骨髓间充质干细胞在小鼠肝脏局部分化为肝细胞的实验研究

目的 探讨小鼠骨髓间充质干细胞（bone marrow mesenchymal stem cells，BMSCs）在肝内分化为肝细胞的可行性。方法腹腔注射丙烯醇致雌性C57BL／6小鼠全肝损伤后局部接受雄性同品系绿色荧光蛋白（green fluorescence protein，GFP）转基因小鼠骨髓间充质干细胞移植。移植后3周取骨髓嵌合小鼠肝脏做冰冻切片，荧光显微镜下观察GFP阳性细胞在肝内分布，石蜡切片行GFP抗体和白蛋白（albumin，Alb）抗体免疫组化双标法检测GFP阳性细胞在受体小鼠肝内的分化情况。结果移植后3周小鼠肝内可见GFP阳性细胞，并可见GFP和Alb双阳性细胞。结论异体骨髓间充质干细胞可在小鼠肝内分化为表达Alb的肝细胞，为肝组织的再生和修复提供了新思路，显示了其在肝病治疗中的应用潜力。     

供体骨髓细胞在放射损伤肠道中的迁移定植和促修复作用研究

目的研究供体骨髓细胞(donor bone marrow cells,d BMCs)在全身放射损伤小鼠肠道内的迁移定植规律和对肠道放射损伤的促修复作用。方法将60只6~8周龄C57BL/6小鼠进行10 Gy全身照射后随机分为移植组和对照组。比较两组小鼠的存活率,免疫组化分析d BMCs在骨髓和肠道内的定植规律,免疫荧光双染鉴定早期植入肠道内的d BMCs表型。HE染色和Brdu免疫组化分析d BMCs对放射损伤肠道的促修复作用。结果实验后第14天,移植组小鼠的存活率为95%。d BMCs同时向受体骨髓和肠道迁移定植。移植早期d BMCs首先定植于肠道固有层底部并向上迁移,最终填充全部固有层。早期植入的骨髓细胞中含有Sca-1阳性造血干细胞。d BMCs可缓解放射所致肠黏膜固有层水肿,促进肠道干细胞增殖并增加肠隐窝密度。移植早期d BMCs不形成肠上皮细胞,稳定嵌合后可形成骨髓来源肠上皮细胞。结论 d BMCs在移植后早期即向肠道迁移定植并促进放射所致肠道损伤的修复重建过程。     

骨髓干细胞转分化为肝细胞的研究进展

目前，有关骨髓干细胞的转分化现象已成为国际上干细胞的研究热点之一。1997年，Eglitis等发现供体来源的骨髓干细胞能分化为神经胶质细胞，表明骨髓干细胞在一定条件下可以跨胚层分化成其他类型的细胞，这种现象被称为转分化／横向分化（transdifferentiation）。1999年，Petersen等首次报道骨髓干细胞可以转分化为肝脏细胞后，人们希望骨髓干细胞能成为治疗急、慢性肝衰竭的一个新方法。但是目前对于骨髓干细胞转化为肝细胞的研究仍存在许多谜团和争议。因此，本文着重对近年来骨髓来源的肝细胞的研究进行综述。     

骨髓间充质干细胞诱导分化后脾内移植治疗急性肝损伤的研究

背景：近年干细胞的研究为肝脏疾病的治疗提供了新契机。研究证实骨髓间充质干细胞（MSCs）不仅可分化为与其组织来源一致的细胞．且具有跨系或跨胚层分化为不同组织来源细胞的潜能。目的：探讨骨髓MSCs诱导分化后脾内移植治疗急性肝损伤的可行性。方法：密度梯度离心法联合贴壁培养分离、扩增BALB／c小鼠骨髓MSCs。体外诱导向肝系细胞分化，逆转录聚合酶链反应（RT-PCR）、免疫细胞化学法检测肝系细胞的标志。制备BALB／c小鼠ccL急性肝损伤模型．脾内移植DAPI标记的诱导分化21d的MSCs。荧光显微镜下观察细胞的迁徙、定位．行血清肝功能指标、肝脏组织病理学检测移植效果。结果：诱导后7、14d，均检测到甲胎蛋白（AFP）mRNA和蛋白的表达，14、21d检测到白蛋白（ALB）mRNA和蛋白的表达。移植后实验组肝脏和脾脏均发现DAPI标记细胞。与对照组相比，实验组肝功能显著改善（P〈0．05），肝脏病变程度减轻。结论：脾内移植诱导分化的骨髓MSCs对急性肝损伤具有修复作用。     

