胰腺干细胞移植治疗1型糖尿病的动物实验分析

山东人学博I：学位论文符号阐明缩略语英文名称中文名称13-actinbeta-actin内参照基因bpbase pair碱基对bFGFbasic fibroblast growth factor碱性成纤维细胞生长因子BrdU5-bromodeoxyuridine5溴脱氧尿苷CK19Cytokeratin-1 9细胞角蛋白19ConAConcanavalin A伴刀豆球蛋白aDNADeoxyribonucleic acid脱氧核糖核酸DEPCDiethylpyrocarbonate焦碳酸二乙酯D”Dithiothreitol二硫苏糖醇DABDiaminobenzidine二氨基联苯胺DMSODimathyl sulfoxide二甲基亚砜DTZDithizon二苯基硫卡巴腙；双硫腙ECMExtracellular matrix细胞外基质EGFEpidermal growth factor表皮细胞生长因子FITCFluorescein isothiocyanate异硫氰酸荧光素FBSFetal bovine serum胎牛血清GluGlucose葡萄糖GITGuanidinium isothiocyanate异硫氰酸胍 HEPESHydroxyethyl piperazine ethanesulfonic acid 羟乙基哌嗪乙磺酸 HGFHepatocyte growth factor肝细胞生长因子IGFInsulinlike growth factor胰岛素样生长因子IgImmunoglobulin免疫球蛋白KGFKinetics gain factor(细胞)动力增益因子kbKilobases千碱基LBLuria-bertani mediumLB培养基MMIMoloney murine leukemia virus reverse逆转录酶trascriptase10山东大学博士学位论文mRNAmessenger Ribonucleic Acid信使核糖核酸Ngn3Neurogenin 3神经原素3PCRPolymerase chain reaction聚合酶链反映PBSPhosphate buffer solution磷酸盐缓冲液PDX1Pancreaticduodenal homeobox1胰十二指肠同源异型盒基因RNaseRibonuclease核糖核酸酶RT-PCRReverse transcription-polymerase chain逆转录聚合酶链反映reactionRPMll640Roosevelt Park Memorial Institute mediumRPMll640培养基rpmRevolution per minute每分钟转数S-PStreptaviclin-peroxidase链霉素生物素一过氧化物酶SPSSStatistical package for social science社会科学记录程序包STZStreptozotocin链脲佐菌素确SIris(hydroxymethyl)aminomethane三(羟甲基)氨基甲烷 TRITCTetramethyl rhodamine isothiocyanate四甲基若丹明异硫氰酸盐 TaqThermos aquaticus水生栖热酶原创性声明本人郑重声明：所呈交的学位论文，是本人在导师的指引下， 独立进行研究所获得的成果。除文中已经注明引用的内容外，本 论文不涉及任何其她个人或集体已经刊登或撰写过的科研成果。 对本文的研究作出重要奉献的个人和集体，均已在文中以明确方 式标明。本声明的法律责任由本人承当。论文作者签名： 敛El期：避王：望有关学位论文使用授权的声明本人完全理解山东大学有关保存、使用学位论文的规定，同 意学校保存或向国家有关部门或机构送交论文的复印件和电子 版，容许论文被查阅和借阅；本人授权山东大学可以将本学位论 文的所有或部分内容编入有关数据库进行检索，可以采用影印、 缩印或其她复制手段保存论文和汇编本学位论文。(保密论文在解密后应遵守此规定)论文作者签名：I蚁导师签名玎镪三幺么日山东人学博士学位论文胰腺千细胞移植治疗1型糖尿病的动物实验研究 中文摘要研究背景 糖尿病是影响人类健康的重要疾病之一。其治疗措施涉及老式药物、胰岛素注射以及胰腺器官移植和胰岛移植等。胰腺器官移植技术复杂，围手术期并发症 发生率较高，手术适应证仍局限于糖尿病合并肾功能衰竭需同步行胰肾联合移植 者，目前仅在世界上比较大的移植中心开展。以胰岛移植为代表的细胞替代治疗 具有手术易操作，创伤小的长处；并且与胰岛素注射治疗相比，胰岛移植物对机 体血糖可实时监控，并及时释放胰岛素，维持机体的血糖稳定状态，避免糖尿病 肾病、视网膜病变等并发症的发生。1999年埃德蒙顿(Edmonton)方案的实行 使胰岛移植的疗效得到了明显提高，标志着糖尿病治疗进入了现代胰岛移植的全 新阶段。但胰岛移植同样面临着供体来源受限的问题，加上移植围手术期技术和 环境变化等因素导致的胰岛细胞大量死亡和功能损害，最后限制了胰岛移植在临 床更大范畴的应用。故扩大胰岛细胞来源成为近年胰岛移植研究领域的热点。有 学者曾尝试采用异种胰岛移植(如猪)解决胰岛供体局限性的矛盾，并且觉得有也许 避免1型糖尿病的自身免疫性袭击，但由于存在猪源性逆转录病毒感染的潜在危 险，目前多数科学家觉得只有在掌握人兽共患病机制之后，此项技术才干开展。 为解决胰岛移植供体短缺的难题，近年来干细胞成为研究的热点之一。