胚胎干细胞向心肌细胞分化研究进展(综述)

胚胎干细胞(embryonic stem cells,ES)在体外分化培养条件下可以分化出各种组织细胞，其中包括心肌细胞。ES细胞在体外向心肌细胞分化与体内完整胚胎心肌发育过程相符合。该细胞在体外分化过程中顺序表达心肌细胞特有结构蛋白和离子通道，如肌球蛋白轻链和重链、特异性肌动蛋白、电压依赖性Ca^2 通道、K^ 通道等。ES细胞分化来源的心肌细胞具有体内心肌细胞的生理学特点，如产生的动作电位、表现自发性收缩等。因此，ES细胞是研究心肌细胞发育分化机制及鉴定其关键基因的理想模型。     

Differentiation of embryonic stem cells transfected by ibeB gene

We have previously identified an E. coli determinant, ibeB gene locus contributing to invasion of human brain microvascular endothelial cells. In the present study, we established embryonic stem (ES) cell lines overexpressing IbeB and found that exogenic ibeB gene could start-up expression of a neural stem cell specific marker, nestin, and give rise to polar changes. In analysis of IbeB location, it was found that GFP-IbeB fusion protein targeted at the ES cell nucleus. These data suggests that ibeB gene may play an important role in the regulation of nestin expression.     

胚胎干细胞及其在人类疾病治疗中的应用

胚胎干细胞是一类多能性干细胞,近年来已成为生命科学研究领域的热点之一．尤其在人类疾病治疗方面有着诱人的应用前景．本文主要介绍了胚胎干细胞在几种疑难疾病治疗上的应用及其前景,胚胎干细胞与异种器官移植。以及目前存在的一些问题．     

Properties and applications of embryonic stem cells

Mouse embryonic stem (ES) cells are pluripotent cells derived from the early embryo and can be propagated stably in undifferentiated state in vitro. They retain the ability to differentiate into all cell types found in the embryonic and adult body in vivo, and can be induced to differentiate into many cell types under appropriate culture conditions in vitro. Using these properties, people have set up various differentiated systems of many cell types and tissues in vitro. Through analysis of these systems, one can identify novel bioactive factors and reveal mechanisms of cell differentiation and organogenesis. ES cell-derived differentiated cells can also be applied to cell transplantation therapy. In addition, we summarized the features and potential applications of human ES cells.     

Induction of embryonic stem cells to hematopoietic cells in vitro

In order to get hematopoietic cells from embryonic stem (ES) cells and to study development mechanisms of hematopoietic cells, the method of inducing embryonic stem cells to hematopoietic cells was explored by differenciating mouse ES cells and human embryonic cells in three stages. The differentiated cells were identified by flow cytometry, immunohistochemistry and Wright's staining. The results showed that embryoid bodies (EBs) could form when ES cells were cultured in the medium with 2-mercaptoethanol (2-ME). However, cytokines, such as stem cell factor (SCF), thrombopoietin (TPO), interleukin-3 (IL-3), interleukin-6 (IL-6), erythropoietin (EPO) and granular colony stimulating factor (G-CSF), were not helpful for forming EBs. SCF, TPO and embryonic cell conditional medium were useful for the differentiation of mouse EBs to hematopoietic progenitors. Eighty-six percent of these cells were CD34+ after 6-d culture. Hematopoietic progenitors differentiated to B lymphocytes when they were cocultured with primary bone marrow stroma cells in the DMEM medium with SCF and IL-6. 14 d later, most of the cells were CD34-CD38+. Wright's staining and immunohistochemistry showed that 80% of these cells were plasma-like morphologically and immunoglubolin positive. The study of hematopoietic cells from human embryonic cells showed that human embryonic cell differentiation was very similar to that of mouse ES cells. They could form EBs in the first stage and the CD34 positive cells account for about 48.5% in the second stage.     

