【作者】 刘倩琦；

【导师】 陈荣华；

【作者基本信息】 南京医科大学 ， 儿科学， 2001， 博士

【摘要】 【研究目的】 观察重组leptin侧脑室注射后，营养性肥胖大鼠模型体重、摄食量、体内脂肪含量（腹膜后和生殖器周脂肪组织湿重）和血脂（TC、TG）的变化、腹膜后脂肪组织obese基因表达、血清leptin含量和血清胰岛素、C肽的变化、脑组织leptin受体（OB-R和OB-Rb）的表达、STAT3及其磷酸化蛋白表达和活性以及重组leptin干预后新生大鼠下丘脑神经元原代培养细胞STAT3蛋白及其磷酸化蛋白表达的变化，以探讨leptin对肥胖大鼠的减重、降脂作用及其对leptin、胰岛素抵抗的改善作用，以及leptin作用的中枢信号转导机制；同时给营养性肥胖大鼠侧脑室注射NPY Y5受体反义寡核苷酸，阻断NPY的生物学效应，观察其减重、降脂作用，从而进一步证实leptin-leptin受体（尤其是OB-Rb）-STAT3（磷酸化）-NPY（NPY Y5受体）-胰岛素通路在肥胖发生中的作用，为今后更加深入的研究肥胖症发生的中枢作用机制以及leptin、NPY Y5反义基因用于肥胖症的治疗提供有益的信息和手段。【方法】1．建立高营养饮食诱导的营养性肥胖大鼠模型：正常对照组（n=40）和 肥胖模型组（n=60），分别喂以普通饲料和高营养饲料，共7周；2．侧脑室插管：饲养7周后，每组再随机分成5组（即对照组、leptin 干预组、NPY Y5受体反义寡核苷酸干预组、错义干预组和生理盐 水组），除正常和肥胖对照组外，其余大鼠均行右侧脑室插管；3．重组leptin和NPY Y5受体反义寡核苷酸干预：恢复7天后，侧 南京医科大学博土学位论文 脑室分别注射重组 leptin＊ …听o生理盐水各印，每天 1次，连 续 5天；分别注射NPY YS受体反义、错义寡核苦酸＊ 小l）和生’理盐水各 10pl，每天 3次，连续 2天。每天第一次给药前称体重和 剩余饲料量：处死大鼠前，取其所有腹膜后脂肪组织及生殖器周脂 肪组织，并称湿重； 4．腹膜后脂肪组织中讪基因表达水平的检测：TRIZOI法抽提上述动 物模型腹膜后脂肪中的总RNA，应用RT—PCR技术检测各组大鼠 脂肪组织中。bese基因的表达水平，并分析毗基因表达水平与血清 lePtin含量、大鼠体重、腹膜后脂肪湿重的相关性； 5．血脂、血清胰岛素、C肽以及血清hptin含量的测定：处死前心脏 穿刺取血，分离血清，分别用酶法、放兔法和 ELISA双抗体夹心法 测定以上指标，分析血清leptin含量与胰岛素、C肽的相关性以及 血脂与大鼠体重的相关性； 6．大鼠脑组织OB－R和OB－Rb mRNA的表达；大鼠处死前，经升主 动脉 4％多聚甲醛灌注固定，制作石蜡切片、制备地高辛标记的 RNA 探针、应用原位杂交技术及APNBT书CIP免疫学检测观察leptin受 体（OB－R和 OB－Rb）mRNA的表达水平以及与肥胖发生的关系； 7．大鼠脑组织STAT3蛋白（胞浆）及其磷酸化蛋白（胞核）含量的测定： 分别用脑组织石蜡切片免疫组化和 Western blot 的方法进行检测， 并分析STAT3磷酸化程度与肥胖发生和减重的关系； 8．大鼠脑组织STAT3蛋白磷酸化活性检测：提取大鼠脑组织核蛋白， 应用EMSA的方法检测磷酸化STAT3蛋臼的活性，观察其活性与肥 胖发生和程度的关系； 9．新生大鼠下丘脑神经元的原代培养及leptin干预后磷酸化STAT3 蛋白的表达：取新生5～7天SD大鼠下丘脑细胞分散培养，观察下 丘脑神经元的形态和 NSE兔疫染色鉴定活性；培养 7天后，leptin口 ng加l培养液）干预24小时，免疫细胞化学染色检测下丘脑神经元 STAT3和磷酸化STAT3蛋白的表达。 2 南京医科大学博士学位论文【结果】1．模型制备前，两组大鼠体重无明显差异。喂以高营养饮食2周后， 肥胖组大鼠体重增长即明显大于正常组。7 周后，肥胖组体重和增 重与正常组之间均有显著性差异；2．注射重组 leptin和 NPY YS受体反义寡核苦酸的肥胖大鼠，体重和 进食量明显减少，与注射前比较差异有显著性；错义、生理盐水及 对照组体重和进食量未见明显下降。正常leptin干预组体重和摄食 量在给药2天后也有所下降，但效应较迟且无肥胖组明显。3．经lePtin和NPY YS受体反义寡核昔酸干预的肥胖大鼠腹膜后脂肪 湿重均较肥胖对照组及肥胖错义、生理盐水干预组显著减少；leptin 干预生殖器周脂肪亦出现明显减少，而 NPY YS受体反义寡核昔酸 干预组则下降不如 leptin干预组明显。4．肥胖 leptin更多还原

