【作者】 闫志风；

【导师】 李亚里；

【作者基本信息】 中国人民解放军军医进修学院 ， 妇产科学， 2008， 博士

【摘要】 胰岛移植或β细胞替代足糖尿病最理想的治疗手段,但细胞来源严重匮乏及免疫排斥反应限制了临床的广泛应用。将人胚胎干细胞(human embryonicstem cell,hESCs)诱导为胰岛素分泌细胞,成为β细胞替代物的新资源,为解决细胞来源匮乏带来了希望。已有多种方案由ESCs获得胰岛素分泌细胞,但均存在各种限制,目前仍缺乏将ESCs诱导分化为有功能的胰岛素分泌细胞的最佳方案。探索诱导人hSCs分化为成熟且有功能的胰岛素分泌细胞,并制定出有效而高效的分化方案,成为本课题的主要研究目的。第一,本研究摸索了在体外培养、扩增、冻存、复苏hESCs细胞株(PKU1)的条件,并对其特异性标志物进行了检测。结果显示,hESCs经多次传代后仍然表达OCT-4和Nanog基因,免疫荧光法显示呈OCT-4、SSEA-4阳性,流式细胞术分析显示,OCT-4、SSEA-4阳性细胞比例分别达89.38%、83.44%;染色体核型分析表明为正常女性染色体核型(46,XX)。第二,采用酶消化法分离出胎龄14-18W的胎儿胰岛,经悬浮培养、双硫腙(DTZ)染色后种植到组织培养皿中,再以免疫荧光法检测一些细胞标志物的表达。结果显示,悬浮培养条件下,胎龄14-18W的胎儿胰岛细胞聚集成球形细胞簇,呈DTZ阳性,培养液中能检测到一定量的胰岛素和C-肽,贴壁培养后还能检测到胰岛细胞标志物insulin和glucagons及前体细胞标志物PDX-1、CK-19和Nestin的表达,证实14-17W胎儿胰岛已具备一定的胰岛特性和前体细胞的特征。第三,模拟胰腺发育分化的生理过程,研究设计出五阶段诱导方案,具体如下:(1).撤除分化抑制因素使hESCs形成拟胚体(EBs),悬浮培养48h;(2).将EBs转移至明胶包被的组织培养皿后,加入含Activin A和bFGF及LY294002的诱导培养基Ⅰ诱导5天,向定形内胚层诱导分化。RT-PCR法鉴定SOX17和FOXA2的基因表达,与同期自发分化的EBs进行比较,免疫荧光法检测SOX17的表达。(3).将细胞转移至laminin包被的培养皿,加入含bFGF、KGF、EGF、GLP-1、尼克酰胺的低血清诱导培养基Ⅱ作用14d,向胰腺前体细胞诱导。RT-PCR法和免疫荧光化学法分别检测前体细胞标志物PDX-1及Ngn3表达。(4).添加含IGF-1、betacellulin、尼克酰胺、GLP-1、laminin的低血清诱导培养基Ⅲ作用14d,促进胰岛细胞分化,RT-PCR法检测细胞胰岛素基因的表达,免疫荧光化学法检测分化细胞胰岛素和C-肽的表达。(5).将细胞转移至低黏附性培养皿中,撤除IGF-I继续培养3天,促进细胞成熟。应用双硫腙(DTZ)染色液对获得的细胞团进行染色,应用RT-PCR法检测了胰岛素和胰高血糖素基因表达,应用RIA法分别测定了获得的细胞团培养液上清和胞浆中的胰岛素、C-肽含量,并与胎儿胰岛培液上清进行比较,静态培养胰岛素释放试验测定胰岛素与C-肽分泌是否呈葡萄糖反应性。结果(1).hESCs悬浮培养2d后,形成类球形EBs;(2).经第二阶段诱导后,细胞表达SOX17和FOXA2基因,且表达量高于同期自发分化的EBs,基本不表达外胚层标志物SOX1,随诱导时间延长,FOXA2表达逐渐增强;免疫荧光化学法显示,诱导5天的细胞胞核呈SOX17。(3).经第三阶段的诱导,细胞呈上皮样集落,RT-PCR法检测到细胞表达PDX-1、Ngn3及微弱的Pax4基因,免疫荧光法显示细胞呈PDX-1阳性。(4)经第四阶段的诱导,集落中一部分细胞聚集成多层,RT-PCR显示,不同诱导时期均有胰岛素基因的表达,且随诱导时间延长表达水平逐渐升高;免疫荧光显示,部分细胞呈胰岛素和C-肽阳性。(5).经第五阶段诱导后,hESCs来源的细胞呈类似胎儿胰岛的胰岛样细胞簇(ICCs),呈DTZ阳性,RT-PCR检测到Insulin和Glucagon基因表达,RIA法检测到ICCs培液上清中含胰岛素与C-肽,水平与15-17W胎儿胰岛培液上清相近(P＞0.05),胞浆内的胰岛素和C-肽水平分别为1089.2±217.06μIU/ml和181.33±30.12ng/ml,静态培养胰岛素释放试验验证了ICCs分泌的胰岛素呈糖反应性。应用五阶段分化方案,hESCs能够有效的分化为具有部分β细胞特性和糖反应性胰岛素分泌功能的细胞,为DM细胞替代治疗提供了有潜力的细胞来源和良好的临床应用前景。更多还原

