Beneficial effects of BV2 cell on proliferation and neuron-differentiating of mesenchymal stem cells in the circumstance of injured PC12 cell supernatant

1 Introduction The injury of nervous system leads to the irreversible death of neurons. The application of stem cells has shed light on the reconstruction and function recovery of in- jured nervous tissues[1].The proliferation and neuron-dif- ferentiating potential of stem cells bring approaches to the issue that “injured neurons are not able to be revitalized”. People have realized that the proliferation and dif- ferentiation of stem cells are closely related to the micro- environment they …     

血小板裂解液对脐带间充质干细胞增殖与分化的影响

背景：脐带间充质干细胞是近年来干细胞生物学研究中发现的重要细胞之一,在细胞治疗领域有着良好的应用前景。血小板裂解液对于干细胞的增殖及诱导分化都存在不同程度的有利作用,在一定程度上甚至可以代替甚至优于血清在培养基中的作用。目的：对血小板裂解液结合脐带间充质干细胞的研究作一综述。方法：于万方等中文数据库和PubMed等英文数据库中,以“脐带,间充质干细胞,血小板裂解液,分化,增殖”为关键词检索文献,总结脐带间充质干细胞及血小板裂解液的生物学特性和相关临床应用,并汇总有关血小板裂解液对脐带间充质干细胞增殖活性和诱导分化能力影响的研究结果。结果与结论：血小板裂解液对脐带间充质干细胞的增殖活性及诱导分化有正面影响,这使血小板裂解液在干细胞移植治疗中将会起到无可比拟的作用。     

Neural cell co-culture induced differentiation of bone marrow mesenchymal stem cells into neuronal-like cells★

BACKGROUND: It has been previously demonstrated that the neural cell microenvironment has the ability to induce differentiation of bone marrow mesenchymal stem cells (BMSCs) into the neural cells. OBJECTIVE: To establish a co-culture system of human BMSCs and neural cells, and to observe effects of this co-culture system on differentiation of human BMSCs into neural cells. DESIGN, TIME AND SETTING: A comparative observation experiment, performed at the Center Labora-tory of the Affiliated Hospital of Medical College Qingdao University from October 2006 to December 2007. MATERIALS: Neural cells were obtained from human fetal brain tissue. BMSCs were harvested from fe-male patients that underwent autonomous stem cell transplantation. METHODS: BMSCs in the co-culture group consisted of BMSCs and third passage neural cells. BMSCs in the control group were solely cultured in vitro. MAIN OUTCOME MEASURES: Morphological changes of BMSCs were observed, and expression of the neuronal specific marker, neuron-specific enolase (NSE), was analyzed by immunofluorescence staining after 4–5-day co-culture. RESULTS: The number of neural cells in the co-culture group increased and the cells spread on the culture bottle surface. Radial dendrite formed and connected with each other. NSE-immunoreactive cells were also detected. The positive ratio of NSE-positive cells reached (32.7±11.5)%, with morphological characteristics similar to neuronal cells. Human BMSCs did not express NSE in the control group. CONCLUSION: The microenvironment provided by neurons induced differentiation of BMSCs into neu-ronal-like cells. Key Words: bone marrow mesenchymal stem cells; stem cell transplantation; cell differentiation; neurons     

枸杞多糖对大鼠骨髓间充质干细胞增殖及向内皮谱系分化的影响

背景：枸杞多糖可促进生精细胞的增殖,诱导大鼠骨髓间充质干细胞、人脐血间充质干细胞向神经元样细胞分化,枸杞多糖对骨髓间充质干细胞的生物学特性有何影响,有待深入研究。 目的：观察枸杞多糖对大鼠骨髓间充质干细胞增殖及向内皮分化的影响。 方法：采用密度梯度离心法分离SD大鼠骨髓间充质干细胞,MTT法检测细胞增殖率。用HG-DMEN+EGM-2诱导大鼠骨髓间充质干细胞向内皮谱系分化,检测细胞Ⅷ因子相关抗原和荆豆凝集素表达。在培养液中加入或不加入枸杞多糖,分析枸杞多糖对大鼠骨髓间充质干细胞增殖和向内皮谱系分化的影响。 结果与结论：枸杞多糖未改变大鼠骨髓间充质干细胞形态,不影响细胞表面抗原CD29、CD45、CD106的表达,细胞Ⅷ因子相关抗原检测呈阴性反应。但明显提高了大鼠骨髓间充质干细胞的增殖率,延长细胞增殖的高峰时间。大鼠骨髓间充质干细胞用含EGM-2的内皮诱导培养基或内皮诱导培养基+枸杞多糖诱导,未加枸杞多糖诱导组大鼠骨髓间充质干细胞在第10天时已转化为具有内皮特征的细胞;而含有枸杞多糖的内皮诱导液需诱导至第12天,细胞才转化为具有内皮特征的细胞,提示枸杞多糖具有延缓EGM-2诱导的大鼠骨髓间充质干细胞向内皮谱系分化的作用。     