Forced expression of theOct-4 gene influences differentiation of embryonic stem cells

By transfecting an Oct-4 expression plasmid into embryonic stem cells (ES cells), the ES-O cell line was constructed, which sustained the expression of Oct-4 gene when induced by retinoic acid. Forced expression of Oct-4 gene could not sustain the stem property of ES-O cells without the differentiation inhibiting factor LIF, but if LIF exists, forced expression of Oct-4 gene could enhance the ability to sustain the undifferentiation state and inhibit cell differentiation induced by retinoic acid. It was indicated that Oct-4 must cooperate with LIF to sustain the undifferentiation state of ES cells. During the cell differentiation, ES-O cells tend to differentiate into neural cells, suggesting that forced expression of Oct-4 gene may be in relation with the differentiation of neuroderm.     

Therapeutic applications of bone marrow-derived stem cells in liver transplantation for end-stage liver diseases

Today, liver transplantation (LT) is the only established treatment for end-stage liver diseases. The de- velopment of LT, including OLT, cadaveric LT, split LT, living donor LT (LDLT), brings hopes to patients with these diseases. However, increasing donor shortage, rejection and life-long immunosuppression with its side effects are the major limitations of this therapy strategy. Bone marrow-derived stem cells (BMDSCs) are capable of differentiating into hepatocyte-like cells and contribute to liver injury repair. The microenvironment of liver injury caused by rejection, ischemia/reperfusion, loss of liver mass, recurrence of HCV and "small-for-size syndrome" after LT can attract a variety of bone marrow-derived stem cell population to the peripheral circulation and then migration to the injury liver to promote the hepatic function restoration. Additionally, BMDSCs can also take part in the functional regeneration of living donor liver after LDLT. This participation in liver regeneration may be associated to the interac- tion between SDF-1and its receptor CXCR4, involving HGF, IL-8, MMP9, and VEGF/VEGFR-2. BMDSC with its bio-characteristics could maintain the allograft tolerance from different angles and in different ways. In conclusion, BMDSCs transplantation, as a new assistant therapeutic method for LT, will ex- pand the space of LT, and provide more survival opportunities for the patients suffering liver diseases in the future.     

Induction of embryonic stem cells to hematopoietic cellsin vitro

In order to get hematopoietic cells from embryonic stem (ES) cells and to study development mechanisms of hematopoietic cells, the method of inducing embryonic stem cells to hematopoietic cells was explored by differenciating mouse ES cells and human embryonic cells in three stages. The differentiated cells were identified by flow cytometry, immunohistochemistry and Wright’s staining. The results showed that embryoid bodies (EBs) could form when ES cells were cultured in the medium with 2-mercaptoethanol (2-ME). However, cytokines, such as stem cell factor (SCF), thrombopoietin (TPO), interleukin-3 (IL-3), interleukin-6 (IL-6), erythropoietin (EPO) and granular colony stimulating factor (G-CSF), were not helpful for forming EBs. SCF, TPO and embryonic cell conditional medium were useful for the differentiation of mouse EBs to hematopoietic progenitors. Eighty-six percent of these cells were CD34⁺ after 6-d culture. Hematopoietic progenitors differentiated to B lymphocytes when they were cocultured with primary bone marrow stroma cells in the DMEM medium with SCF and IL-6. 14 d later, most of the cells were CD34^－CD38⁺. Wright’s staining and immunohistochemistry showed that 80% of these cells were plasma-like morphologically and immunoglubolin positive. The study of hematopoietic cells from human embryonic cells showed that human embryonic cell differentiation was very similar to that of mouse ES cells. They could form EBs in the first stage and the CD34 positive cells account for about 48.5% in the second stage.     

昆明鼠胚胎干细胞的分离培养与鉴定

目的：从昆明系小鼠的早期胚胎分离和培养胚胎干细胞(ES细胞)．方法：收集小鼠3．5d胚龄的囊胚，将其培养在小鼠胚胎成纤维细胞饲养层上，5—6d后取隆起生长的内细胞团块分离后再培养，观察集落的生长情况并通过碱性磷酸酶染色、原位杂交、细胞核型分析等对细胞集落进行鉴定．结果：KS细胞集落性生长，符合小鼠胚胎干细胞的一系列特性．结论：昆明系小鼠囊胚在胚胎成纤维细胞饲养层上可以发育成ES细胞，并能进行传代培养．     

