干细胞研究概述

干细胞具有很强的可塑性,可用于治疗各种外伤、病理损伤、组织缺陷性疾病、免疫缺陷性疾病和遗传疾病等.两类主要的干细胞--胚胎干细胞和成体干细胞分别在不同的研究领域可用于各种医学实验,几乎涉及所有医学范畴,具有广阔发展前景,但是干细胞应用上还存在许多尚待解决的问题.对干细胞的研究进展及存在的相关问题进行适当的总结概述.     

Ex vivo expansions and transplantations of mouse bone marrow-derived hematopoietic stem/progenitor cells

To examine the effects of co-culture with bone marrow mesenchymal stem cells on expansion of hematopoietic stem/progenitor cells and the capacities of rapid neutrophil engraftment and hematopoietic reconstitution of the expanded cells, we expanded mononuclear cells (MNCs) and CD34⁺/c-kit⁺ cells from mouse bone marrow and transplanted the expanded cells into the irradiated mice. MNCs were isolated from mouse bone marrow and CD34⁺/c-kit⁺ cells were selected from MNCs by using MoFlo Cell Sorter. MNCs and CD34⁺/c-kit⁺ cells were co-cultured with mouse bone marrow-derived mesenchymal stem cells (MSCs) under a two-step expansion. The expanded cells were then transplanted into sublethally irradiated BDF1 mice. Results showed that the co-culture with MSCs resulted in expansions of median total nucleated cells, CD34⁺ cells, GM-CFC and HPP-CFC respectively by 10.8-, 4.8-, 65.9- and 38.8-fold for the mononuclear cell culture, and respectively by 76.1-, 2.9-, 71.7- and 51.8-fold for the CD34⁺/c-kit⁺ cell culture. The expanded cells could rapidly engraft in the sublethally irradiated mice and reconstitute their hematopoiesis. Co-cultures with MSCs in conjunction with two-step expansion increased expansions of total nucleated cells, GM-CFC and HPP-CFC, which led us to conclude MSCs may create favorable environment for expansions of hematopoietic stem/progenitor cells. The availability of increased numbers of expanded cells by the co-culture with MSCs may result in more rapid engraftment of neutrophils following infusion to transplant recipients. Project supported by NIH-Blood, Heart & Lung (National Institute of Health, USA, IR 01 4L70593-01) and Zhejiang Provincial Science Foundation (No. 011103397), China     

Immunomodulative effects of mesenchymal stem cells derived from human embryonic stem cells in vivo and in vitro

Objective  

Human embryonic stem cells (hESCs) have recently been reported as an unlimited source of mesenchymal stem cells (MSCs). The present study not only provides an identical and clinically compliant MSC source derived from hESCs (hESC-MSCs), but also describes the immunomodulative effects of hESC-MSCs in vitro and in vivo for a carbon tetrachloride (CCl₄)-induced liver inflammation model.     

尼克酰胺与exendin-4诱导胰腺干细胞向β细胞定向分化研究

目的体外分离胰腺干细胞,选择最佳培养条件探索胰腺干细胞在体外环境下向β细胞的分化情况.方法取SD大鼠胰腺组织,胶原酶消化,密度梯度离心获得纯化的胰腺导管上皮细胞.采用分步诱导法诱导胰腺导管上皮细胞向胰岛β细胞分化;用胰岛素释放实验检测胰岛功能,免疫荧光法检测nestin,PDX-1,CK-19,CK-20胰岛素及胰高血糖素等的表达.结果胰腺消化培养6～12h,可看到胰腺导管上皮细胞贴壁生长,通过4步诱导培养,nestin阳性细胞快速生长,并可分泌胰岛素.结论体外分离的成人胰岛前体细胞,在诱导因子的作用下,胰腺干细胞可定向分化为β细胞,有望应用于糖尿病的治疗.     

神经干细胞及其在神经系统疾病治疗中的应用

概述了神经干细胞的生物学特性、分布以及影响其分化和增殖的因素，并探讨了神经干细胞移植和基因治疗在神经系统疾病治疗中的应用.     

大鼠胚胎神经干细胞的分离、培养和鉴定

目的:探讨胚胎神经干细胞的分离、培养和鉴定方法.方法:原代培养、培养细胞生长状况观察、组织化学鉴定、流式细胞仪进行细胞凋亡分析.结果:准确分离出了神经干细胞,了解其生长规律及细胞凋亡的情况.结论:用此方法分离胚胎神经干细胞实用、便捷和可行.     

