人胚胎干细胞系NS1的建立

目的:探讨利用长期冷冻的废弃胚胎建立人胚胎干(hES)细胞系.方法:将临床体外受精(IVF)及精子卵浆内注射(ICSI)周期中剩余冷冻5年以上的废弃胚胎复苏后继续培养至扩张囊胚,链霉蛋白酶去除透明带后采用全囊胚培养法获取原代类胚胎干细胞,再用机械法传代分离纯化hES细胞,命名为NS1.取不同代次的培养细胞,进行碱性磷酸酶染色、转录因子OCT-4、阶段特异性胚胎抗原SSEA-4、肿瘤排斥抗原TRA-1-60、TAR-1-81、核型及体内分化实验对NS1进行全能性鉴定.结果:12枚废弃胚胎在体外继续培养后,有4枚发育为囊胚,最终得到1个干细胞系,经鉴定,这些细胞都具有hES细胞的生物学特性.结论:长期冷冻的废弃胚胎可用来建立hES细胞系.     

利用月经血建立的经血源性基质干细胞系

背景：经血源性基质干细胞被证实是一种新型的成体干细胞,目前国内关于其系统的建系方法报道极少。 目的：建立经血源性基质干细胞系,并鉴定其表型和多能性。 方法：收集健康成年女性的月经血,通过密度梯度离心法分离出经血源性基质干细胞进行培养,观察细胞的形态及增殖特性;采用CCK-8法检测各株细胞的增殖能力,定期收集细胞进行核型分析,采用流式细胞术检测细胞表面抗原CD34,CD38,CD44,CD45,CD73,CD90,CD105,Oct-4的表达,对细胞进行体外成脂和成骨诱导分化并鉴定。 结果与结论：成功从6名健康志愿者的经血中分离获得4株经血源性基质干细胞系。细胞呈现典型的梭状结构,具有46,XX核型,能在体外稳定迅速增殖,传代后大约48 h进入对数生长期,表达CD44,CD73,CD90,CD105及Oct-4,不表达CD38,CD34,CD45;成脂诱导22 d后油红O染色阳性,成骨诱导2周后茜素红染色阳性。实验表明经血源性基质干细胞具备扩增迅速、核型稳定、多潜能性等优点,通过建立经血源性基质干细胞系,为进一步的临床治疗应用以及科学研究奠定了基础。  中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

以4种基因诱导产前诊断绒毛细胞建立诱导性多能干细胞

背景：诱导性多能干细胞因具有多能性特征,可以诱导分化为特定的细胞,包括神经细胞、造血细胞等。 目的：建立产前诊断绒毛细胞来源的诱导性多能干细胞。 方法：运用反转录病毒介导4种基因hOct4、hSox2、hc-Myc、hKlf4诱导产前诊断绒毛细胞,对建立的诱导性多能干细胞进行多能性、体内外分化能力、核型等鉴定。 结果与结论：建立的诱导性多能干细胞能维持自我更新状态,在蛋白和mRNA水平上高表达全能性的标志基因,具有体内、外分化潜能;在体外长期培养能维持正常核型。说明4种全能性基因转入绒毛细胞可获得具有多能性的诱导性多能干细胞,这为胎儿的细胞自体移植治疗提供理想来源,为产前诊断疾病机制研究提供很好的细胞模型。     

人诱导性多潜能干细胞系的建立

背景：诱导性多潜能干细胞与肿瘤干细胞的发生过程极其相似,而且具有的干细胞特性极其接近人胚胎干细胞。因此,研究诱导性多潜能干细胞有利于人们进一步认识并了解人类发育以及肿瘤的发生过程。 目的：掌握建立人诱导性多潜能干细胞系的技术,以便为特异性疾病细胞的重编程建立技术平台,从而利用重编程技术研究疾病的发病机制。 方法：将含有Oct4、Sox2、Klf-4和c-Myc 4个转录因子的反转录病毒感染人皮肤成纤维细胞(HS27细胞),在人胚胎干细胞培养条件下诱导产生人胚胎干细胞样的克隆。挑取并进一步扩增,通过克隆形态、碱性磷酸酶活性、免疫荧光检测是否有人胚胎干细胞标记物Oct4、Sox2、c-Myc、Klf-4的表达,悬滴法检测HS27细胞来源的克隆形成畸胎瘤的能力和验证向3个胚层的分化能力。 结果与结论：经病毒感染诱导产生的胚胎干细胞样克隆呈绿色荧光蛋白阴性,克隆在细胞形态方面与人胚胎干细胞克隆相似,进一步扩增经碱性磷酸酶检测克隆呈阳性,免疫荧光检测克隆表达Oct4、Sox2、c-Myc、Klf-4,并且HS27细胞来源的克隆注入免疫缺陷小鼠体内可以形成畸胎瘤并经苏木精-伊红染色显示具有向三胚层分化能力。实验成功构建了人诱导性多潜能干细胞系,为下一步开展疾病细胞特异性重编程研究奠定了良好的实验基础。中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程全文链接：     

