肿瘤干细胞与肝癌干细胞

肿瘤起源于干细胞的假说正在各种人类肿瘤中得到证实,肿瘤不单是一种基因病,而且是一种干细胞病,基因突变作用于干细胞,干细胞突变成为肿瘤干细胞,这是肿瘤发生、再生、转移和复发的关键。肝细胞癌是最常见的恶性肿瘤之一,其中是否存在“肝癌干细胞”的问题一直倍受人们关注。该文介绍肿瘤干细胞与肝癌干细胞的研究情况。     

间充质干细胞及其作用的研究新进展

间充质干细胞(MSCs)可以成功地从所有哺乳动物组织细胞内分离出来,它的多向分化潜能在再生医学中有很大应用潜力.随着对MSCs的生物学特征、MSCs与其周围内环境之间的相互作用、MSCs归巢的分子调节机制及多向分化能力的深入研究,MSCs将作为细胞修复或外源性基因转染和表达的载体在临床试验中发挥重要作用.     

Characterization of Two Monoclonal Antibodies Raised Against Human Testicular Cells/Charakterisierung von zwei monoklonalen Antikörpern gegen menschliche Hodenzellen

Summary: Human testicular cells isolated from biopsy tissue were used for generation of monoclonal antibodies. Two hybridoma supernatants C3 and D4 were selected according to their reaction with sperm-precursor cells (immature sperms) in an enzyme-linked immunosorbent assay (ELISA). C3 reacted with testicular but no other tissue. D4 did not reveal any pattern of testicular staining in spite of its similarity to C3 in binding to sperm-precursor cells in ELISA and microcytotoxicity test.
Zusammenfassung: Für die Bildung von monoklonalen Antikörpern wurden menschliche Keimzellen verwendet, die aus dem Gewebe von Hodenbiopsien stammten. Zwei Hybridom-Überstand-Lösungen, C3 und D4, wurden hinsichtlich ihrer Reaktion mit Spermatozoen-Precursorzellen (unreife Spermatozoen) in einem ELISA-Test selektiert. C3 reagierte mit Hoden-, aber nicht mit anderem Gewebe. D4 deckte keine weiteren Muster in der Hodenfärbung auf, ungeachtet der Ähnlichkeit zu C3 hinsichtlich der Bindung der Spermatozoen-Precursorzellen an ELISA und an den Mikrocytotoxizitätstest.     

热损伤与碱性成纤维细胞生长因子联合处理诱导表皮细胞去分化的实验研究

目的：体外建立表皮细胞去分化模型,为深入阐明表皮细胞去分化过程奠定基础。方法：离体条件下选择热损伤与碱性成纤维细胞生长因子（bFGF）作为联合诱导因素处理成熟表皮细胞。通过Giemsa染色和电镜观察细胞形态的改变;通过细胞培养和滋养层培养体系观察细胞的增殖能力,包括克隆形成和复层生长情况;采用器官培养体系体外构建三维皮肤,观察细胞再分化形成表皮情况;应用基因芯片技术观察细胞基因表达的改变。结果：经联合诱导后,细胞形态发生改变,表现为体积减小,细胞器数目减少,核浆比增大。增殖和再分化能力的改变体现在诱导后的表皮细胞能形成克隆,在滋养层培养条件下可以形成复层生长,并能够在体外构建出包括基底层和基底上层在内的表皮结构。基因水平的改变表现为与角质化相关基因下调和与有丝分裂相关基因上调。结论：在热损伤与bFGF联合诱导下,已分化的表皮细胞会发生形态、功能和基因表达的改变,表现为干细胞的特性。     

Cell proliferation studies in the rat prostate: II. The effects of castration and androgen-induced regeneration upon basal and secretory cell proliferation

The changes over short and prolonged periods (up to three months) after castration on the proliferative activity of basal and secretory epithelial cells in the rat prostate were studied. Although castration induced widespread apoptosis of the secretory cells, no compensatory hyperplasia of the basal cells in response to this was noted. Instead, observations of the cell kinetics and ultrastructure suggested that both the basal and secretory cells entered a quiescent state as a result of castration. The proliferative potential of secretory cells was not diminished up to three months after castration. During androgen-induced regeneration of the prostate the pattern of basal and secretory cell proliferation was found to be similar to that observed during normal growth, although it was more rapid and of shorter duration.     