论脐带血干细胞的所有权归属

探讨脐带血干细胞的所有权归属,不仅具有学术意义,也牵动着与该高科技产物相关法律行为的效力判断。在质疑关于脐带血干细胞的所有权归于新生儿这一通说的同时,以民法和物权法理论为基础,并配合脐带血干细胞特有的生物解剖学特性,通过分析胎儿分娩过程中的不同阶段,主张母体才是脐带血干细胞的真正所有权人。     

用密度梯度离心法富集源于小鼠胚胎干细胞的运动神经元样细胞前体

目的:我们的目标是从源于胚胎体(EBs)的小鼠胚胎干细胞(mESCs)中分离运动神经元样细胞前体(MNLCPs),以用于发展针对运动神经元疾病的药物筛选试验和移植疗法.天然Shh蛋白(或Shh通路激动剂)和维甲酸诱导的胚胎体中,MNLCPs和未分化细胞的含量是不确定的.如果不把未分化的细胞从细胞培养中充分去除,其可能会干涉药物筛选试验或在移植后增生.我们开发了一种以密度梯度离心法为基础的富集MNLCPs的方法.方法:我们用Wichterle等人2008年改进的方法,将mESCs( HBG3:eGFP:HB9)扩大和分化.通过化学和酶学的无研磨处理,含有绿色荧光蛋白的MNLCPs和未分化细胞的胚胎体被小心轻轻地离解成单细胞.利用OptiprepTM8％～20％逐步梯度离心技术将MNLCPs回收.拥有绿色荧光蛋白的MNLCPs的含量由流式细胞仪检测.结果:我们的结果表明,在胚胎体形成前,mESCs在明胶包被的培养板上生长,其分化为MNLCPs的能力减少.比较mESCs在明胶,明胶与PMEFs,及PMEFs包被的培养板上的生长发现,mESCs在PMEFs包被的培养板上产生含绿色荧光蛋白的MNLCPs的得率为(54.1±11.0)％(x-±s;n=12),在明胶包被的培养板上的得率为(2.8±1.1)％(x-±s;n=9).用密度梯度离心法获得的含绿色荧光蛋白的MNLCPs的平均含量为(87.7±5.5)％(x-±s;n=3).结论:我们的数据表明,不使用细胞分选器,无研磨解离和密度梯度离心法也能用于富集具有高存活率的MNLCPs.有必要对MNLCPs在体外、体内和表型上进行进一步的生理学意义(如神经轴突的生长及形成神经肌肉接头的能力)上的鉴定.     

体外诱导实现猪的ips细胞向神经干细胞分化

项目的主要目标是实现猪的诱导多能干细胞(i PSCs)向神经干细胞(NSCs)体外诱导。在整个实验探索的过程中,我们使用了传统的诱导分化形成神经干细胞(NSCs)的方法,即通过拟胚体(EB)的形成并添加维甲酸(RA)进行诱导。猪的诱导多能干细胞(i PSCs)经历了传代培养、拟胚体(EB)时期、诱导分化时期三个重要阶段,生成一类新的细胞。最后我们使用免疫荧光法检测到新的细胞中存在神经干细胞的特异性标志物Nestin、神经元标志物β-tubulin III以及神经胶质细胞标志物GFAP表达,从而确定这类新的细胞为神经干细胞。可见,猪的诱导多能干细胞(i PSCs)经过这种传统诱导分化方法培养可分化为具备基础特征的神经干细胞(NSCs),能够表达出神经干细胞(NSCs)特异的标志分子nestin。     

Induced pluripotent stem cells: origins, applications, and future perspectives

Embryonic stem(ES)cells are widely used for different purposes,including gene targeting,cell therapy,tissue repair,organ regeneration,and so on.However,studies and applications of ES cells are hindered by ethical issues regarding cell sources.To circumvent ethical disputes,great efforts have been taken to generate ES cell-like cells,which are not derived from the inner cell mass of blastocyst-stage embryos.In 2006,Yamanaka et al.first reprogrammed mouse embryonic fibroblasts into ES cell-like cells called induced pluripotent stem(iPS)cells.About one year later,Yamanaka et al.and Thomson et al.independently reprogrammed human somatic cells into iPS cells.Since the first generation of iPS cells,they have now been derived from quite a few different kinds of cell types.In particular,the use of peripheral blood facilitates research on iPS cells because of safety,easy availability,and plenty of cell sources.Now iPS cells have been used for cell therapy,disease modeling,and drug discovery.In this review,we describe the generations,applications,potential issues,and future perspectives of iPS cells.     

