igf-ⅱ及雷帕霉素的联合治疗在胰腺干细胞分化中的保护机制研究

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1、IGF-II及雷帕霉素的联合治疗在胰腺干细胞分化中的保护机制研究IGF-IIRestoresRapamycin-inducedSuppressionofβ-cellDifferentiationandExpansionofAdultPancreasStemCells作者姓名：范佳文领域（方向）：内科学指导教师：孙成林副教授学位类别：临床医学硕士论文答辩日期：2015年5月29日摘要目的：本课题研究雷帕霉素对猪新生胰腺细胞簇（NPCCs）分化和增殖的有害影响，探索能有效降低雷帕霉素毒副作用的治疗方法。方法：体外实验，雷帕霉素与IGF-II联合处理猪NPCCs，检测caspase-33ass

2、ay和H-thymidine摄取分析细胞凋亡和增生，RT-PCR及Westernblot分析胰岛细胞特定基因胰岛素、PDX-1、NeuroD/Beta2的表达。体内实验，猪NPCCs植入正常裸小鼠的肾脏被膜下，进行雷帕霉素及IGF-II联合处理，评估体外实验结果。结果：雷帕霉素治疗能够导致β-细胞数量、胰岛素含量、单层人工培养的猪NPCCs细胞的生存发育能力显著降低，但IGF-Ⅱ联合治疗可以预防雷帕霉素的毒副作用，抑制β-细胞数量及胰岛素含量的减少，保护NPCCs细胞的增殖能力预防凋亡，并且IGF-Ⅱ联合治疗可以抑制胰腺特异性基因胰岛素、PDX-1、NeuroD/Beta2表达的降低。结

3、论：IGF-Ⅱ联合雷帕霉素治疗，可以最大限度地降低雷帕霉素对猪NPCCs分化和增殖的有害影响。I关键词：猪胰新生细胞簇，雷帕霉素，IGF-II，β-细胞，干细胞IIAbstractBackground：Theroleofrapamycininpancreasstemcellsremainstobeclearlyelucidated.Herein,weevaluatedtheeffectsofrapamycinonporcineneonatalpancreascellclusters(NPCCs),whichprimarilycomprisedpancreaticprecursors,an

4、dattemptedtofindanintracellularmechanismabouttheharmfuleffectsofrapamycin.Methods：PorcineNPCCsweretreatedwithrapamycininamonolayer,andtheapoptosis3andproliferationweredeterminedviacapase-3assayandH-thymidineuptakeanalysis.Theexpressionoftranscriptionfactorswasassessedviareverse-transcriptasepolym

5、erasechainreactionandWesternblotting.Forthevivostudy,theporcineNPCCsweretransplantedintothekidneysubcapsulesofnormalnudemiceandtreatedwithrapamycin.Results:Rapamycintreatmentsignificantlyreducedthenumberofβcells,glucose-stimulatedinsulinsecretion,andtheinsulincontentsinthemonolayer-culturedporcin

6、eNPCCs.Furthermore,rapamycintreatmentincreasedtheapoptosisandinhibitedtheNeuroD/Beta2genesweredown-regulatedviarapamycintreatment.Theexpressionofinsulin-likegrowthfactor-IIwassignificantlydown-regulated,buttheexpressionofFoxo1wassimultaneouslyinverselyincreased,andthetranslocationofFoxo1fromthecy

7、toplasmtothenucleuswasinducedbyrapamycintreatment.Moreover,rapamycintreatmentinducedamarkedreductionintherelativevolumeandabsolutemassofβcellsintheporcineNPCCsgraftsat8weeksaftertransplantationinthenormalnudemice.Concl