人支气管上皮不典型增生与浸润癌组织的比较蛋白质组学研究

为筛选支气管上皮鳞状不典型增生进展的分子标志物,采用改良的脱氧胆酸-三氯醋酸(deoxycholate-trichloroaetic acid, DOC-TCA)法提纯支气管上皮总蛋白质进行双向电泳（two-dimensional electrophoresis,2-DE）,应用ImageMaster 2D分析软件、Student’s t-检验识别差异蛋白质点,基质辅助激光解吸电离飞行时间质谱（matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, MALDI-TOF-MS）得到相应的肽质指纹图（peptide mass fingerprint,PMF）,搜索数据库鉴定差异蛋白质.由此获得人支气管上皮不典型增生和浸润癌组织的2-DE图谱及其凝胶的平均蛋白质点数（1 273.00±43.31,1 326.00±66.63）,且两阶段间平均差异蛋白质点数为 56.00±8.96.取38个差异蛋白质点进行PMF分析,鉴定出一些与细胞生长、分化或肿瘤发生等有关的蛋白质,随即应用免疫组化检测差异蛋白质EGFR、c-Jun、Mdm2在两类组织中的表达,其结果也显示了类似的表达差异.支气管上皮不典型增生恶性转化过程中存在蛋白质的差异表达,这些差异蛋白质可能以不同的方式参与了癌变过程,且EGFR、c-Jun、Mdm2的免疫组化验证结果与质谱结果的一致性表明,比较蛋白质组学是一种筛选癌变相关分子标志物的可靠方法之一.     

大鼠不同发育时期胰腺相关蛋白的差异表达

探讨大鼠胰腺不同发育时期相关蛋白的差异表达,应用显微技术分离了大鼠孕15.5天,孕18.5天胚胎胰腺和新生鼠及成年鼠的胰腺,提取其蛋白质后,用固相pH梯度双向聚丙烯酰胺凝胶电泳和质谱分析等蛋白质组学方法,得到了4个不同发育时期的蛋白质表达谱.对其中的6个在孕18.5天胚胎胰腺中有高丰度表达,而在成年鼠胰腺中缺失的蛋白质点,4个在成年胰腺中特异表达的蛋白质点, 8个在成年胰腺中表达明显下调的蛋白质点和1个在成年中表达上调的点,进行了肽质量指纹分析和蛋白质鉴定,共获得18个点的肽质量指纹图.经BIOWORK等软件搜索大鼠非冗余蛋白质数据库来鉴定其身份,发现其中7个点为大鼠甲胎蛋白（AFP）、5个点为胰脂酶相关蛋白1前体、1个点为微管蛋白β、2个点为蛋白二硫异构酶、1个为FLN29基因产物的类似物、1个为胰蛋白酶V-A前体、1个为过氧化物氧化还原酶4.其中AFP为特异表达于大鼠胚胎期及新生期胰腺的蛋白质,在孕18.5天的胰腺中表达量最高,在成年胰腺中极低表达.对它们的功能和与胚胎胰腺代谢调节功能完善过程的可能关系进行了初步探讨.     

大鼠胰腺胚胎发育功能相关基因表达谱的分析

目的:研究大鼠胰腺胚胎发育不同阶段的基因表达谱,对比其功能相关基因随大鼠胰腺发育的变化.方法:采用显微分离及提取技术获得胚胎发育不同阶段胰腺组织并提取RNA,采用高密度寡核普酸芯片(Affemetrix芯片)对胚胎发育至第12.5天、15.5天、18.5天胚胎胰腺及成年胰腺进行基因转录水平分析,用生物信息学方法分析具体基因的表达情况.结果:胰腺的生物学功能尤其beta细胞功能相关基因insulin RNA,amylopsin RNA,GLUT-2 RNA等在胚胎15.5及18.5天显著高表达.结论:E15.5到E18.5直至出生是胰腺功能完善和成熟的阶段,这个时期以细胞功能成熟为主.     