微波消融联合树突状细胞瘤内注射对荷瘤小鼠T淋巴细胞亚群的影响

目的 观察微波消融联合树突状细胞(DC)瘤内注射对荷瘤小鼠T淋巴细胞亚群的影响.方法 建立BALB/C小鼠皮下骨髓瘤模型,分为对照组、微波消融组、微波消融联合不成熟DC组、微波消融联合成熟DC组,分别于微波消融后第1、3、5、7、9及11天采用流式细胞仪检测各组小鼠外周血T淋巴细胞亚群.结果 对照组小鼠的CD3~+、CD4~+及CD4~+/CD8~+值逐渐下降(P<0.05);微波消融组小鼠的CD3~+、CD4~+及CD4~+/CD8~+无明显改变(P>0.05);微波消融联合不成熟DC组小鼠的CD3~+、CD4~+及CD4~+/CD8~+在术后第3、5、7天明显减低(P<0.05),第9、11天恢复;微波消融联合成熟DC组小鼠CD3~+、CD4~+及CD4~+/CD8~+则在术后第3、5、7天明显增高(P<0.05),第9、11天下降.结论 微波消融联合成熟DC在一定时间内(约1周)能够增强实验小鼠的抗肿瘤免疫反应.     

Germline-competent stem cell in avian species and its application

Germ cells are the only cell type in the body that can transfer genetic information to the next generation. Germline-competent stem cells can self-renew and contribute to the germ cell lineage giving rise to pluripotent stem cells under specific conditions. Hence far, studies on germline-competent stem cells have contributed to the generation of avian model systems and the conservation of avian genetic resources. In this review, we focus on previous studies on germline-competent stem cells from avian species, mainly chicken germline-competent stem cells, which have been well established and characterized. We discuss different sources of germline-competent stem cells and recent advances for the future applications in birds.     

Stem cells: novel players in the treatment of erectile dysfunction

Stem cells are defined by their capacity for both self-renewal and directed differentiation; thus, they represent great promise for regenerative medicine. Historically, stem cells have been categorized as either embryonic stem cells (ESCs) or adult stem cells (ASCs). It was previously believed that only ESCs hold the ability to differentiate into any cell type, whereas ASCs have the capacity to give rise only to cells of a given germ layer. More recently, however, numerous studies demonstrated the ability of ASCs to differentiate into cell types beyond their tissue origin. The aim of this review was to summarize contemporary evidence regarding stem cell availability, differentiation, and more specifically, the potential of these cells in the diagnosis and treatment of erectile dysfunction (ED) in both animal models and human research. We performed a search on PubMed for articles related to definition, localisation and circulation of stem cells as well as the application of stem cells in both diagnosis and treatment of ED. Strong evidence supports the concept that stem cell therapy is potentially the next therapeutic approach for ED. To date, a large spectrum of stem cells, including bone marrow mesenchymal stem cells, adipose tissue-derived stem cells and muscle-derived stem cells, have been investigated for neural, vascular, endothelial or smooth muscle regeneration in animal models for ED. In addition, several subtypes of ASCs are localized in the penis, and circulating endogenous stem cells can be employed to predict the outcome of ED and ED-related cardiovascular diseases.     

