神经节苷脂-1与β-巯基乙醇体外联合诱导脂肪间充质干细胞向神经元样细胞分化的实验研究

目的探讨脂肪间充质干细胞（ADSCs）向神经元样细胞分化的规律,以及神经节苷脂-1（GM- 1）在诱导分化过程中的作用。方法取成年兔颈后脂肪组织采用胶原酶消化法原代培养脂肪间充质干细胞,取第5代脂肪间充质干细胞分别采用β-巯基乙醇及GM-1与β-巯基乙醇联合作用的方式向神经元样细胞诱导。免疫细胞化学法检测神经元特异性烯醇化酶（NSE）的表达,细胞化学染色检测尼氏小体的生成。结果成功分离出脂肪间充质干细胞,通过β-巯基乙醇及GM-1与β-巯基乙醇联合作用均可使ADSCs定向诱导为神经元样细胞,联合诱导组NSE及尼氏小体表达的阳性率均高于单纯β-巯基乙醇诱导组,差异有统计学意义（P＜0．05）。结论从兔脂肪组织中能够分离出脂肪间充质干细胞,通过诱导可向神经元样细胞分化,GM-1具有促进脂肪间充质干细胞向神经元样细胞分化的能力。     

脂肪源性干细胞的临床转化研究

脂肪源性干细胞又称脂肪间充质干细胞,是一类处于未定向分化状态并具有自我更新、高度增殖和多向分化潜能的细胞群体,具有一般干细胞的特点,是一种理想的组织工程种子细胞.近年来,对脂肪间充质干细胞的应用研究已经取得了初步进展,其特有的生物学特性为多种疾病的治疗开辟了新的思路.本文就脂肪间充质干细胞的生物学特性及其在临床上的转化研究进行阐述.     

大鼠脂肪间充质干细胞的分化潜能鉴定及XIAP基因修饰*

目的分离培养扩增大鼠脂肪源间充质干细胞(ADSCs),以活体标记并鉴定其分化潜能,了解ADSCs的X连锁凋亡抑制蛋白（XIAP）基因修饰的可行性。方法无菌条件下取大鼠一侧腹股沟脂肪组织,Ⅰ型胶原酶消化法分离培养ADSCs,胰酶消化法传代扩增。检测细胞分化为脂肪细胞、软骨细胞及成骨细胞的潜能,转染XIAP表达质粒进入ADSCs,通过Western blotting等方法检测XIAP的表达能力。结果 ADSCs呈长梭形漩涡样生长,细胞流式鉴定显示CD29、CD44、CD90、CD105均呈高表达,并在特定诱导剂下分化为脂肪细胞、软骨细胞或成骨细胞。XIAP转染后显像经XIAP基因修饰的脂肪间充质干细胞在PVDF膜的相应分子质量区域出现相应的条带。结论脂肪源干细胞易于培养和传代扩增,并可活体标记,具有多向分化潜能,可作为组织工程的种子细胞。     

大鼠脂肪间充质干细胞体外诱导成骨分化的实验性研究

目的探讨大鼠脂肪间充质干细胞体外分离培养和成骨诱导分化的可行性。方法取6周龄大鼠双侧腹股沟处脂肪，酶鹪法分离培养原代脂肪间充质干细胞，成骨诱导培养18d，检测细胞成骨表型转化：碱性磷酸酶、骨钙素的表达以及细胞矿化细胞外基质的能力。结果大鼠脂肪中能分离培养出一定量的脂肪间充质干细胞，在成骨诱导培养液作用下，可特异性表达成骨分化的标志：碱性磷酸酶、骨钙素，同时也具有矿化细胞外基质的能力。结论大鼠脂肪间充质干细胞可能是一种理想的骨组织工程种子细胞的来源。     