骨髓源性肝细胞的研究进展

经典发育生物学认为,成体干细胞的分化能力有限,并具有器官特异性,主要用于维持细胞功能的稳态[1],一般不能分化为其他细胞类型.但在1997年,Eglitis等[2]用亚致死剂量放射性核素照射雌性小鼠,破坏其骨髓系统,然后将雄性小鼠的骨髓干细胞移植给雌鼠,结果发现这些细胞在受体内重新分化为神经胶质细胞,表明骨髓干细胞在一定条件下可以跨胚层分化成其他类型的细胞,这种现象被称为横向分化(transdifferentiation).近5年来,有关骨髓干细胞横向分化为肝细胞的研究已成为国际上干细胞研究的热点之一,这些研究发现了骨髓干细胞的新特性,有学者乐观地认为将来可以移植患者自身骨髓干细胞治疗急、慢性肝衰竭.     

小鼠骨髓干细胞分化为小胶质细胞的在体研究

将增强型绿色荧光蛋白转基因小鼠的骨髓细胞通过股静脉移植入实验小鼠制作成嵌合鼠模型,正规饲养8周后,通过免疫组织化学和免疫荧光染色法鉴定移植细胞在嵌合鼠脑内的分布和分化情况。结果利用免疫组织化学方法在嵌合鼠脑内发现了移植细胞,但只存在于小脑。利用免疫荧光双染方法证明了移植细胞仅表现为小胶质细胞表型。认为小鼠骨髓干细胞能够进入血脑屏障完整的正常小鼠小脑,并分化为小胶质细胞。     

小肠干细胞与其微环境

人类和啮齿类动物的小肠上皮终生更新不断，这种更新是由位于小肠隐窝底部的多潜能干细胞驱动的。小肠上皮细胞的凋亡脱落及受损坏死与干细胞增殖、分化之间的动态平衡，在维持肠道屏障结构和功能的完整以及损伤后的修复中扮演着重要角色。小肠上皮是单层柱状上皮结构，在人类、小鼠和大鼠，肠上皮在从功能上可分为两个不连续的单位——绒毛分化单位和隐窝增殖单位，干细胞增殖、分化移行路线清晰，使之成为研究成体干细胞体内分化机制的良好模型。定位和分析小肠干细胞的生存环境并阐明细胞分化具体的分子机制，将有助于理解环境因素对干细胞生物学性状的影响。本文就小肠干细胞的分化机制与微环境的关系作一综述。     

缺血性心脏病细胞移植疗法的理想工具--骨髓基质干细胞

骨髓基质干细胞是一种由骨髓中分离获得的具有多种分化潜能的间质干细胞。在体外培养条件下，它可以分化为成骨细胞、软骨细胞、脂肪细胞、甚至于成肌细胞。因为骨髓基质干细胞具有易于获取、分高方便和良好的分化特性等特点，在细胞移植和基因治疗方面具有非常巨大的应用前景。对于缺血性心脏病而言，骨髓基质干细胞是非常好的细胞移植供体，对于改善心功能帮助巨大。本文献骨髓基质干细胞的生物学特性和在缺血性心脏病治疗中的应用前景进行简要综述。     