干细 胞最明显的生物学特性是既具有自我更新、高度增殖的能力，又具有多向分化的 潜能。按其来源可将它们分为胚胎干细胞(Embryonic stem cells，ESCs)和成体干细胞(Adult sommic stem cells，ASSCs)。胰腺干细胞(Pancreatic stem cell，PSCs)属于ASSCs，目前还没有明确的定义。一般是指未达终未分化状态，能产 生胰岛组织或来源于胰岛，具有自我更新复制能力的未定型细胞。PSCs由于与 胰腺内分泌细胞属于同一细胞谱系，分化成目的细胞的生物学环节最短，被觉得 是最有但愿作为临床胰岛移植的干细胞来源之一。其他长处涉及：(1)具有高度 增殖和多向分化潜能，能极大解决胰岛细胞来源局限性的难题；(2)可通过胰腺活山东人学博：学位论文检获得自身的PSCs、干细胞建库、基因工程改造或修饰同种异体PSCs的措施避 免或减少免疫排斥反映的发生；(3)避免了ESCs弓起的无限增值而导致的肿瘤 生长；(4)避免了应用ESCs带来的社会伦理问题。目前已有较多的研究对PSCs的细胞来源、培养、体外诱导分化、鉴定等进 行了探讨。由于PSCs的研究尚处在起步阶段，多种措施差别较大，还没有一套 公认的的培养、诱导分化及鉴定方案。此外，由于体外条件下无法模拟促使干细 胞发育成熟的体内环境，致使大多数研究不能获得真正成熟的胰岛细胞，不可以 完全逆转动物的高血糖状态。因此，我们设计本实验，确立一种简朴有效的培养、 鉴定PSCs的方案；鉴于体内微环境有增进PSCs发育成熟的刺激信号、营养物 质和因子，我们也试图寻找促使PSCs发育成熟的最佳体内环境，也就是使PSCs 发挥最佳的治疗糖尿病的效果。目的 (1)摸索从新生Wistar大鼠胰腺组织中分离、培养胰腺干细胞并进行鉴定以及 体外诱导分化的措施。 (2)探讨并比较不同分化状态的胰腺干细胞(未分化细胞和经体外诱导方案诱 导分化的细胞)经两种移植途径(分别经门静脉和肾包膜下移植)对糖尿病大鼠 的治疗效果。措施 (1)取新生Wistar大鼠的胰腺组织，经胶原酶消化成组织碎片，转入具有 RPMll640高糖培养基(含333mM的Glu，并添加了10FBS、20ngml EGF， 20ngml bFGF)的培养瓶(预先用鼠尾胶原解决)中进行原代和传代培养；使用 无血清RPMll640低糖培养基(含111mM的Glu，撤去EGF、bFGF和FBS， 另添加lOmM尼克酰胺，715uM p一疏基乙醇)对培养细胞诱导分化。以扫描与透射电镜观测细胞的超微构造形态；免疫荧光法检测胰岛素、胰高血糖素、PDX一1、CK-19以及Ngn3等抗原的体现；双硫腙染色检测干细胞分化前后D细 胞比率；放免法测定细胞胰岛素分泌水平；PCR技术检测PDX-1 mRNA、CK19 mRNA、Ngn3 mRNA和胰岛素mRNA的体现状况。 (2)取Wistar大鼠50只，经尾静脉一次性注射链脲佐菌素(60 mgkg body weight)，随机血糖167mM并持续稳定1周为造模成功。然后将成模大鼠随机2山东人学博十学位论文分为5组：A组(n=5，对照组，经门静脉注射生理盐水)、B组(n=10，经门静 脉移植未分化PSCs)、C组(n=10，经门静脉移植已分化细胞)、D组(n-10， 肾包膜下移植未分化PSCs)和E组(肾包膜下移植已分化细胞)。移植术后监测 大鼠的随机血糖、体重和胰岛素的变化；移植后13天和28天行经腹腔注射的葡 萄糖耐量实验；术后14天和30天分两批处死所有大鼠，取A、B、C三组大鼠 的肝脏和D、E两组大鼠的肾脏做病理切片和免疫组织荧光染色，测量体内“类 胰岛构造”的直径、观测其形态以及胰岛素体现的状况。成果 (1)胶原酶消化法联合使用高糖培养基可有效分离得到纯化的胰腺导管上皮细 胞。培养细胞具有高度增值能力，电镜下呈未分化细胞特性，同步体现PDX1 和CK-19，随着传代次数增长，部分细胞体现Ngn3；但该种细胞不体现胰岛素 和胰高血糖素，双硫腙染色和胰岛素分泌实验未证明有胰岛素阳性细胞。分化培 养后，部分细胞可体现胰岛素和胰高血糖素，双硫腙染色阳性细胞比率为25+6， 胰岛素反映能力提高，对10-30mM浓度的糖浓度刺激具有反映。 (2)50只Wistar大鼠48只造模成功，成模率为96。细胞移植术后第6天至 第18天，B组糖尿病大鼠血糖与对照组相比差别有明显性意义，术后14天时血 糖曾降至正常范畴；C、E两组血糖变化趋势基本相似，与对照组两两相比，第 4天时血糖浓度差别有明显性意义，但持续时间较短，且整个过程中未见血糖恢 复至正常范畴。D组与对照组相比，直到实验结束时两组血糖差别无明显性意义。 经腹膜腔注射的葡萄糖耐量实验显示B组细胞移植物移植后13天具有较强的抗 高血糖效应。形态学观测发现未分化胰腺干细胞可以在大鼠肝脏内(B组)诱导 分化为较大的“类胰岛样构造”，直径多在150-200肛m左右，胰岛素染色阳性 细胞较密集，而C、E两组以直径50 Ixm左右较小的“类胰岛样构造”居多，胰岛 素染色阳性细胞较稀疏。免疫组织荧光染色显示B组类胰岛样构造中有较多的 BrdU阳性细胞。结论 (1)本研究运用胶原酶消化法得到的细胞具有高度增值能力和分化成胰岛细胞 的潜能，经多种实验技术措施证明是来源于导管上皮的胰腺干细胞。低糖培养基 的条件下，有助于胰腺干细胞的分化成熟。山东人学博1：学位论文(2)通过体外条件诱导分化的胰腺细胞移植入肝脏内和肾包膜下能进一步分化 成熟，以较小的“类胰岛样构造”为主；而未分化的胰腺干细胞在肝内微环境和高 血糖刺激下可以诱导分化为更为成熟的较大的“类胰岛样构造”，并且能短暂的完 全逆转糖尿病大鼠的高血糖。