【Abstract】 Effects of recombinant leptin and NPY Y5 antisense oligonucleotide on the signal pathway after leptin receptor in the obesity and the centralmechanism of Ieptin administrationPh. D. CandidateLIU QianqiSupervisorCHEN RonghuaDepartment of Pediatrics, The Second Clinical Medical College,Nanjing Medical University, Nanjing, 210029, ChinaAbstractObjective:1.To study the effects of intracerebroventricular injection of leptin and neuropeptide Y Y5 receptor antisense oligonucleotide on (1) food intake and body weight and the weights of retroperitoneal adipose tissue and paragenital adipose tissue; (2) the expression of obese gene in retroperitoneal adipose tissue; (3) the levels of blood lipids (TC, TG), serum leptin and serum insulin, C-peptide;2.To observe the expression of OB-R and QB-Rb mRNA and STAT3 protein and phospho-STAT3 protein and the activity of phospho-STAT3 in the brains in dietinduced obese rats with leptin administration in vivo; and elucidate the cellular mechanism of the tyrosine phosphoiylation of STAT3 protein in the cultured hypothalamic neurons of newly born SD rats with leptin administration in vitro;3.To investigate the relation between the levels of serum leptin and insulin and C-peptide, between the levels of TC, TG and the body weight, and between the expression of QBRb and phospho-STAT3 and obesity so as to identil~?the effects of leptin-leptin receptor (OB-R and QB-Rb) -STAT3(phosphorylation)-NPY(Y5 receptor)-insulin pathway on the obesity;4.To study the mechanism of obesity at the level of central nerve system further and the future clinical therapy of recombinant leptin and NPY Y5 receptor antisense gene, so as to provide useful message and means of preventing and treating the obesity.Methods:1. PmparN of obese rat modeh: Weanling SPrague-Dawey male rats were edInlydlythed into two grOUs (the W grOuP and the Obese grOUP). Forty rats in thenormai grouPs wer gta a standar diet and stw in the Obese grouP wer fed wh ahighnUtrion diet for seven week5.2. Rgut wt venM annbo: arer seven weeks, rats in the nonnal andobese grouP were randoml divided into flve grouPs again, respectiv1y All rats excePthe nonnal and the obese contrOl grOuP wer twlanted wh stainlss steal cannula intoright lW ventriculax Unde soddri pentoarbhal anesthesin.3. ANtw: Rats were allowed to recover for aPPforly one week. They thenwer reCehed an intraCerebroventricular injection of 5Ug lgh in 5Ul salin or saljnealone at l0:00 per day fOr five consecutive day or 50pg neurpePtide Y Y5 recePtorantisenSe ollgOnUCleotid and ndStnWd OligOnUleotide in l0Ul salin or salinealon thee tiInes per day for two days. On each eXPerimen day the body weigh andfood intak wer measured before the first injection. Before sacrifice, the weights oftwtwal ampose tissue and paxagW adiPose hssue wer measured.4. Btoodpe (TC TG), serum forthe CT~ and pe measumeent At the timof sHe, b1Ood samPles were collected by cardiopUnCtUr direetly and cenWd at200for for 20 thes. Serum was stored at -20C. They wer detected by USin theHlysis, ndiolerassay and murin lePtin ELSA kit, reSPethely Mewver,the relationshiPs betWen the level of seruxn lePtjn and the concentalons of seruxnndin anct CTePticte were analped.5. tw of W and Mb M in the bthes: Before sacrifice, the brainswee mOUnted with 4% paIafOnndthede pchion and qulckly removed. Secton in acortical plane at l0Pm wer mounted on We-free slides and treated with 4%parafnnalW. NonedoaCtiv in situ hybriHon with DIGlabeled OB~R andOB-Rb W Prthe and trUe-color medical tw Syho wer used to eXwhd theeXPressiOn of OBH and OB-Rb InRNA in the brains from rats, Which wee7bostratd wh ltw.6. Nurement of STAT3 pwt andPhOwiMT3 Proteto in the brbo: STAT3and phoSPhO-STAT3 protein in the brains wer detected by edstOchdricalstaining and WeStem blotting tecboques, and analyZd by true-color medical hagusvstern.2 Hurement ofacdvty ofPosPhO8TAT3PrOtde in the brwh: The nuclear proteinfrOIn t更多还原