人脂肪组织来源的基质细胞分化为神经元样细胞的体外研究

目的 探讨人脂肪组织来源的基质细胞（adipose tissue-derived stromal cells，ADSCs）向神经元样细胞分化的可能性，为神经移植探索新的细胞来源。方法 采用胶原酶消化法分离培养成人的ADSCs，含血清的培养基进行培养，胰蛋白酶消化传代，采用第3～9代的ADSCs进行诱导。应用异丁基甲基黄嘌呤、消炎痛、胰岛素和地塞米松，诱导其向神经元样细胞和脂肪细胞分化，采用苏丹黑B和免疫细胞化学方法对ADSCs进行鉴定。结果 成功培养出ADSCs来源的基质细胞，细胞在体外生长形态类似成纤维细胞，可维持在未分化状态并稳定增殖，体外扩增可达20代。细胞表达波形蛋白和巢蛋白，大部分细胞还表达平滑肌肌动蛋白和βⅢ管蛋白；应用异丁基甲基黄嘌呤、消炎痛、胰岛素和地塞米松，可诱导ADSCs向神经元样细胞和脂肪细胞分化，其中0．1％～0．2％的细胞分化为神经元样细胞，40％～50％分化为脂肪细胞。分化的神经元样细胞具有典型的神经元形态，并能表达神经元标志物；部分分化的神经元样细胞仍然表达平滑肌肌动蛋白。结论 脂肪组织中存在能分化为神经元样细胞的基质细胞，并能克服间充质细胞的限制，分化为神经元样细胞，但这种细胞是否为有功能的神经元，还需深入研究。     

The effect of organ preservation solutions on kidney tubular and endothelial cells

Organ preservation solutions have primarily been tested in whole organ animal models. In the current study, we have examined the effect of commonly used organ preservation solutions on both kidney tubular and endothelial cells. Primary human endothelial and kidney tubular cells were incubated at 4 °C in the following solutions: 0.9 % saline (NS), EuroCollins (EC), University of Wisconsin (UW), or Hank's balanced salts with 5 % polyethylene glycol (PEG). Cell viability was assessed by colorometric measurement of mitochondrial reduction of 3 (4,5-dimethylthiazol-2-yl)-2-,5-diphenyltetrazolium bromide (MTT) to purple 1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan. After hypothermic storage, cells were incubated at 37 °C in media with MTT, and the amount of reduced formazan present was quantified. Endothelial cells preserved in PEG displayed the best viability (P < 0.05). UW provided better cellular viability than EC or NS (P < 0.05). Control endothelial cells preserved in culture media at 37 °C displayed the highest absorbance values (P < 0.01).For kidney tubular cells, UW and PEG provided the best cellular protection (P < 0.05). Control kidney tubular cells cultured in complete media at 37 °C displayed the highest absorbance values (P < 0.01). Although the model presented here was not part of a truly morphological study, it may be more reliable for the rapid assessment of preservation-induced cell injury than models presented in previous morphological studies and may help in the development of improved preservation techniques. Received: 11 March 1997 Received after revision: 20 June 1997 Accepted: 1 August 1997     

Multinucleate epithelial cells in the ductuli efferentes of human epididymis: Mehrkernige Epithelzellen in den Ductuli efferentes des menschlichen Nebenhodens

The histological and ultrastructural study of the ductuli efferentes in epididymides from 40 adult men revealed the occurrence of multinucleate epithelial cells in all specimens. These cells appeared in the luminal protrusions of epithelial folds and correspond to either principal or ciliated cells. The ultrastructure of their cytoplasm did not differ from that of their respective mononucleate cells. Multinucleate cells contained 3-20 closely juxtaposed nuclei, thus appearing much more irregularly outlined than those of the mononucleate epithelial cells. Multinucleation four times more frequent in the principal cells than in the ciliated cells. The number of multinucleate cells increased progressively from the age of 60 years onwards. The average number of nuclei per cell increased in the fourth decade of life, was maintained up to the eighth decade, and then increased again.     

The Characterisation of Non-Sperm Cells in the Ejaculates of Fertile Men Using Transmission Electron Microscopy/Charakterisierung von nicht-Spermatozoen-Zellen in den Ejakulaten von fruchtbaren Männern unter Verwendung der Transmissions-Elektronen-Mikroskopie