人胚胎干细胞系HUES4的培养和染色体核型分析

目的建立人胚胎干细胞无动物源性饲养层培养方法,同时对长时间体外培养的人胚胎干细胞核型变化进行分析。方法人胚胎干细胞系HUES4细胞分别培养于小鼠胚胎成纤维细胞和人包皮成纤维细胞饲养层,并对其干细胞特性进行鉴定;在培养传代过程中,收获P27、P34、P41和P44细胞进行染色体核型分析,P27细胞还进行DNA短串联重复序列多态性分析。结果生长于人包皮成纤维细胞饲养层的HUES4细胞碱性磷酸酶染色以及SSEA-4、TRA-1-60和TRA-1-81抗原阳性,SSEA-1抗原阴性。所检测的4代细胞中均见46,XY/46,XY,t（9;15）（q22;q26）核型嵌合现象,且异常核型百分比随传代次数增加有上升的趋势。结论培养人胚胎干细胞的饲养层细胞可由无动物源性的饲养层细胞替代;长期体外培养有增加细胞染色体核型异常的风险。     

人脂肪干细胞的提取和鉴定

背景：脂肪干细胞是存在于脂肪中的全能干细胞,具备自我更新能力与多向分化潜能,遗传背景相当稳定,体内植入后免疫排斥少,是一种比较理想的种子细胞。目的：提取人大网膜脂肪干细胞,并进行成脂和成骨分化能力鉴定。方法：收集手术患者大网膜的脂肪组织,经Ⅰ型胶原酶消化、过滤、离心后进行原代培养,观察细胞生长状态;用细胞计数法绘制人脂肪干细胞增殖曲线,计算处于对数生长期的倍增时间;用相应的定向诱导液诱导人脂肪干细胞向脂肪细胞及骨细胞方向分化,并用油红O染色、茜素红染色进行鉴定。结果与结论：从手术患者大网膜脂肪组织中成功分离人脂肪干细胞,贴壁生长的人脂肪干细胞为长梭形,形态类似成纤维细胞。第3代人脂肪干细胞生长曲线呈S形,对数生长期人脂肪干细胞的倍增时间为45.90 h。人脂肪干细胞经成脂、成骨定向诱导分化2,3周后,油红O染色显示细胞内有大小不等的橘红色脂肪滴,茜素红染色可见着橘红色的典型钙化结节,提示所培养的脂肪干细胞具有向脂肪细胞、骨细胞系分化的能力。 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

人胚胎干细胞不同传代方法的比较

目的探讨不同传代方法对人胚胎干细胞生长和分化情况的影响。方法用1mg/mlⅣ型胶原酶和机械切割两种不同的方法传代人胚胎干细胞,比较这两种方法传代的人胚胎干细胞克隆生长和分化情况。结果 1.机械切割法传代的人胚胎干细胞克隆较传统酶消化法生长快速且不易分化。2.传代后的人胚胎干细胞仍然维持胚胎干细胞的特有形态：表达胚胎干细胞表面标记,具有正常核型和多潜能分化能力（在体外形成拟胚体在体内形成畸胎瘤）。结论采用机械切割法传代人胚胎干细胞更利于细胞的培养扩增,且传代后细胞在继续培养中仍可保持干细胞的所有特性。     

鸡胚胎干细胞分离方法和培养体系的优化

背景：胚胎干细胞是从动物早期胚胎的内细胞团或原始生殖细胞分离出来的具有发育全能性的一种未分化的无限增殖细胞系。而鸡胚胎干细胞则是从X期鸡胚的胚盘分离而来。 目的：优化鸡胚胎干细胞分离方法和离体培养体系。 方法：采用滤纸纸环-发环的方法从X期鸡胚分离胚盘细胞,并采用STO细胞作为饲养层和大鼠肝细胞(BRL)条件培养基(CM)+细胞因子作为离体培养体系对分离的胚盘细胞进行培养。 结果与结论：滤纸纸环-发环法获得的完整胚盘率为75%~85%,克隆形成率约为50%。BRL-CM+饲养层培养体系,鸡胚胎干细胞可传至7代,而BRL-CM+饲养层+细胞因子培养体系,鸡胚胎干细胞可传至25代。分离到的鸡胚胎干细胞,经碱性磷酸酶染色、SSEA-1染色鉴定,表明鸡胚胎干细胞处于未分化状态。提示,实验不仅优化了鸡胚胎的分离方法,获得完整且杂质少的胚盘,而且进一步优化了鸡胚胎干细胞体外培养体系。     

