人胚胎干细胞系NJGLLhES1的建立

目的:使用人类冷冻胚胎建立新的人胚胎干细胞系。方法:将人类冷冻胚胎解冻后培养至囊胚,用免疫外科法分离内细胞团,以ICR小鼠胚胎成纤维细胞作为饲养层,机械法传代。将此细胞系命名为NJGLLhES1,检测其核型,对其碱性磷酸酶和特异性标记物(Oct-4,SSEA-3,SSEA-4,TRA-1-60,TRA-1-81)进行鉴定,并进行类胚体形成实验及畸胎瘤成瘤实验,对其体内外分化能力进行检测。结果:NJGLLhES1已在体外培养逾1年时间。细胞呈人胚胎干细胞克隆样,具正常人类核型,碱性磷酸酶和胚胎特异性标记物的表达为阳性,可形成类胚体和畸胎瘤。结论:成功建立人胚胎干细胞系NJGLLhES1,此细胞系具人胚胎干细胞形态特征,可长期稳定增殖且保持不分化状态,核型正常,在体内外具有多向分化潜能。     

应用钙离子载体A23187及6-甲基嘌呤对人卵母细胞进行孤雌激活

目的 :观察钙离子载体 A2 3 1 87( Ca-A2 3 1 87)及 6-甲基嘌呤 ( 6-DMAP)对人类卵母细胞的孤雌激活作用。方法 :收集体外受精周期中不适于进行卵胞浆内单精子注射的生发泡期或第一次减数分裂中期的不成熟卵母细胞 2 44个在体外培养成熟 ,其中 1 55个体外成熟卵母细胞按不同激活方案分组 :5μmol/ L Ca-A2 3 1 87组、1 0 μmol/ L Ca-A2 3 1 87组、对照组、6-DMAP组及 Ca-A2 3 1 87+6-DMAP组。激活处理后 1 2～ 1 8h观察第二极体排出及原核形成情况。结果 :5及 1 0 μmol/ L Ca-A2 3 1 87组卵母细胞激活率分别为 40 .7% ( 1 1 / 2 7)和 3 7.1 % ( 1 3 / 3 5) ,较对照组的 9.5% ( 2 / 2 1 )明显增加 ( P<0 .0 1 ) ;6-DMAP组的激活率 ,1 1 .5% ( 3 / 2 6)较 Ca-A2 3 1 87+6-DMAP组 ,58.7% ( 2 7/ 46)明显下降。 Ca-A2 3 1 87激活后卵母细胞主要表现为一原核二极体 ,而 Ca-A2 3 1 87+6-DMAP激活的卵母细胞以一原核一极体占多数 ( 1 9/ 2 7,70 .4% )。结论 :卵母细胞孤雌激活的发生及原核形成类型与激活方案有关 ,单独 Ca-A2 3 1 87或与蛋白激酶抑制剂 6-DMAP联合应用都能使人类卵母细胞发生孤雌激活 ,二者联合应用更易于诱导卵母细胞发生孤雌激活     

化学激活在小鼠圆形精子细胞体外受精中的应用

目的:通过对生精细胞体外培养所获圆形精子细胞显微注射(ROSI)后的小鼠卵母细胞进行单一和联合化学激活,寻找小鼠圆形精子细胞体外受精的最佳激活方案。方法:采用不同浓度的乙醇(EH)、离子霉素(Ion)、钙离子载体A23187(CIA)、氯化锶(SrCl2)、6-二甲基苯胺嘌呤(6-DMAP)及放线菌酮(CHX)对ROSI后的卵母细胞进行不同作用时间的激活,并根据不同的激活途径,联合两种最佳激活浓度和时间的激活剂对ROSI后的卵母细胞行联合激活,以受精率、卵裂率和桑囊胚形成率为观察指标,比较单一及联合方案对卵母细胞的激活效果。结果:①单一激活时,各激活剂的最佳方案分别为7%EH作用6 min,5μmol/L CIA作用5 min,5μmol/L Ion作用5 min,2 mmol/L 6-DMAP作用2 h,10 mmol/L SrCl2作用1.5 h,10μg/ml CHX作用1.5 h,其中10 mmol/L SrCl2作用1.5 h桑囊胚率最高,显著优于CHX方案(P0.05),但与其它各组无显著性差异;②EH+6-DMAP组桑囊胚率(29.63%)显著高于其它联合激活组以及除SrCl2以外所有单一激活方案(P0.05),且正常受精率、卵裂率及桑囊胚率均高于SrCl2组,但无显著性差异。结论:单一激活方案中10 mmol/L SrCl2作用1.5 h,联合方案中7%EH作用6 min+2 mmol/L 6-DMAP作用2 h对体外培养所获圆形精子细胞ROSI后的卵母细胞激活效果最佳。EH+6-DMAP的联合激活方案优于单一激活。     