蛋白质组技术及其在胰腺疾病研究中的应用

蛋白质组技术由于具有大通量研究蛋白质组成和蛋白质差异的作用,已有研究将其应用于胰腺疾病的相关研究中,并为胰腺癌、急慢性胰腺炎、胰腺纤维瘤等研究提供了新的思路.介绍了蛋白质组样品提取技术、蛋白质组分离技术、蛋白质组分析技术、生物信息学及其在胰腺疾病的差异表达蛋白、诊断标记物、相关疾病鉴别标记物、发病机制、治疗敏感性研究中的应用.     

激光捕获显微切割技术结合^18O标记定量蛋白质组技术在胃癌标志物筛查中的应用研究

建立一种更加精确地分离鉴定胃癌特异肿瘤标志物的定量蛋白质组学技术.首先采用激光捕获显微切割技术(LCM)纯化胃腺癌细胞及胃黏膜良性上皮细胞,将裂解的样本总蛋白经过1D SDS-PAGE预分离,然后采用17O/16O分别标记两种样本酶切后的多肽混合物.结合纳升级液相色谱(Nano-HPLC-MS/MS)定量地鉴定胃癌细胞和胃黏膜良性上皮细胞的差异表达蛋白.共筛选出78个差异表达蛋白,其中42个蛋白质在胃癌组织中表达上调,36个蛋白质下调.Western blot技术验证了其中几个差异蛋白(moesin,periostin,annexin A2,annexin A4)的表达,与蛋白质组学研究的结果一致.LOM技术结合18O稳定同位素标记的定量蛋白质组学技术,为研究胃癌发生机制、筛选胃癌的分子标志物提供了新的思路,亦为诸如胃癌等复杂体系蛋白质的分离鉴定提供了新的技术选择.     

人肺鳞癌组织的血清蛋白质组学的比较分析

采用以肿瘤免疫学与蛋白质组学(proteomics)研究技术有机地结合为基础的血清蛋白质组学研究体系(serologicproteomeanalysis ,SERPA)筛选肺癌分子标志物.对10例人肺鳞癌组织,应用双向凝胶电泳(two dimensionalelectrophoresis ,2 DE)技术对同一肺鳞癌组织的细胞总蛋白同时进行电泳后获得3张相同的凝胶,其中一块2 DE凝胶经银染显色作为平行胶,其余两块2 DE凝胶经电转膜将凝胶中的蛋白质转至硝酸纤维素(NC)膜上,然后分别与肺癌患者的自身血清以及正常对照血清进行Western印迹分析,获取Western印迹反应图谱.经计算机图像分析识别差异反应的蛋白质,然后与平行胶比较找出相应的差异反应蛋白质点.获得了分辨率较高的人肺鳞癌组织与患者的自身血清以及正常对照血清的Western印迹反应图谱;图像分析共识别36±8个差异反应的蛋白质;在平行胶上找到了匹配的差异反应蛋白质点.对2 0个差异蛋白质点进行了肽质指纹图分析,鉴定出14个与细胞生长增殖、细胞代谢、细胞周期调控、信号转导等有关的肺鳞癌相关抗原.通过血清蛋白质组技术对肺鳞癌组织进行的研究,建立了分辨率较高的人肺鳞癌组织与患者的自身血清以及正常血清的Western印迹反应图谱,成功鉴定14个肺鳞癌相关抗原,为进一步筛选用于肺鳞癌诊断、治疗和预后评估     

