稳定转染DMBT1对胆囊癌细胞株GBC-SD糖链表达的影响及机制

目的 应用已建立的稳定表达候选抑癌基因DMBT1的胆囊癌细胞株GBC-SD,探讨转染前后细胞糖链的变化及其机制.方法 针对已成功建立的稳定表达DMBT1的胆囊癌细胞株GBC-SD,细胞分组:GBC-SD/DMBT1,转染DMBT1组;GBC-SD/Vector,转染空载体组;GBC -SD,未处理组;标记2种凝集素探针,即蔓陀萝凝集素(DSA)、刀豆素A(ConA),检测转染前后细胞表达糖链类型变化;采用流式细胞仪检测生物素标记的两种凝集素DSA与ConA在细胞表面的结合;逆转录－聚合酶链反应(RT-PCR)及免疫印迹进一步检测N－乙酰氨基葡萄糖转移酶V(GnT-V)表达.结果 凝集素探针实验及生物素标记表明实验组多表达ConA结合型糖链,对照组多表达DSA结合型糖链;RT-PCR及免疫印迹进一步发现实验组表达GnT-V相对灰度值分别为0.32,0.47,显著低于对照组(0.62,0.75),差异均有统计学意义(P＜0.01).结论 DMBT1在体外过表达促进胆囊癌细胞由表达3、4天线、偏2天线转而向2天线型转变,至少部分依赖于GnT-V活性的降低.     

不同支架材料体外构建组织工程软骨的研究

目的:体外构建组织工程软骨,筛选更为适合组织工程软骨构建的支架材料。方法:体外获取SD大鼠肋软骨细胞。采用第一代软骨细胞作为种子细胞,接种于壳聚糖/明胶和BMG/生物蛋白胶支架,体外培养的不同时间对其进行HE、甲苯胺蓝染色、Mas s on染色、免疫学检测、扫描电镜观察。结果:在培养2周时,BMG/生物蛋白胶各种染色结果显示软骨细胞在其表面以及内部分布均匀,蛋白多糖和Ⅱ型胶原染色阳性;壳聚糖/明胶表面细胞稍多于前者,但内部细胞数量极少且分布不均,染色结果不如前者明显。随着培养时间的延长各种检测均显示有大量的软骨细胞特异性的蛋白多糖和Ⅱ型胶原的表达,壳聚糖/明胶凸显出明显的优势。结论:体外成功构建组织工程软骨,软骨细胞在BMG/生物蛋白胶上的生长、增殖和分泌基质情况优于壳聚糖/明胶支架。     

人卵泡壁层颗粒细胞源性非整合诱导多能干细胞的制备

目的探索将卵泡液中壁层颗粒细胞诱导为非整合的诱导多能干细胞(iPS细胞),并检测其颗粒细胞方向的分化能力。方法在取卵的操作过程中,收集废弃的壁层颗粒细胞,在颗粒细胞培养至第4天时,加入iPS仙台病毒,经过20余天的维持,挑取iPS细胞克隆,并扩增培养。对其多能基因表达情况、外源基因整合情况、自然分化能力、颗粒细胞定向分化能力进行鉴定,并与皮肤细胞来源的iPSC系进行平行分化能力比较。结果成功建立了壁层颗粒细胞来源的iPS细胞系,其通过了类似胚胎干细胞的多能性检测及体外三胚层的分化能力检测,尤其在定向分化为颗粒细胞时,分化出了大量FOXL2、CYP19A1和FSHR阳性的细胞,经ELISA试剂盒检测,发现该分化细胞可以分泌AMH并且能够将雄激素转化成雌激素;且颗粒细胞源iPS系较皮肤细胞源的iPS系在颗粒细胞方向的分化效率更高。结论提供了一种从人颗粒细胞建立iPSC的方法,并验证了其颗粒细胞方向分化的优势。该系统不仅可以用于建立生殖不孕疾病的iPSC库,还为颗粒细胞功能障碍不孕的患者提供了一种细胞治疗的新思路。     