Citalopram increases the differentiation efifcacy of bone marrow mesenchymal stem cells into neuronal-like cells

Several studies have demonstrated that selective serotonin reuptake inhibitor antidepressants can promote neuronal cell proliferation and enhance neuroplasticity both in vitro and in vivo. It is hypothesized that citalopram, a selective serotonin reuptake inhibitor, can promote the neuronal differentiation of adult bone marrow mesenchymal stem cells. Citalopram strongly enhanced neuronal characteristics of the cells derived from bone marrow mesenchymal stem cells. The rate of cell death was decreased in citalopram-treated bone marrow mesenchymal stem cells than in control cells in neurobasal medium. In addition, the cumulative population doubling level of the citalopram-treated cells was signiifcantly increased compared to that of control cells. Also BrdU incorporation was elevated in citalopram-treated cells. These ifndings suggest that citalopram can improve the neuronal-like cell differentiation of bone marrow mesenchymal stem cells by increasing cell proliferation and survival while maintaining their neuronal characteristics.     

间充质干细胞生物学特性及其分化潜能

背景：随着再生医学的兴起,细胞疗法正得到不断发展,间充质干细胞因为来源广泛,分离培养容易,增殖和多向分化能力强,已成为细胞治疗领域的研究热点。 目的：概述国内及国际上关于间充质干细胞生物学特性及多向分化能力的新进展。 方法：应用计算机检索2000-01/2010-09 PubMed数据库、Springer Link数据库及万方数据库有关间充质干细胞发现、命名、生物学特性及分化潜能等方面的文献,英文检索词为“mesenchymal stem cells,differentiation,transdifferentiation”,中文检索词为“间充质干细胞,分化,转分化”。检索文献量总计132篇。 结果与结论：间充质干细胞发现较久,命名较多,在体内分布广泛,增殖能力受多种因素影响,可分泌多种细胞因子,产生一系列细胞外基质分子,表达多种表面标志物,但不是单一性的,属免疫缺陷细胞,分离纯化方法主要有全骨髓贴壁分离筛选法和密度梯度离心法,其鉴定需综合几个方面内容。除向中胚层的各种成熟细胞分化外,间充质干细胞还可转分化为神经细胞、肝细胞、胰岛细胞、上皮细胞等,转分化机制主要有异质学说、核重编程学说和胚胎干细胞残留学说。     

骨碎补总黄酮可促进兔骨髓间充质干细胞的增殖和分化*

背景：骨碎补促进骨损伤愈合疗效确切,但具体作用机制尚不清楚。 目的：观察骨碎补总黄酮对兔骨髓间充质干细胞增殖、分化的影响。 方法：冲洗兔股骨和胫骨骨髓腔获取骨髓,采用密度梯度离心法联合贴壁培养法体外纯化扩增兔骨髓间充质干细胞;以四甲基偶氮唑蓝法测定不同浓度骨碎总黄酮对兔骨髓间充质干细胞增殖的影响;经骨碎补总黄酮体外诱导后,行扫描电镜形态学观察,碱性磷酸酶染色、茜素红染色和Von Kossa银染色了解细胞钙化情况。 结果与结论：兔骨髓间充质干细胞可以通过密度梯度离心法联合贴壁培养法扩增和纯化;含骨碎补总黄酮血清培养后,四甲基偶氮唑蓝测定结果显示,骨碎补总黄酮浓度为10-6 mmol/L时可明显促进兔骨髓间充质干细胞增殖。经骨碎补总黄酮诱导液诱导,扫描电镜下可见成骨细胞样形态和钙结节形成;细胞碱性磷酸酶染色、钙结节茜素红染色和Von Kossa银染色均呈阳性,提示骨碎补总黄酮可促进兔骨髓间充质干细胞增殖和分化。     