胰腺干细胞定向分化为内、外分泌细胞的潜能有助 于内分泌细胞的进一步成熟。核心词：胰腺干细胞；细胞培养；糖尿病模型；干细胞移植4山东人学博上学位论文THE ANIM认L EMPIRICAL STUDY 0F PANCREATIC STEMPROGENIToR CELLS TRANSPLANTATION FOR THE TREATMENT oF TYPE 1 DIABETES MELLITUSABSTRACTBackgroundDiabetes mellitus is one of the principal diseases affecting mankindThe therapies include the traditional drugs，injection of insulin，pancreatic organ transplantation and islets transplantationThe pancreas transplantation is a complicated technique， perioperative complications incidence is hi曲，and the operation indication is still limited to the patients suffered from diabetes in combination with renal failure who need to receive combined pancreas and kidney transplantation，which is currentlyperformed in the large center of transplantion across the worldThe cells substitutiontherapy represented by islets cells transplantation has the advantages of simple operation and minimal invasionCompared to the injection of insulin，islets grafts Can momitor the blood glucose timely,and release the insulin in time，which keep the body glucostasis and prevent diabetic nephropathy and retinopathy from happening In 1 999，the applications ofEnmonton Protocolsignificantly increase the therapeutic effect of islets tranplantaion，which is the milestone of modern islets tansplantionHowever islets transplantation confronts the problem of limited donors， plus the mass mortality and functional lesion of islets damaged by perioperative technique and change of circumstance，which finally limited the wider application of islets transplantationSo it becomes the hot topic recently in the field of islets transplation to enlarge the sources of islets cellsSome authors tried to adopt heterogeneity islets transplantation(1ike pigs)to resolve the insufficiency of islets donors，and they also believed this strategy may avoid the autoimmune attack of type1 diabetes mellitusHowever most scientistes consider this technique Call bedeveloped only human understand the mechanism of amphixenosis because it has the5山东人学博1j学位论文 potential dangers of re仃o virus infectionTo resolve the tough problem of islets donorsshortage，the research of stemcells are becoming the hot topic recentlyThe most obvious biological characteristics of stem cells arc to possess the self-renewal and proliferative ability,and also