成人胰腺导管干细胞的体外分离、纯化、培养及鉴定

目的:建立人胰腺导管干细胞的体外分离、纯化、培养及鉴定的方法.方法:胶原酶分次消化剪切的人胰腺组织,经过Ficoll密度梯度离心后去除胰岛组织,培养于含,10%胎牛血清的CMRL1066培养液中,7-10天可行成单层细胞,用0.25%胰酶-EDTA消化并传代培养.取2-3代细胞利用免疫荧光染色和RT-PCR方法检测CK19、Pdx-1、Nestin、Insulin及Glucagon的表达.结果:经过胶原酶消化、Ficoll密度梯度离心及后续的培养,去除了胰岛组织及外分泌腺,可获得较纯化的鹅卵石样的胰腺导管细胞.免疫荧光结果示:胰腺导管细胞CK19、Pdx-1和Nestin染色呈阳性,阳性细胞率分别为(87.5±6.2)%、(77.5±8.6)%和(50.9±9.5)%,而Insulin染色为阴性.RT-PCR结果显示该细胞表达CK19、Pdx-1和Nestin基因,而未观察到Insulin及Glueagon基因的表达.结论:该方法可较好的分离纯化出人胰腺导管细胞,经鉴定获得细胞具有胰腺干细胞的特性.     

利用谱系追踪方法探究Lgr5在胰腺组织及其类器官中的表达

富含亮氨酸重复序列G蛋白偶联受体5 (leucine-rich repeat containing G protein-coupled receptor 5, Lgr5)在体内分布广泛,可以作为多种上皮组织(包括小肠、结肠、胃和毛囊)中干细胞的标记物。为了探究小鼠(Mus musculus)胰腺发育过程中导管上皮细胞及体外培养的胰腺导管类器官中Lgr5的表达情况,本研究利用Lgr5-Cre^ERT2+/–和Rosa26-mTmG杂交后的转基因小鼠,经Tamoxifen(他莫昔芬)诱导后,观察不同发育阶段胰腺组织切片的荧光表达情况,并通过三维培养建立成体小鼠胰腺导管类器官,观察诱导后类器官细胞中的荧光变化。结果显示：Tamoxifen诱导的正常成体转基因小鼠胰腺导管内未检测到表达Lgr5的细胞;通过对孕鼠及哺乳母鼠注射Tamoxifen,在胚胎发育15.5d和新生小鼠胰腺中也未发现Lgr5阳性细胞;但是将4-hydroxyTamoxifen (4-羟基–他莫昔芬)添加到培养基中,在Lgr5-Cre^ERT2+/–;Rosa26-mTmG转基因小鼠胰...     

血清蛋白质组分析方法

蛋白质组学一经出现 ,人们便开始尝试将其用于疾病特定分子标志物的研究当中 ,比如通过比较疾病与正常生理状况下蛋白质表达谱的差异来寻找与疾病密切相关的蛋白质 .但采用该方法有一明显的缺点 ,即需要分析大量同一肿瘤组织的二维凝胶电泳 (two dimensionalgelelectrophoresis ,2 DE)图谱 ,否则难以得到有统计学意义的差异蛋白质 .另外 ,由于细胞内的蛋白质表达是不均匀的 ,这就导致了高表达和易溶解的蛋白质远较其他低表达、难溶解的蛋白质容易出现在2 DE图谱中 ,因而明显地降低了计算机匹配分析发现有生物学意义的差异蛋白质的灵敏性…     

亚健康便秘人群结肠黏膜蛋白质组的筛选鉴定与临床应用

筛选亚健康便秘人群结肠黏膜变化的分子标志物,为亚健康便秘人群的结肠黏膜改变机制提供理论依据.采用双向凝胶电泳(two-dimensional electrophoresis,2-DE)对亚健康便秘人群及健康志愿者结肠黏膜组织进行蛋白质分离,ImageMaster2D Elite分析软件进行图像分析,基质辅助激光解吸电离飞行时间质谱(matrix-assisted laser desorption/ionization time of flightmass spectrometry,MALDI-TOF-MS)得到相应的肽质量指纹图(peptide mass fingerprint,PMF),搜索数据库鉴定差异蛋白.建立了亚健康便秘人群及健康志愿者结肠黏膜组织2-DE图谱,分析出其凝胶的平均蛋白质点数(501.00±37.16,536.00±41.63),两者平均差异蛋白质点数为46.00±7.82,取20个表达量明显改变的蛋白质点进行质谱分析,鉴定出17个蛋白质.其中7个蛋白质点表达下调,10个蛋白质点表达上调.差异蛋白质点包括蛋白质合成与分解、分子伴侣、氧化还原调节及信号传导等相关蛋白质.随即应用免疫印迹(Western blot)技术分析差异蛋白β-actin、YWHAZ及PBP-Ⅰ(phosphatidylethanolamine-binding proteinⅠ)在两类组织中的表达水平及临床意义.结果表明,亚健康便秘人群和健康志愿者的结肠黏膜组织蛋白表达存在差异,β-actin、YWHAZ表达下调及PBP-Ⅰ上调参与了亚健康便秘的发生,对此状态进行合理干预,可使身体向健康转化.     

