用低形态学评分D3胚胎建立人胚胎干细胞系

目的 寻找人胚胎干细胞(hESC)建系材料来源.方法 选用IVF低形态学评分的D3胚胎行序贯囊胚培养,用免疫外科的方法去除滋养细胞,将得到的内细胞团(ICM)接种于丝裂霉素C灭活的小鼠胚胎成纤维细胞(MEFs)上培养5～8 d,每4～7 d传代1次,分别取不同代的hESC进行碱性磷酸酶(AKP)染色、转录因子OCT-4、阶段特异性胚胎抗原(SSEA)SSEA-4、SSEA-1、肿瘤排斥抗原(TRA)TRA-1-60、TAR-1-81、核型及体内外分化全能性鉴定.结果 130枚废弃的D3低形态学评分(评分＜16)的胚胎培养出囊胚19枚,获得原代克隆5个,成功培养出两株hESC系,它们具有hESC的共同的生物学特性.结论 部分低形态学评分的D3废弃胚胎可发育成囊胚.囊胚形成率与形态学评分相关,这些胚胎可作为建立hESC系的材料来源之一.     

慢病毒介导绿色荧光蛋白转染人胚胎干细胞及其培养

目的 稳定培养人胚胎干细胞,并通过慢病毒载体对其进行绿色荧光蛋白标记.方法 利用小鼠胚胎成纤维细胞作为饲养层或Matrigel作为基质培养人胚胎干细胞,包装带有GFP序列的慢病毒转染人胚胎干细胞.对转染前后的人胚胎干细胞进行了碱性磷酸酶和SSEA-3免疫组化鉴定.结果 在MEF饲养层和Matrigel上均可培养出呈克隆样生长,表达标志抗原的人胚胎干细胞,经慢病毒转染及抗生素筛选后仍可稳定表达GFP.结论 成功地培养了人胚胎干细胞系,并进行了GFP标记.     

稳定表达GFP的胚胎干细胞株的建立及其生物学特性研究

目的建立稳定表达绿色荧光蛋白(GFP)的胚胎干细胞株,为探索胚胎干细胞及其衍生细胞移植后在体内的分化、迁移及整合提供细胞模型。方法构建含Neo抗性基因质粒pCX-EGFP-Neor并用脂质体转染胚胎干细胞系ES-D3,G418筛选得到稳定表达的细胞株。细胞计数检测其倍增时间、流式细胞仪分析细胞周期,酶组织化学检测碱性磷酸酶(AKP)的表达,免疫组化检测阶段特异性胚胎抗原-1(SSEA-1)并观察其体外分化能力。结果该细胞株可在体外形成拟胚体及各种形态的成熟细胞且均可发出绿色荧光。该细胞株的倍增时间约为12h,G0/G、G2/M、S期分别为22.16±2.03%、18.46±1.54%、59.38±5.76%。倍增时间和细胞周期与未转染GFP的ES-D3相比没有显著性差异(P>0.05)。碱性磷酸酶(AKP)和SSEA-1呈强阳性表达。结论我们成功地建立了稳定表达GFP的胚胎干细胞株,且其生物学特性未受到GFP的影响。     

食蟹猴胚胎干细胞的培养及向神经细胞诱导分化

目的 建立食蟹猴胚胎干细胞系体外培养体系,并诱导其向神经细胞分化,为在体移植实验奠定基础。方法 用小鼠胚胎成纤维细胞作为滋养层,长期培养食蟹猴胚胎干细胞。在无血清培养基中添加小鼠重组Noggin的方式诱导其向神经细胞分化,并对分化各阶段细胞进行免疫组化染色检测分化效果。结果 食蟹猴胚胎干细胞在滋养层上成克隆样生长,可以长期扩增超过20代并保持胚胎干细胞的特性。诱导向神经细胞分化约14d,即可形成呈玫瑰花环样的神经前体细胞结构,可见大量Nestin阳性细胞,及部分Tuj-1阳性细胞;分化约21d时,可见大量Nestin阳性细胞以及大量Tuj-1阳性细胞;分化超过35d,可见GFAP阳性细胞,而Tuj-1阳性细胞减少。结论 成功建立食蟹猴胚胎干细胞的培养体系,在此基础上诱导其分化可获得大量神经前体细胞,尤其是早期神经细胞。     

