玻璃化冷冻技术临床应用的安全性

随着体外受精一胚胎移植（IVF—ET）及其衍生技术的不断发展,低温冷冻保存配子、胚胎以及卵巢或睾丸组织已经成为人类辅助生殖技术（ART）中一个重要的环节。近年来,玻璃化冷冻技术成功用于冻存人类卵母细胞和胚胎。然而,玻璃化冷冻要达到普及性应用之前仍有许多安全性问题有待解决,包括高浓冷冻保护剂毒性、开放式冷冻载体交叉污染、液氮病原微生物潜在污染以及复苏后胚胎发育潜能及损伤鉴定等。度     

应用玻璃化冷冻技术保存人未成熟卵母细胞的研究

目的利用玻璃化冷冻保存技术合理利用常规促排卵周期获得的未成熟卵母细胞。方法1．对注射hCG后38h、43h、60h获得GV期卵母细胞进行玻璃化冷冻保存,冻融后的未成熟卵母细胞行体外成熟培养（IVM）,用ICSI技术对获得的成熟卵母细胞完成授精,并体外胚胎培养至囊胚期。比较各组间的冻融存活率、体外成熟率、受精率、卵裂率及囊胚形成率。2．对GV卵分别行先玻璃化冻融后IVM和先IVM后玻璃化冻融,比较它们的冻融存活率、体外成熟率、受精率、卵裂率及囊胚形成率。结果1．38h与43h卵龄GV卵在各项指标比较上没有差异,60h卵与前2组有相似的冻融存活率,但在体外成熟率、受精率和卵裂率上较前2组出现明显下降趋势。3组均无囊胚形成。2．先玻璃化冻融后IVM和先IVM后玻璃化冻融在未成熟卵母细胞的利用率上没有差异。结论1．冷冻卵龄较短的GV期卵母细胞能获得更好的冷冻保存效果。2．玻璃化冻融和IVM先后不影响未成熟卵母细胞的利用。     

玻璃化冷冻法保存关节软骨

背景：同种异体骨软骨移植技术是治疗关节软骨缺损有效的方法之一,但由于移植物保存方法不理想,明显制约着该技术的临床应用。 目的：探讨玻璃化冷冻法保存关节软骨组织的可行性和优越性。 方法：切取成年猪骨软骨,制成约5 mm×6 mm(直径×长度)大小的圆柱形骨软骨块。以新鲜软骨组为对照,分别采用0.5 mol/L甘油 、1 mol/L二甲基亚砜、1 mol/L玻璃化溶液3种方法预处理软骨块,再行冷冻法保存软骨块8周,采用组织化学染色、免疫荧光染色观察并比较软骨细胞活性的变化。 结果与结论：玻璃化溶液预处理组的关节软骨细胞存活率达到74.5%,明显高于甘油和二甲基亚砜预处理组,软骨基质成分仅少量丢失。3种方法相比较,玻璃化溶液预处理后慢速梯度降温冷冻保存法可以明显提高冻存关节软骨组织的活性。     

诱导多能干细胞与p53基因的研究

背景：诱导多能干细胞可以绕过胚胎干细胞存在的伦理学问题,已成为目前干细胞研究的热点问题。 目的：探讨诱导多能干细胞的研究进展以及需要解决的问题。 方法：回顾分析诱导多能干细胞的发现,近年来的研究过程,目前存在的问题。检索汤森路透Web of Science数据库关于诱导多能干细胞和p53基因研究相关的文献进行分析。 结果与结论：近年来国内外学者对诱导多能干细胞进行大量研究。例如,日本正在筹备建立干细胞库,将为视网膜等疾病治疗提供基础。但是,在诱导多能干细胞能广泛应用之前,还面临许多安全问题,其中p53相关的功能性研究是必不可少的紧迫问题,p53基因中的其他成员p73也参与诱导多能干细胞的产生和分化也需要进一步深入。关于p53功能的发现将伴随着干细胞研究,促进再生医学的深入和发展。     

不同玻璃化冷冻方案对小鼠卵巢组织的影响

目的比较不同组合的冷冻保护剂对小鼠卵巢组织的影响,为人类卵巢组织玻璃化冷冻的保护剂选择提供实验依据。方法将20只小鼠的卵巢进行玻璃化冷冻,组织标本随机分为4组,新鲜的和冻融后的卵巢皮质分别进行光学显微镜观察、TUNEL实验和免疫组织化学分析。结果光镜观察发现各实验组的各级卵泡形态正常率均明显低于对照组,B组的始基卵泡形态正常率和初级卵泡形态正常率均高于A组和C组,差异有统计学意义（P〈0．05）。A组和C组冻融后卵泡凋亡率、卵母细胞凋亡率和颗粒细胞凋亡率均高于B组,有统计学差异（P〈0．05）。另外,B组卵泡Bax蛋白阳性表达率低于A组和C组,而B组Bcl-2蛋白阳性表达率高于A组和C组,差异具有统计学意义（P〈0．05）。结论实验结果提示：与其它组合相比,EG与PROH的组合更适合小鼠卵巢组织的玻璃化冷冻。     