用囊胚内细胞团建立小鼠胚胎干细胞系的尝试

目的建立小鼠129品系胚胎干细胞(ES)系。方法分离囊胚内细胞团接种至小鼠胚胎成纤维细胞,培养、传代以获得细胞系,通过形态观察、碱性磷酸酶检查、体内外分化实验予以鉴定。结果共获取12个囊胚,培养后有9个内细胞团增殖,消化传代,分离出2株克隆;形态观察具有明显的ES细胞形态,染色体核型检查为XY,碱性磷酸酶检查细胞呈阳性,体外分化实验表明可自发分化为外、中和内胚层细胞,体内分化实验获得典型的畸胎瘤,并具备小鼠特异性表面标志物Oct-4、SSEA-1。结论应用本方法建立了两株129品系胚胎干细胞系,并证实其符合小鼠胚胎干细胞的特征。     

卵母细胞激活技术在辅助生殖技术中的临床应用

目的观察辅助激活技术对辅助生殖技术中受精低下或失败患者成熟卵母细胞的激活效果及其胚胎发育情况。方法回顾性分析2014年10月至2016年10月在山西省儿童医院妇幼保健院生殖医学中心助孕治疗、至少有1次ICSI受精失败或受精低下(受精率30%),再次行ICSI助孕时采用成熟卵母细胞激活处理的12名患者资料。12名患者共收集105枚成熟卵母细胞,全部采用钙离子载体A23187联合6-甲基氨基嘌呤(6-DMAP)进行激活处理,观察卵母细胞激活后的原核形成及胚胎发育情况。结果激活周期的受精率及卵裂率分别为63.81%(67/105)和88.06%(59/67),较既往周期的受精率[23.96%(23/96)]和卵裂率[69.57%(16/23)]均显著升高(P0.05);激活周期中可移植胚胎及优质胚胎数量较既往周期均显著提高,分别为(3.02±2.51)vs.(1.41±0.58)枚和(1.70±1.36)vs.(0.31±0.52)枚(P0.05);既往ICSI受精低下或失败的12例患者中,卵母细胞激活处理后5例患者获得临床妊娠。结论辅助激活技术可以改善ICSI受精失败或受精低下患者的受精率及胚胎发育质量。     

小鼠慢性连续取卵及孤雌激活后发育

小鼠是生命科学研究中最为重要的实验动物之一。许多模型首先在小鼠中建立或只在小鼠中获得成功。例如胚胎干细胞系的建立，生长素转基因模型以及体细胞克隆治疗模型。通常情况下，经过超促排卵处理或未经处理的雌鼠仅在处死后取1次卵。至今还未见到有关在小鼠活体取卵的报道。本研究旨在建立一种可重复从同一个体小鼠收集卵母细胞或受精卵的方法。本实验通过对收集的卵母细胞进行孤雌激活和体外培养观察比较发育情况以检验卵母细胞的质量。     

钙离子载体A23187联合卵细胞胞质内单精子注射治疗圆头精子症2例并文献复习

目的:探讨圆头精子症的病因、圆头精子的受精能力,以及人工激活技术在圆头精子症患者卵细胞胞质内单精子注射(ICSI)治疗中的应用价值。方法:回顾性分析2例圆头精子症ICSI治疗病例,患者配偶的卵子经ICSI处理后,随机分为2组,其中1组卵子接受钙离子载体A23187激活处理,另外1组不接受任何处理。结合国内外文献对圆头精子症的发病原因、受精能力和治疗方案进行复习和讨论。结果:A23187激活处理组均获得优质胚胎,而常规处理对照组的卵子则未受精、未卵裂、无优质胚胎、无囊胚形成。2例患者均移植A23187激活组的优质胚胎,获得妊娠并分娩健康婴儿。结论:A23187可以用于圆头精子症患者的ICSI治疗,并获得较好的临床结局,但还应密切关注A23187的安全性问题。     

钙对人卵母细胞皮质颗粒排放的调节及与经典型蛋白激酶C关系

目的探讨经药物处理人卵母细胞,钙引起人卵母细胞皮质颗粒排放的机制。方法分别用蛋白激酶C (PKC)激活剂、抑制剂及钙离子载体处理人卵母细胞,使用激光共聚焦扫描显微镜观察PKC的转位及皮质颗粒的排放。结果使用PKC激活剂处理后,卵母细胞出现了明显的PKCα和γ的转位,但未观察到皮质颗粒的排放。钙离子载体 A23187可以引起明显的皮质颗粒的排放,但未出现PKC的转位。结论PKC不介导人卵母细胞内钙离子升高引起的皮质颗粒的释放。     

