人主动脉-性腺-中肾区基质细胞诱导胚胎干细胞分化为造血干细胞及体内造血重建

背景：研究证实多种造血生长因子、基质细胞饲养层及其条件培养液可促进胚胎干细胞向造血干细胞分化。目的：以人主动脉-性腺-中肾（aorta-gonad-mesonephros,AGM）区基质细胞为饲养层体外诱导小鼠胚胎干细胞分化为造血干细胞,并比较不同移植途径对造血干细胞体内造血重建能力的影响。方法：将小鼠E14胚胎干细胞诱导为拟胚体,采用Transwell非接触共培养体系在人AGM区基质细胞饲养层上诱导6d,接种NOD-SCID小鼠检测体内致瘤性。再将诱导后的拟胚体细胞移植经致死量60Coγ射线辐照的BALB/C雌鼠,受鼠随机分为静脉移植组、骨髓腔移植组、照射对照组及正常对照组。结果与结论：拟胚体细胞经人AGM区基质细胞诱导后Sca-1＋c-Kit＋细胞占（13.12±1.30）%。NOD-SCID小鼠皮下接种经人AGM区基质细胞诱导的拟胚体细胞可出现畸胎瘤,经骨髓腔接种未见肿瘤形成。静脉移植组动物全部死亡,骨髓腔移植组生存率为55.6%,移植后21d外周血象基本恢复,存活受鼠检测到供体来源Sry基因。提示小鼠胚胎干细胞经人AGM区基质细胞诱导分化的造血干细胞通过骨髓腔移植安全并具有一定的造血重建能力。     

人主动脉-性腺-中肾区基质细胞诱导胚胎干细胞分化为造血干细胞及体内造血重建

背景:研究证实多种造血生长因子、基质细胞饲养层及其条件培养液可促进胚胎干细胞向造血干细胞分化.目的:以人主动脉-性腺-中肾(aorta-gonad-mesonephros,AGM)区基质细胞为饲养层体外诱导小鼠胚胎干细胞分化为造血干细胞,并比较不同移植途径对造血干细胞体内造血重建能力的影响.方法:将小鼠E14 胚胎干细胞诱导为拟胚体,采用Transwell非接触共培养体系在人AGM区基质细胞饲养层上诱导6 d,接种NOD-SCID小鼠检测体内致瘤性.再将诱导后的拟胚体细胞移植经致死量60Co γ射线辐照的BALB/C雌鼠,受鼠随机分为静脉移植组、骨髓腔移植组、照射对照组及正常对照组.结果与结论:拟胚体细胞经人AGM区基质细胞诱导后Sca-1+c-Kit+细胞占(13.12±1.30)%.NOD-SCID小鼠皮下接种经人AGM区基质细胞诱导的拟胚体细胞可出现畸胎瘤,经骨髓腔接种未见肿瘤形成.静脉移植组动物全部死亡,骨髓腔移植组生存率为55.6%,移植后21 d外周血象基本恢复,存活受鼠检测到供体来源Sry基因.提示小鼠胚胎干细胞经人AGM区基质细胞诱导分化的造血干细胞通过骨髓腔移植安全并具有一定的造血重建能力.     

