipogenesis such as sterol regulatory element-binding protein( SREBP-1) without any changes in phosphorylation of insulin related protein kinase B(PKB) and in insulin induced glucose transportion. Another study indicated that the mice specially knock out PTP1B-/- in adipose tissue got weight gain. So what is the exact effect of PTP1B on adipose tissue?This time we first use 3T3-L1 preadipocytes and investigate the expression level of PTP1B during the differentiation of adipocytes, to elucidate the effect of PTP1B in the differentiation of adipocytes and in the course of lipids accumulation.In this study we use 3T3-L1 preadipocytes cultured in vitro and induced them with regular inducer(MIX+DEX+INS). Oil Red O stain was used to clarified matural adipocytes. RT-PCR and Real Time FQ-PCR were used to assess the level of PTP1B mRNA, and western blotting was used to detect the protein level of PTP1B. Our study shows that with the relatively high level in 3T3-L1 preadipocytes, both the expression of mRNA and protein level of PTP1B went down with the differentiation of adipocytes, and reached the lowest level in matured adipocytes. So we can conclude that the declining of PTP1B level during the differentiation of adipocyte maybe a promoter for adipocyte maturation. Part III The Impacts of TNF-alpha And Rosiglitazone on The Expression of Protein Tyrosine Phosphatase 1B During The Differentiation of 3T3-L1 PreadipocytesAdipose tissue has been accepted as an important endocrinal organ which can secrete so many hormones and cytokines such as leptin, resistin, adiponectin and tumor necrosis factor participating in more and more metabolic regulation. It has been confirmed that it is the abnormal adipocyte differentiation, but not the adiposity, is the culprit of metabolic syndrome such as type 2 diabetes, hypertension and coronary heart disease. Central obesity which is closely related with diabetes and coronary heart disease It is due to the dysfunction of adipocyte differentiation, which can not produce enough adipocytes to storage more energy, that causes ectopic aggradation of lipids in liver or muscles, inducing so many metabolic disorders.PTP1B, that can dephosphorylate the tyrosine in InsR and IRS-1, has been thought as the negative regulator of insulin signal transducting. But there is obvious tissue specificity of the effect of PTP1B. In liver and skeletal muscles, the lower level of PTP1B, the more of phosphorylation in InsR and IRSs, and the higher level of insulin stimulated whole-body glucose disposal. But to adiopose tissue, the exact effect is still not so clear, just as the relationship between PTP1B and insulin sensitivity.So we use in vitro cultured 3T3-L1 adipocytes to investigate the expression of PTP1B protein level and the phosphorylation level of insulin receptor, and their changes under the influences of tumor necrosis factor-alpha and rosiglitazone during the differentiation of 3T3-L1 adipocytes. We hope to get out the real effect of PTP1B in adipogenensis and the possible mechanisms of rosiglitazone’s and tumor necrosis factor-alpha’s influence on insulin sensitivity adipocyte.3T3-L1 preadipocytes were cultured in vitro and induced with three types of inducers, regular inducer( MIX+DEX+INS), regular inducer plus 20 ug/l TNF-α(CT) and regular inducer plus 10-5mol/l rosiglitazone(CR). Western blotting was used to detect the level of PTP1B protein in each group during the differentiation of adipocytes.Immunoprecipitation and western blot were used to detect the expression and phosphorylation level of insulin receptor in matured adipocytes of the three groups.Our study showed that compared with regular inducers, CR could promote the differentiation of adipocytes, while CT could retard this programme. Each group showed the relatively high level of PTP1B in 3T3-L1 preadipocytes, going down with the differentiation of adipocytes, and reaching the bottom in fully-matured adipocytes. Comparing the late period of differentiation in these three groups, CT one were sluggishly differentiated with more PTP1B protein, and CR group showed active differentiation with the lowest level of PTP1B. The expression of insulin receptor showed no difference in the three groups, but the phosphorylation level of tyrosine in insulin receptor decreased obviously in CT group, and increased dramatically in CR group.So we can conclude that the declining of PTP1B level and its activity during the differentiation of adipocytes is in favor of lipid accumulation and maybe a promoter for adipocytes maturation. The effects of TNF-αand rosiglitazone on adipocytes’sensitivity to insulin perhaps partly depended on their influences on PTP1B level and its activity in adipocytes.更多还原