骨髓干细胞分化为心肌表型需要与肌细胞间的信息交流

背景 :骨髓干细胞 (BMSCs)有分化为各种细胞的潜能 ,若细胞环境适宜 ,可望横向分化为肌细胞 ,但这一过程的分子信号转导尚未完全明了。作者报告BMSCs的分化取决于它们微环境中的诸细胞的信息交谈。方法及结果 :由绿色荧光蛋白 (GFP) 转基因小鼠分离出BMSCs。以 1∶4 0比例与肌     

骨髓细胞参与甘油引起的小鼠急性肾小管坏死修复的实验观察

目的：观察骨髓来源的细胞能否分化成肾小管上皮细胞。方法：15只绿色荧光蛋白（GFP）标记的C57BL／6转基因小鼠提供骨髓细胞，64只同种无荧光标记的C57BL／6小鼠随机分为正常对照组（N组）、全身照射组（TBI组）、骨髓移植组（BMT组）、骨髓移植＋甘油注射组（B＋G组），每组16只。各组小鼠于不同时间点取血检测血常规、尿素氮及血肌酐，并取肾行H-E染色检查肾脏病理变化。流式细胞仪可以明确受体鼠骨髓细胞中CFP阳性细胞的比例，利用荧光显微镜及激光共聚焦显微镜采用荧光组织化学、免疫组织化学等方法观察GFP阳性细胞在受体鼠肾脏的分布及数量。结果：致死剂量^60Co照射虽然引起血常规三系减少，但并未对肾脏的组织结构及功能造成损伤。BMT组受体鼠在骨髓移植后第56天、84天时，肾小管中有少量GFP阳性细胞的存在，B＋G组受体鼠于上述同样时间点时肾小管中的GFP阳性细胞增多。激光共聚焦显微镜进一步证实了这些GFP阳性细胞位于肾小管上皮，并且荧光组织化学显示，这些GFP阳性细胞表达肾小管上皮细胞特异性功能蛋白Megalin。结论：骨髓细胞可以向肾小管上皮细胞分化．参与肾小管上皮细胞的更新。并且损伤可以使骨髓细胞的肾向分化率增加。     

Hematopoietic stem cell transplantation prevents diabetes in NOD mice but does not contribute to significant islet cell regeneration once disease is established

The treatment of type I diabetes by islet cell transplantation, while promising, remains restricted due to the incomplete efficacy and toxicity associated with current immunosuppression, and by limited organ availability. Given reports suggesting bone marrow derived stem cell plasticity, we sought to determine whether such cells could give rise to pancreatic islet cells in vivo. In the context of autoimmune diabetes, we transplanted unfractionated bone marrow from beta-gal trangenic donor mice into NOD mice prior to, at, and two weeks beyond the onset of disease. Successful bone marrow engraftment before diabetes onset prevented disease in all mice and for 1 year after transplant. However, despite obtaining full hematopoietic engraftment in over 50 transplanted mice, only one mouse became insulin independent, and no beta-Gal positive islets were detected in any of the mice. To test whether tolerance to islets was achieved, we injected islets obtained from the same allogeneic donor strain as the hematopoietic cells into 4 transplant recipients, and 2 had a reversion of their diabetes. Thus allogeneic bone marrow transplantation prevents autoimmune diabetes and tolerizes the recipient to donor islet grants, even in diabetic animals, yet the capacity of bone marrow derived cells to differentiate into functional islet cells, at least without additional manipulation, is limited in our model.     

骨髓来源细胞向肺内迁移分化的实验研究

目的：观察骨髓来源细胞能否向肺内迁移及其特点。方法：野生型（257BL／6J雌性小鼠作为受体接受10Gy的射线照射后,经尾静脉植入同等背景的转增强型绿色荧光蛋白（EGFP）基因的C57BL／6J雄性小鼠（绿鼠）骨髓细胞1×10^7个／只。移植受体稳定1年后检测肺组织中EGFP的表达。结果：野生型小鼠肺组织中EGFP的表达率为0,绿鼠各组织中EGFP的表达率为100％。骨髓移植受体鼠肺组织有广泛的EGFP阳性细胞分布,主要分布在肺间质中,在支气管上皮、肺泡上皮中也有EGFP阳性细胞存在,表达率为100％,与野生型相比差异有显著性（P〈0．01）。结论：受放射线照射后的C57BL／6J小鼠,接受同种异基因小鼠骨髓来源细胞能向肺内迁移并分化为肺的组织细胞。     