间充质干细胞靶向转运胸苷激酶基因联合更昔洛韦杀伤鼻咽癌细胞

背景：研究证实,间充质干细胞能通过基因修饰成为肿瘤治疗的靶向载体。目的：观察转染胸苷激酶基因的胸苷激酶-间充质干细胞联合更昔洛韦对鼻咽癌细胞的靶向杀伤作用。方法：应用LipofectamineTM2000将表达胸苷激酶基因的重组质粒pGL3-EGFP-胸苷激酶转染至SD大鼠间充质干细胞,观察其增殖能力。应用Transwell小室观察胸苷激酶-间充质干细胞的归巢特性;将胸苷激酶-间充质干细胞植入裸鼠,观察其致瘤性;用胸苷激酶-间充质干细胞/更昔洛韦干预人鼻咽癌细胞5-8F,观察其对细胞的杀伤作用及旁观者效应。结果与结论：荧光显微镜观察及RT-PCR检测结果提示实验成功将胸苷激酶基因转染至间充质干细胞,CCK-8检测结果显示胸苷激酶-间充质干细胞与间充质干细胞增殖能力差异无显著性意义（P〉0.05）。Transwell小室迁移实验显示胸苷激酶-间充质干细胞具有归巢特性,裸鼠移植瘤实验显示胸苷激酶-间充质干细胞无致瘤性。CCK-8检测检测发现胸苷激酶-间充质干细胞/更昔洛韦具有旁观者效应,可明显降低5-8F的生存率（P〈0.01）。提示胸苷激酶-间充质干细胞/更昔洛韦对鼻咽癌细胞具有靶向迁移及杀伤作用,间充质干细胞可作为治疗鼻咽癌的理想靶向转运载体。     

人类胚胎干细胞的研究现状与趋势

胚胎干细胞是来源于早期胚胎内细胞团或原始生殖细胞的一种多潜能性细胞，具有自我更新的多向分化的潜能。胚胎干细胞在临床移植医学，细胞治疗，组织工程，生物学基础研究等领域具有重要的科学意义和巨大的应用前景。     

人类胚胎干细胞的研究现状与趋势

胚胎干细胞是来源于早期胚胎内细胞团或原始生殖细胞的一种多潜能性细胞,具有自我更新和多向分化的潜能。胚胎干细胞在临床移植医学、细胞治疗、组织工程、生物学基础研究等领域具有重要的科学意义和巨大的应用前景。     

肝干细胞研究进展

198 1年英国的Evans和Kaufman用延缓着床的胚泡首次成功地分离了小鼠胚胎干细胞 ,从而在全球掀起了有关干细胞的研究热潮。 1998年 11月 ,美国Thomson和Gearhart分别用不同的方法获得人胚胎干细胞及胚胎生殖细胞 ,此后 ,干细胞的研究便进入了一个新的时代。所谓干细胞 (stemcell) ,是指那些处于分化过程之中 ,具有分裂增殖能力 ,可自我更新 ,有多向分化潜能即能分化产生一种以上“专业”细胞的原始细胞。干细胞依其分化潜能的大小可分为 :①胚胎干细胞 (embryonicstemcell,ES细胞 ) ,即具有分化为机体任何一种组织器官潜能的细胞 ,如囊胚…     

干细胞的研究及应用前景

干细胞是存在于生命个体中的一类具有高度自我更新能力和多向分化潜能的细胞群体。它们不仅存在于胚胎发育时期而且在成体内也广泛分布于各种组织器官的特定部位,故宏观上将其分为胚胎干细胞(ES细胞)和成体干细胞。其中ES细胞具有分化全能性,可以分化成任一机体组织细胞,它的主要功能是参与机体的发育;成体干细胞的分化潜能则相对有限,其功能主要是维持组织器官的新陈代谢。随着细胞替代治疗的发展,干细胞移植治疗已成为临床上治疗某些疾病的重要手段,利用干细胞在体外扩增培养并诱导成所需要的细胞后移植入患者体内,用于组织损伤的修复、退行性器官的替代及改善遗传性缺陷组织器官的功能。而成体干细胞可塑性分化的发现,为细胞替代治疗提供了新的种子细胞。     

干细胞的研究及应用前景

干细胞是存在于生命个体中的一类具有高度自我更新能力和多向分化潜能的细胞群体。它们不仅存在于胚胎发育暑期而且在成体内也广泛分布于各种组织器官的特定部位，故宏观上将其分为胚胎干细胞（ES细胞）和成体干细胞。其中ES细胞具有分化全能性，可以分化成任一机体组织细胞，它的主要功能是参与机体的发育；成体干细胞的分化潜能则相对有很，其功能主要是维持组织器官的新陈代谢。随着细胞替代治疗的发展，干细胞移植治疗已成为临床上治疗某些疾病的重要手段，利用干细胞在体外扩增培养并诱导成所需要的细胞后移植入患体内，用于组织损伤的修复、退行性器官的替代及改善遗传性缺陷组织器官的功能。而成体干细胞可塑性分化的发现，为细胞替代治疗提供了新的种子细胞。     