The non-spermatozoal cells (NSC) in the semen samples of 20 fertile human males have been studied by transmission electron microscopy (TEM), in order to accurately distinguish between the different types of cell present and to give a quantitative profile of their relative proportions. The main seminal constituents of the average fertile man were found to be the germinal elements, accounting for 84.0% of the total NSC population. Of this percentage the anucleate bodies, designated "CM" in this study, were the greatest component (43.0%, SEM +/- 4.7). Spermatids were the next commonly occurring cell (22.2%, SEM +/- 2.9), the anucleate cellular masses with organelles, CM(O)'s and the spermatocytes making up the remaining 18.8%. Leucocytes accounted for 13% of the total NSC, respectively: neutrophils - 12% (SEM +/- 4.5); macrophages - 0.9% (SEM +/- 0.3) lymphocytes - 0.1% (SEM +/- 0.1). The remaining 3% consisted of 2.3% (SEM +/- 0.7) epithelial cells and 0.7% (SEM +/- 0.4) Sertoli cells. The principal conclusions reached were: that some normal ejaculates host active phagocytosis and possibly macrophage activation; that anucleate bodies which form a major component of the ejaculate merit further quantitative study and that the shedding of spermatids is an interesting and important aspect of ejaculation.     

人脂肪间充质干细胞向表皮细胞表型转化的研究

目的:探索人脂肪间充质干细胞(adipose derived mesenchymal stem cells,ADSCs)向表皮细胞表型转化的方法。方法:以手术中剩余的人皮下脂肪组织为材料来源,利用胶原酶消化法分离ADSCs,流式细胞仪检测细胞表面标记CD29、CD90、CD105的表达,成脂诱导后油红O染色鉴定。实验组利用第四军医大学西京医院烧伤与皮肤外科实验室已成功构建的pc DNA3.1(+)/SP+EGF质粒经脂质体介导转染的Ha Cat细胞,与ADSCs在Transwell小室中共培养,对照组为ADSCs加入10%FBS培养基,14天后Realtime-PCR检测两组ADSCs中CK19、integrin-β的m RNA表达量。结果:实验组ADSCs中CK19、integrin-β的m RNA表达量较对照组明显升高,且差别有统计学意义。结论:转染EGF的Ha Cat细胞可诱导ADSCs向表皮细胞表型分化,从而为其成为组织工程理想的种子细胞提供了进一步的支持。     

大鼠脐血来源单个核细胞诱导为内皮祖细胞的免疫荧光检测

目的脐血（umbilical cord blood,UCB）中含有大量内皮祖细胞（endothelial progenitor cells,EPCs）,内皮祖细胞（EPCs）是血管内皮细胞的前体细胞,有分化为血管内皮细胞的能力,为骨组织工程血管化带来新的途径。本实验研究大鼠脐血来源的单个核细胞体外诱导为EPCs的可能性。方法分离大鼠脐血单个核细胞,血管内皮诱导液（添加10％胎牛血清,1％青霉素,1％链霉素,VEGF20ug／L,IGF-12ug／L,bFGF2ug／L,EGF20ug／L）培养分化,于3周、6周进行免疫荧光染色检测贴壁细胞CD34、CD133、Flk-1、vWF表面标志。结果培养三周单个核细胞CD34、CD133、Flk-1、vWF抗体免疫荧光检测均为阳性;六周免疫荧光染色可见CD34、Flk-1、VWF组为阳性,CD133组部分为阳性,阴性对照组未发现阳性细胞。结论大鼠脐血来源的单个核细胞在体外诱导3周可以分化为EPCs,6周部分细胞分化为成熟血管内皮细胞。     

间充质干细胞来源微囊泡对卵巢颗粒细胞的作用

目的探讨间充质干细胞来源微囊泡(MVs)对卵巢颗粒细胞的作用。方法取传代第8代(P8)前的人脐带间充质干细胞进行无血清培养,超速离心结合蔗糖密度梯度离心法从培养液上清中分离纯化MVs,透射电镜形态学观察,Bradford法蛋白定量。将PKH26标记的不同浓度MVs(0、10、15、20、25μg/ml)与3周龄雌性SD大鼠卵巢颗粒细胞共培养,3h后荧光显微镜观察颗粒细胞对MVs的内化作用;48h检测培养细胞周期各时相的变化及培养液上清中E2浓度。结果随培养时间的延长,MVs释放量显著增加,8×105个细胞大约释放10μg左右的MVs,电镜下MVs呈圆形或椭圆形,直径介于30~1 000nm之间,中间为低电子密度区。与MVs共培养3h后,颗粒细胞胞浆中可见呈红色荧光信号的MVs;不同浓度的MVs均可显著增加颗粒细胞的增殖指数(PI)、S期细胞比率(SPF)及E2水平,且呈先上升后下降趋势(P0.05)。结论MVs能够被颗粒细胞摄取;人脐带间充质干细胞来源的MVs能够促进颗粒细胞增殖及分泌E2功能。     