the potency of multi-directinal differentiationStem cells can be divided into embryonic stem cells and adult somatic stem cells according to the sourcePancreatic stem cells belong to adult somatic stem cells and dont have the exact definition yetIt generallyrefers to the indeterminate cells before terminal differentiation that Can generate islets tissue or origin from islets and possess self-renewalPancreatic stem cells belong to the same cell lineage with pancreatic endocrine cells，SO the process of differentiatinginto target cells is the shortest，which get pancreatic stem cells the promising sourcoof islets transplantionOther advantages include(1)high proliferation and potency of multidirectional differentiation，which Can completely resolve the problem of islets shortage，(2)avoidance of immunological rejection via pancreatic biopsy to gainautologous pancreatic stem cells，establishment of stem cells treasury,and geneticengineering to reform or modify homologous pancreatic stem cells，(3)avoidance of tumor resulted from embryonic stem cells proliferation，and(4)avoidance of social ethical issues brought by using embryonic stem cellsAt present many authors have explored the cells source，cells culture，in vitro induction and differentiation， identification of pancreatic stem cells Since the research of pancreatic stem cells is still in the early phase，there are lots of differentmethods and viewpoints，that is，no accepted strategy of culture，induction and idectification existIn addition，since in vitro conditions Cant mimic in vivo microenviroment that can make stem cells differentiated，the really mature islets cellsCant be gotten in vitro and Cant completely reverse the hyperglycemiaTherefore，we design this study in order to set up a simple and effective culture and identified protoc01Due to spikes， nutrient substance and factors exsiting in vivo microenviroment,we try to look for the best niche for pancreatic stem cells todifferentiate，that is，a niche Can make pancreatic stem cells to play an optimal role inthe treatment of diabetes6山东大学博一l：学位论文Objective(1)To explore the methods of isolation,culture,identification and in vitro differentiation of pancreatic stem cells from newborn Wistar rats(2)To explore the therapeutic efficacy of pancreatic stem cells with differentdifferentiated status(such觞undifferentiated