人精原干细胞特异性标志的初步筛选

目的:寻求可能用于人精原干细胞(SSC)分离和纯化的特异性表面标志。方法:通过免疫组化方法,应用造血干细胞(HSC)表面标志c-kit、Thy-1,人胚胎干细胞(ES)表面标志阶段特异性胚胎抗原SSEA-3、SSEA-4、碱性磷酸酶(ALP),原始生殖细胞(PGC)标志SSEA-1以及小鼠SSC表面标志α6和β1整合素对成人及胎儿睾丸SSC的特异性表达进行筛选和鉴定。结果:在成人睾丸组织中,α6整合素在生精小管生殖细胞表面存在较广泛而显著阳性表达,而β1整合素主要在生精小管基底部细胞存在显著阳性染色,Thy-1在成人睾丸生精小管基底部细胞可见散在阳性染色,少量间质细胞中亦可见阳性染色。上述3种抗原标志在成人生殖细胞表面的表达具有一定的特异性。在胎儿睾丸生精小管中,可见SSEA-1在生殖细胞表面存在显著阳性表达,具有明显特异性。结论:α6、β1整合素和Thy-1可作为阳性标志用于人SSC的分选。SSEA-1可作为识别胎儿SSC的特异性标志。     

胚胎干细胞标志CD31、SSEA-1在小鼠胚胎发育和成体组织中表达的初步研究

目的观察胚胎干细胞表面标志CD31、SSEA-1在小鼠胚胎发育过程中不同阶段的表达及在新生鼠、成体骨髓、睾丸中的表达情况,为进一步利用它们作为分子标志从胚胎及成体组织中分离多潜能性干细胞奠定基础。方法本实验选用129/SV系小鼠。孕10.5天、14.5天、18.5天的胚胎各取3例,新生鼠、6周龄成年鼠骨髓、成体睾丸各取3例,0.3%胶原酶消化获得单细胞悬液,经抗体染色标记后,通过流式细胞仪分析CD31、SSEA-1的表达情况。结果胚胎第10.5天、14.5天、18.5天和新生鼠中均有CD31、SSEA-1的表达,阳性细胞量随胚胎发育呈下降趋势。成年鼠骨髓和睾丸组织中也有CD31、SSEA-1阳性细胞存在,含量低于胚胎中的数量。结论 CD31、SSEA-1在小鼠个体发育过程以及成体组织中都有不同程度的表达,阳性细胞比率随个体发育而降低。     

Embryonic stem cell derived and adult hematopoietic stem cell transplantation for tolerance induction in a renal allograft recipient:--a case report

We generated an human embryonic stem cell (hESC) line to augment chimerism-associated tolerance. A 40-year-old African with chronic glomerulonephritis-chronic renal failure with 100% G6PD enzyme deficiency presented for renal transplantation with a 27-year-old, 6/6 HLA-matched sister as a willing donor. METHOD: We generated an hESC line from the donor's oocytes using long ovarian stimulation protocol simultaneously with tolerance induction protocol. A nuclear transfer (NT)-hESC line was derived by transferring a donor cumulus cell into an enucleated oocyte, subjected to electrical fusion, and cultured for 5 days. ESCs hatched from the blastocyst on day 6 were cocultured with her unmodified bone marrow for 2 days and suspended in Ringer's lactate. Five milliliters of suspension were collected for cell counting, viability, pluripotency, flow cytometry, and karyotyping. The remaining suspension was infused into the periphery of the recipient. Transplantation was performed 1 week later following a negative lymphocytotoxicity cross-match test using no immunosuppression. Peripheral blood chimerism (PBC) was studied using fluorescent in situ hybridization technique. Allograft biopsy was performed on day 7. RESULTS: NT-hESC CD34+ count was 7.6%, viability 100%, karyotyping normal, pluripotency markers: SSEA-1, SSEA-4, OCT-3/4, TRA-1/60:positive; 12% PBC was noted at 1 week after transplantation. Serum creatinine was 1.2 mg%, graft biopsy was unremarkable, and G6PD enzyme deficiency was corrected to 0% at 100 days posttransplant. Liver function tests and hematology profile were unremarkable for graft-versus-host disease. CONCLUSION: This is the first report of tolerance induction using NT-hESC-induced hematopoietic chimerism with synergistic use of adult bone marrow. It was safe and effective.     

Induction of Insulin-Producing Cells From Human Pancreatic Progenitor Cells

Introduction

We previously established a mouse pancreatic stem cell line without genetic manipulation. In this study, we sought to identify and isolate human pancreatic stem/progenitor cells. We also tested whether growth factors and protein transduction of pancreatic and duodenal homeobox factor-1 (PDX-1) and BETA2/NeuroD into human pancreatic stem/progenitor cells induced insulin or pancreas-related gene expressions.

Materials and method

Human pancreata from brain-dead donors were used for islet isolation with the standard Ricordi technique modified by the Edmonton protocol. The cells from a duct-rich population were cultured in several media, based on those designed for mouse pancreatic or for human embryonic stem cells. To induce cell differentiation, cells were cultured for 2 weeks with exendin-4, nicotinamide, keratinocyte growth factor, PDX-1 protein, or BETA2/NeuroD protein.

