【作者】 鲁云霞；

【导师】 李俊；

【作者基本信息】 安徽医科大学 ， 药理学， 2009， 博士

【摘要】 蛋白酪氨酸磷酸酶1B(PTP1B)属于蛋白质酪氨酸磷酸酶(PTPs)家族,以跨膜受体样蛋白和胞内酶2种形式存在,催化蛋白质的磷酸化酪氨酸残基的去磷酸化反应,是哺乳动物体内最早被鉴定、纯化的PTPs[1,2]。PTP1B作用于胰岛素受体(IR),胰岛素受体底物1、2(IRS-1,IRS-2),生长因子受体结合蛋白2(Grb2),磷脂酰肌醇3激酶(PI-3K)等与胰岛素信号转导相关的蛋白,使它们的磷酸化酪氨酸残基脱磷酸,衰减胰岛素信号转导,从而产生受体后的胰岛素抵抗[3]。最近有人向小鼠的肝细胞瘤中导入针对PTP1B的siRNA,结果发现胰岛素信号转导增强[4],再次证实了PTP1B在胰岛素信号通路中的作用。PTP1B的高表达除了引起胰岛素抵抗外还可引起瘦素抵抗,从而引发2型糖尿病(T2DM)及肥胖[5,6]。PTP1B基因敲除鼠不仅胰岛素敏感性增强,而且去除了PTP1B对胰岛素和瘦素信号转导的抑制,使小鼠在高脂饮食条件下也不发生肥胖,且发育良好,具有正常的繁殖能力,癌症的发生率也未见提高[7],这提示了PTP1B作为胰岛素信号通路的主要负调控因子,其小分子抑制剂可能是有效的抗糖尿病/肥胖症药物,而且不产生副作用。事实上PTP1B抑制剂已成为胰岛素增敏剂的候选药物之一[8],全世界有不少实验室正在从天然中草药或人工合成的药物中以PTP1B作为靶点筛选高特异性的抑制剂。PTP1B与肥胖症、T2DM和肿瘤关系密切[7],因此它的大规模诱导表达对研究这些疾病有着重要的意义。本研究将PTP1B基因作为T2DM合并肥胖症的重要候选基因,结合豹皮樟总黄酮(TFLC)对T2DM大鼠模型的疗效及降糖机制研究,为TFLC应用于临床治疗肥胖合并2型糖尿病提供前期试验依据。主要研究内容包括以下四个方面:1.人PTP1B基因cDNA全长的克隆和原核系统表达取2型糖尿病人(BMI>28kg·m-2)外周抗凝静脉血,用淋巴细胞分离液分离淋巴细胞,提取总RNA,以Oligo dT为引物,合成出cDNA第一链。以逆转录(RT)产物为模板,PCR扩增出PTP1B插入片段,连接入克隆载体pMD-18T Vector中构建pMD-hPTP1B。设计带酶切位点(BamHⅠ,EcoRⅠ)的引物,以pMD-18T Vector作模板进行亚克隆,双酶切后构建原核表达载体pET28a(+)-hPTP1B,氯化钙法转化入BL21(DE3),IPTG诱导表达并对诱导剂的浓度、时间及诱导温度等条件进行了优化。从2型糖尿病患者外周血中扩增得到1301bp PTP1B cDNA全长序列,经双酶切鉴定及测序分析,表明已成功构建了原核表达载体pET28a(+)-hPTP1B;IPTG的最适诱导浓度为0.05 mmol·L-1,最适诱导时间为5 h,最适诱导温度为37℃;Western blot表明表达的重组融合蛋白具有与天然hPTP1B相同的结合抗体活性。2. rhPTP1B蛋白的分离纯化、酶活性测定以及抑制剂实验亲和层析法纯化rhPTP1B,复性后用于酶活性测定及抑制剂的实验。取细菌裂解上清液经Ni2+亲和层析柱纯化后,利用PTP1B水解特异性底物4-硝基苯基磷酸二钠盐(pNPP-Na2)的磷酸基团而产生颜色反应来测定rhPTP1B的活性,分析其与rhPTP1B浓度、底物浓度及反应温度的关系,确定钒酸钠(Na3VO4,VAN)的抑制类型和豹皮樟总黄酮(TFLC)对rhPTP1B的抑制作用和浓度依赖关系,并研究2型糖尿病常用治疗药物吡格列酮和二甲双胍对rhPTP1B有无抑制作用,探讨其有无新的降血糖机制。结果表明诱导表达纯化的rhPTP1B融合蛋白可用于抑制剂的研究:1.rhPTP1B活性随酶浓度及底物浓度的增加而增加;2. 35℃时rhPTP1B的活性最大;3.VAN的抑制作用呈浓度依赖性增强,可能为非竞争性抑制作用;4.TFLC对rhPTP1B有明显的抑制作用;5.治疗2型糖尿病的常用药物二甲双胍和吡格列酮对PTP1B酶并无明显抑制作用,说明这两种药物不是通过对PTP1B的抑制起作用的。3.人PTP1B基因的真核系统表达及在HepG2细胞的胰岛素信号通路中的作用构建真核表达载体pcDNA3.1-hPTP1B,电穿孔法转染至人肝癌HepG2细胞,G-418筛选2周后得到稳定转染的细胞株,检测rhPTP1B在转录水平和翻译水平的表达,免疫荧光法确定表达的rhPTP1B主要定位在细胞浆,MTT法初步研究rhPTP1B可能对细胞增殖和细胞周期无显著影响,免疫沉淀法结合Western blot检测其对IRS-1、IRβ的去磷酸化作用,结果表明rhPTP1B可显著减少IRS-1、IRβ的磷酸化程度,即明显衰减胰岛素刺激时胰岛素的信号转导通路;构建了真核沉默载体pBI-dsPTP1B,瞬时转染HepG2细胞,Western blot检测PTP1B的被抑制表达可达约50%。4.新型2型糖尿病大鼠模型的建立、TFLC的治疗及与PTP1B表达的关系用高糖高脂乳剂灌胃雄性SD大鼠5个月,小剂量STZ注射构建T2DM模型,分别给予TFLC (400mg/kg)和阳性对照药钒酸钠(VAN,10mg/kg)灌胃治疗6周,观察疗效。结果表明:糖尿病大鼠经TFLC治疗后空腹血糖(FBG)、糖化血红蛋白(HbA1c)、胰岛素(FINS)、游离脂肪酸(FFA)、总胆固醇(TC)、甘油三酯(TG)和低密度脂蛋白(LDL-C)、胰岛素抵抗指数(HOMA-IR)显著下降,高密度脂蛋白(HDL-C)和胰岛素敏感指数(ISI)明显提高,胰岛β细胞显著增生,肝匀浆丙二醛(MDA)含量下降,超氧化物歧化酶(SOD)活性升高;肾、心肌和肝脏的病理学明显改善。RT-Real time PCR、Western blot结果表明T2DM组靶组织中PTP1B的表达明显升高,TFLC组中PTP1B的表达明显降低。与VAN相比,TFLC可明显降低血糖和血脂,减轻肝脏组织的自由基氧化应激,其改善胰岛素抵抗,可能与其显著促进胰岛β细胞的增生,明显降低靶组织中PTP1B的表达,增强胰岛素信号通路有关。更多还原

【Abstract】 Protein tyrosine phosphatase 1B (PTP1B) belong to family of protein tyrosine phosphatases(PTPs), exists as two forms such as receptor-like and cytoplasmic signal transducing enzymes, dephosphorylate the phosphorylated tyrosine residues of substrate proteins and is the first identified and purified PTPs. PTP1B acted on the phosphorylated insulin receptor(IR), insulin receptors substrate-1 and -2(IRS-1, IRS-2), growth factor receptor binding protein 2(Grb-2) and phosphatidylinositol 3 kinase(PI-3K), which all related with insulin signal transduction, thus attenuated insulin signal transduction and resulted in post-receptor