The Molecular Mechanism Of Insulin Resistance In Intrauterine Growth Retardation Rats

[Objective]1. To investigate the expression of Insulin/Fox01/Pdx1/MafA genesduring pancreatic development in intrauterine growth retardation (IUGR) rats.2. Toinvestigate the expression of microRNA (miRNA) during pancreatic development inIUGR rats.[Methods]Part 1:(1)The establishment of rat model: female and male SD rats werecopulated by 1:3, then were divided into the following two groups: IUGR group andnormal group. The former were treated by 50% calorie compared with normal fromgestational day 1 until term. (2)The collection of samples: pancreas of rat embryonicday14.5(E14.5), 19.5(E19.5) and new born were dissected under microscoperespectively. (3)The detection of pancreatic morphological changes: HE staining andlight microscope were used. (4)The expression of Insulin/Fox01/Pdx1/MafA genes:after abstraction of total RNA, the technique of RT-PCR was used to study theexpression of insulin/Fox01/Pdx1/MafA genes.(5)The expression of Pdx1/MafAprotein : western blot was used to study the expression of Pdx1/MafA protein. Part 2:(1)The establishment of IUGR model: using the same way of part 1. (2)The collectionof samples: pancreas of rat embryonic day14.5, day19.5 and new born were dissectedunder microscope respectively.Then total RNA are harvested using TRIzol andRNeasy mini kit according to manufacturer's instructions. (3)The expression ofmiRNA: the samples are labeled using miRCURY. Hy3./Hy5. Power labelingkit and hybridized on the miRCURY. LNA Array(v.11.0).(4)The confirmation of theresults: the real-time PCR was used to confirm the miRNA array results.[Results]Part 1:(1)Compared with normal, the pancreatic construction, islet area and quantities had abnormal morphological changes during pancreatic development.(2)The gene of Insulin/Fox01/Pdx1/MafA began to be expressed from E14.5, and theexpressions of Insulin and MafA were increased as the fetus grew while Fox01 andPdx1 were decreased. (3)Compared with the normal, the expressions of Fox01 andMafA were lower than normal rats(P<0.05) while Insulin and Pdx1 had no evidentchange. (4) Compared with the normal, the expressions of MafA protein were lower(P<0.05) while Pdx1 protein had no evident change in IUGR group. Part 2:(1) In thenormal model, comparing with embryonic day14.5, 48 miRNAs of embryonicday19.5 were up-regulated while 28 were down-regulated; 64 miRNAs of new bornwere up-regulated while 55 were down-regulated.Comparing with embryonic day19.5,40 miRNAs of new born were up-regulated while 52 were down-regulated.Comparing with normal, in the IUGR model, 10 were up-regulated while 26 weredown-regulated in embryonic day14.5, 22 were up-regulated while 15 weredown-regulated in embryonic day19.5, 9 were up-regulated while 13 weredown-regulated in new born. (2) miR-124,let-7d* were be chosen to confirm: innormal group, miR-124 of the new born was increased comparing with embryonicday14.5 and embryonic day14.5 (FoldChange>=1.5), and let-7d* was increased as thefetus grew (FoldChange>=1.5); in IUGR group, miR-124 had no evident changecomparing with normal (0.67