异种肝前体细胞移植治疗急性肝损伤大鼠

背景：成体肝前体细胞可在受体肝脏内定植并分化为肝细胞。不过,异种肝前体细胞移植能否促进急性肝损伤的恢复,脾脏微环境能否促进移植物的存活和向肝细胞分化,尚没有研究。目的：评价异种肝前体细胞移植治疗急性肝损伤的作用;监测移植肝前体细胞在大鼠脾脏实质内的定植及向肝细胞的分化。方法：体外培养雄性小鼠来源的肝前体细胞系肝上皮样前体细胞。通过CCl4腹腔注射联合2/3肝切除构建急性肝损伤大鼠模型,进行肝上皮样前体细胞脾脏移植。在肝切除后1,5,14和21 d,苏木精-伊红染色观察肝脏病理改变,全自动生化分析仪监测血清转氨酶变化,PCR反应检测脾脏组织Y染色体特异性序列Sry,脾脏CK-19和Alb免疫组织化学追踪移植肝上皮样前体细胞的植入和肝细胞分化。结果与结论：肝上皮样前体细胞可在体外长期培养,保持增殖能力和双向分化潜能。肝上皮样前体细胞脾脏移植后,肝损伤大鼠肝细胞肿胀明显减轻,丙氨酸转氨酶和天门冬氨酸转氨酶下降更明显。移植后1,5,14和21 d,脾脏DNA中均能检测到Sry序列。在整个实验期间CK-19阳性细胞在大鼠脾脏实质内始终存在。Alb阳性细胞在移植后5 d在脾脏实质中出现,随后阳性细胞数逐渐增多。实验表明,移植肝前体细胞能在大鼠脾脏实质中植入,并分化为肝细胞,能有效促进CCl4腹腔注射联合2/3肝切除诱导的大鼠急性肝损伤的修复过程。     

成体干细胞及其分化机理

成体干细胞是存在于胎儿和成人组织器官中具有自我更新，高度增殖和多向分化潜能的细胞，在适当的诱导条件下。可变成不同类型的细胞。成体干细胞的分化是指成体干细胞具有可在体内、体外分化成不同类型细胞的能力，将其分化物植入体内后在各种情况下都能稳定地存活。目前认为分化的可能机制是：多种成体干细胞可能在不同的器官中存在，包括出生后体内仍存在的多能性干细胞。美国Minnesota大学干细胞研究所一系列的研究证明成体骨髓中确实存在具有多分化潜能的成体干细胞，命名为MAPC，能在体外由单细胞分化为具有中胚层系、外胚层系或内胚层系特征的细胞；将其注入胚泡，能分化成各种组织细胞。多能的成体干细胞在今后能用于多个不同器官的变性或遗传性疾病的治疗。     

Molecular and Magnetic Resonance Imaging of Human Embryonic Stem Cell�CDerived Neural Stem Cell Grafts in Ischemic Rat Brain

Real-time imaging of transplanted stem cells is essential for understanding their interactions in vivo with host environments, for tracking cell fate and function and for successful delivery and safety monitoring in the clinical setting. In this study, we used bioluminescence (BLI) and magnetic resonance imaging (MRI) to visualize the fate of grafted human embryonic stem cell (hESC)–derived human neural stem cells (hNSCs) in stroke-damaged rat brain. The hNSCs were genetically engineered with a lentiviral vector carrying a double fusion (DF) reporter gene that stably expressed enhanced green fluorescence protein (eGFP) and firefly luciferase (fLuc) reporter genes. The hNSCs were self-renewable, multipotent, and expressed markers for neural stem cells. Cell survival was tracked noninvasively by MRI and BLI for 2 months after transplantation and confirmed histologically. Electrophysiological recording from grafted GFP⁺ cells and immuno-electronmicroscopy demonstrated connectivity. Grafted hNSCs differentiated into neurons, into oligodendrocytes in stroke regions undergoing remyelination and into astrocytes extending processes toward stroke-damaged vasculatures. Our data suggest that the combination of BLI and MRI modalities provides reliable real-time monitoring of cell fate.     