insulin resistance. Recently, someone imported siRNAs which targeted against PTP1B into mouse hepatoma and found that the insulin signal transduction was increased, thus proved the role of PTP1B in insulin signal transduction again.Overexpression of PTP1B could also cause leptin resistance, thus initiated the development of obesity and type 2 diabetes mellitus. Mice deficient with PTP1B exhibited increased insulin sensitivity as well as resistance to weight gain when fed with a high-fat diet, which resulted from an increased basal metabolic rate and total energy expenditure. They growed very well and had normal breeding capability and low cancer development ratio. All these results indicated that small molecular inhibitors of PTP1B would be potential effective anti-diabetic/anti-obese drugs and had no side effects. In fact, inhibitors against PTP1B had already become candicate drugs as one of insulin sensitizers; many laboratories in the world are screening inhibitors of PTP1B from Chinese traditional and herbal drugs or artificial systhesised drugs.PTP1B has close relationship with obesity, type 2 diabetes and tumors, so its large-scale induction expression is very important for studying these diseases. This research took PTP1B as an important candidate gene of T2DM, investigated the antidiabetic effects and possible mechanisms of TFLC on T2DM model rats. The main contents are divided into four sections as follows:1. Cloning and expression of human PTP1B cDNA in E.coliIn order to express the hPTP1B protein effectively in E.Coli, total RNA was isolated from the peripheral blood mononuclear cells of type 2 diabetes mellitus. PTP1B full length cDNA sequence was amplified using two-step RT-PCR. The cloning plasmid pMD-hPTP1B was constructed, then PTP1B was subcloned with primers containing restriction endonucleases recognition sites of BamHⅠand EcoRⅠand ligated into pET-28a(+), transducted into DE3, induced with IPTG and expressed proteins were detected with SDS-PAGE and Western blot. Induction conditions of high level expression were experimented. Human PTP1B was sequenced and the plasmid pET28a(+)-hPTP1B was cut with two enzymes. PTP1B was expressed successfully in the form of insoluble inclusion body in E.Coli, SDS-PAGE analysis showed the molecular weight was about 50 kD. Western blot result showed the highly expressed protein was really PTP1B. The best induction concentration of IPTG was 0.05 mmol·L-1, the best induction time was 5h and the best induction temperature was 37℃. The rhPTP1B and corresponding prokaryotic expression plasmids were obtained, thus established a basis for screening highly specific small molecular inhibitors.2. High expression of rhPTP1B and in vitro inhibition experiments of TFLCThe rhPTP1B was expressed effectively in E.coli DE3 transducted with pET28a(+)-hPTP1B, purified by affinity chromatography, then renatured, the activity was measured with different reactive conditions and inhibitors, such as Na3VO4 ,TFLC(a vulgar extract from traditional Chinese medicine called Laoying tea). The tests of enzyme activity and studies of the inhibitor were showed as following: 1. The activities of rhPTP1B increased with enzyme concentration and substrate concentration; 2. The optimal temperature was 35℃at which the enzyme activity was maximum; 3. Inhibitory manner of Na3VO4 was dependant on its concentration and possibly act as uncompetitive inhibitor. 