类胚体中残留胚胎干细胞数量与其致瘤性相关性的初步研究

目的 探讨类胚体(EBs)中残留未分化胚胎干细胞(ESCs)的数量与其致瘤性的相关性.方法 小鼠R1胚胎干细胞株,体外类胚体诱导分化10d,流式细胞仪检测残留未分化ESCs表面标志SSEA-1阳性率.将第10天EBs消化打散后重新给予ESCs常规培养体系培养,观察EBs中残留未分化ESCs形态,流式细胞仪检测残留细胞表面标志物;第10天EBs消化打散后以104～2×106细胞量分别注射至裸鼠四肢肌肉内,观察不同细胞数量与畸胎瘤形成的相关性.结果 ESCs分化为EBs 10 d后有(13.5±0.75)%的细胞表达SSEA-1,提示存在残留未分化ESCs.残留未分化细胞生长形态呈克隆样,高表达SSEA-1等未分化ESCs标志.EBs消化打散后仅在注射2×l06个细胞的部位形成畸胎瘤,瘤体组织中存在成熟的内胚层、中胚层和外胚层组织,其余各组均未见畸胎瘤的形成.结论 胚胎干细胞分化为类胚体后仍存在残留未分化胚胎干细胞,并需要一定细胞数量才具有致瘤性.     

序贯培养法诱导小鼠胚胎干细胞向神经细胞分化的研究

目的诱导小鼠胚胎干细胞向神经细胞分化。方法通过"序贯培养法"诱导小鼠胚胎干细胞分化为神经细胞,并应用RT-PCR、免疫荧光、端粒酶活性及基因芯片等对诱导结果进行检测。结果分化细胞表达神经元特异性烯醇化酶(NSE)、低分子量神经微丝蛋白(NF-L)、神经细胞粘附分子1(NCAM1)、微管相关蛋白Tau等多种神经元标志物,免疫荧光显示NSE阳性率为(81±9.2)%,而神经胶质细胞原纤维酸性蛋白(GFAP)几乎不表达。基因芯片分析结果显示,在3张芯片中有8179个基因共表达,另有998个基因的表达完全不同,且有多种神经元基因的标志物表达明显升高。结论通过"序贯培养法"能有效地诱导小鼠胚胎干细胞向神经元分化。     

小鼠胚胎干细胞的体外培养及诱导分化成酪氨酸羟化酶阳性神经元

探索小鼠胚胎干细胞 (ES)在体外培养及向酪氨酸羟化酶阳性神经元诱导分化的可能性。将小鼠胚胎干细胞在含有白血病抑制因子 (LIF)的ES培养基中扩增 ,并通过以下几个步骤 :胚胎体的形成、巢蛋白 (Nestin)阳性细胞的筛选、Nestin阳性细胞的体外扩增及撤除碱性成纤维细胞生长因子等后观察向酪氨酸羟化酶阳性神经元的分化。结果表明小鼠胚胎干细胞在含有LIF的特定培养基中能够稳定传代并保持不分化状态 ,经过无血清培养基的筛选和培养 ,在SonicHedgehog(SHH)及成纤维细胞生长因子 (fibroblastgrowthfactor 8,FGF8)等细胞因子的作用下能定向分化成酪氨酸羟化酶阳性神经元。这种方法有望为帕金森病等神经变性病的细胞移植治疗提供充足的细胞来源。     

维甲酸诱导小鼠胚胎干细胞向神经样干细胞分化

目的探讨维甲酸（RA）诱导体外培养的小鼠胚胎干细胞（ESC）向神经样干细胞定向分化。方法在无白血病抑制因子（LIF）存在的条件下,将体外培养的ESC悬浮培养4天,形成拟胚体（EB）,再经RA诱导4天后进行细胞冰冻切片,行免疫细胞化学染色计数生殖特异性基因Oct-4和神经干细胞特异性标志物Nestin的表达百分率。结果经诱导的ESC呈现Oct-4阳性细胞百分率为67．34％,而Nestin阳性细胞百分率为36．52％。结论体外培养的小鼠ESC经RA诱导后有部分细胞定向诱导分化为神经样干细胞。     