间充质干细胞对CD4+T细胞亚群的作用研究进展

CD4⁺T细胞亚群是一类重要的免疫细胞，是决定自身免疫疾病发展的关键免疫细胞，比如哮喘、类风湿性关节炎和多发性硬化症等。由于间充质干细胞具有强大的免疫调节功能，以间充质干细胞为基础的治疗方法已被广泛应用于多种炎症性疾病的治疗。据相关文献研究其内在原因是间充质干细胞对CD4⁺T细胞亚群的平衡调节作用，因此了解间充质干细胞的具体作用机制是十分重要的。在这篇综述中，讨论了间充质干细胞对CD4⁺T细胞的调节作用及其机制并展望间充质干细胞的应用前景。     

脂肪来源间充质干细胞与免疫调节因子间 作用的研究进展

间充质干细胞（MSCs）是中胚层中具有高度自我更新和多向分化潜能的非造血多能干细胞,可以分化为成 脂细胞、成骨细胞、成软骨细胞、肝细胞、心肌细胞、神经元干细胞、胰岛样细胞等。MSCs 具有免疫调节特性,可通过 分泌吲哚胺-2,3-双加氧酶（IDO）和前列腺素 E2（PGE2）来发挥免疫调节作用,同时又能在炎性因子如干扰素 γ （IFN-γ）的预刺激下增强其免疫调节功能。研究发现,在脂肪组织中存在丰富的,使 MSCs 成为成年干细胞非常有吸 引力的来源。本文对目前脂肪来源间充质干细胞（ADSCs）和以上 3 种免疫调节因子之间相互作用的研究进展作一 综述。     

人脂肪来源的间充质干细胞的生物学研究

目的探索从人脂肪中获取间充质干细胞的方法,通过观察人脂肪来源间充质干细胞(hADAS)的形态学、生长动力学、细胞表面标志,揭示其生物学特性。方法分离培养脂肪间充质干细胞并观察其形态学变化;四甲基偶氮唑盐(MTT)比色法测细胞活性;流式细胞仪测细胞周期和间充质干细胞表面抗原。结果脂肪干细胞呈成纤维细胞样;MTT比色法证实其有很强的增殖活性;流式细胞周期显示处于增生活跃期的细胞占很大比例;间充质干细胞表面抗原CD29、CD44随着传代表达逐渐增高,不表达内皮细胞标志CD31,造血干细胞标志CD34随细胞培养时间延长表达逐渐降低。结论通过酶消化法可从人脂肪中获取间充质干细胞,具有很强的增殖能力,表达干细胞表面标志。     

脂肪细胞分化过程中的分子事件

脂肪的发育表现为组织体积增加，其原因可为细胞体积增大或数量增多，他们各自或同时起作用。脂肪细胞由起源于中胚层的间充质干细胞逐步分化形成，按间充质干细胞→脂肪母细胞→前脂肪细胞→不成熟脂肪细胞→成熟脂肪细胞的过程发展。前脂肪细胞在多种转录因子调控下，激活脂肪组织相关基因，并在这些基因的顺序性调控下，经一系列复杂的步骤分化为成熟脂肪细胞。     

人脐带来源间充质干细胞在神经系统疾病中的研究进展

人脐带血来源充足，免疫原性弱，其间充质干细胞更为原始，扩增能力更强，优于骨髓及外周血，故其作用越来越突出，可作为一种新的替代细胞来源用于各系统疾病的细胞移植及基因治疗，尤其是在神经系统疾病治疗中作用更为明显。现将人脐带血间充质干细胞在神经系统疾病中研究现状综述如下。1干细胞与间充质干细胞干细胞（Stemcells）是一类具有自我更新能力，一定条件下能向多种功能细胞分化的多潜能细胞。根据发育阶段的不同，     