Results

The cells in serum-free media showed morphologies similar to a mouse pancreatic stem cell line, while the cells in the medium for human embryonic stem cells formed fibroblast-like morphologies. The nucleus/cytoplasm ratios of the cells in each culture medium decreased during the culture. The cells stopped dividing after 30 days, suggesting that they had entered senescence. The cells treated with induction medium differentiated into insulin-producing cells, expressing pancreas-related genes.

Conclusion

Duplications of cells from a duct-rich population were limited. Induction therapy with several growth factors and transduction proteins might provide a potential new strategy for induction of transplantable insulin-producing cells.     

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。     

胆囊癌细胞株GBC-SD类肿瘤干细胞筛选的研究

目的 筛选胆囊癌GBC-SD系中具有干细胞特性的细胞克隆.方法 在肿瘤干细胞培养基加入不同剂量的顺铂悬浮培养GBC-SD,将悬浮细胞收集后注射裸鼠,并持续瘤内注射顺铂,成瘤后取瘤内细胞继续原培养基培养,获得悬浮生长的克隆,分别测定其干细胞标志物(CD133、CD44、CD24)、耐药基因ABCG2细胞株MDR-1和转录因子OCT-4、Nanog的表达.结果 在肿瘤干细胞培养基和顺铂的筛选压力下,GBC-SD细胞能有效形成悬浮生长的克隆球,流式检测结果表明克隆球高表达CD133+(97.6%)、CD44+(77.9%),低表达CD24+(2.3%),同时表达耐药基因ABCG2和MDR-1,定量聚合酶链反应(PCR)及免疫荧光方法检测发现,与普通胆囊癌细胞株GBC-SD比较,克隆球干细胞基因OCT-4、Nanog表达分别增强266、284倍.结论 顺铂结合无血清培养基悬浮培养法筛选GBC-SD,作为一种新的干细胞分选方法,可以分离出具有肿瘤千细胞特征的胆囊癌克隆球.     

孤雌生殖的胚胎干细胞研究进展

人类胚胎干细胞可以在体外诱导分化成为人体的各种细胞 ,用于医学临床和科学研究。由于胚胎干细胞来源于胚胎 ,在某些国家和地区受到宗教、道德和伦理的制约。孤雌生殖的胚胎干细胞具有与胚胎干细胞相同的全能性和增殖性 ,可以在体外诱导分化成为体内的各种细胞。本文就孤雌生殖的胚胎干细胞的体外建系、定向分化的研究进展作一综述     

Long-term culture of Japanese human embryonic stem cells in feeder-free conditions

Human pluripotent embryonic stem cells (hESCs) have great promise for research into human developmental biology, development of cell therapies for the treatment of diseases, toxicology, and drug discovery. Traditionally, undifferentiated hESCs are maintained on mouse embryonic fibroblasts (MEFs), which impede the clinical applications of hESCs. Here we have examined the long-term stability of the Japanese hESC line (KhES-1) in feeder-free culture. KhES-1 cells were cultured with MEF conditioned medium (CM) and different doses of basic fibroblast growth factor (bFGF) in six-well-plates of which the surface was coated with Matrigel. KhES-1 cells were maintained for at least 40 passages. In this culture system, the cells maintained stable proliferation rates and steadily expressed Oct-4, Nanog, and alkaline phosphatase. In addition, KhES-1 cells maintained without direct feeder contact formed embryonic bodies with expression of markers from the three germ layers. Here we demonstrated that Japanese human embryonic stem cells KhES-1 were cultured long term in a feeder-free method, while retaining pluripotency in vitro.     

Evidence for stem cells in cultures of mouse prostate epithelial cells.

BACKGROUND: Primary cultures of mouse prostatic epithelial cells were studied to identify cells having stem cell characteristics. METHODS: Culture conditions supporting the growth of mouse prostatic epithelial cells were identified. Immunostaining identified cells expressing cytokeratin 5 (K5), cytokeratin 8 (K8), and bcl-2. Cells with clonogenic capacity were identified by replating dissociated cells from primary colonies. RESULTS: Only 2-4% of prostatic epithelial cells that were plated formed colonies. Most cells cultured for 2-4 days expressed bcl-2, K5, and K8. After 6 days in culture, colonies had two distinct phenotypes, star- or disc-shaped. In colonies of both phenotypes, only a few cells expressed K5 and bcl-2, while all cells expressed K8. Upon replating, two-thirds of star-shaped colonies gave rise to a single colony; of 29 disc-shaped colonies, none gave rise to new colonies. CONCLUSIONS: Colonies deriving from putative prostatic epithelial stem cells are identified. These cells express K5 and bcl-2 and are clonogenic.