体外分化胚胎干细胞为造血干细胞重建造血功能的研究

目的：探讨体外定向分化胚胎干细胞（ESCs）为造血干细胞（HSCs）对体内造血功能的重建作用。方法：将小鼠E14.1胚胎干细胞采用“三步诱导法”在体外分化发育为HSCs，造血克隆形成(CFU)实验观察其体外造血集落形成情况，免疫磁珠分选纯化HSCs移植给经亚致死剂量γ射线照射的雌性SCID小鼠，观察其植入及小鼠造血功能恢复情况。结果: 经过分阶段诱导，多种造血刺激因子联合应用能有效促进ESCs定向分化发育为HSCs,流式细胞仪检测HSCs特异性表面标志物CD34+/Sca-1+表达最高为（58.64±4.20）%，CFU培养能形成较多的红系、粒系/巨噬细胞系及混合细胞集落, Wright-Giemsa 染色显示为原始的造血细胞。此阶段的HSCs经分选纯化后移植给经γ射线照射后的小鼠，移植组小鼠+10 d造血功能开始恢复，观察40 d后除血小板恢复较慢外，白细胞、红细胞、血红蛋白等指标已接近正常，植入率为71.4%，存活率为43.0%，染色体检测证实已由受体鼠的XX转为供体鼠的XY。结论: 采用分阶段诱导的方法，可在体外定向诱导小鼠ESCs分化发育为HSCs，此来源的HSCs可以有效重建体内造血功能。     

体外定向诱导胚胎干细胞分化为表皮样干细胞的研究

目的　探索体外定向诱导小鼠胚胎干细胞 (embryonicstemcell,ES)分化为表皮样干细胞的条件 ,为胚胎干细胞来源的表皮样干细胞的临床应用 ,及ES细胞的定向分化机制的研究奠定基础。　方法　采用与人羊膜共培养法对小鼠胚胎干细胞进行定向诱导。实验分 3组 :1 羊膜上皮面向上 ,铺布全孔底 ;2 羊膜上皮面向上 ,铺布半孔底 ;3 以不加羊膜组为对照。用流式细胞仪、免疫组织化学技术对分化后的细胞进行鉴定。　结果　共培养 3～ 4d后 ,在第 1、2实验组中的人羊膜上皮面上 ,小鼠ES细胞形成表皮样干细胞集落 ,表达高水平的表皮干细胞特异标志物整合素 β1 、CK19和CK15。流式细胞仪检测结果显示 :整合素 β1 、CK19和CK15阳性细胞比率均较对照组有显著差异 (P <0 0 1)。而在第 2实验组中无羊膜覆处 ,细胞贴壁生长 ,形成表皮样细胞单层 ,细胞呈多边形 ,排列紧密 ,表达整合素 β1 ,仅见少数CK19和CK15阳性细胞散布于表皮样细胞之间。流式细胞仪检测结果显示 :整合素 β1 阳性细胞率与对照组有显著差异 (P <0 0 1) ,而CK19和CK15阳性细胞率与对照组差异不明显。　结论　人羊膜可诱导小鼠胚胎干细胞向表皮样细胞定向分化 ,并提示生长在羊膜上皮面上的细胞克隆可能是表皮样干细胞 ,而贴壁生长的细胞可能是表皮样细     

Development of novel markers for the characterization of chicken primordial germ cells

This study was undertaken to develop novel markers for chicken primordial germ cells (PGCs), which are of potentially enormous value in transgenic research. Gonadal cells collected from 5.5-day-old chicken embryos were cultured in a Dulbecco's minimal essential medium and the PGC colonies formed during the primary culture period were subcultured three times. Characterization of the PGCs with the candidate marker reagents was performed on the mixed cell population 2 hours after seeding, after the primary culture period (day 10), and after the third passage (day 40). Mouse embryonic stem (ES) cells were used as controls. The cytochemical reagents investigated included periodic acid-Schiff (PAS) stain, antibodies to stage-specific embryonic antigens (SSEA-1, SSEA-3, and SSEA-4), antibody to epithelial membrane antigen (EMA)-1, antibodies to integrins alpha6 and beta1, several lectins (Solanum tuberosum agglutinin [STA], Dolichos biflorus agglutinin [DBA], concanavalin A agglutinin [ConA], and wheat germ agglutinin [WGA]), and double staining with antibodies to SSEA-1, SSEA-3, SSEA-4, integrin alpha6, or integrin beta1 and then with the lectin STA. Densitometric quantification was used to identify PGC-specific markers. The results showed that chicken PGCs were stained selectively by PAS and by antibodies to SSEA-1, SSEA-3, SSEA-4, EMA-1, integrin alpha6, and integrin beta1. The control mouse ES cells reacted with PAS, anti-SSEA-1, and anti-EMA-1 antibodies, as well as with antibodies to integrins alpha6 and beta1, but not with antibodies to SSEA-3 and SSEA-4. Chicken PGCs reacted with the lectins STA and DBA, but mouse ES cells reacted with STA and WGA. The results of double staining of PGC colonies subcultured three times showed that the intensity of staining was not altered by concomitant use of the marker reagents. This study demonstrated that, in addition to PAS and antibodies to SSEA-1 and EMA-1, new specific markers of chicken PGCs are recognized by the lectins STA and DBA and by antibodies to SSEA-3 and SSEA-4 and integrins alpha6 and beta1. Double staining using these newly developed markers might be the method of choice for rapid characterization of chicken PGCs.     