四倍体滋养层干细胞的分离培养与鉴定

目的建立可在体外培养的小鼠四倍体滋养层干细胞(TTSCs)系,为研究胎盘发育提供新的细胞模型。方法用电融合的方法获得四倍体胚胎并进行体外培养,挑取克隆并传代后获得细胞系。通过中期染色体计数检测染色体数目; RT-qPCR、Western blot、免疫荧光检测标志基因表达;显微注射检测细胞的体内发育潜能。结果建立了可以传代培养并且可在体内发育的TTSCs。分化条件下,TTSCs分化基因的表达量与二倍体滋养层干细胞(TSCs)存在差异(P0.001)。结论可传代的TTSCs系可以在体外建立,但其维持自我更新及分化的机制与二倍体TSCs有所不同。     

骨髓基质细胞对共培养条件下的脊髓源性神经干细胞分化为胆碱能神经元的诱导

目的：研究骨髓基质细胞对共培养条件下脊髓源性神经干细胞分化为胆碱能神经元的情况。方法：从孕龄13 d的胚胎大鼠脊髓组织中分离神经干细胞,采用含EGF及bFGF的无血清限定性培养基培养,并通过与骨髓基质细胞进行共培养,观察脊髓源神经干细胞向胆碱能神经元分化的情况,用细胞免疫荧光染色鉴定分化结果。结果：从胚胎脊髓中分离得到大量的神经干细胞,通过限定性培养基培养可获得干细胞球,与骨髓基质细胞共培养可被诱导分化,用细胞免疫荧光染色鉴定,可见有胆碱能神经元生成。结论：胚胎大鼠脊髓源神经干细胞在添加EGF与bFGF的限定性培养基中可以增殖并保持稳定的性状,在与骨髓基质细胞共培养时,可以被诱导分化为胆碱能神经元。     

A variant Klinefelter syndrome patient with an XXY/XX/XY karyotype studied by GTG-banding and fluorescence in situ hybridization.

Klinefelter syndrome is the first human sex chromosomal abnormality to be reported. The majority of Klinefelter syndrome patients have the XXY karyotype. Approximately 15% of Klinefelter patients, however, are mosaics with variable phenotypes. Among the variant Klinefelter genotypes are such karyotypes as XY/XXY and XX/XXY. The variation in phenotypes most likely depends on the number of abnormal cells and their location in body tissues. In this paper we report the case of a 42-year-old patient with Klinefelter syndrome and a rare variant mosaic XXY/XX karyotype initially identified by GTG-banding. This was confirmed by fluorescence in situ hybridization (FISH) using a dual-color X/Y probe. The patient presented with erectile dysfunction and few other physical findings. Thus, this case illustrates a rare variant of Klinefelter syndrome with a relatively mild phenotype. It also illustrates the utility of FISH as an adjunct to conventional cytogenetics in assessing the chromosome copy number in each cell line of a mosaic. In our case, FISH also detected the presence of a small population of cells with the XY karyotype not previously detected in the initial 30-cell GTG-banding analysis. Thus, through a combination of GTG-banding and FISH, the patient was determined to be an XXY/XX/XY mosaic. Given that most individuals with Klinefelter syndrome are infertile, and that these individuals may wish to reproduce with the aid of modern reproductive technology, such as testicular fine needle aspiration and intracytoplasmic sperm injection, it is important that accurate estimation of the frequency of abnormal cells be obtained for accurate risk estimation and genetic counseling, as recent studies in patients with mosaic Klinefelter syndrome revealed that germ cells with sex chromosomal abnormalities were nevertheless capable of completing meiosis.     

Rare XXY/XX mosaicism in a phenotypic male with Klinefelter syndrome: case report

Klinefelter syndrome represents the most commonly found human sex chromosomal abnormality. It is characterized by small, firm testes with hyalinization of the seminiferous tubules, elevated gonadotropins and azoospermia. Males with Klinefelter syndrome may have a 47,XXY or a mosaic 47,XXY/46,XY constitutional karyotype and varying degrees of spermatogenic failure. Mosaicism 47,XXY/46,XX with clinical features suggestive of Klinefelter syndrome, is very rare and so far only 10 cases have been described in literature [1,2,5,8,10,15,22,23,25,44]. We report here a case of a mosaic 47,XXY/46,XX infertile male in whom detailed cytogenetic, histological and molecular studies were performed. Cytogenetic analysis revealed 80% and 50% mosaicism for the 46,XX cell line in blood lymphocytes and in skin fibroblasts, respectively, and the presence of 47,XXY cells only, in cultured testicular tissue. Testicular histopathology revealed atrophy of the testes with no spermatogenesis and absence of germ cells. Molecular analysis showed paternal inheritance of the extra X chromosome.     