stem cells and differentiated cells under the in vitro induction conditions)transplantation by two pathways，that is，portal vein and beneath renal capsule for the type 1 diabetes mellitusMethods(1)The pancreatic tissue WaS removed form neonatal Wistar rats，digested into fragments by collagenase，then transferred into the culture flasks pretreated with collagen that contained RPMI 1 640 high glucose media(added 333mM Glu,1 0 FBS，20ngml EGF,and 20ngml bFGF)，where pancreatic tissue Was parimafilycultured and serially subculturedThe stem cells were induced and differentiated、衍mRPMI 1 640 low glucose media without serum(reduced concentration of Glu to 1 11 mM，removed EGE bFGF and FBS；and then added 10mM niacinamide and 7151tM 13-mercaptoethan01)Ultrastructure of these cells WaS observed by scanning andtransmission microscope electronThe expression of the antigens such as insulin,glucagon，PDX-1，CK-19 and Ngn3 Was detected with immunofluorenscenceThe ratio of p cells was detected with DTZ stainingThe insulin secreted by cells was determined with RIPCR Was performed to detecte the expression of PDX-1 mRNA， CK-1 9 mRNA，Ngn3 mRNA and insulin mRNA(2)50 Wistar rats were injected through caudal vein with streptozotocinThe successful diabetes models Can be concluded when random blood glucose was continuously more than 1 67 mM during one weekThen all rats were randomly divided into five groups：A group(n=5，嬲control，PBS WaS injected via portal vein)， B group(n=1 0，undifferentiated pancreatic stem cells were transplanted via portal vein)，C group(n=1 0，differentiated cells were transplanted via portal vein)，D group (n=l O，undifferentiated cells were transplanted beneath renal capsule)and E group (n=1 0，differentiated cells were transplanted beneath renal capsule)The random blood glucose，weight，plasma insulin and introperitoneal glucose tolerance test was7山东大学博l：学位论文detected after transplantationAll the rats were sacrificed respectively on 1 4 and 30 days postoperativelyThe liver specimen of A，B and C group，and the kidney specimen of D and E group were removed to make pathological sections andimmunohistofluorescence,measure the diameter of islet-like stmc眦s，and observemorphous and insulin expression Results(1)Collagcrmse digestion in combination with high glucose media Can effectively isolate and get p谢fied