诱导性多能干细胞与心血管疾病

BACKGROUND:Induced pluripotent stem cells have great prospects in tissue repair, due to the characteristic of self-renewal, multi-directional differentiation, no immunological rejection and ethics controversy. OBJECTIVE:To summarize the differentiation of induced pluripotent stem cells towards cardiomyocytes and endothelial cells, and their applications in the cardiovascular diseases. METHODS:The first author performed a data retrieval of PubMed and CNKI databases from 2000 to 2015 to search articles addressing the differentiation of induced pluripotent stem cells towards cardiomyocytes and endothelial cells, and reviewed the literatures systematically. Finally, 78 articles were chosen for further analysis. RESULTS AND CONCLUSION:Induced pluripotent stem cells can differentiate into cardiovascular cells through a variety of methods. Factors such as cyclosporin A and ascorbic acid C may improve myocardial differentiation of induced pluripotent stem cells, while vascular endothelial growth factor and basic fibroblast growth factor may improve the endothelial differentiation of induced pluripotent stem cells. Cardiovascular cells derived from induced pluripotent stem cells can be applied to build disease models in vitro, transplantation in vivo and drug screening.     

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。     

冷冻方法对人卵裂期冷冻胚胎移植周期妊娠结局的影响

目的探讨慢速冷冻和玻璃化冷冻方法对人卵裂期冷冻胚胎移植周期妊娠结局的影响。方法回顾性分析2011年1月~2015年12月在北京大学第一医院进行卵裂期冷冻胚胎移植的1331个周期,包括慢速冷冻周期431个和玻璃化冷冻周期900个,比较两种不同冷冻方法的胚胎复苏率、完整胚胎率、临床妊娠率、种植率、流产率等各项指标,并分析卵裂球损伤对胚胎发育潜能的影响。结果玻璃化冷冻的胚胎复苏率(92.53%)、完整胚胎率(75.43%)、临床妊娠率(45.72%)、种植率(27.41%)高于慢速冷冻(76.93%、48.46%、39.52%、19.88%,P0.05);而流产率分别为12.20%、12.56%(P0.05);周期取消率分别为3.71%、1.33%(P0.05)。慢速冷冻移植0、1、2、3个无卵裂球损伤胚胎的临床妊娠率分别为:36.2%、37.7%、42.0%、44.3%(P0.05);玻璃化冷冻移植0、1、2、3个无卵裂球损伤胚胎的临床妊娠率分别为:20.5%、42.3%、48.8%、54.1%(P0.05)。结论玻璃化冷冻法更适合于人卵裂期胚胎冷冻保存,其冷冻胚胎移植周期的妊娠结局要优于慢速冷冻法;卵裂球损伤对玻璃化冷冻复苏胚胎的发育潜能影响较大。     

超低温冰箱转液氮阶梯降温法与传统程序降温法保存造血干细胞的比较

背景：在造血干细胞整个冷冻保存过程中,受到降温速率、储存温度深浅、冷冻保护剂组合等因素影响,各国学者在提倡选择何种保存方法上面存在不同的主张。 目的：比较-80 ℃低温冰箱转液氮阶梯降温法与传统程序降温法保存外周造血干细胞的效果。 方法：将采集的造血干细胞分两组,第一组细胞浓度为1×10¹¹ L^-1,加入10%二甲基亚砜,放入程序冷冻仪内程序设置为室温至-4 ℃按1 ℃/min的速率降温,按35 ℃/min快速降至-45 ℃,再以15 ℃/min升至-21 ℃,然后以5 ℃/min降至-90 ℃,取出冷冻管置-196 ℃液氮罐内。第二组细胞浓度为1×10¹¹ L^-1,加入5%二甲基亚砜、3%羟乙基淀粉、4%人血白蛋白,将冷冻管置-80 ℃冰箱过夜后取出,置-196 ℃液氮罐内的气相,再过夜后置液氮罐液相内。 结果与结论：两组冷冻方法保存细胞的锥虫蓝拒染率、回收率、凋亡率和死亡率差异无显著性意义(P > 0.05)。结果显示用5%二甲基亚砜、4%白蛋白、3%羟乙基淀粉组成的冷冻保护剂,通过-80 ℃低温冰箱转液氮阶梯降温法冷冻保存造血干细胞与传统10%二甲基亚砜作为冷冻保护剂用程序降温的方法取得一样效果,操作简便易于临床应用。     