In vivo molecular imaging of murine embryonic stem cells delivered to a burn wound surface via Integra? scaffolding

It has been demonstrated that restoration of function to compromised tissue can be accomplished by transplantation of bone marrow stem cells and/or embryonic stem cells (ESCs). One limitation to this approach has been the lack of noninvasive techniques to longitudinally monitor stem cell attachment and proliferation. Recently, murine ESC lines that express green fluorescent protein (GFP), luciferase (LV), and herpes simplex thymidine kinase (HVTK) were developed for detection of actively growing cells in vivo by imaging. In this study, the authors investigated the use of these ESC lines in a burned mouse model using Integra? as a delivery scaffolding/matrix. Two different cell lines were used: one expressing GFP and LV and the other expressing GFP, LV, and HVTK. Burn wounds were produced by application of a brass block (2 × 2 cm kept in boiling water before application) to the dorsal surface of SV129 mice for 10 seconds. Twenty-four hours after injury, Integra? with adherent stem cells was engrafted onto a burn wound immediately after excision of eschar. The stem cells were monitored in vivo by measuring bioluminescence with a charge-coupled device camera and immunocytochemistry of excised tissue. Bioluminescence progressively increased in intensity over the time course of the study, and GFP-positive cells growing into the Integra? were detected. These studies demonstrate the feasibility of using Integra? as a scaffolding, or matrix, for the delivery of stem cells to burn wounds as well as the utility of bioluminescence for monitoring in vivo cellular tracking of stably transfected ESC cells.     

多模态成像对间充质干细胞移植后活体示踪的应用研究

目的 探讨生成像（BLI）和MRI相结合对移植入大鼠损伤肝脏内的间充质干细胞（MSCs）的实时、无创示踪的能力及移植的MSCs对大鼠损伤肝脏的功能修复作用。方法 对急性肝损伤大鼠模型移植标记了荧光素酶和SPION的MSCs,分析大鼠的血浆转氨酶水平及肝脏病理学改变,观察MSCs对肝功能损伤的修复作用,并利用BLI和MRI观察MSCs在大鼠肝脏内积累的时间进程,最后进行大鼠肝脏组织切片的原位免疫组织化学染色。结果 MSCs移植后的第5、9、10天检测的大鼠血浆天冬氨酸转氨酶和丙氨酸转氨酶水平较对照组显著降低（P均<0.05）。MSCs移植后第1天,大鼠肝脏区域的生物发光强度显著增强,T2^*WI标准化信号强度明显减低,于移植后第10天才逐渐恢复到移植前水平。并且随着大鼠肝脏生物发光强度的逐渐下降,荧光素酶阳性的MSCs数目亦随时间进程逐渐减少。结论 移植MSCs对大鼠损伤肝脏的功能具有良好的修复作用;且将BLI和MRI相结合可实时、无创示踪移植入大鼠损伤肝脏内的MSCs。     

Human embryonic stem cell-derived mesenchymal stem cells as cellular delivery vehicles for prodrug gene therapy of glioblastoma

Mesenchymal stem cells (MSCs) possess tumor-tropic properties and consequently have been used to deliver therapeutic agents for cancer treatment. Their potential in cancer therapy highlights the need for a consistent and renewable source for the production of uniform human MSCs suitable for clinical applications. In this study, we seek to investigate whether human embryonic stem cells can be used as a cell source to fulfill this goal. We generated MSC-like cells from two human embryonic stem cell lines, HuES9 and H1, and observed that MSC-like cells derived from human embryonic stem cells were able to migrate into human glioma intracranial xenografts after being injected into the cerebral hemisphere contralateral to the tumor inoculation site. We engineered these cells with baculoviral and lentiviral vectors, respectively, for transient and stable expression of the herpes simplex virus thymidine kinase gene. In tumor-bearing mice the engineered MSC-like cells were capable of inhibiting tumor growth and prolonging survival in the presence of ganciclovir after they were injected either directly into the xenografts or into the opposite hemisphere. Our findings suggest that human embryonic stem cell-derived MSCs may be a viable and attractive alternative for large-scale derivation of targeting vehicles for cancer therapy.     

