Effects Of Recombinant Leptin And NPY Y5 Antisenes Oligonucleotide On The Signal Pathway After Leptin Receptor In The Obesity And The Central Mechanism Of Leptin Administration

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...