人成熟卵母细胞玻璃化冷冻技术的临床应用

目的探讨玻璃化冷冻技术应用于人成熟卵母细胞冷冻保存的临床效果。方法使用开放式载体cryotop进行人成熟卵母细胞的玻璃化冻存,对10例患者的冷冻卵子复苏后进行卵母细胞浆内单精子（ICSI）注射后行胚胎移植,观察复苏卵母细胞受精、卵裂、胚胎发育及临床妊娠情况。结果 10例患者共86枚玻璃化冻存的成熟卵母细胞复苏后77枚存活,69枚正常受精,获得65枚胚胎,共移植17枚胚胎,3例患者获临床妊娠,其中2例双胎妊娠。结论玻璃化冷冻技术是有效地保存人成熟卵母细胞的方法,有一定的临床应用价值。     

Cryopreservation of embryonic chick myogenic lineage-committed stem cells

Summary Undifferentiated embryonic chick stem cells provide a good in vitro test system to examine the effects of recombinant growth factors on the resultant phenotypic expression of these cells. One of the major difficulties in using freshly isolated cells is variation in the proportion of stem cells recovered from different cell harvests. Variation is of particular concern when multiple cell harvests are necessary to provide cells for assaying a single growth factor. As an attempt to minimize this variation, we have examined the efficacy of cryopreserving myogenic lineage-committed stem cells derived from embryonic chick leg muscle. Percent recovery, viability, differentiation morphology, and cellular proliferation rates were compared between sister cultures of freshly isolated and cryopreserved stem cells. Embryonic chick stem cells retained their capacity to differentiate myogenically in culture when slowly frozen in and recovered from 7.5% dimethyl sulfoxide medium supplemented with horse serum and stage-specific embryo extract.     

Cryopreservation of human embryonic stem cells without the use of a programmable freezer

BACKGROUND: An effective freezing-thawing technique is crucial for the clinical application of human embryonic stem (ES) cells. The aim of this study was to find an optimal cryopreservation protocol for human ES cells using slow freezing-rapid thawing without a programmable freezer. METHODS: The human ES cell line, SNUhES-3, was cultured on an STO feeder layer in gelatin-coated tissue culture dishes. All cryopreservation steps were performed using a simple commercial freezing container. The survival rate of cryopreserved-thawed human ES cells was estimated by counting colony numbers under a stereomicroscope. Initially, we compared the survival rates of cryopreserved human ES cells using three cryoprotectants: dimethylsulphoxide (DMSO), ethylene glycol (EG) and glycerol. In this experiment, 5% DMSO/95% fetal bovine serum (FBS) (vol/vol) showed the highest survival rate. We next tested the impact of various concentrations of FBS (95, 50 and 5%) with 5% DMSO, and then examined the effects of adding EG or glycerol to 5% DMSO + optimal FBS. RESULTS: No significant difference in survival rate was observed between 95 and 50% FBS in the presence of 5% DMSO. A significant improvement in survival rate was obtained by adding 10% EG to 5% DMSO+50% FBS. After thawing, surviving cells were found to maintain the inherent characteristics of human ES cells. CONCLUSION: 5% DMSO+50% FBS+10% EG may be an optimal cryoprotectant for the slow freezing-rapid thawing of human ES cells.     

干细胞若干定义的相关探讨

背景：近10年来干细胞研究所取得的巨大进展,不仅影响和促进了生物学及其相关基础科学,而且还在医学、药物开发、农业等许多领域得到广泛的应用,目前已成为研究的热点问题。 目的：在干细胞的定义上还存在一些需要探讨的问题,明确定义将有利于干细胞研究的快速发展。 方法：回顾干细胞的发展和概念提出,特别是通过中英文权威定义的比较,提出作者的看法。 结果与结论：干细胞的定义上还存在一些问题值得探讨,特别是一些中英文表述不同影响了理解。例如,“多能干细胞”对应译成两个单词：Pluripotent Stem Cell和Multipotent Stem Cell,然而Pluripotent和Multipotent两个单词的英文意义不同。为了学术交流和沟通,作者提出将Pluripotent Stem Cell称为“万能干细胞”,而保留Multipotent Stem Cell为“多能干细胞”的定义。以上结论供广大同仁探讨,以利于抛砖引玉。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：     