Allograftic bone marrow-derived mesenchymal stem cells transplanted into heart infarcted model of rabbit to renovate infarcted heart

Objective: To investigate the directed transplantation of allograftic bone marrow-derived mesenchymal stem cells (MSCs) in myocardial infarcted (MI) model rabbits. Materials and Methods: Rabbits were divided into 3 groups, heart infarcted model with MSCs transplanted treatment (MSCs group, n=12), heart infarcted model with PBS injection (control group, n=20), sham operation with PBS injection (sham group, n=l 7). MSCs labelled by BrdUrd were injected into the MI area of the MSCs group. The same volume of PBS was injected into the MI area of the control group and sham group. The mortality, LVIDd, LVIDs and LVEF Of the two groups were compared 4 weeks later. Tropomyosin inhibitory component (Tn I) and BrdUrd immunohistochemistry identified the engrafted cells 4 weeks after transplantation. Result: The mortality of the MSCs group was 16.7% (2/12), and remarkably lower than the control group's mortality [35% (7/20) (P＜0.05)].Among the animals that survived for 4 weeks, the LVIDd and LVIDs of the MSCs group after operation were 1.17±0.21 cm and 0.74±0.13 cm, and remarkably lower than those of the model group, which were 1.64±0.14 cm and 1.19±0.12 cm (P＜0.05); the LVEF of the MSCs group after operation was 63±6%, and remarkably higher than that of the model group,which was 53±6% (P＜0.05). Among the 10 cases of animals that survived for 4 weeks in the MSCs group, in 8 cases (80%),the transplanted cells survived in the non MI, MI region and its periphery, and even farther away; part of them differentiated into cardiomyocytes; in 7 cases (70%), the transplanted cells participated in the formation of blood vessel tissue in the MI region. Conclusion: Transplanted allograftic MSCs can survive and differentiate into cardiomyocytes, form the blood vessels in the MI region. MSCs transplantation could improve the heart function after MI.     

大鼠骨髓间充质干细胞的分离、培养及生物学鉴定

目的:探讨成体大鼠的间充质干细胞(MSCs)的分离、体外培养,为其应用提供理论依据.方法:用密度梯度离心结合贴壁培养法分离纯化大鼠骨髓MSCs,传代扩增,对分离的细胞进行碱性磷酸酶的检测.结果:取得较高纯度的成体大鼠骨髓MSCs,并保持细胞的活性;成体大鼠骨髓MSCs在体外培养中为贴壁生长的单个核球形细胞,培养3～4d后开始大量增殖,并形成形态均一的细胞增殖集群,对分离后所获得的细胞进行AKP染色为强阳性.结论:采用密度梯度离心结合贴壁培养法可获得较高纯度的MSCs,是实用、便捷和可行的方法.并且在体外培养条件下能大量增殖,形成形态均一的细胞集落,可以成为进一步进行细胞扩增或其他实际应用的基础.     

Allograftic bone marrow-derived mesenchymal stem cells transplanted into heart infarcted model of rabbit to renovate infarcted heart

Objective: To investigate the directed transplantation of allograftic bone marrow-derived mesenchymal stem cells (MSCs) in myocardial infarcted (MI) model rabbits. Materials and Methods: Rabbits were divided into 3 groups, heart infarcted model with MSCs transplanted treatment (MSCs group, n=12), heart infarcted model with PBS injection (control group, n=20), sham operation with PBS injection (sham group, n=17). MSCs labelled by BrdUrd were injected into the MI area of the MSCs group. The same volume of PBS was injected into the MI area of the control group and sham group. The mortality, LVIDd, LVIDs and LVEF of the two groups were compared 4 weeks later. Tropomyosin inhibitory component (Tn I) and BrdUrd immunohistochemistry identified the engrafted cells 4 weeks after transplantation. Result: The mortality of the MSCs group was 16.7% (2/12), and remarkably lower than the control group's mortality [35% (7/20) (P<0.05)]. Among the animals that survived for 4 weeks, the LVIDd and LVIDs of the MSCs group     

Ex vivo expansion and pluripotential differentiation of cryopreserved human bone marrow mesenchymal stem cells