Proteome analysis of rat pancreas induced by pancreatectomy

The previous study demonstrated that the streptozotocin (STZ)-induced diabetic mice can be cured by injecting the regenerating pancreatic extract (RPE) of the partially pancreatectomized Wistar-Kyoto rats. In this study, to characterize the complex pattern of protein expression in RPE, the proteins of altered expression level after the pancreatectomy were identified by 2-dimensional electrophoresis (2-DE) and mass spectrometry. Of 76 significantly up- or down-regulated protein spots, 61 were identified by MALDI-TOF/MS. Moreover, the whole RPE was fractionated into 4 groups using an anion-exchange chromatography and each fraction's cell proliferating activity was measured by MTT assay. Compared to the normal pancreatic extract, fraction 3 and 4 of RPE showed the maximal cell proliferating activity. On 2-DE of 3 and 4 fractions, a total of 10 spots, which are differentially expressed after the pancreatectomy, were identified by MS/MS. Of these identified proteins, Reg III which might be functionally associated with well known regenerating factor (Reg I) was found. Taken together, our results demonstrated that the differential protein expression associated with pancreas regeneration could be sought by 2-DE and mass spectroscopy and suggested that the pre-fractionation method combined with in vitro cell proliferation assay is effectively used to pinpoint the active components for pancreas regeneration.     

Impaired β-cell regeneration after partial pancreatectomy in the adult Goto-Kakizaki rat, a spontaneous model of type II diabetes

In the Goto-Kakizaki (GK) rat, a genetic model of type II diabetes, there is a restriction of the beta-cell mass as early as fetal age, which is maintained reduced in the adult animal. In order to investigate the beta-cell growth potential in the adult hyperglycemic GK rat, and to determine whether it differs from non-diabetic Wistar (W) rats, we have performed 90% pancreatectomy (Px) in 8- to 10-week-old male animals. Spontaneous beta-cell regeneration and involvement of beta-cell replication, beta-cell neodifferentiation from ductal precursor, and beta-cell apoptosis were evaluated by immunocytochemistry and morphometry at different time points: day 0 (D0), D2, D7, and D14 after Px. In GK rats, deterioration of the diabetic state with severe and chronic hyperglycemia was evident as soon as D2, while in W/Px, normoglycemia to moderate hyperglycemia was observed. In W/Px rats, the total beta-cell mass gradually increased on D2, D7, and D14, as compared to non-Px W rats. By contrast, in GK/Px rats, there was only a non-significant tendency to increased total beta-cell mass, as compared to related non-Px group. Adult GK rats displayed lower beta-cell proliferation rates compared to W. In response to Px, early increase of beta-cell proliferation was present in both W/Px and GK/Px rats on D2, but it returned to non-Px values in GK rats on D7 and D14, while in W/Px rats beta-cell proliferation was maintained increased as compared to non-Px W rats. The very low apoptotic beta-cell frequency on D0, D2, D7, and D14, in both W and GK, either non-Px or Px, did not allow us to conclude that any significant differences exist between the different groups. beta-cell neoformation from ducts, and more specifically from foci of regeneration, was found to be less activated in GK/Px rats as compared to W/Px. Together, these results suggest that in the adult hyperglycemic GK rat undergoing Px, beta-cells still have the capacity to regenerate, but with a lower efficiency as compared to non-diabetic W rats. This defect in the GK rat is the result of both genetic predisposition contributing to an altered beta-cell neogenesis potential already present in the neonatal period, and environmental factors (chronic hyperglycemia) leading to a reduced beta-cell proliferative capacity specific to the adult animals.     