Maintenance of human embryonic stem cells on gelatin

Matrigel is routinely used as a coating material in the feeder-free culture system of human embryonic stem cells （hESCs）. However, matrigel is costive and inconvenient to use. In this study, the possibility of using gelatin as an alternative coating material was investigated. The results showed that, after trypsinization, hESCs were maintained undifferentiated on gelatin. These hESCs expressed pluripotent markers, formed teratoma and maintained a normal karyotype. As measured at passage 10, the hESCs expressed a high level of Oct4 on both gelatin and Matrigeh hESCs growing on gelatin formed AP-positive colonies in similar size and number to those growing on Matrigel （P〉 0.05）. Moreover, hESCs growing on gelatin contained a comparable percentage of SSEA-4-positive cells to those growing on Matrigel （95.1% vs.94.3%, P〉 0.05）. H-1 hESCs were maintained undifferentiated on gelatin for 20 passages and remained the stable normal karyotype. This gelatin-based culture protocol may allow us to propagate hESCs in large scale, with less cost.     

胎肝间充质干细胞治疗实验性糖尿病小鼠的研究

目的:研究胎肝间充质干细胞向胰岛β样细胞分化和治疗1型糖尿病的可行性.方法: 用贴壁筛选法从正常C57BL/6j胎鼠(孕11.5～15.5 d)肝脏中分离出胎肝间充质干细胞,用高浓度葡萄糖、碱性成纤维细胞生长因子(bFGF)和尼克酰胺体外诱导胎肝间充质干细胞向胰岛β样细胞分化;制作1型糖尿病小鼠模型,并将诱导后的细胞移植到1型糖尿病小鼠肾被膜下,连续6周观察血糖变化;用组织学分析移植细胞体内的发育.结果:胎肝间充质干细胞来源的胰岛β样细胞移植到糖尿病小鼠肾被膜下后,具有一定的降低血糖的作用,经过6周以后,接近正常水平;取出移植物,小鼠高血糖重新出现,并很快死亡;移植物作免疫组织化学染色,可以观察到肾被膜下区含有大量胰岛素阳性细胞.结论:胎肝间充质干细胞有望成为1型糖尿病胰岛移植的种子细胞.     

哺乳动物胚胎干细胞的特性及利用

哺乳动物胚胎干细胞（ES细胞）是由动物早期胚胎发育的内细胞团（ICM）或原始生殖细胞（PGC）分离得到的。人们利用ES细胞所具有的全能性、体外分化以及稳定的遗传性能等特点，展示了ES细胞在建立哺乳动物的早期胚胎体外分化模型、转基因动物模型、器官和组织的修复和移植治疗、克隆动物的生产、发育生物学的研究等方面广阔的应用前景。但是，由于哺乳动物错综复杂的基因调控和环境因素的影响，对于胚胎干细胞的研究还存在诸多问题，还需作更深入细致的研究。     

人胚胎脑神经干细胞分离纯化及体外培养的研究

为了探讨人胚神经干细胞体外培养条件下的生物学特性,为其应用于临床治疗奠定基础.取胎龄16周的人流产胚胎,胰酶消化结合机械法分离成单细胞悬液,以2×106个细胞/mL接种到含hEGF和h-bFGF的DMEM/F12、N2培养基进行体外培养;观察细胞生长情况,用10% FBS诱导神经干细胞球分化,免疫细胞化学鉴定. 结果显示从人胚大脑分离出的细胞经悬浮培养可以形成细胞球,表达Nestin蛋白.经诱导分化后具有表达神经元,神经胶质细胞的特异性抗原. 说明人胚神经干细胞在体外可以稳定生长,并能分化成为神经原及胶质细胞.     

成纤维细胞和ES细胞在小鼠和兔去核卵母细胞内发育

以昆明白小鼠成纤维细胞和胚胎干（ES）细胞作为供核细胞,以昆明白小鼠和日本大耳白兔的MⅡ期去核卵母细胞作为受体,采用核移植方法,构楚了克隆胚胎．在同种克隆中,以ES细胞为供核细胞的克隆胚胎卵裂率明显低于以成纤维细胞为供核细胞的克隆胚胎卵裂率（24．4％相对于56．9％,P〈0．05）,1．8％的ES细胞克隆胚胎发育到囊胚阶段,而成纤维细胞克隆胚胎没能发育到囊胚阶段;在异种克隆中,以ES细胞为供核细胞的克隆胚胎卵裂率（89．6％）和囊胚发育率（18．8％）明显高于以成纤维细胞为供核细胞的克隆胚胎卵裂率（54．2％）和囊胚发育率（4．2％）．     