4.TFLC had a definite inhibitory action on rhPTP1B, this maybe as one of mechanisms of its hypoglycemic effect. 5. Commonly used therapeutic drugs such as pioglitazone and metformin had no apparent inhibitory action on rhPTP1B, this result showed that the two drugs exhibited their hypoglycemic effect not through rhPTP1B.3. Expression and participation of human PTP1B in insulin signal transduction in human hepatoma HepG2 CellsHuman PTP1B full length cDNA was subcloned into pcDNA3.1/His-myc-B, then transfected into the HepG2 cells. After screening with 400mg·L-1G-418 for 2 weeks, a HepG2 cell line that stably expressed hPTP1B was obtained and testified by double enzyme cutting, RT-PCR and Western blot. The ectopically expressed human PTP1B cDNA was located in cytosol by immumofluorescence method, it had no influence on cellular proliferation by MTT assay. We also showed that expressed PTP1B affected the phosphorylation of known PTP1B targets such as insulin receptor substrate-1 (IRS-1) and insulin receptor (IR) after insulin stimulation, thus participated in insulin signal transduction pathway in HepG2 cells. We also constructed RNAi plasmid called pBI-hPTP1B and found out that inhibited expression of PTP1B extent could be reached about 50%. Maybe this RNAi plasmid was used to inhibit PTP1B expression in thearapy of type 2 diabetic rats in future. 4. Antidiabetic effect of total flavonoids from Litsea Coreana leve in high-fat emulsion/streptozotocin induced diabetic SD ratsA study was undertaken to evaluate the hypoglycemic activity and mechanism of TFLC on a new SD rat model of type 2 diabetes mellitus. Male Sprague–Dawley rats were fed high-fat emulsion via gavage for 22 weeks, then injected with 12 mg/kg streptozotocin (STZ) and followed by 0.5ml/kg CFA injection on next day to induce type 2 diabetes mellitus. The rat models were treated with TFLC (400 mg/kg) or Na3VO4 (10 mg/kg) via gavage for 6 weeks. HFE/STZ rats became obese, insulin resistant, exhibited increased levels of fasting blood glucose(FBG), glycated hemoglobin(HbA1c), fasting insulin(FINS), free fat acids (FFA), total cholesterol (TC), triglyceride (TG), malondialdehyde (MDA) and reduced superoxide dismutase (SOD) activities. Treatment with TFLC or Na3VO4 showed a significant decrease in FBG, HbA1c, FFA, TC, TG, MDA, HOMA-IR and a significant increase in ISI and SOD activities. Pancreatic immunohistochemistry indicated that TFLC or VAN could ameliorate auantic islets. Morphological changes in tissues of model rats included inflammation, mitochondrial lesions, steatosis, vaculous degeneration and hydropic degeneration in liver, heart and kidney while changes attenuated in two treatment groups. Expression of PTP1B were elevated in target tissues of model rats, but decreased in TFLC or Na3VO4 treatment rats. When used in a new T2DM rat model, TFLC could have anti-inflammation, anti-oxidation, insulin-sensitizing and hypoglycemic activity possibly by decreasing the elevated expression of PTP1B in the target tissues of T2DM rats.更多还原