Potential for Pancreatic Maturation of Differentiating Human Embryonic Stem Cells Is Sensitive to the Specific Pathway of Definitive Endoderm Commitment

This study provides a detailed experimental and mathematical analysis of the impact of the initial pathway of definitive endoderm (DE) induction on later stages of pancreatic maturation. Human embryonic stem cells (hESCs) were induced to insulin-producing cells following a directed-differentiation approach. DE was induced following four alternative pathway modulations. DE derivatives obtained from these alternate pathways were subjected to pancreatic progenitor (PP) induction and maturation and analyzed at each stage. Results indicate that late stage maturation is influenced by the initial pathway of DE commitment. Detailed quantitative analysis revealed WNT3A and FGF2 induced DE cells showed highest expression of insulin, are closely aligned in gene expression patterning and have a closer resemblance to pancreatic organogenesis. Conversely, BMP4 at DE induction gave most divergent differentiation dynamics with lowest insulin upregulation, but highest glucagon upregulation. Additionally, we have concluded that early analysis of PP markers is indicative of its potential for pancreatic maturation.     

胎儿胰岛样细胞团源上皮样细胞分离、纯化和鉴定

旨在优化胰腺干细胞分离、鉴定的方法和体系,为糖尿病研究和治疗提供种子细胞。采用胶原酶消化法,分离培养出胰岛样细胞团(ICCs),对其进行贴壁培养,从中纯化出上皮样细胞。采用MTT法测定其生长情况并绘制生长曲线。采用免疫组织化学染色检测分离得到细胞的PDX-1、PCNA、CK-7、CK-19、Nestin、Glut2、Vimentin、Insulin表达情况。应用流式细胞仪检测其表面标志。由分离培养的ICCs成功纯化出上皮样细胞;传40代,每代冻存106~108个细胞;生长曲线显示其传代第3天进入对数生长期,第5天进入平台期;免疫组织化学染色显示其表达PDX-1、PCNA、CK-7、CK-19、Nestin、Glut2、Vimentin;不表达Insulin;流式细胞仪分析表明其表达CD29、CD44、CD166,不表达CD11a、CD14、CD34、CD45、CD90、CD105、CD117。由胎儿胰腺能分离出具有自我更新能力的上皮样细胞,为导管来源,具干细胞特性。     

Stellate Cells from Rat Pancreas Are Stem Cells and Can Contribute to Liver Regeneration

The identity of pancreatic stem/progenitor cells is still under discussion. They were suggested to derive from the pancreatic ductal epithelium and/or islets. Here we report that rat pancreatic stellate cells (PSC), which are thought to contribute to pancreatic fibrosis, have stem cell characteristics. PSC reside in islets and between acini and display a gene expression pattern similar to umbilical cord blood stem cells and mesenchymal stem cells. Cytokine treatment of isolated PSC induced the expression of typical hepatocyte markers. The PSC-derived hepatocyte-like cells expressed endodermal proteins such as bile salt export pump along with the mesodermal protein vimentin. The transplantation of culture-activated PSC from enhanced green fluorescent protein-expressing rats into wild type rats after partial hepatectomy in the presence of 2-acetylaminofluorene revealed that PSC were able to reconstitute large areas of the host liver through differentiation into hepatocytes and cholangiocytes. This developmental fate of transplanted PSC was confirmed by fluorescence in situ hybridization of chromosome Y after gender-mismatched transplantation of male PSC into female rats. Transplanted PSC displayed long-lasting survival, whereas muscle fibroblasts were unable to integrate into the host liver. The differentiation potential of PSC was further verified by the transplantation of clonally expanded PSC. PSC clones maintained the expression of stellate cell and stem cell markers and preserved their differentiation potential, which indicated self-renewal potential of PSC. These findings demonstrate that PSC have stem cell characteristics and can contribute to the regeneration of injured organs through differentiation across tissue boundaries.     