诱导多能干细胞体外向生精细胞自发分化潜能的研究

目的:探讨诱导多能干细胞(iPS)体外培养自发分化过程中生精细胞相关基因的表达,评估iPS体外向生精细胞自发分化的潜能。方法:经类胚体(EB)形成,体外诱导iPS向生精细胞分化,实时定量PCR和PCR检测生精细胞相关基因的表达。结果:实时定量PCR和PCR结果显示iPS经EB形成诱导分化后生精细胞不同时期的相关基因均有不同程度表达。iPS体外培养自发分化后生精细胞相关基因出现4种时间表达特征:Oct4基因表达量呈波浪状上升;Dppa3和Stra8基因表达量随诱导时间延长而下降;Dazl基因表达量呈波浪状下降;减数分裂前期基因Tex14、Msy2,减数分裂期基因Scp1、Scp3以及单倍体基因Akap3随着诱导时间延长先表达增加,而后表达下降。结论:iPS在经EB自发分化过程中表达生精细胞不同时期的相关基因,并且表达雄性配子单倍体基因,具有向雄性配子的分化潜能。     

骨髓来源血管内皮祖细胞的体外分离培养和鉴定

目的:探索体外分离培养骨髓来源血管内皮祖细胞的方法。方法:将骨髓细胞接种至明胶涂层的培养皿内,用含10%胎牛血清、添加50μg/ml ECGS的M199培养液,置37℃、体积分数为5%的CO2饱和湿度恒温培养箱培养,0.05%胰酶-EDTA消化传代。通过CD31免疫荧光染色、荆豆凝集素免疫细胞化学染色及毛细血管腔形成能力检测进行鉴定。结果:培养5～7天,内皮祖细胞的早期克隆形成,2周后细胞表现出典型的"鹅卵石"状。可与荆豆凝集素特异性相结合;内皮细胞特异性表面标志CD31荧光染色呈阳性表达;培养过程中可形成管腔状结构。结论:自骨髓可以获取足量的EPCs。     

骨髓来源血管内皮祖细胞的体外分离培养和鉴定

目的:探索体外分离培养骨髓来源血管内皮祖细胞的方法。方法:将骨髓细胞接种至明胶涂层的培养皿内,用含10%胎牛血清、添加50μg/ml ECGS的M199培养液,置37℃、体积分数为5%的CO2饱和湿度恒温培养箱培养,0.05%胰酶-EDTA消化传代。通过CD31免疫荧光染色、荆豆凝集素免疫细胞化学染色及毛细血管腔形成能力检测进行鉴定。结果:培养5～7天,内皮祖细胞的早期克隆形成,2周后细胞表现出典型的"鹅卵石"状。可与荆豆凝集素特异性相结合;内皮细胞特异性表面标志CD31荧光染色呈阳性表达;培养过程中可形成管腔状结构。结论:自骨髓可以获取足量的EPCs。     

诱导的多潜能干细胞类胚体分化过程中残留未分化细胞的研究

目的：探讨诱导的多潜能干细胞（induced pluri potent stem cells,iPS cells）通过类胚体长期分化后残留未分化细胞的特性。方法：小鼠iPS细胞株,体外类胚体分化20天后消化打散,重新给予i PS细胞常规培养液培养。观察扩增的残留细胞形态;流式细胞仪和免疫荧光染色检测和观察残留细胞表面标志物及体外再次分化能力。将残留细胞扩增后注射入裸鼠背部皮下,6周后注射部位取材进行大体和组织学检查。结果：分化20天的类胚体中存在残留未分化的细胞,呈克隆样生长,高度表达SSEA-1、CD-9和OCT-4等多潜能性标志。残留细胞能反复传代,并可在体外再次分化和残留。残留细胞注射部位形成畸胎瘤,瘤体组织中存在成熟的内胚层、中胚层和外胚层组织。结论：iPS细胞分化为类胚体后残留部分未分化细胞,残留细胞在体内、外可再次分化,并能在体外分化中再次残留。     