Lectin binding characteristics of mouse placental cells

The lectin binding characteristics of mouse placental cells were examined. Wax embedded tissue sections of placentae from d 14 pregnant mice were stained with 26 lectins, with a wide range of sugar specificities. Cell cultures prepared from d 14 mouse placentae and cultured for 24 h were stained with 7 of the lectins to determine if they could be used as markers for the different trophoblast cells in culture. In tissue sections all placental cell populations bound lectin but no lectin bound specifically to any single trophoblast population. All the lectins which bound to layer 1 cytotrophoblast lining the maternal blood spaces of the labyrinthine placenta also bound to the fetal endothelium of the labyrinthine placenta. Binding of lectin appeared strongest on the adluminal membrane of these cell populations suggesting a role for the carbohydrate moieties in nutrient transfer. Few lectins bound to junctional zone trophoblast. Overall, the binding of lectin to cultured cells did not correlate exactly with lectin binding to the cell populations in tissue sections. The value of lectins as markers for placental cells in culture was therefore found to be limited. Our findings indicate that carbohydrate expression by at least some placental cells may vary in culture from that expressed by the cells in vivo with obvious concerns for the validity of functional in vitro studies.     

Antibodies against surface antigens common to spermatogenic and F9 teratocarcinoma cells in mice immunized with blood group substances from human saliva

Significant inhibition of spermatogenesis and production of antibodies against membrane antigens of spermatogenic and F9 teratocarcinoma cells were observed in mice of the strain 129/Sv after immunization with human blood group substances from saliva of A, B or H secretors. Absorption of the mouse anti-ABH sera with appropriate human erythrocytes did not change their reactivity with testicular and F9 cells, whereas absorption with F9 cells eliminated the reactivity with both F9 and spermatogenic cells. This pattern of reactivity, together with higher binding of the anti-ABH sera to the cells expressing stage-specific embryonic antigen 1 (SSEA-1), suggests that these antisera contain antibodies against developmentally regulated carbohydrate antigens. SSEA-1 was found in the blood group substances used for immunization. The results support the hypothesis that the oncofetal F9 antigens and spermatogenic differentiation antigens are similar carbohydrate structures.     

Light and electron microscopic studies of lectin binding on the glycocalyx of rat pancreatic cells. I. Normal tissue and isolated cells.

Lectin binding of the glycocalyx of pancreatic tissue sections as well as of isolated pancreatic acini and acinar cells was studied of healthy wistar rats by light and electron microscopy. For light microscopy, we used FITC (WGA, RCAI, LCA) or peroxidase marked (WGA, RCAI, PNA, PHA, LCA, UEAI, LPA) as well as unmarked lectins (Con A, VAA I). Gold marked lectins were used for electron microscopy (WGA, RCAI, LCA, HPA, PNA, VAAI-B). Intact acinar cells in pancreatic tissue sections and isolated acini showed a strong binding of WGA, RACI, and HPA on the apical cell surface, whereas VAAI, UEAI, LCA, and Con A reacted strongly with the basolateral glycocalyx, but not with the apical surface. The 2 main domains of the glycocalyx of pancreatic cells showed their specific lectin binding so long as the junctional complexes between the cells are intact. The polarity of the cell surface of pancreatic acinar cells is discussed in regard to the possible function of the 2 domains.     

Characterisation of glycoconjugate sugar residues in the vomeronasal organ of the armadillo Chaetophractus villosus (Mammalia, xenarthra)