Proteome analysis on an early transformed human bronchial epithelial cell line,BEP2D,after alpha-particle irradiation

To probe the mechanism of carcinogenesis of lung cancer at the molecular level and to find potential protein markers involved in the early phase of tumorgenesis, differential proteome analysis on primary passage cell line R15H, and early transformed cell line R15H20 derived from (238)Pu alpha-particle irradiation of human papillomavirus (HPV) 18-immortalized human bronchial epithelial cell line (BEP2D), was carried out using two-dimensional electrophoresis (2-DE) and peptide mass fingerprinting (PMF) with matrix-assisted laser desorption/ionisation-time of flight mass spectrometry. Image analysis and Student's t-test (p < 0.05) showed that three protein spots were only expressed in R15H, intensities of 43 protein spots on the gels were altered between R15H and R15H20. Two of the three spots that were only expressed in R15H were identified as high mobility group protein 1. Two proteins decreased in abundance in R15H20 were identified as maspin precursor, a tumor suppressor and aminoacylase-1. Ornithine aminotransferase and peptidyl-prolyl cis-trans isomerase A that were increased in R15H20, were also identified. Relationships between these differentially expressed proteins and the carcinogenesis mechanism of lung cancer are discussed. The protein expression profile of the R15H cell line was also constructed during the study as a reference map for further comparative proteome analysis of the irradiation induced BEP2D cell line. Of the 90 spots analyzed with PMF in the 2-DE gel of R15H cell line, 50 proteins were identified by searching the nonredundant protein database SWISS-PROT/TrEMBL.     

Expression and localization of regenerating gene I in a rat liver regeneration model

Regenerating gene (Reg) I has been identified as a regenerative/proliferative factor for pancreatic islet cells. We examined Reg I expression in the regenerating liver of a rat model that had been administered 2-acetylaminofluorene and treated with 70% partial hepatectomy (2-AAF/PH model), where hepatocyte and cholangiocyte proliferation was suppressed and the hepatic stem cells and/or hepatic progenitor cells were activated. In a detailed time course study of activation of hepatic stem cells in the 2-AAF/PH model, utilizing immunofluorescence staining with antibodies of Reg I and other cell-type-specific markers, we found that Reg I-expressing cells are present in the bile ductules and increased during regeneration. Reg I-expressing cells were colocalized with CK19, OV6, and AFP. These results demonstrate that Reg I is significantly upregulated in the liver of the 2-AAF/PH rat model, accompanied by the formation of bile ductules during liver regeneration.     

再生胰腺提取物诱导人羊膜间充质干细胞定向分化为胰岛素分泌细胞

利用天然生物诱导剂大鼠再生胰腺提取物(Rgenerating pancreatic extract,RPE)定向诱导人羊膜间充质干细胞(Human amniotic mesenchymal stem cells,hAMSCs)向胰岛素分泌细胞分化。切除大鼠60%胰腺刺激胰腺再生,而后制备RPE,以终浓度为20 mg/L的RPE诱导hAMSCs。实验通过形态学鉴定、双硫腙染色、免疫荧光分析、RT-PCR基因检测和高糖刺激胰岛素分泌等实验鉴定细胞诱导结果。实验结果显示P3代hAMSCs经RPE诱导后形态变化明显,诱导15 d后细胞呈簇状生长,经双硫腙染色可见棕红色细胞团;免疫荧光染色结果显示诱导细胞呈胰岛素阳性表达;RT-PCR实验证明诱导细胞阳性表达人胰岛相关基因Pdx1和insulin;高糖刺激实验证明培养液中有胰岛素成分产生,且分泌量随刺激时间的延长先增加而后趋于稳定。实验结果表明hAMSCs在体外经RPE诱导可以分化为胰岛素分泌细胞。