金黄地鼠前列腺分泌蛋白对输卵管液糖蛋白的影响

目的:在整体水平探讨前列腺分泌蛋白对金黄地鼠输卵管液中糖蛋白的影响。方法:金黄地鼠雄鼠依据手术方式的不同分为3组,分别为假手术组(SH)、附属性腺全去组(TX)和腹前列腺组(VP)(仅存前列腺)。收集与各手术组交配后不同时间点(交配后0.5、2、4、6h)的输卵管液(每一时间点及每一组,n=3),输卵管液蛋白经SDS-聚丙烯酰胺凝胶电泳,考马斯亮蓝或阿仙蓝染色分析,应用蛋白电泳印迹后与系列某一种特异性糖基专一性结合凝集素反应,分析糖蛋白的变化。结果:不同组雄鼠交配及不同时间点收集输卵管液蛋白电泳谱类似,约15条主要条带。凝集素结合谱示,麦胚凝集素(WGA)结合的相对分子质量(Mr)为32000、35500、47000、52000糖蛋白见于6h VP组输卵管液,而6h TX组可见Mr为81000、128000条带;与豌豆凝集素(PSA)结合Mr为37500、32000糖蛋白仅见于6h VP组,而6h TX组缺乏;仅6h VP组可见与双花扁豆凝集素(DBA)结合Mr为52000、47000糖蛋白,而6h TX组缺乏。而0.5、2、4h时间点收集的输卵管液各凝集素结合谱相似。结论:前列腺分泌蛋白可影响修饰交配6h后的输卵管液中含乙酰氨基葡萄糖、N-乙酰半乳糖胺/半乳糖和甘露糖糖链的糖蛋白。这些糖蛋白可能在胚胎的发育过程中起作用。     

大鼠骨髓内皮祖细胞SPIO体外标记的实验研究

目的探索体外超顺磁性氧化铁(superparamagnetic iron oxide,SPIO)标记大鼠骨髓内皮祖细胞(endothelial progenitor cells,EPCs)及其条件优化,为下一步EPCs活体示踪实验奠定基础。方法采用密度梯度离心法分离培养大鼠骨髓EPCs,不同浓度的SPIO体外标记EPCs,普鲁士蓝染色测定细胞标记率,MTT法检测细胞增殖力,台盼蓝染色检测细胞活力。结果 EPCs培养约7 d逐渐生长呈单层排列,SPIO浓度为50μg/m L时普鲁士蓝染色显示标记率达到90%,标记后的EPCs生长状态良好,能正常贴壁生长并传代,台盼蓝染色及MTT法显示此时细胞活力及增殖能力最强。结论 SPIO浓度为50μg/mL时标记EPCs其标记率高,不影响细胞的活力及增殖能力,可为下一步EPCs活体示踪实验奠定基础。     

新型猪诱导多潜能干细胞的成功培育

诱导多潜能（inducedpluripotentstem,iPS）干细胞是通过在分化的体细胞中表达特定的几个转录因子,以诱导体细胞重编程而获得的可不断自我更新（self-renewal）且具有多向分化潜能的细胞。由于iPS细胞在细胞形态、生长特性、表面标志物和形成畸胎瘤等方面与胚胎干细胞非常相似,具有避免免疫反应又不涉及伦理道德问题等优势,因此具有广泛且重要的临床应用价值。自从2006年Takahashi和Yamanaka报道通过特定的基因组合与转染可以将已分化的体细胞诱导重编程成功地建立小鼠的iPS细胞以来,全世界众多实验室开始了iPS细胞研究,并取得了巨大进展。     

Carbohydrate residues in non-malignant prostatic epithelium as revealed by lectins

Summary Non-neoplastic prostatic epithelium from 39 patients obtained at transurethral resection for outflow tract obstruction and 5 normal prostates from men under 35 years of age obtained at postmortem were formalinfixed and paraffin-embedded. The distribution of 8 lectin receptors were studied using a peroxidase anti-peroxidase method and an avidin-biotin method. Con A, WGA, and PNA bound to most epithelial cells. Con A,and WGA also showed major stromal binding. Approximately 5% to 10% of cells bound UEA1, GS1, DBA, and BPA. No major differences in lectin receptor expression were observed between normal and hyperplastic epithelium with either of the immunohistochemical techniques except that hyperplastic cells stained more strongly than normal epithelium.     

Comparative analysis of endoderm formation efficiency between mouse ES cells and iPS cells

