【作者】 陈锦鹏；

【导师】 钱海鑫；

【作者基本信息】 苏州大学 ， 普通外科（专业学位）， 2013， 博士

【摘要】 目的研究胚胎干性相关基因Oct4和Nanog在人胰腺癌肿瘤干细胞(PCSCs)和普通肿瘤细胞中表达情况，然后再通过特异shRNA沉默PCSCs细胞中Oct4和Nanog的表达，研究PCSCs干性特征的变化及其机制。方法本实验分为两大部分。第一部分：用流式细胞仪在人胰腺癌Panc-1细胞系中以CD44+CD24+ESA+为表面标记筛选PCSCs，RT-PCR法和细胞免疫荧光法检测PCSCs细胞和Panc-1细胞中Oct4和Nanog基因的表达情况。用CCK8法检测其体外增殖能力，描制生长曲线，用吉西他滨分别干预普通肿瘤细胞和PCSCs细胞，研究PCSCs的干性特征。第二部分：用慢病毒载体介导的shRNA沉默PCSCs中Oct4和Nanog的表达，并用FQRT-PCR和Western blot检测干扰效率。单克隆形成实验、Transwell小室模型和CCK8法检测Oct4和Nanog基因对PCSCs增殖、迁移、侵袭和药物敏感性的影响；FQRT-PCR和Western blot探讨其作用机制。通过NOD/SCID小鼠致瘤实验观察Oct4和Nanog对PCSCs致瘤性的影响。结果分选出的CD44+CD24+ESA+三阳性PCSCs细胞约占细胞总量的0.1%-0.8%，Oct4及Nanog基因在PCSCs细胞中表达高于Panc-1细胞，Oct4在PCSCs细胞中的表达高于Panc-1细胞8.141±1.378倍；Nanog在PCSCs细胞中的表达高于Panc-1细胞7.945±0.652倍。Oct4和Nanog基因在两种干细胞中均为核表达。Panc-1细胞PCSCs细胞体外培养，Panc-1细胞第2天进入对数生长期，肿瘤干细胞体外无血清培养后第5天仍未进入对数生长期，呈干细胞球样缓慢生长。吉西他滨刺激后细胞生长被抑制，第48H，测Panc-1细胞IC50值为982μg/mL，PCSCs细胞IC50值为1981μg/mL。第72H测Panc-1细胞IC50值为670μg/mL，PCSCs细胞IC50值为1484μg/mL，差异有统计学意义(P<0.05)；慢病毒载体介导shRNA可明显降低PCSCs中Oct4、Nanog的表达(P<0.05)。沉默Oct4和Nanog后，PCSCs的单克隆形成、增殖、迁移、侵袭力较对照组显著下降，药物敏感性增强并诱导凋亡(P<0.05)。致瘤实验表明PCSCs致瘤性在沉默Oct4和Nanog基因后，较对照组明显下降(P<0.05)。结论1．通过流式细胞仪在胰腺癌Panc-1细胞株中分选出CD44+CD24+ESA+三阳性细胞，在体外培养中表现缓慢生长及为耐受化疗药物的干细胞特性。2．Oct4和Nanog基因在CD44+CD24+ESA+阳性PCSCs细胞中的表达高于普通胰腺癌细胞，说明这两种基因与胰腺癌干细胞的干性特征相关。3．慢病毒介导的shRNA可以沉默PCSCs中Oct4和Nanog基因的表达，可显著抑制其干细胞性特征，增强药物敏感性并诱导凋亡。更多还原

【Abstract】 ObjectiveTo study the gene expression of Oct4and Nanog that related toembryonic stem cell in human pancreatic cancer stem cells（PCSCs）and study the proliferation and drug-resistence ability of pancreaticcancer stem cell in vitro. Silence Oct4and Nanog expression in PCSCsby specific shRNA, study the biological characteristics of PCSCs andthe related mechanisms.MethodsPancreatic cancer stem cells of CD44+CD24+ESA+phenotypesisolated by a FACS Aria II in human cell line Panc-1. Detect theexpression of Oct4and Nanog gene expression in Panc-1cells andPCSCs cells by immunofluorescence and RT-PCR. To study theproliferation ability of cancer stem cell, we draw a growth curve bythe ways of CCK8staining. To detect the ability of drug-resistence ofPCSCs, we stimulated both cells by gemcitabine and study thedifference of their IC50. Nanog and Oct4in PCSCs silenced byLentiviral vector-GFP(LV) mediated shRNA. Observe themigration,invasion, proliferation and the drug sensitivity of PCSCsinhibited by Oct4and Nanog by monoclonal formation as well as theCCK8assay, transwell chamber model. Investigate related mechanismsby FQRT-PCR and Western blot and NOD/SCID mice tumorigenicityexperiments was done to study the tumorigenic potential of PCSCsinfluenced by Oct4and Nanog. ResultsThe isolated PCSCs is0.1%-0.8%of all cells. Oct4and Nanoggenes have a higher expression in sorted cells than unsorted ones.The△CT of Nanog in PCSCs and Panc-1cell is7.945±0.652. The△CT of Oct4in PCSCs and Panc-1cell is8.141±1.378; Both twogenes were expressed in nucleus and were restrained by gemcitabine.The48th hour’s IC50of Panc-1cell is982μg/mL, IC50of PCSCsis1981μg/mL. The72th hour’s IC50of Panc-1cell is670μg/mL,IC50of PCSCs cell is1484μg/mL.LV mediated shRNA have stronginhibitory effects for Nanog and Oct4expression(P<0.05).Compared tothe control group,The proliferation, monoclonal formation, invasionability and migration of PCSCs decreased significantly if silencing Oct4and Nanog expression, while enhance drug sensitivity and induceapoptosis(P<0.05). Tumorigenicity experiments showed PCSCstumorigenic capacity decreased significantly than that of control groupif Oct4and Nanog gene silenced.(P<0.05).Conclusions1. Cancer stem cell perform a stem cell-like characteristic of highlydrug-resistence and declined proliferation.2. Oct4and Nanog gene have a higher expression in PCSCs thanin Panc-1cells.3. LV mediated shRNA effectively reduce the expression of Oct4and Nanog gene in PCSCs.4. Silence Nanog and Oct4can inhibit the biological characteristicsof PCSCs，while enhance drug sensitivity and induce apoptosis.更多还原