Generation of a Chimeric Mouse Reconstituted with Green Fluorescent Protein-Positive Bone Marrow Cells: A Useful Model for Studying the Behavior of Bone Marrow Cells in Regeneration In Vivo

Studies have indicated that bone marrow contains both hematopoietic stem cells and mesenchymal stem cells that can differentiate into a variety of mesenchymal tissues, such as bone, cartilage, muscle, and adipose tissue. Therefore, bone marrow cells are thought to be very useful for cell and gene therapy for various diseases. However, the multipotentiality of these cells remains unclear. To address this issue, we established a chimeric model mouse stably reconstituted with green fluorescent protein (GFP)-marked bone marrow cells. We injected bone marrow cells from GFP-transgenic C57BL/6 mice into the tail veins of recipient wild-type C57BL/6 mice that had been irradiated with a lethal dose of 10 Gy from a cesium source. Microscopic examination and fluorescence-assisted cell sorter (FACS) analysis showed that bone marrow cells, including mesenchymal cells, were almost completely reconstituted with GFP+ cells 5 weeks after transplantation. FACS analysis with lineage-specific antibodies confirmed that the GFP+ cells could differentiate into all types of blood cells. To confirm the usefulness of this mouse model, we studied the role of circulating bone marrow-derived cells in healing of damaged intestine. We performed amputation and anastomosis of the jejunum 10 cm from the pyloric region of the stomach. On the third day after operation, a large number of GFP+ cells were infiltrated in the area of anastomosis, and these cells were positive for CD45 and F4/80 antigens. In 7 days, several cells became negative for CD45 and F4/80 and positive for alpha smooth muscle actin antigen, which is specific for smooth muscle. This finding suggested that bone marrow-derived cells had differentiated into smooth muscle. Because reconstituted bone marrow cells as opposed to injected bone marrow cells, behave naturally, this model is ideal for studying the multipotentiality of bone marrow cells in vivo.     

Heterogeneity of engrafted bone-lining cells after systemic and local transplantation

The outcome of various osteoprogenitor-cell transplantation protocols was assessed using Col1a1-GFP reporter transgenic mice. The model requires the recipient mice to undergo lethal total body irradiation (TBI) followed by rescue with whole bone marrow. When the mice are rescued with total bone marrow from a Col1a1-GFP transgenic mouse, green fluorescence protein (GFP)-positive donor cells can be observed on most endosteal and trabecular bone surfaces. Although the cells express an osteoblast-restricted GFP, they fail to progress to osteocytes, do not form a mineralized matrix, and do not generate bone nodules in vitro. However when calvarial progenitor cells derived from the same transgenic mice are injected into the bone marrow space, osteogenesis by the donor cells is observed. Using different GFP colors that distinguish the donor and recipient osteoblasts, commingling of the 2 cells types is observed along the mineralizing osteoblast surface as well as within the osteocyte population of the endosteal bone. Despite the ability of the injected progenitor cells to produce bone within the injected bone, they lack the ability to form mineralized bone nodules when explanted to primary osteoblast culture. These reagents and imaging protocols will be useful in evaluating other cells having a better progenitor potential than calvarial-derived stromal cells.     

Osteoblast-specific gene expression after transplantation of marrow cells: implications for skeletal gene therapy.