pancreatic ductal epithelial cellsThe cultured cells had the ability of high self-duplication,which show the characteristics of undifferentiated cells and expressed PDX一1 and CK一19 simultaneously、im the passage increased some of these cells expressed Ngn3，but didnt express the antigens，like insulin and glucagonBoth DTZ staining and insulin secretion tests didnt show insulingenerated cellsOnec the cells differentiated，some cells Can express insulin and glucagon,and ratio of DTZpositive cells increased to 25+6，which had an increased ability of insulin respondence to glucose with a range of 1 030 mM(2)48 out of 50 Wistar rats Were successfully made to diabetes models and the successful rate Was 96Between 6 and 1 8 days after transplantation，the randomblood glucose of B group has significant difference compared to that of control group， which once reduced to the normal rangeC and E groupS blood glucose had the similar tendencyCompared respectively to the control group，the difference of blood glucose on 4th day after transplantation was significant，but the time duration lasted too shortDuring the experiment，the blood glucose in both groups didnt reduce to the normal rangeCompared to the control group，the blood glucose of D group didnt have statistical significance during the processConsidering the introperitonealglucose tolerance test，the grafts in B group have better antihyperglycemic effect 1 3day after transplantationThe undifferentiated pancreatic stem cells can be induced into the larger isletlike structurcs in liver,the diameters of which ranged from 1 50-200 pm or SoThe intensity of insulin-generated cells WaS strongIn botll C and E group，small islet-like structures were usually 50 pm in diameter,and the intensity WaS weakImmunohistofluorescence examination showed BrdU positive cells of BR山东大学博学位论文group that surrounded the islet-like structures were more than those of C and E group Conclusions(1)In this study,collagenase digestion was used to get the cells with ability tO self-duplicate and potency to differentiate into insulingenerated cells，which Can be proved to originate from ductal epithelium through many different kinds of experimental techniquesThe condition of low glucose media contributes to the maturity of pancreatic stem cells(2)The differentiated pancreatic cells under in vitro induced