Definitive endoderm (DE) derived from stem cells holds potential to differentiate into hepatocytes. Stem cell therapy using those cells has potential for a treatment of liver disease. To date, various ways of inducing hepatocytes from embryonic stem (ES) cells have been reported by researchers. However, it has not been proved enough that induced pluripotent stem (iPS) cells behave in the same manner as ES cells in endoderm differentiation. The purpose of this study was to establish an efficient method to induce DE from iPS cells, through comparatively analyzing the efficacy of endoderm formation from mouse ES cells. Furthermore, the efficiency of a serum-free medium in the differentiation into DE was investigated. Mouse ES cells and iPS cells were floated in culture medium for 2 or 5 days and embryoid bodies (EB) were formed. Subsequently, DE was induced with 100 ng/ml activin A and 100 ng/ml basic fibroblast growth factor (bFGF). RT-PCR and real-time PCR analyses were carried out at each step to determine the gene expression of EB markers. The difference in cellular proliferation between serum-containing and serum-free media was examined by an MTS assay in EB and DE induction. iPS cells showed the paralleled mRNA expression to ES cells in each step of differentiation into EB, but the levels of expression of Sox17 and Foxa2 were relatively higher in ES cell-derived DE, whereas Cxcr4 expression was higher in iPS cell-derived DE. The utilization of serum-free medium for iPS cells showed significantly favorable cellular proliferation during EB formation and subsequent DE induction. Forming EB for 5 days and subsequently DE induction with activin A and bFGF with serum-free medium was an appropriate protocol in iPS cells. This may represent an important step for generating hepatocytes from iPS cells for the development of cell therapy.     

Generation of male germ cells from induced pluripotent stem cells (iPS cells): an in vitro and in vivo study

Recent studies have reported that induced pluripotent stem (iPS) cells from mice and humans can differentiate into primordial germ cells. However, whether iPS cells are capable of producing male germ cells is not known. The objective of this study was to investigate the differentiation potential of mouse iPS cells into spermatogonial stem cells and late-stage male germ cells. We used an approach that combines in vitro differentiation and in vivo transplantation. Embryoid bodies (EBs) were obtained from iPS cells using leukaemia inhibitor factor (LIF)-free medium. Quantitative PCR revealed a decrease in Oct4 expression and an increase in Stra8 and Vasa mRNA in the EBs derived from iPS cells. iPS cell-derived EBs were induced by retinoic acid to differentiate into spermatogonial stem cells (SSCs), as evidenced by their expression of VASA, as well as CDH1 and GFRα1, which are markers of SSCs. Furthermore, these germ cells derived from iPS cells were transplanted into recipient testes of mice that had been pre-treated with busulfan. Notably, iPS cell-derived SSCs were able to differentiate into male germ cells ranging from spermatogonia to round spermatids, as shown by VASA and SCP3 expression. This study demonstrates that iPS cells have the potential to differentiate into late-stage male germ cells. The derivation of male germ cells from iPS cells has potential applications in the treatment of male infertility and provides a model for uncovering the molecular mechanisms underlying male germ cell development.     

Introduction to induced pluripotent stem cells: advancing the potential for personalized medicine

Induced pluripotent stem (iPS) cell technology has enormous potential to advance medical therapy by personalizing regenerative medicine and creating novel human disease models for research and therapeutic testing. Before this technology is broadly used in the clinic, we must realistically evaluate its disease modeling and therapeutic potential. Recent advances including the use of iPS cells to successfully model spinal muscular atrophy in vitro, as well as new techniques in generating iPS cells with recombinant proteins have accelerated the prospects of iPS cells for clinical use in regenerative therapy. This review explores the development and limitations of iPS cell technology, presents a critical comparison of iPS cells and embryonic stem cells, and discusses potential clinical applications and future research directions.     

Highly efficient generation of definitive endoderm lineage from human induced pluripotent stem cells

Background

Although hepatocytes can be an option for liver transplantation, the shortage of donor organs continues to worsen. Since the development of induced pluripotent stem (iPS) cell technology, it is eagerly anticipated to produce functional elements from pluripotent stem cells. These functional cells differentiated from iPS cells could be used for transplantation, drug screening, and in vitro toxicology.

Methods

Human iPS cells are maintained on Mitomycin C-treated mouse embryonic fibroblast layers in DMEM-Ham F12-based medium supplemented with Knockout Serum Replacement, nonessential amino acids, 2-mercaptoethanol, and Glutamax. Differentiation of human iPS cells into a definitive endodermal lineage was induced with PRMI 1640 medium supplemented with B27 and 100 ng/mL human activin A. Two B27 supplements were examined with and without insulin. Furthermore, the PI3 kinase inhibitor LY294002 was used to examine the effect of inhibiting insulin signaling.

Results and Discussion

We established efficient induction of definitive endodermal differentiation from iPS cells. Quantitative analysis revealed efficient (93.03 ± 2.74%) differentiation of human iPS cells into definitive endoderm cells using B27 minus insulin. This protocol may contribute as a fundamental technique to promote human iPS studies to develop cellular sources for transplantation.     