【Abstract】 Islet transplantation orβcells replacement therapy are taken as a promising approach for diabetes treatment,but widespread application of this treatment is restricted by absence of islet source and immunological rejection.Progresses made in human embryonic stem cells(hESCs)field brought hope for settling the shortage of islet source.Many reports have obtained insulin-producing cells from ESCs using different strategies,however,these all have various limitations and there is lack of an optimal protocol producing functionalβcells from ESCs.To explore a high-performance differentiation protocol that can obtaine mature and functional insulin-producing cells from hESCs is the major problem that will be settled in this study.At first,this study explored the condition to culture,amplificate,freeze and reanimate the hESCs line PKUl and the expression of several specific markers of hESCs were detected.Results showed,after high passage,hESCs expressed OCT-4 and Nanog gene,remained positive for OCT-4 and SSEA-4,and the percentage of OCT-4- and SSEA-4-positive cells was respectively 89.38%and 83.44%.Chromatosome karyotype analysis showed the 114 generation hESCs karyotype is normal female(46,XX).Thus,the study successfully established the hESCs culture system that could maintain their stem cell features.In order to realize the morphology and character of islets during embryo development and provide reference for identification of the insulin-producing cells derived from hESCs,the fetal islets gestation age between 14-18 weeks were isolated by enzymatic digestion and cultured in suspension for 3 days.After DTZ staining,fetal islets were changed to tissue culture dish,then some islet markers and precursor cells markers were identified by immunofluorescence.The insulin and C-peptide content in culture supernatant was measured by radioimmunoassay (RIA).Results showed,fetal islets were spherical clusters and were positive for DTZ,insulin and C-peptide were detected in the culture supernatant,and some of the cells in the epithelioid cell colonies were positive for insulin and glucagon, and several precursor cell markers such as PDX-1、CK19 and Nestin.From this, fetal islets of gestation age between 14-17W have some features of islet and some precursor cells.This study designed a proposal of five stages minic embryo pancreas development.Firstly,withdraw the inhibitor of differentiation and hESCs formed embryonic bodies(EBs),after 2 days the EBs were planted to 0.1%gelatin-coated culture dishes and cultured in DF12 medium containing serum replacement(SR) supplemented with bFGF,Activin A and LY294002 for 5 days(stage 2),then the cells were dissociated with trypsin and planted to lminin-coated culture dishes in DF12 medium containing low fetal calf serum(FBS)supplemented with bFGF, KGF,EGF,GLP-1 and nicotinamide(stage 3).After 14 days,bFGF,EGF and KGF were withdrew and replaced by IGF-1 and BTC and cultured for another 14 days(stage 4).The cells were dissociated with collagenaseⅣand transferred into low-attachment plates and cultured in RPMI-1640 medium supplemented with GLP-1 and nicotinamide for 3-5 days(stage 5).Ihe induction effects were identified by RT-PCR,immunofluorescence,DTZ staining and RIA.The indexes markers included definitive endoderm(DE)markers such as SOX17 and FOXA2, pancreatic markers PDX-1 and Ngn3 and islet markers insulin,glucagons and C-peptide,and the insulin and C-peptide content in the last stage culture supernatant and glucose-stimulated insulin and C-peptide release analysis were carried out by RIA.The results showed,the cells induced in the stage 2 for 5d expressed SOX17 and FOXA2 genes,their expression levels were higher than that of the same days EBs,and the FOXA2 level increased with the induction time.The immunofluorescence displayed the cells were positive for SOX17 in nuclei.After stage 3,the cells were positive for PDX-1 and expressed pancreatic precursor marker PDX-1 and islet precursor marker Nng3 gene,even a very low expression of Pax4.After stage 4,the induced cells expressed Insulin gene and expression level raised with induction days,part of them were positive for insulin and C-peptide.At last stage,the hESC- derived cell clusters were spherical just like fetal islets,and were positive for DTZ.RT-PCR showed,the cells expressed Insulin and Glucagon gene.By RIA,insulin and C-peptide content in culture supernatant were similar with that were detected in fetal islets culture supernatant. Intracellular insulin and C-peptide content was 1089.2±217.06μIU/ml and 181.33±30.12ng/ml,and insulin release from the ICCs was regulated by glucose.Thus,results presented in this study provide credence that the five stages protocol differentiated hESCs into mature insulin-producing cells and can offers a promising source of cells for diabetes cell transplantation therapy.更多还原