condition Can further become mature and differrentiate many small islet-like structuresWhile in the circumstances of in vivo，ie，the liver,undifferentiated pancreatic stem cells Candifferentiate into more fullgrown large islet-like structures，which call transientlytotally rev6Tse the high blood glucose of Wistar ratsThe potency of pancreatic stemcells to differentiate into endocrine and excrocrine cells might contribute to the further maturity of insulin-generated cellsKey Words：Pancreatic stem cells,Cell culture,Diabetic animal models,Stem cellstransplantation9山东人学博上学位论文第一部分新生Wistar大鼠胰腺干细胞的分离、培养及定向诱导 分化的研究刖舌近年来，干细胞成为糖尿病研究领域的热点之一。其中，胰腺干细胞 (Pancreatic stem cells，PSCs)由于具有其他干细胞无法比拟的优势，成为最有希 望替代临床胰岛移植的干细胞之一。由STZ诱导的糖尿病大鼠模型【11、胰腺导管 结扎【21以及胰腺部分切除等【3】的动物模型中发现的胰岛新生(Islet neogenesis)， 提供了PSCs存在的客观证据。目前较多数的观点觉得成体胰腺导管上皮细胞可作为PSCs的来源。支持此 观点的证据涉及胚胎发生中胰岛的生长与导管上皮具有密切的有关性【4】，胰腺导 管内有散在的内分泌细胞【51，新生胰岛从小导管“发芽”长出等【61。近年来更是有 学者直接从胰腺导管上皮组织中培养出了PSCs，如Bonner-weir等【7】用基质胶培养 人类成体胰腺导管组织后发既有“类胰岛样芽”(Islet1ike buds)形成，这些“类胰 岛样芽”由CK19(导管上皮细胞的标志物)阳性的导管上皮细胞和胰岛素阳性细胞所构成。Ramiya等【8】消化NOD成年小鼠胰腺组织获得单一胰腺导管上皮细 胞，这些细胞经无糖、高氨基酸培养液培养形成上皮样PSCs单层，诱导后形成 小而圆的胰岛祖细胞(Islet progenitor cells，IPCs)；继续培养，这些小细胞迅速 扩增形成胰岛性细胞集落(IPCderived islets)。体内移植实验证明运用体外培养得 到的类胰岛样细胞可以逆转糖尿病模型鼠的病变，进一步验证了PSCs的功能。持续予以大鼠胰岛细胞营养因子如胰高血糖素样多肽1(Glucagon1ike peptide。1，GLP1)或葡萄糖48d时可见胰岛数量成倍增长，这表白PSCs也存在 于胰岛自身。Zulewski等【9】证明可以自纯化的胰岛内分离培养出Nestinl；fl性细 胞，最lJNestin阳性的胰岛来源的干细胞(Nestinpositive isletderived progenitor， NIP)。NIP可以克隆性增生并具有多向分化潜能，产生具有胰腺内分泌、外分泌、 导管及肝细胞表型的细胞。尚有某些观点觉得胰腺内的干细胞不仅仅局限于上述的两种PSCs，如 Petropavlovskaia等10】觉得PSCs有也许像造血干细胞和肝脏的卵圆细胞，大部分12山东大学博上学位论文时间处在静止休眠并保持未分化状态。她们描述了一种存在于人和犬类的新的 PSCs，占到总胰岛细胞的l，直径710岬并且体现出相称不成熟的形态，称 之为“小细胞”。这种小细胞PDX-1、突触素、胰岛素、胰高血糖素以及生长抑素等抗体反映阳性，但是CK19和Nestin呈阴性反映，区别于此前报道的PSCs。 综上所述，PSCs的组织来源具有多样性，尚无特异的细胞标志物，体外培养和诱导方案也存在较大差别【Il】。本实验使用容易获得的新生Wistar大鼠的胰腺 导管上皮作为PSCs的组织来源，使用胶原酶消化法分离、培养PSCs；培养出的 细胞采用联合标志物鉴定的措施，得到了CK-19和PDX-1双阳性的细胞；体外实 验证明培养细胞可以诱导分化成胰岛素分泌细胞，并且对不同浓度的葡萄糖刺激 显示胰岛素分泌反映。通过本实验我们可以理解所培养细胞向内分泌方向分化的 潜力以及作为移植细胞来源的也许性。材料与措施一实验动物取新生(出生2472小时内)的Wistar大鼠作为胰腺供体，购自山东大学动物 实验中心。有关动物的实验操作得到了该中心实验动物伦理委员会的承认和批 准。二重要试剂及配制措施 l细胞培养试剂RPMll640培养基、胰蛋白酶、DMSO、尼克酰氨(Nicotinamide)、 bFGF、EGF、胶原酶V型等购自美I垂ISigma公司；DTZ、FBS等购I刍Hyclone公司； DGlu购自上海生物工程有限公司；HEPES购自北京KAI生物医学科学公司；丙 酮酸、【广谷氨酰胺、B巯基乙醇购自北京化学试剂公司(分析纯)。 