This study is aimed at investigating the potentials of ex vivo expansion and pluri-differentiation of cryopreservation of adult human bone marrow mesenchymal stem cells （hMSCs） into chondrocytes, adipocytes and neurocytes. Cryopreserved hMSCs were resuscitated and cultured for 15 passages, and then induced into chondrocytes, adipocytes and neurocytes with corresponding induction medium. The induced cells were observed for morphological properties and detected for expressions of type II collagen, triglyceride or neuron-specific enolase and nestin. The result showed that the resuscitated cells could differentiate into chondrocytes after exposure to transforming growth factor 61 （TGF-~0, insulin-like growth factor I （IGF-I） and vitamin C （Vc）, and uniformly changed morphologically from a spindle-like fibroblastic appearance to a polygonal shape in three weeks. The induced cells were heterochromatic to safranin O and expressed cartilage matrix-procollagenal （If） mRNA. The resuscitated cells cultured in induction medium consisting of dexamethasone, 3-isobutyl-l-methylxanthine, indomethacin and IGF-I showed adipogenesis, and lipid vacuoles accumulation was detectable after 21 d. The resuscitated hMSCs were also induced into neurocytes and expressed nestin and neuron specific endolase （NSE） that were special surface markers associated with neural cells at different stage. This study suggested that the resuscitated hMSCs should be still a population ofpluripotential cells and that it could be used for establishing an abundant bMSC reservoir for further experiment and treatment of various clinical discases.     

Progress in Safe Induced Pluripotent Stem Cells for Clinical Applications

诱导性多能干细胞（induced pluripotent stem cells,iPSCs）是被重新编程的、具有多能性的成体细胞。最近的研究证实,iPSCs能够达到与胚胎干细胞同样的全能性。源于成体的iPSCs对药物或毒性筛选、医疗研究以及疾病专一性治疗提供了极其有价值的细胞来源,然而,在应用于临床之前,必须克服潜在的风险,包括iPSCs诱导中病毒的整合、基因的改变。iPSCs潜在的风险涉及外源因子的转入、目标细胞的改变及诱导因子的表达和重新激活等。本文回顾iPSCs的应用前景和面临的挑战,同时对诱导安全性iPSCs的方法进行了讨论。     

神经干细胞分化相关的一个新基因的研究

1IntroductionNeural stemcells(NSCs)are a subtype of progenitorcells in the nervous systemthat can differentiate intoneurons and glia[1-3].Due to their feature of self-re-newal,NSCs have expectations for treatment of ner-vous system diseases such as Parkin…     

人胚胎干细胞研究的伦理学问题

文章考查、分析了与人胚胎干细胞研究密切相关的几个伦理学问题:人胚胎地位的伦理学争议、治疗性克隆与生殖性克隆的伦理学问题、世界各国克隆技术的政策与法规,等等,特别提到了生殖性克隆对社会伦理造成的巨大冲击。结果表明,各国政府普遍支持治疗性克隆的研究而反对生殖性性克隆(克隆人)的研究。最后论及了一些学者对“克隆人”伦理学问题作出的相关预测。     

Growth factors induce the improved cardiac remodeling in autologous mesenchymal stem cell-implanted failing rat hearts

Therapeutically delivered mesenchymal stem cells (MSCs) improve ventricular remodeling. However,the mechanism underlying MSC cardiac remodeling has not been clearly determined. Congestive heart failure (CHF) was induced in rats by cauterization of the left ventricular free wall. MSCs were cultured from autologous bone marrow and injected into the border zone and the remote myocardium 5 d after injury. Ten weeks later,when compared with sham operation,CHF significantly increased nucleus mitotic index,capilla...     

芹菜水提物促进神经干细胞的体外分化

1IntroductionNeural stem cells have great potential for use intreating neural damage and neurodegenerative disor-ders such as Parkinson’s Disease and Alzhei mer’s Di-sease.They are undifferentiated elements found inboth the embryonic and adult brain[1].Growth factor-responsive cells fromthe embryonic and adult controlnervous system(CNS)were isolated and culturedinvitrointhe early1990’s[2-4].Thelocation of CNS stemcells in the adult brain were therefore identified[5]mainlyinthe striatum,…     

脐带血干细胞捐赠与自存契约之效力分析

与脐带血储存相关的契约以其储存目的为标准,称当事人以自存脐带血干细胞为目的所缔结的为"自存契约",而以他捐为目的者,称之为"捐赠契约"。基于脐带血干细胞所有权归属解释上的差异,比照法律行为之生效要件,检视我国现有的脐带血捐赠与自存契约的效力,并得出结论即宜以母亲为脐带血所有权人以及契约当事人的地位订立契约,借此契约的订立,能让母亲为子女预存脐带血的意志得以完整实现,脐带血干细胞的利用也会更有效。