Bulk vitrification of human embryonic stem cells

BACKGROUND: The traditional vitrification method cannot keep up with the increased culture and propagation efficiency required to cryopreserve large quantities of vigorously proliferating human embryonic stem (HES) cells. In this study, we describe a newly invented vitrification carrier for cryopreserving large amount of HES cells and evaluate whether this bulk vitrification (BV) method is as effective as the popular open-pulled straw (OPS) vitrification method. METHODS: HES cell clumps were harvested after passage and transferred to a cell strainer; only those clumps with a diameter more than 70 microm were included in the study and randomly selected to be cryopreserved by the BV method, OPS vitrification or slow freezing method. HES cell survival, growth and pluripotency were analyzed after thawing. RESULTS: Bulk vitrification method with cell strainer could cryopreserve 136 +/- 23.4 cell clumps at one time (round), which was 30 times as high as those for OPS method (4 +/- 1.5). After thawing, bulk-vitrified HES cells exhibited high survival rate up to 94.3%, comparable with the OPS method. All surviving cell clumps generated HES cell colonies. Teratomas comprising all three primordial germ layers were formed in severe combined immunodeficient mice after subcutaneous injection of post-thawed, bulk-vitrified HES cell clumps, confirming pluripotency. CONCLUSIONS: This new BV method could cryopreserve a large quantity of HES cell clumps at one time, which not only would satisfy routine cryopreservation of HES cell during daily culture process but also guarantee researchers have large quantity of efficiently cryopreserved HES cells ready for a scheduled study at any time.     

钠钙交换体启动子促进诱导多能干细胞向心肌细胞转化

背景：诱导多能干细胞治疗缺血性心脏病被认为是极具前景的方法,但目前仍未找到一种理想的诱导方式。 目的：探讨钠钙交换体(NCX1)启动子诱导多能干细胞体外分化为心肌细胞的作用。 方法：构建含钠钙交换体1启动子的pLVX-IRES-ZsGreen1慢病毒质粒,使用ViraPowerTM慢病毒包装系统共转染293FT细胞。收集病毒测定病毒滴度后感染小鼠诱导多能干细胞,并使用嘌呤霉素进行筛选。观察胚状体数量以及诱导多能干细胞的分化效率,使用RT-PCR、免疫荧光分析法检测心肌特异标记物。 结果与结论：成功构建了含钠钙交换体1启动子的pLVX-IRES-ZsGreen1慢病毒,感染小鼠诱导多能干细胞后使用流式细胞仪分选NCX1⁺/GFP⁺细胞。与NCX1^-/GFP^-细胞相比,NCX1⁺/GFP⁺细胞在4 d即出现细胞分化和跳动,NCX1+细胞GATA4/MEF2c/Nkx2.5基因表达分别是NCX1-细胞的4.2,7.5,2.5倍,免疫荧光显示CX43和心肌肌钙蛋白I呈阳性表达。结果表明,钠钙交换体1启动子可以促进小鼠诱导多能干细胞向心肌细胞分化。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：     

人诱导性多能干细胞向神经干细胞分化的方法探讨

目的:探讨人诱导性多能干细胞(iPS细胞)体外定向分化为神经干细胞(NSCs)的方法。方法:人iPS细胞悬浮培养4 d形成的拟胚体(EB)经维甲酸(RA)诱导4 d,诱导后的EB在无血清培养基中筛选并扩增培养。倒置显微镜下观察诱导后细胞的形态变化,RT-PCR检测iPS细胞多能性基因Nanog、Oct4和Sox2的表达,免疫荧光法检测NSCs特异性标志物Nestin、神经元标志物β-tubulinⅢ和神经胶质细胞标志物GFAP的表达。结果:(1)人iPS细胞在饲养层细胞上稳定传代,表达多能性基因Nanog、Oct4和Sox2,去除饲养层后悬浮培养能形成球形EB;(2)经RA诱导后的EB在无血清培养基中筛选培养第1 d贴壁生长,周围爬出细胞;第3 d,在高倍镜下可见中央聚集的细胞呈环形的rosette结构,rosette结构增多到第7 d达高峰。对照组未观察到rosette结构。随后大部分rosette细胞脱离瓶壁悬浮生长,形成神经球样克隆。免疫荧光显示神经球呈nestin阳性表达。贴壁培养的神经球能分化成β-tubulinШ和GFAP阳性细胞。结论:RA诱导结合无血清培养基筛选法能诱导iPS细胞高效分化为NSCs。     

Spider silk for xeno-free long-term self-renewal and differentiation of human pluripotent stem cells

Human pluripotent stem cells (hPSCs) can undergo unlimited self-renewal and have the capacity to differentiate into all somatic cell types, and are therefore an ideal source for the generation of cells and tissues for research and therapy. To realize this potential, defined cell culture systems that allow expansion of hPSCs and subsequent controlled differentiation, ideally in an implantable three-dimensional (3D) matrix, are required. Here we mimic spider silk – Nature's high performance material – for the design of chemically defined 2D and 3D matrices for cell culture. The silk matrices do not only allow xeno-free long-term expansion of hPSCs but also differentiation in both 2D and 3D. These results show that biomimetic spider silk matrices enable hPSC culture in a manner that can be applied for experimental and clinical purposes.