骨髓间充质干细胞促进中脑前体细胞增殖及定向分化的实验研究

目的 观察BMSCs对胚胎腹侧中脑前体细胞(VMP)体外扩增和定向分化的影响,并分析其可能的营养机制.方法 分别取胎龄11d大鼠胚胎VMP、成年大鼠BMSCs进行体外培养,并建立二者的共培养体系.体外扩增7 d的VMP分为对照组、BMSCs分化液组、BMSCs+VMP共培养分化液组,分别加入普通分化液、BMSCs分化液、BMSCs+VMP共培养分化液进行诱导分化.观察细胞生长情况.分化期末行免疫荧光染色,分析比较3组细胞中酪氨酸羟化酶(TH)阳性细胞占总细胞数的比例.结果诱导分化7 d后,对照组、BMSCs分化液组和BMSCs+VMP共培养分化液组中细胞数分别较培养前扩增(44.13±4.75)倍、(60.63±5.25)倍、(64.00±7.63)倍,TH阳性细胞比例分别为(18.76±5.20)%、(23.49±4.10)%、(28.08±5.42)%,比较差异有统计学意义(P<0.05).结论 BMSCs能够通过分泌营养因子有效促进VMP增殖并定向分化为多巴胺能神经元.     

PEX基因修饰骨髓间质干细胞对C6胶质瘤细胞增殖和凋亡的影响

目的探讨PEX基因修饰的骨髓间质干细胞（mesenchymal stem cells,MSC）对C6胶质瘤细胞的作用及机制。方法分子克隆技术构建PEX基因真核表达载体并转染MSC,G418筛选获取稳定表达PEX基因的MSC（MSC-PEX）,将不同数量的MSC-PEX细胞与C6胶质瘤细胞进行共培养试验,用水溶性四氮唑法（WST-1）观察MSC-PEX对C6胶质瘤细胞增殖的影响,用Annexin-Ⅴ-FITC/PI双染荧光观察C6胶质瘤细胞凋亡的形态学变化,并用Annexin-Ⅴ-FITC/PI双标记法通过流式细胞仪检测胶质瘤细胞凋亡率。结果成功获得稳定表达PEX基因的MSC-PEX,MSC-PEX抑制C6胶质瘤细胞的生长作用较明显,Annexin-Ⅴ-FITC/PI双染荧光发现C6胶质瘤细胞发生凋亡形态改变;流式细胞仪检测显示：MSC-PEX转染组和DMEM对照组的凋亡率分别为16.7%和1.3%,差异有统计学意义（P〈0.05）。结论PEX基因修饰的骨髓间质干细胞抑制胶质瘤细胞增殖并诱导其凋亡,为胶质瘤的治疗奠定理论基础。     

高海拔地区骨髓源神经干细胞及BDNF联合移植对大鼠缺血再灌注模型的治疗

目的 探讨高海拔地区大鼠骨髓源神经干细胞(bone marrow mesenchymal stem cells-derived neural stem cells,BMSCs-NSCs)及脑源性神经生长因子(Brain-derived neurotrophic factor,BDNF)联合移植对大鼠脑缺血再灌注模型的疗效及其相关机理。方法 60只Wistar雄性大鼠,置西宁地区正常饲养,制备大鼠脑缺血-再灌注损伤模型; 模型制备完毕后立体定向下进行细胞移植治疗,将大鼠分为3组,即A组:大鼠骨髓源性神经球组(BMSCs-NSCs组,n=20); B组:大鼠骨髓源性神经球联合BDNF组(BMSCs-NSCs+BDNF组,n=20),注射大鼠骨髓源性神经球细胞的同时,联合注射100 ng BNDF; C组:对照组(仅注射DMEM/F12培养基,n=20); 术后对其神经功能进行评定,并于术后24 d取脑组织,行Nestin、GFAP、Map2免疫荧光检测。结果 细胞移植后第3 d,各组间神经功能评分无显著性差异; 细胞移植后第14 d BMSCs-NSCs+BDNF组神经功能评分显著优于BMSCs-NSCs组,BMSCs-NSCs组优于对照组; 免疫组化检测发现,BMSCs-NSCS+BDNF组Nestin、GFAP、Map2的IOD值均显著高于BMSCs-NSCs组; BMSCs-NSCs+BDNF组、BMSCs-NSCs组各检测指标水平均高于对照组; Nestin、GFAP、Map2的表达主要集聚于脑梗死灶与正常脑组织交界处。结论 在西宁地区联合移植大鼠骨髓源神经干细胞及BDNF可显著促进大鼠大脑中动脉闭塞再灌注损伤模型的神经功能恢复。     