诱导多能干细胞与心脏再生

诱导多能干细胞(induced pluripotent stem cells, iPSCs)研究的快速发展为心血管转化医学研究领域提供了新的策略. 诱导多能干细胞不仅具有与胚胎干细胞类似的多能性, 且巧妙地回避了胚胎干细胞面临的伦理学问题和免疫排斥反应. 心肌细胞等成体心血管细胞在发生心血管疾病后增殖能力有限, 而iPSCs 来源的心血管细胞在心脏再生治疗中颇具应用前景,是理想的细胞来源, 因此在基础医学和转化医学研究领域受到广泛关注. 就iPSCs 的发展过程及其在心脏再生中的应用作一综述, 并探讨目前iPSCs 在心脏再生临床转化中亟待解决的问题.     

小鼠胚胎干细胞对病毒性心肌炎的影响

目的使用小鼠验证这样一个假设:外界病毒浸入诱发心肌炎时,机体的干细胞将进入心脏提高心肌的抗病毒能力。方法雄性BALB/c小鼠分为三组:小鼠胚胎干细胞对照组(ES),心肌炎病毒组(EM CV)及EM-CV加ES治疗组。通过尾静脉注射,令小鼠立即感染病毒。小鼠死亡率,炎性细胞浸润及心肌坏死等为观察指征。干细胞的游走及分化等通过免疫荧光法来验证。结果给予干细胞后的小鼠的存活率明显高于生理盐水对照组,炎性细胞侵润及心肌坏死亦明显低于生理盐水对照组。免疫荧光法表明,干细胞进入心肌并分化成新的心肌细胞。结论干细胞能明显提高心肌炎小鼠的存活率,减少心肌组织的坏死。同时,亦证明当心脏遭受病毒的侵入后,干细胞通过某种机理修复或再生心肌细胞,从而提高组织的抗病毒能力。     

Preliminary study on human fibroblasts as feeder layer for human embryonic stem cells culture in vitro

To avoid the direct contact with mouse cells and possible heterogeneous pathogen in future application ,we need to replace mouse embryonic fibroblasts with human fibroblasts as the feeder layer to maintain human embryonic stem cells growth in the undifferentiated state,We Success-fully use human fibroblasts derved from aborted fetus and adult prepuces as feeder layer to maintain human embryonic stem cells growth ,During the passage and growth on this feeder layer,the human embryonic stem cells can keep their undifferentiated state.     

人骨髓间充质干细胞不同亚群

目的 比较不同亚群的人骨髓间充质干细胞(human bone mesenchymal stem cell, hBMSC)自我更新和分化能力。方法 通过获赠的人髂骨骨髓标本,联合运用密度梯度离心和差异贴壁法分离MSCs , 用10μm 滤膜将不同群体细胞分离,倒置相差显微镜观察不同亚群细胞的形态;流式细胞仪检测BMSC不同亚群细胞的表型;在地塞米松、Vit C、β-磷酸甘油钠作用下将不同亚型细胞向成骨细胞诱导分化,分别观察其分化能力。结果 成熟的MSC,即mMSCs 细胞(mature cells)呈纤维样梭形, RS 细胞(rapidly MSC self-renewing cells)呈圆形。RS细胞增殖能力明显强于mMSCs。经定向诱导分化后,RS细胞向成骨细胞分化能力较mMSCs细胞强。结论 RS 细胞较之mMSC细胞可能是一种更原始的中胚层前体细胞,具有更强的自我更新和分化潜能。     

On the “plasticity”of adult stem cells and its application in regenerative medicine

In recent years, with the increasingly further studies on embryonic stem cells and the recognition of the biologic characteristics of adult stem cells, it has been discovered that adult stem cells have another phenomenon of “plasticity” in addition to the characteristics of strong potential for self-renewal, proliferation and multi-differentiation, which brings us the hope for regenerative medicine—renewing new organ or tissue cells to replace those damaged by injury or diseases. Although the mechanism of “plasticity” and its application in the regenerative medicine are still in doubt, thorough exploration in these subjects would open up broad prospects for the use in cell and tissue engineering in the near future.