Somatic gene therapies require targeted transfer of the therapeutic gene(s) into stem cells that proliferate and then differentiate and express the gene in a tissue-restricted manner. We have developed an approach for gene therapy using marrow cells that takes advantage of the osteoblast specificity of the osteocalcin promoter to confine expression of chimeric genes to bone. Adherent marrow cells, carrying a reporter gene [chloramphenicol acetyltransferase (CAT)] under the control of a 1.7-kilobase rat osteocalcin gene promoter, were expanded ex vivo. After transplantation by intravenous infusion, engrafted donor cells in recipient mice were detected by the presence of the transgene in a broad spectrum of tissues. However, expression of the transgene was restricted to osteoblasts and osteocytes, as established by biochemical analysis of CAT activity and immunohistochemical analysis of CAT expression at the single cell level. Our data indicate that donor cells achieved long-term engraftment in various tissues of the recipients and that the CAT gene under control of the osteocalcin promoter is expressed specifically in bone. Thus, transplantation of multipotential marrow cells containing the osteocalcin promoter-controlled transgene provides an efficacious approach to deliver therapeutic gene expression to osteoblasts for treatment of bone disorders or tumor metastasis to the skeleton.     

Bone marrow stem cells do not repopulate the healthy upper respiratory tract

Recent studies reported differentiation of both bone marrow and tissue-specific stem cells into cells of other organs. The demonstration that bone marrow stem cells differentiate into human hepatocytes in vivo has raised the possibility of new therapeutic approaches for liver disease. For diseases such as cystic fibrosis (CF), correction of the respiratory epithelium is being attempted by gene therapy. Differentiation of bone marrow stem cells into epithelium of the lung and airway was recently reported in an animal model, and would provide an alternative approach. We examined the nasal epithelium of female patients up to 15 years after gender-mismatched bone marrow transplantation. Donor-derived epithelial cells were sought with a combination of Y-chromosome fluorescence in situ hybridization and anti-cytokeratin antibody.In nasal brushing samples from 6 transplant-recipients, a median of 2.5% (range, 0.7-18.1%) of nuclei was male and identified as being of donor-origin. However, a complete absence of staining with anti-cytokeratin antibodies confirmed that these were not epithelial cells, but were likely to be either intraepithelial lymphocytes or mesenchymal cells.Following whole bone marrow transplantation, bone marrow progenitor cells do not differentiate into respiratory epithelium of the healthy upper airway. The differences between this and other studies could relate to the cells transplanted, to differential rates of turnover, or to the requirement for specific triggers to stimulate migration and differentiation. In the absence of such conditions, whole bone marrow transplantation is unlikely to provide a route for correction of the CF airway.     

Little evidence of transdifferentiation of bone marrow-derived cells into pancreatic beta cells

Aims/hypothesis Bone marrow cells contain at least two distinct types of stem cells which are haematopoietic stem cells and mesenchymal stem cells. Both cells have the ability to differentiate into a variety of cell types derived from all three germ layers. Thus, bone marrow stem cells could possibly be used to generate new pancreatic beta cells for the treatment of diabetes. In this study, we investigated the feasibility of bone marrow-derived cells to differentiate into beta cells in pancreas.Methods Using green fluorescent protein transgenic mice as donors, the distribution of haematogenous cells in the pancreas was studied after bone marrow transplantation.Results In the pancreas of green fluorescent protein chimeric mice, green fluorescent protein-positive cells were found in the islets, but none of these cells expressed insulin. Previous data has suggested that tissue injury can recruit haematopoietic stem cells or their progeny to a non-haematopietic cell fate. Therefore, low-dose streptozotocin (30 or 50 mg/kg on five consecutive days) was injected into the mice 5 weeks after bone marrow transplantation, but no green fluorescent protein-positive cells expressing insulin were seen in the islets or around the ducts of the pancreas.Conclusions/interpretation Our data suggests that bone marrow-derived cells are a distinct cell population from islet cells and that transdifferentiation from bone marrow-derived cells to pancreatic beta cells is rarely observed.Abbreviations STZ streptozotocin - EGFP enhanced green fluorescent protein - GP guinea-pig - vWF von Willebrand Factor - BrdU bromodeoxyuridine - GFP green fluorescent protein - IPGTT Intraperitoneal glucose tolerance test