Beneficial role of adipose-derived mesenchymal stem cells from microfragmented fat in a murine model of duchenne muscular dystrophy

Introduction: No etiologic therapy is available for Duchenne muscular dystrophy (DMD), but mesenchymal stem cells were shown to be effective in preclinical models of DMD. The objective of this study is to investigate the effect of microfragmented fat extracted on a murine model of DMD. Methods: Fat tissue was extracted from healthy human participants and injected IM into DMD mice. Histological analysis, cytokines, and force measurement were performed up to 4 weeks after injection. Results: Duchenne muscular dystrophy mice injected with microfragmented fat exhibited an improved muscle phenotype (decreased necrosis and fibrosis), a decrease of inflammatory cytokines, and increased strength. Discussion: Administration of microfragmented fat in key muscles may improve muscular phenotype in patients with DMD. Muscle Nerve, 2019     

人骨髓间充质干细胞的体外培养及向神经细胞的分化研究

目的探讨人骨髓间充质干细胞(hMSC)的体外培养生长特性及向神经细胞分化的诱导条件。方法采用密度梯度离心方法分离hMSC,体外扩增生长传代培养,倒置于显微镜下观察其生长特性。采用阿魏酸钠对传至3～6代的hMSC进行诱导培养,并以β-巯基乙醇作为对照,观察诱导培养不同时间后细胞的形态变化,应用免疫细胞化学方法鉴定诱导培养后细胞表面神经细胞特异性标志物神经丝蛋白(NF)及神经元特异性烯醇化酶(NSE)的表达。结果hMSC在体外培养条件下,能保持旺盛的增殖能力,阿魏酸钠诱导培养后6 h即可见细胞形态发生明显变化,24h后表现为典型的神经细胞样形态,NF和NSE呈阳性表达。结论hMSC在体外具有自我更新能力及多分化潜能;阿魏酸钠具有诱导体外培养的hMSC向神经细胞分化的作用。     

骨髓间质干细胞诱导分化为神经细胞的研究进展

骨髓间质干细胞具有较强的自我复制和多向分化能力,近几年发现在一定的条件下,能够诱导分化为神经细胞。由于其具有取材方便,回植后不会发生免疫排斥反应,体外基因转染率高并能稳定高效表达外源基因等优点,将为神经系统疾病的治疗提供新的思路。本文着重对骨髓间质干细胞向神经细胞诱导分化方面的研究进行了综述。     

人骨髓间充质干细胞向多巴胺神经元分化的体外研究

目的探讨人骨髓间充质干细胞(hMSC)向神经元和多巴胺神经元分化的潜能。方法分离和纯化hMSCs;在体外以WHI-P131预处理和碱性成纤维细胞生长因子预诱导后,全反式维甲酸和胶质细胞源性神经营养因子联合诱导hMSCs向神经元和多巴胺神经元分化。光镜下观察其分化过程中hMSCs的形态变化,免疫组化检测诱导前后细胞是否表达神经元和多巴胺能神经元标志蛋白。结果诱导后的hMSCs能分化成为具有典型神经元形态的细胞,并明显表达抗人神经巢蛋白(nestin)[(54.2±3.7)%]和神经元特异性烯醇化酶(NSE)[(77.0±5.7)%],低表达胶质纤维酸性蛋白(GFAP)[(8.8±2.4)%];对照组细胞这些表达均为阴性;而且相当部分hMSCs表达酪氨酸羟化酶(TH)[(36.5±15.8)%]和多巴胺转运体(DAT)[(26.0±14.2)%]。结论在适当条件下,hMSCs可分化成为神经元样细胞和多巴胺神经元样细胞。     