Conventional carbohydrate histochemistry and the binding patterns of 21 lectins were analysed to characterise the glycoconjugate content in the components of the vomeronasal organ of the armadillo Chaetophractus villosus . The mucomicrovillous complex of the sensory epithelium bound most of the lectins studied. No reaction was observed with Con A, PSA, S-Con A and SBA, and the sustentacular cells were stained with UEA-I, DSL, LEL, STL and Con A. The vomeronasal receptor neurons were labelled with S-WGA, WGA, PNA, UEA-I, STL, Con A, S-Con A, ECL and RCA₁₂₀. The basal cell layer reacted with S-WGA, WGA, LCA, UEA-I, DSL, LEL, STL, Con A, JAC and VVA. The nonsensory epithelium exhibited a differential staining in relation to the different components. The mucociliary complex stained with ECL, DBA, JAC, RCA₁₂₀, STL, LCA, PHA-E, PHA-L, LEL, BSL-I and VVA. However, SJA and UEA-I stained the mucus complex lining a subpopulation of columnar cells. The cytoplasm and cell membranes of columnar cells was labelled with DBA, DSL and LCA. The apical region of these cells exhibited moderate reactivity with LEL and SJA. None of the lectins bound specifically to secretory granules of the nonsecretory cells. Basal cells of the nonsensory epithelium were labelled with DSL, LEL, LCA, BSL-I and STL. The vomeronasal glands showed a positive reaction with WGA, DSL, LEL, LCA, DBA, PNA, RCA₁₂₀ and SBA. Subpopulations of acinar cells were observed with ECL, S-WGA, Con A, S-Con A and DBA. PNA and RCA₁₂₀ stained the cells lining the glandular ducts. In comparison with previous results obtained in the olfactory mucosa of the same group of armadillos, the carbohydrate composition of the vomeronasal organ sensory epithelium differed from the olfactory sensory epithelium. This is probably related to the different nature of molecules involved in the perireceptor processes.     

Expression of cell-surface antigens and embryonic stem cell pluripotency genes in equine blastocysts

Embryonic stem-like (ES-like) cells have now been derived from the inner cell mass (ICM) of horse embryos at the blastocyst stage. Because they have been shown to express cell-surface antigens found in both human and mouse ES cells, the present study investigated gene expression patterns in day-7 horse blastocysts from which the horse ES-like cells had been derived originally. The genes studied included Oct-4, stage-specific embryonic antigen-1 (SSEA-1), SSEA-3, SSEA-4, tumor rejection antigen-1-60 (TRA-1-60), TRA-1-81, and alkaline phosphatase activity, and whereas all three of the SSEA antigens were expressed in both the ICM and the trophoblast on day 7, Oct-4, TRA-1-60, TRA-1-81, and alkaline phosphatase activity were localized mostly in the ICM. Upon in vitro differentiation of the horse ES-like cells, their expression of the stem cell markers was abolished. Therefore, the species-specific expression pattern of stem cell markers in horse ES-like cells reflects gene expression in the blastocysts from which they are derived.     

Simple, efficient, and reproducible gene transfection of mouse embryonic stem cells by magnetofection

Embryonic stem (ES) cells are recognized as an excellent cell culture model for studying developmental mechanisms and their therapeutic modulations. The aim of this work was to define whether using magnetofection was an efficient way to manipulate stem cells genetically without adversely affecting their proliferation or self-renewal capacity. We compared our magnetofection results to those of a conservative method using FuGENE 6. Using enhanced green fluorescent protein (eGFP) as a reporter gene in D3 mouse ES (mES) cells, we found that magnetofection gave a significantly higher efficiency (45%) of gene delivery in stem cells than did the FuGENE 6 method (15%), whereas both demonstrated efficient transfection in NIH-3T3 cells (60%). Although the transfected D3 (D3-eGFP) mES cells had undergone a large number of passages (>50), a high percentage of cells retained ES markers such as Oct-4 and stage-specific embryonic antigen-1 (SSEA-1). They also retained the ability to form embryoid bodies and differentiated in vitro into cells of the three germ layers. eGFP expression was sustained during stem cell proliferation and differentiation. This is the first transfection report using magnetofection in ES cells. On the basis of our results, we conclude that magnetofection is an efficient and reliable method for the introduction of foreign DNA into mouse ES cells and may become the method of choice.     

Glycoprotein abnormalities in colonic carcinomata, adenomata, and hyperplastic polyps shown by lectin peroxidase histochemistry.

A technique for lectin-peroxidase histochemistry was adapted for the study of formalin fixed paraffin embedded colonic tissue. Ten lectins with differing carbohydrate binding specificity were tested against 20 normal rectal biopsy specimens and tissue from 19 colonic carcinomata, 19 tubular or tubulovillous adenomata, and 19 hyperplastic polyps. None of the normal rectal biopsy specimens bound the lectins peanut agglutinin (PNA), Griffonia simplicifolia II (GSII), and Ulex europaeus I (UEAI), whereas 18 carcinomata, 12 adenomata, and 18 hyperplastic polyps showed affinity for one or more of these lectins. Hyperplastic colonic polyps are shown to possess similar abnormalities in glycoprotein structure to malignant and adenomatous colonic tissue. This may simply indicate a non-specific reaction to changed rates of cell proliferation but might represent a more fundamental association between hyperplastic polyps and adenocarcinomas.