2免疫组化及放免试剂胰岛素抗体(兔抗大鼠)、胰高血糖素抗体(兔抗大鼠)、 CK19抗体(小鼠抗大鼠)、PDX1抗体(山羊抗大鼠)、Ngn3抗体(小鼠抗大鼠) 购自美I亘Sigma公司；山羊抗兔TRITC标记二抗、羊抗小鼠FITC标记二抗、兔抗 山羊TRITC标记二抗购自北京中山生物制品有限公司；125I胰岛素放免试剂盒购 自中国原子能研究所。13山东人学博J：学位论文3 RNA提取试剂DEPC、GIT、2M醋酸钠(NaAC)、Tris饱和酚、氯仿、异丙醇、 无水乙醇(以上均购自上海试剂总厂)。4 PCR及电泳试剂细胞裂解液(TRIzol，Gibco公司)、TaqDNA聚合酶(Fermentas 公司)、dNTP(Fermentas公司)、1Kb DNA Ladder(Fermentas公司)、Oligo dT (Fermentas公司)、MMLV(Invitrogen公司)、DTT(Invitrogen公司)、6xloading buffer(Takara公司)、溴化乙锭(EB，Sigma公司)、琼脂糖(Sigma公司)、超纯 水(-蒸水再f-空Millipore纯水器纯化)。5引物设计通过Genbank网站查找mRNA序列，根据引物设计原则，运用Primer Premier 50软件设计特异性PCR弓I物，以pactin为内参照，引物均由上海博亚生物技术公司合成。6重要试剂的配制 (1)高浓度DGlu(333mmoH)培养基的配制新鲜双蒸馏水溶解RMPll640粉剂， hllX,NaHC03(29L)，HEPES，充足溶解。随后力HXIO灭活胎牛血清，20ngml EGF，20ngml bFGF，lmmolL的丙酮酸钠，10万UL青霉素、10万UL链霉素， 4冰箱保存。 (2)低浓度DGlu(11ImmolL)培养基的配制新鲜双蒸馏水溶解RMPll640粉剂， JJHX,NaHC03(29L)，HEPES，充足溶解。随后加入2灭活胎牛血清，10mmolLNicotinamide，715umolL D一疏基乙醇，lO万UL青霉素、lO万UL链霉素，4C 冰箱保存。O)O25胰酶消化液的配制称取025碳酶粉剂溶于100ml DHanksPBS液中， 在磁力搅拌器上充足搅拌溶解，用一次性针头抽滤器过滤除菌，无菌条件下分装，-20保存，4备用。(4)不同浓度Glu缓冲液一方面配S JPBS，此后将此溶液配制为30raM糖浓度的溶 液，梯度稀释为30，25，20，15，10，5mM糖浓度的溶液，用022岬的滤器 过滤除菌；4冰箱储存。(5)DHanks液的配制在电子天平上分别称取NaCI 8009，KCl 0409，NaHC03 0359，Na2HP041 2H20 0069，KH2P04 0069，NaI-12P042H20 0069于烧杯中充分溶解于800ml超纯水，用容量瓶定容至1000ml，调pH至tJ74，分装后高压灭菌， 4冰箱保存备用。14山东人学博上学位论文(6)056mgml DTZ溶液临用前配制。10mgDTZ溶于3ml无水乙醇及50L浓氨水， 充足溶解后加入148ml HankS液，混匀后56。C水浴30分钟。过滤除菌，装入棕 色密封试剂瓶，4。C避光保存。用时按3：l比例染色(样本液DTZ溶液，V)比例染色，终浓度为140mg1。 (7)rBs液的配制在电子天平上分别称取NaCl 909，Na2HP0412H20409， NaH2P04。2H20 049后在定容瓶中充足溶解亍：1000ml新鲜制备的去离子三蒸水， 用1N HCl或1N NaOH调节pH值到74，常温保存备用。(8)细胞冻存液的配制RPMll640培养基、FBS和DMSO按6：3：1的体积比混合而 成，配备后用一次性针头抽滤器过滤除菌，4C备用。三重要仪器及用品80超低温冰箱，37C02恒温培养箱(日本SANYO公司产品)， Eppendor借、吸头(江苏三和申华公司)，25rtl、10I-tl、20I-tl、200Irtl、10009l可 调微量移液器(Gilson公司产品)，DU 530紫外分光光度计(美国BECKMAN公司产品)，XK96一A型迅速混匀器(江苏省新康仪器厂产品)，l 8l 0B型 自动双重纯水蒸馏器(上海玻璃仪器一厂产品)，Tgradient96型PCR仪(德国Biometra公司产品)，Biofuges 28RS低温高速离心机及配套转子(Heraeus公司)，ER120A型电子天平(同本岛津公司产品)，台式离心机(上海医疗器械厂)， 水平电泳槽(北京六一实验仪器厂产品)，EC25090多功能电泳仪(美国 EC公司产品)，Fluorchem 9900凝胶成像系统(德Alpha innotech公司产品)， 恒温水浴箱(Boehrmger Mannhem德国)，超净工作台(北京半导体设备一厂)， 一般冰箱(青岛海尔集团)，倒置相差显微镜，荧光显微镜(日本OLYMPuS)， 扫描电镜(JSM6000F)，透射电镜(JEM1010)(同本JEOL)，层流大鼠独立笼 具饲养系统(苏州冯氏实验动物设备有限公司)，数字凝胶成像系统(Flurochem 990050)(美Alpha Innotech公司)，pH计(920)(美国Orion公司)，病理图像分 析系统(JEDA801)(江苏省捷达科技公司产品)，眼科弯剪、组织镊、不锈钢筛 网(200目)，三角烧瓶(20ml、50m1)、50ml离心管、弯头吸管、注射管，培养 皿、培养瓶，细胞计数板(均购自美国Falcon公司) 四大鼠PSCs的培养及诱导分化l PSCs的培养开腹后辨认胰腺(出生后1-3天的胰腺组织为白色，与脾脏毗邻)15山东人学博l：学位论文(附图1A)，完整取出胰腺，采用胶原酶消化法原代培养P