真核表达载体介导的ICOSIg基因在大鼠脂肪间充质干细胞中表达的实验研究#br#

目的　构建含可诱导共刺激分子（ICOS）融合蛋白（ICOSIg）基因的真核表达载体,探讨其能否在大鼠脂肪间充质干细胞（ADSCs）中表达。方法　克隆编码大鼠ICOS的胞外片段,将其与编码人免疫球蛋白IgG Fc段的基因融合,构建ICOSIg融合基因及其分泌型真核表达载体pcDNA3.1（+）/ICOSIg。经测序鉴定后转染ADSCs,Western blot法检测ICOSIg在ADSCs中的表达。结果　经测序鉴定验证pcDNA3.1（+）/ICOSIg质粒构建成功,且能在ADSCs中成功表达。结论　ICOSIg在ADSCs中成功表达,为进一步研究免疫耐受机制提供了实验基础。     

Human mesenchymal stem cells from adipose tissue: Differentiation into hepatic lineage.

Adipose tissue represents an accessible source of mesenchymal stem cells (ADSCs), with similar characteristics to bone marrow-derived stem cells. The aim of this work was to investigate the transdifferentiation of ADSCs into hepatic lineage cells in vitro. ADSCs were obtained from human adipose tissue from lipectomy. Cells were grown in medium containing 15% AB human serum. Cultures were serum deprived for two days and exposed to a two-step protocol with two different media using growth factors and cytokines. Hepatic differentiation was assessed by RT-PCR of liver-marker genes. ADSCs exhibited a fibroblastic morphology that changed to a cuboidal shape when cells differentiated. Expression of liver genes increased when using one of the two studied media consisting of DMEM supplemented with HGF, bFGF and nicotinamide for 14 days. The results indicate that, under certain specific inducing conditions, ADSCs can be induced to differentiate into hepatic lineage in vitro. Adipose tissue may be an ideal source of high amounts of autologous stem cells.     

Study of lead-induced neurotoxicity in neural cells differentiated from adipose tissue-derived stem cells

Abstract

In recent years, the use of stem cells as a new tool to create an in vitro model for toxicological studies has been considered. Adipose tissue-derived stem cells (ADSCs) are mesenchymal stem cells which have been extracted from adipose tissue by a less invasive method and rapidly propagated in culture medium compared with other sources. These cells have the capacity to differentiate into different cell lineage in vitro including neural cells. The aim of this study was to investigate the effect of lead exposure at various stages of differentiation on the neural differentiation of ADSCs. Third-passaged ADSCs were differentiated to neural cell in differentiation medium during 16?d. The ADSCs were exposed to lead (0.1–100?µg/ml) before differentiation and during differentiation on days 1, 7 and 14. The cell viability was assessed by MTT assay after 48?h. Also expression of β-tubulin III protein and Nestin, NeuN, NF70, Synaptophysin genes were evaluated at the end of differentiation in all treated groups. The results showed that lead had no effect on viability of undifferentiated ADSCs but differentiating cells showed various sensitivities to lead exposure and cells were more vulnerable to lead exposure at early stage of differentiation. Also, lead exposure at different stages of differentiation had various effects on gene expressions. Our study indicated that neural cells differentiated from ADSCs in vitro are sensitive to neurotoxic effect of lead as well-known developmental neurotoxicant, and then ADSCs could be a candidate as an alternative method for assessing neurodevelopmental toxicity potential of chemicals.     

基因修饰间充质干细胞治疗肿瘤研究进展

间充质干细胞（MSCs）是来源于中胚层的成体干细胞,体内分布广泛,可从骨髓、脂肪、牙髓、脐带/胎盘等组织中分离获取,具有高度的可增殖和分化潜能,较低的免疫原性,同时具有向炎症损伤部位微环境的趋向性,在疾病治疗方面可作为基因药物的载体实现精准治疗。癌症被认为是永远无法愈合的伤口,其组织微环境在损伤与修复中呈无休止的动态变化,MSCs在其中扮演了重要角色。利用MSCs具有的向肿瘤组织中归巢及定位的特点,对其进行抗肿瘤药物的基因工程改造可能在癌症的治疗上是一种新的策略。概述MSCs在癌症发生发展中的作用以及基因修饰MSCs治疗癌症的研究进展,旨在为临床使用基因修饰MSCs治疗癌症提供策略和见解。     