Genetically modified donor leukocyte transfusion and graft-versus-leukemia effect after allogeneic stem cell transplantation

Seven patients with acute myeloid leukemia (AML) and two patients with chronic myelogenous leukemia (CML) were transplanted from HLA-identical sibling donors with CD34(+) cell-enriched stem cells (HSCTs) without further immunosuppression. The myeloablative standard transplantation protocol was adapted to include transfusion of gene-modified donor T cells after HSCT. Donor T cells were transduced with the replication-deficient retrovirus SFCMM-3, which expresses herpes simplex thymidine kinase (HSV-Tk) and a truncated version of low-affinity nerve growth factor receptor (ΔLNGFR) for selection and characterization of transduced cells. Transduced T cells were detectable in all patients during follow-up for up to 5 years after transfusion. Proteomic screening for development of acute graft-versus-host disease (aGvHD) was applied to five of the seven patients with AML. No positivity for the aGvHD grade II-specific proteomic pattern was observed. Only one patient developed aGvHD grade I. To date, three of the patients with AML relapsed; one responded to three escalating transfusions of lymphocytes from the original donor and is in complete remission. Two were retransplanted with non-T cell-depleted peripheral blood stem cells from their original donors and died after retransplantation of septic complications or relapse, respectively. In one patient with CML, loss of bcr-abl gene expression was observed after an expansion of transduced cells. Seven of nine patients are alive and in complete remission.     

干细胞移植用于肝再生治疗的研究

背景：肝终末期疾病的常规综合治疗不能达到理想的治疗效果,因此,干细胞移植治疗肝脏疾病成为研究的热点之一,并且已经成为治疗肝脏疾病的新方法。目的：明确干细胞移植用于肝再生治疗的实验研究和临床应用结果。方法：分别对不同来源的干细胞如骨髓干细胞、胚胎干细胞、脂肪干细胞以及外周血造血干细胞移植分化治疗肝脏疾病进行体外和体内实验研究分析,并且应用免疫组化染色、病理学以及血清中各项生化指标检测与正常肝脏细胞的生物特性及功能进行比较分析,确定干细胞移植治疗肝脏疾病的效果。结果与结论：骨髓干细胞、胚胎干细胞、脂肪干细胞以及外周血造血干细胞均可以在特定条件下诱导分化成为肝样细胞,对损伤肝脏不仅有治疗作用而且能够促进肝脏的修复和再生,同时还可以进行干细胞移植转基因治疗。除此之外,干细胞移移植可以避免免疫排斥反应的发生,可用于治疗各种类型的肝脏疾病。     