引物原位标记联合核型分析快速检测克氏综合征

目的探索PRINS技术联合G显带核型分析检测克氏综合征染色体。方法对1034例男性不育患者外周血用常规G显带核型分析方法进行分析,对检出的克氏综合征（Klinefelter综合征）患者用引物原位标记（PRINS）技术进行染色体检测,比较分析染色体异常的检出情况。结果用常规G显带核型分析方法检出核型异常患者134例,核型异常比例为12.96%;其中染色体数目异常70例占异常总数的52.23%（Klinefelter综合征患者56例占41.79%）,余下为染色体结构异常64例占47.77%;采用PRINS技术对Klinefelter综合征患者的染色体进行检测,结果与G显带核型分析结果一致。结论与常规核型分析方法相比,PRINS技术可快速、准确检测染色体数目异常。     

Mosaic status in lymphocytes of infertile men with or without Klinefelter syndrome

BACKGROUND: Gonosomal aneuploidies such as Klinefelter syndrome (47,XXY) are the most frequent chromosomal aberration in infertile men. Normally the chromosomal status of patients is detected by karyotyping of up to 20 metaphase spreads of lymphocyte nuclei, whereby low grade mosaicism may be overlooked. To test whether Klinefelter patients with 47,XXY karyotype or infertile men with 46,XY karyotype represent gonosomal mosaicisms, we performed meta- and interphase fluorescence in situ hybridization (FISH) on 45 men. METHODS AND RESULTS: A total of 400 interphase and 40 metaphase lymphocyte nuclei per patient were scored after hybridization with DNA probes specific for chromosomes X and Y, and chromosome 9 as a control. On the basis of conventional karyotype, hormone levels and clinical appearance, patients were subdivided into 18 Klinefelter syndrome patients with 47,XXY (group I), 11 Klinefelter syndrome-like patients with normal karyotype, 46,XY (group II) and six non-Klinefelter-like infertile patients with normal 46,XY karyotype (group III). Ten normal men (group IV) served as controls. Testicular volume in the Klinefelter group I was smaller compared with group II (P = 0.016), group III (P < 0.001) and group IV (P < 0.001). In addition, testicular volumes in group II were lower compared with group III and group IV (P < 0.004). No significant differences between the aneuploidy rate analysed by FISH in interphase nuclei and metaphases were found in either single patients or groups. Patients with Klinefelter syndrome, 47,XXY (group I) or with symptoms similar to those in Klinefelter patients 46,XY (group II) showed a similar aneuploidy rate (group I 7.1 +/- 4.0% and group II 4.6 +/- 3.4%) and two 47,XXY patients with a high prevalence for normal 46,XY lymphocytes had sperm in their ejaculate. However, in general, no correlations between FISH mosaic status and serum hormone parameters, nor with ejaculate parameters were found. CONCLUSIONS: The results suggest that 47,XXY patients with an increased incidence of XY cells (average of 4.2 +/- 2.3) may have a higher probability of germ cells as we found sperm only in the ejaculate of Klinefelter syndrome patients with mosaic 46,XY cells (6.0 and 7.0%). On the other hand, 46,XY patients with mosaic sex chromosome aneuploidies detected by FISH analysis more often show symptoms of hypogonadism phenotypically resembling Klinefelter syndrome.     

人胚胎干细胞HuES17向造血干细胞的分化

背景：人类胚胎干细胞是来源于着床前囊胚的内细胞团,能在长期培养中无限增殖并保持未分化状态,且具有分化成人体组织各种细胞类型能力的细胞。 目的：进一步验证人胚胎干细胞HuES17细胞株向造血干细胞分化的能力。 方法：人胚胎干细胞HuES17采用与人包皮成纤维细胞二维共培养的方式培养,采用人胚胎干细胞与小鼠骨髓基质细胞(OP9) 二维共培养的方法诱导胚胎干细胞向造血干细胞分化。 结果与结论：人胚胎干细胞与小鼠骨髓基质细胞(OP9) 二维共培养诱导造血分化的第四五天即开始出现OP9细胞逐渐老化,很快死亡;可以观察到人胚胎干细胞分化,然而,随着OP9细胞死亡,分化的人胚胎干细胞亦死亡,不能诱导人胚胎干细胞向造血干细胞分化。提示人胚胎干细胞HuES17细胞株可能不能向造血干细胞分化,或向造血干细胞分化的能力较低。     