干扰素γ增强脂肪间充质干细胞对淋巴细胞的免疫调节作用

摘要： 目的 观察干扰素 （IFN） -γ增强成人自体脂肪间充质干细胞 （ADSCs） 对外周血淋巴细胞免疫调节的作用及机制。方法 取亲体移植供体术中腹部皮下脂肪组织并留取外周血, 分离单个核细胞 （PBMCs）; 分离、 培养 ADSCs;将 IFN-γ预刺激 ADSCs（IFN-γ预刺激组）及未刺激 ADSCs（未刺激组）分别与 PBMCs 在植物血凝素（PHA） +白细胞介素 （IL） -2 刺激条件下共培养 5 d 后, 用 MTT 法检测活化 T 细胞增殖抑制率, 流式细胞术检测 CD4+CD25+调节性 T细胞（Treg）比例; 实时定量 RT-PCR 法检测 ADSCs 内吲哚胺 2,3 双加氧酶（IDO） mRNA 水平; 观察共培养细胞加入1-甲基色氨酸（1-MT; IDO 阻断组）后活化 T 细胞的增殖能力。结果 分离的自体 ADSCs 高表达 CD73、 CD90、CD105; 具有分化为成脂和成骨细胞的能力。IFN-γ预刺激组 ADSCs 显著增强活化 T 细胞增殖抑制率, 呈剂量依赖关系, 高于未刺激组; IFN-γ预刺激组 CD4+CD25+Treg 比例与未刺激组相比显著升高; IFN-γ预刺激组 ADSCs 内 IDOmRNA 表达水平显著高于未刺激组; IDO 阻断组对活化 T 细胞增殖抑制率较 IFN-γ预刺激组显著降低（P < 0.01）。结论 IFN-γ促进 ADSCs 对活化 T 细胞免疫抑制能力, IDO 在 ADSCs 介导的免疫抑制中起重要免疫调节作用。     

人脂肪间充质干细胞分离培养及其生物学特性的实验研究

目的探索人脂肪间充质干细胞（adipose tissue—derived mesenchymal stem cells，ADMSCs）分离培养的方法及体外扩增的条件，观察ADMSCs的生物学特性。方法以腹部手术患者皮下脂肪组织为材料，采用I型胶原酶消化法及贴壁法分离培养ADMSCs，在含10％胎牛血清的低糖DMEM培养基中贴壁培养，倒置显微镜观察，流式细胞仪检测细胞表面标记CD29、CD44、CD105、CD31、CD34、CD106的表达，透射电镜及扫描电镜下观察ADMSCs超微结构，流式细胞仪测定细胞周期。结果原代和传代细胞呈梭形外观，生长增殖能力良好。CD29、CD44、CD105均呈阳性表达，阳性率分别为95.3％、98．6％和86．5％；而CD31、CD34、CD106阳性率分别为3．5％、2．6％、1．3％。透射电镜观察显示ADMSCs表现出早期幼稚细胞形态的特点，流式细胞仪检测显示84．8％的细胞处于G0/G1期。结论酶消化法能有效地从人脂肪组织分离培养人ADSCs，细胞生长稳定，增殖能力活跃，为今后ADMSCs的分离培养提供了更简单有效的方法。     