多因子联合诱导人胚胎干细胞定向分化为肝细胞样细胞

目的 建立有效的体外诱导人胚胎干细胞(hESCs)分化为肝细胞样细胞的培养体系.方法 将H9 hESC细胞株接种到基底膜提取物包被的培养板上,序贯加入含有下列诱导因子的分化培养基诱导分化:100μg/L细胞因子活化素A( activin A)诱导3d,20 μg/L骨形成蛋白4(BMP-4)和10 μg/L碱性成纤维细胞生长因子(bFGF)诱导4d,10μg/L肝细胞生长因子(HGF)诱导5d,最后以含10 μg/L制瘤素M(OSM)及1×10-7 mol/L地塞米松(Dex)的培养液继续诱导4d.于细胞诱导分化第16天,采用逆转录-聚合酶链反应(RT-PCR)及免疫荧光检测分化细胞肝脏特异性基因和蛋白表达水平;过碘酸-雪夫反应(PAS)试验和吲哚氰绿(ICG)摄取试验检测分化细胞是否具备肝细胞功能.结果 activin A、BMP-4、bFGF、HGF、OSM和Dex联合诱导的分化细胞具有类似肝细胞的形态结构,呈多角形或卵圆形;RT-PCR结果显示分化第16天的细胞表达甲胎蛋白(AFP)、细胞角蛋白7(CK7)、细胞角蛋白19(CK19)、肝细胞核因子-4α(HNF4-α)、1-抗胰蛋白酶(AAT)等肝脏特异性基因;免疫荧光化学检测结果显示分化第16天的细胞表达AFP、ALB、CK19、CYP7A1等肝脏特异性蛋白;PAS试验及ICG试验显示分化细胞具备糖原合成和ICG摄取释放等肝细胞功能.结论 体外联合多种诱导因子可诱导hESCs定向分化为肝细胞样细胞.     

大鼠神经干细胞与小鼠雪旺细胞混合培养的研究

外培目的观察小鼠雪旺细胞体养时对大鼠神经干细胞存活及分化的影响。方法获取Wistar鼠坐骨神经并采用组织块法分离和纯化雪旺细胞；体外分离新生乳鼠脑神经干细胞，将雪旺细胞和神经干细胞分别培养扩增后进行共培养。共分5组进行：实验组1（NSC悬浮＋SC悬浮＋DMEM／F12）；实验组2（NSC悬浮＋SC贴壁＋DMEM／F12）；试验对照组1（SC培养基＋NSC＋DMEM／F12）；试验对照组2（EGF／bFGF＋NSC＋DMEM／F12）；试验对照组3（NSC＋DMEM／F12）。倒置相差显微镜对各组培养细胞每天观察形态和计数，免疫组织化学检测混合培养细胞特异性标记物的表达：SC采用P0和S100，NS采用nestin标记，神经干细胞分化神经元分别采用GFAP、GalC、Tubulin-β染色。结果共培养组NF染色阳性神经元样细胞的百分率明显高干其他各组；实验组1克隆球直径明显高于其他各组，其平均直径为8μm；实验组神经元样细胞突起的长度比对照组的长，3周长度差为26．5-67．3μm。结论大鼠神经干细胞与小鼠雪旺细胞共培养使两者不仅能够共生，而且雪旺细胞能显著促进体神经干细胞分化为神经元样细胞；神经干细胞分化神经元突起增长并且有序排列成轴索样结构。