C57BL/6J×129/J杂交小鼠ES细胞系的建株(英)

 目的：建立C57BL/6J×129/J杂交小鼠ES细胞系。 方法： 收集3.5 d.p.c.的囊胚，培养在预先铺有小鼠成纤维细胞（MEFs）的高糖DMEM培养液中。3-4 d后，挑出内细胞团（ICM），消化后重新种到新鲜的有MEFS培养液中。等到有典型的ES样集落长出，即传代以得到永久ES细胞系。通过分析碱性磷酸酶活性，SSEA-1，Oct-4的表达和形成畸胎瘤的能力来鉴定ES细胞的多向分化能力。 结果： 获得的两个C57BL/6J×129/J杂交小鼠ES细胞系绝大多数细胞具有正常的核型（40，XY），碱性磷酸酶染色阳性，SSEA-1，Oct-4表达阳性， ES细胞注入SCID鼠后可获得来自3个胚层的组织。 结论： 建立了两株具有长期自我更新能力和多向分化潜能的C57BL/6J×129/J杂交小鼠ES细胞系。     

广州地区Klinefelter综合征的临床与细胞遗传学分析

目的 对15例Klinefelter综合征进行分析.方法 外周血淋巴细胞培养染色体核型分析.结果 15例Klinefelter综合征中,典型（47,XXY）占86%,嵌合型（46,XY/47,XXY）占13%,均有不孕或性发育异常的临床表现.结论 男性不育与性发育异常应该行细胞遗传学检查.     

Klinefelter综合征的早期诊断(附14例报告)

本文总结了14例克氏综合征，最小2岁，最大38岁，表现型为男性，但阴茎短小，睾丸小，性成熟期不能产生精子，细胞核型以47，XXY为最多，占80％，嵌合型占10％，罕见核型占10％。产生机制是经细胞分裂过程中发生性染色体不分离所致，其原因有待进一步探索。口腔粘膜X染色质检查对早期诊断克氏综合征具有重要意义，强调对男性新生儿本病普查的必要性。     

A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells

BACKGROUND: Human embryonic stem (hES) cell lines were first cultured using fetal mouse fibroblasts as feeder cells. To avoid feeders and to reduce the amount of xeno-components, Matrigel- and laminin-coated dishes, and conditioned mouse feeder cell medium have been used, and hES cells have also been cultured on human fetal muscle and skin, and adult Fallopian tube epithelial cells. METHODS: We used post-natal, commercially available human foreskin fibroblasts as feeder cells. Inner cell masses (ICM) were isolated from five supernumerary blastocysts, obtained as donations from couples undergoing IVF treatment. RESULTS: Two ICM showed continuous growth. One line, HS181, has been in culture for 41 weeks with a doubling time of 24-36 h. It continues to express stem cell markers alkaline phosphatase, Oct-4, stage-specific embryonic antigen (SSEA)-4 and tumour-related antigen (TRA)-1-60. The karyotype is 46,XX. Pluripotency was demonstrated by teratoma formation in immunodeficient mice. In high-density cultures, spontaneous differentiation to beating cells and neuron-like cells was seen. The second line, HS207, was cultured for 9 weeks and cryopreserved, as were samples of line HS181. Both lines began to grow after thawing. CONCLUSIONS: We used successfully human foreskin fibroblasts as feeder cells for derivation and continued undifferentiated growth of hES cells. These feeder cells are convenient for IVF units, because no fetal human tissues or tissue from operations are needed.     

Derivation, characterization, and differentiation of human embryonic stem cells

The derivation of human embryonic stem (hES) cells establishes a new avenue to approach many issues in human biology and medicine for the first time. To meet the increased demand for characterized hES cell lines, we present the derivation and characterization of six hES cell lines. In addition to the previously described immunosurgery procedure, we were able to propagate the inner cell mass and establish hES cell lines from pronase-treated and hatched blastocysts. The cell lines were extensively characterized by expression analysis of markers characteristic for undifferentiated and differentiated hES cells, karyotyping, telomerase activity measurement, and pluripotency assays in vitro and in vivo. Whereas three of the cell lines expressed all the characteristics of undifferentiated pluripotent hES cells, one cell line carried a chromosome 13 trisomy while maintaining an undifferentiated pluripotent state, and two cell lines, one of which carried a triploid karyotype, exhibited limited pluripotency in vivo. Furthermore, we clonally derived one cell line, which could be propagated in an undifferentiated pluripotent state.