胚胎干细胞源性肝干细胞肝内移植对宿主肝功能的影响及安全性评估

背景：体外诱导胚胎干细胞分化为肝细胞已有不少成功的报道，但其体内移植后能否有效整合入宿主肝板、在肝内能否进一步生长分化并表达肝细胞功能以及成瘤的风险等情况目前还不清楚。 目的：应用治疗性肝再生模型进行胚胎干细胞源性肝干细胞肝内移植．观察其在肝组织替代、体内的生长分化及成瘤性情况。 设计：随机对照动物实验。 单位：中山大学附属第二医院小儿外科。 材料：选用BALB／c小鼠24只为受体，鼠龄6～8周，体质量20～352，雌雄不拘购自广州市实验动物中心。实验所用胚胎干细胞源性肝干细胞由作者所在课题组诱导胚胎干细胞分化而成。小鼠胚胎干细胞株E14由本院干细胞中心提供。 方法：实验于2006-07／2007-06在中山大学附属第二医院干细胞研究中心完成。将24只小鼠随机分为2组：肝再生模型＋干细胞移植组和肝切除＋干细胞移植组，每组12只。前组分两次按50mg／kg剂量腹腔内注射倒千里光碱（retrorsine），间隔2周，第2次注射4周后行70％肝部分切除制造肝损伤；然后经门静脉分别移植1×10^5羟基荧光素乙酰乙酸（CFDA-SE）荧光标记的细胞入小鼠肝内进行胚胎干细胞源性肝干细胞移植。后组在行70％肝部分切除制造肝损伤模型后进行胚胎干细胞源性肝干细胞移植。 主要观察指标：荧光显微镜下观察移植细胞组受体鼠肝脏内分布、整合与体内生长分化情况。2周后行白蛋白荧光免疫组化（双荧光染色）、血清白蛋白水平检测其功能状况。将胚胎干细胞源性肝干细胞注入治疗性肝再生小鼠肝内，将未分化的胚胎干细胞移植入小鼠腋区皮下作为对照，观察胚胎干细胞源性肝干细胞体内成瘤情况。 结果：①肝干细胞在受体鼠肝内生长情况：CFDASE标记的胚胎干细胞源性肝干细胞肝内移植1周，受体小鼠肝实质内可见散     

Targeted suicide gene therapy for glioma using human embryonic stem cell-derived neural stem cells genetically modified by baculoviral vectors

Tumor-tropic neural stem cells (NSCs) can be used in the Trojan horse approach as cellular vehicles for targeted delivery of therapeutic agents to distant tumor sites. To realize this cancer therapy potential, it is important to have a renewable source to generate large quantities of uniform human NSCs. Here, we reported that NSCs derived from HES1 human embryonic stem cell line were capable of migrating into intracranial glioma xenografts after systemic injection or after intracranial injection at a site distant from the tumor. To test whether the HES1-derived NSCs can be used for cancer gene therapy, we used a baculoviral vector to introduce the herpes simplex virus thymidine kinase suicide gene into the cells and demonstrated that baculovirus-mediated transgene expression may last for at least 3 weeks in NSCs. After being injected into the cerebral hemisphere opposite the tumor site and in the presence of ganciclovir, NSCs expressing the suicide gene were able to inhibit the growth of human glioma xenografts and prolong survival of tumor-bearing mice. Our findings suggest that human embryonic stem cells could potentially serve as a clinically viable source for production of cellular vehicles suitable for targeted anticancer gene therapy.     

延胡索酰乙酰乙酸水解酶基因敲除大鼠胚胎干细胞株的建立

背景：小鼠肝损伤模型已被广泛应用,大鼠肝损伤模型能在发病机制,肝移植环境等方面更好地模拟人,建立大鼠延胡索酰乙酰乙酸水解酶（fuarylacetoacetat hydrolase,Fah）基因敲除的胚胎干细胞株是大鼠肝损伤模型建立的重点和难点。目的：建立Fah基因缺失的大鼠胚胎干细胞株。方法：根据Genbank检索出的大鼠Fah基因序列构建带有正负双向筛选系统的Fah基因敲除载体pKO-Fah,并进行大鼠胚胎干细胞制备与培养,通过电穿孔转染将线性化质粒转入大鼠胚胎干细胞中,用嘌呤霉素和增强型绿色荧光进行克隆的筛选,再对筛选出来的阳性克隆进行PCR验证。结果与结论：电穿孔转染后观察到绿荧光胚胎干细胞克隆,经过3轮嘌呤霉素药物筛选后约有90%的胚胎干细胞克隆表达绿荧光,成功挑取到2个稳定表达绿荧光的胚胎干细胞克隆,重组胚胎干细胞-Fah,通过PCR鉴定为正确同源重组的阳性克隆。提示大鼠胚胎干细胞稳定建系后,能通过传统的同源重组方法来建立基因敲除胚胎干细胞,并为后期基因敲除动物的建立奠定基础。