Heme oxygenase-1 localization in the rat nephron

BACKGROUND/AIMS: Renal tubules undergo oxidative injury in various nephropathies. It is unknown whether tubular cells possess mechanisms to attenuate this form of injury. Heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme catabolism, may provide such a mechanism by reducing levels of free heme, a prooxidant molecule, and by limiting activity of heme-containing prooxidant enzymes. Determination of the distribution of HO-1 in the nephron may identify those segments where HO-1 can afford protection against oxidative injury. METHODS: Rats were injected subcutaneously with two different inducers of HO-1: Stannous chloride and cobalt protoporphyrin. At completion of injections, frozen sections of kidneys were stained for HO-1 using a biotin-conjugated monoclonal anti-HO-1 antibody. To identify the origin of tubules staining positive for HO-1, Tetragonolobus purpureas (TP)-derived lectin and Arachnis hypogaea (AH)-derived lectin were applied to sequential sections of the kidney cortex. RESULTS: In rats injected with either HO-1 inducer, HO-1 was immunolocalized in tubules but not in glomeruli. Staining of sequential sections with TP-derived lectin, which binds mainly to proximal tubular cells, was negative in the tubules that stained positive for HO-1. Staining of sequential sections with AH-derived lectin, which binds mainly to distal and collecting tubular cells, was positive in those tubules that were also positive for HO-1. CONCLUSIONS: In kidneys of rats injected with inducers of HO-1, distal and collecting tubular cells were identified as the main segments of the nephron that express HO-1. We suggest that the distal nephron, by expressing HO-1, may be less vulnerable to oxidative injury.     

Liver‐Regenerative Transplantation: Regrow and Reset

Liver transplantation, either a partial liver from a living or deceased donor or a whole liver from a deceased donor, is the only curative therapy for severe end‐stage liver disease. Only one‐third of those on the liver transplant waiting list will be transplanted, and the demand for livers is projected to increase 23% in the next 20 years. Consequently, organ availability is an absolute constraint on the number of liver transplants that can be performed. Regenerative therapies aim to enhance liver tissue repair and regeneration by any means available (cell repopulation, tissue engineering, biomaterials, proteins, small molecules, and genes). Recent experimental work suggests that liver repopulation and engineered liver tissue are best suited to the task if an unlimited availability of functional induced pluripotent stem (iPS)–derived liver cells can be achieved. The derivation of iPS cells by reprogramming cell fate has opened up new lines of investigation, for instance, the generation of iPS‐derived xenogeneic organs or the possibility of simply inducing the liver to reprogram its own hepatocyte function after injury. We reviewed current knowledge about liver repopulation, generation of engineered livers and reprogramming of liver function. We also discussed the numerous barriers that have to be overcome for clinical implementation.     

Hepatic differentiation of mouse iPS cells in vitro

Induced pluripotent stem (iPS) cells are pluripotent and are able to unlimitedly proliferate in vitro. This technical breakthrough in creating iPS cells from somatic cells has noteworthy implications for overcoming the immunological rejection and the ethical issues associated with the derivation of embryonic stem cells from embryos. In the current work, we present an efficient hepatic differentiation of mouse iPS cells in vitro. iPS cells were cultured free floating to induce the formation of embryoid bodies (EB) for 5 days. EB were transferred to a gelatin-coated plate and treated with 100 ng/ml activin A and 100 ng/ml basic fibroblast growth factor (bFGF) for 3 days to induce definitive endoderm. Cells were further cultured for 8 days with 100 ng/ml hepatocyte growth factor (HGF) to generate hepatocytes. Characterization was performed by RT-PCR assay. Functional analysis for albumin secretion and ammonia removal was also carried out. iPS cell-derived hepatocyte-like cells (iPS-Heps) were obtained at the end of the differentiation program. Expression levels of a gestational hepatocyte gene and lineage-specific hepatic genes intensified in iPS-Heps. The production of albumin increased in a time-dependent manner. iPS-Heps were capable of metabolizing ammonia. We present here instant hepatic differentiation of mouse iPS cells using combined 3-day treatments of activin A and bFGF with subsequent 8-day HGF. Our study will be an important step to generate hepatocytes from human iPS cells as a new source for liver-targeted cell therapies.