年龄对人脂肪来源间充质干细胞生物学特性的影响

目的 探讨供体年龄对脂肪来源间充质干细胞（ADSCs）体外生物学特性的影响。方法 收集外科腹部术后留取的皮下脂肪组织和外周血,按年龄分为儿童组、成年组和>50岁组,每组10例,分离培养ADSCs;流式细胞术分析ADSCs免疫表型,实时细胞分析系统检测ADSCs增殖细胞指数（CI）及迁移CI,使用不同的诱导分化培养液检测ADSCs向脂肪细胞和成骨细胞的分化能力,实时逆转录-聚合酶链反应检测骨桥蛋白（OPN）和过氧化物酶体增殖物激活受体-γ（PPAR-γ）mRNA表达水平。将ADSCs与植物血凝素刺激的外周血单个核细胞（PBMCs）共培养,酶联免疫吸附试验检测上清液干扰素（IFN）-γ含量。结果 传3代后3组ADSCs形态均为典型的纺锤形,均表达典型间充质干细胞（MSCs）表面标志物CD90、CD105和CD73。与成年组、>50岁组比较,儿童组细胞体外增殖CI、迁移CI及OPN mRNA相对表达水平均增加（P<0.05）。儿童组ADSCs抑制PBMCs的IFN-γ分泌水平优于成年组及>50岁组ADSCs（P<0.05）。结论 儿童来源ADSCs具有更强体外增殖、迁移、多向分化以及免疫抑制能力。     

Stem cell therapy for neurologic disorders: therapeutic potential of adipose-derived stem cells

There is growing evidence to suggest that reservoirs of stem cells may reside in several types of adult tissue. These cells may retain the potential to transdifferentiate from one phenotype to another, presenting exciting possibilities for cellular therapies. Recent discoveries in the area of neural differentiation are particularly exciting given the limited capacity of neural tissue for intrinsic repair and regeneration. Adult adipose tissue is a rich source of mesenchymal stem cells, providing an abundant and accessible source of adult stem cells. These cells have been termed adipose derived stem cells (ASC). The characterization of these ASCs has defined a population similar to marrow-derived and skeletal muscle-derived stem cells. The success seen in differentiating ASC into various mesenchymal lineages has generated interest in using ASC for neuronal differentiation. Initial in vitro studies characterized the morphology and protein expression of ASC after exposure to neural induction agents. Additional in vitro data suggests the possibility that ASCs are capable of neuronal activity. Progress in the in vitro characterization of ASCs has led to in vivo modeling to determine the survival, migration, and engraftment of transplanted ASCs. While work to define the mechanisms behind the transdifferentiation of ASCs continues, their application to neurological diseases and injuries should also progress. The subject of this review is the capacity of adipose derived stem cells (ASC) for neural transdifferentiation and their application to the treatment of various neurologic disorders.     

Immunomodulatory properties of mesenchymal stem cells and their therapeutic applications

Mesenchymal stem cells (MSCs) are adult stem cells that can be isolated from most adult tissues, including bone marrow, adipose, liver, amniotic fluid, lung, skeletal muscle and kidney. The term MSC is currently being used to represent both mesenchymal stem cells and multipotent mesenchymal stromal cells. Numerous reports on systemic administration of MSCs leading to functional improvements based on the paradigm of engraftment and differentiation have been published. However, it is not only difficult to demonstrate extensive engraftment of cells, but also no convincing clinical results have been generated from phase 3 trials as of yet and prolonged responses to therapy have been noted after identification of MSCs had discontinued. It is now clear that there is another mechanism by which MSCs exert their reparative benefits. Recently, MSCs have been shown to possess immunomodulatory properties. These include suppression of T cell proliferation, influencing dendritic cell maturation and function, suppression of B cell proliferation and terminal differentiation, and immune modulation of other immune cells such as NK cells and macrophages. In terms of the clinical applications of MSCs, they are being tested in four main areas: tissue regeneration for cartilage, bone, muscle, tendon and neuronal cells; as cell vehicles for gene therapy; enhancement of hematopoietic stem cell engraftment; and treatment of immune diseases such as graft-versus-host disease, rheumatoid arthritis, experimental autoimmune encephalomyelitis, sepsis, acute pancreatitis and multiple sclerosis. In this review, the mechanisms of immunomodulatory effects of MSCs and examples of animal and clinical uses of their immunomodulatory effects are described.