Neuronal-like cell differentiation of non-adherent bone marrow cell-derived mesenchymal stem cells*

Non-adherent bone marrow cell-derived mesenchymal stem cells from C57BL/6J mice were separated and cultured using the "pour-off" method.Non-adherent bone marrow cell-derived mesenchymal stem cells developed colony-forming unit-fibroblasts,and could be expanded by supplementation with epidermal growth factor.Immunocytochemistry showed that the non-adherent bone marrow cell-derived mesenchymal stem cells exposed to basic fibroblast growth factor/epidermal growth factor/nerve growth factor expressed the neuron specific markers,neurofilament-200 and NeuN,in vitro.Non-adherent bone marrow cell-derived mesenchymal stem cells from β-galactosidase transgenic mice were also transplanted into focal ischemic brain (right corpus striatum) of C57BL/6J mice.At 8 weeks,cells positive for LacZ and β-galactosidase staining were observed in the ischemic tissues,and cells co-labeled with both β-galactosidase and NeuN were seen by double immunohistochemical staining.These findings suggest that the non-adherent bone marrow cell-derived mesenchymal stem cells could differentiate into neuronal-like cells in vitro and in vivo.     

黄芪诱导大鼠骨髓间充质干细胞向神经类细胞分化

BACKGROUND： Chemical induction has been shown to be effective at promoting the differentiation of bone marrow-derived mesenchymal stem cells （MSCs）. However, these inductors have cytotoxicity side effects that may damage cells over time. Traditional Chinese medicines avoid this disadvantage while still producing effective induction.
OBJECTIVE： To investigate the influence of RadixAstragafi （Huangql） on the differentiation of MSCs.
DESIGN, TIME AND SETTING： In vitro study of traditional Chinese medicine in neural stem cell differentiation. The experiment was performed at the Central Laboratory of Hebei North University between April and June 2007.
MATERIALS： Radix Astragafi solution （lot No. 060105; license No. Z53021585） was purchased from Dali Pharmaceutical Co., Ltd., China; rabbit anti-rat nestin, rabbit anti-rat neuron-specific enolase （NSE）, mouse anti-rat microtubule-associated protein 2, and rabbit anti-rat glial fibrillary acidic protein were purchased from Wuhan Boster, China.
METHODS： Whole bone marrow was isolated from the femur and tibia of 6-week-old male Wistar rats and subcultured. The fourth passage of MSCs were harvested and induced by different concentrations （50, 100, 200, 400 g/L） of Radix Astragali.
MAIN OUTCOME MEASURES： Hematoxylin-eosin staining was used to observe MSC morphology after 24 hours of induction. Immunocytochemistry was employed to observe the expression of NSE （specific neuronal marker）, nestin （marker of neural stem cell）, glial fibrillary acidic protein and microtubule-associated protein 2 （markers of astrocytes）.
RESULTS： Following Radix Astragali treatment, changes occurred in cell morphology including： cell body pyknosis; thin and long processes formed in some cells, with growth corresponding to drug concentration and induction time; and the formation of network-like connections between some cells. With increasing drug concentration and induction time, nestin expression was upregulated, and the number of positive cells increased; cells produced NSE, glial fibrillary acidic protein and microtubule-associated protein 2; nestin was expressed earlier than glial fibrillary acidic protein and microtubule-associated protein 2 expression. In addition, the number of NSE-positive cells was increased significantly more than glial fibrillary acidic protein-positive cells.
CONCLUSION： Radix Astragafi promoted process formation in stem cells. It may induce the differentiation of MSCs into neural stem cells, and subsequently into neuronal- and glial-like cells. Radix Astragafi exhibits stronger inductive effect on neuronal differentiation than glial differentiation of MSCs.     

高频脉冲电磁场对骨髓间充质干细胞增殖和成骨分化的影响

背景：采用低频脉冲电磁场干预骨髓间充质干细胞增殖分化的研究很多,但采用高频(> 300 MHz)脉冲电磁场干预的研究国内未见报道。 目的：观察> 300 MHz高频脉冲电磁场照射能否促进骨髓间充质干细胞增殖,并向成骨分化。 方法：分离培养SD大鼠骨髓间充质干细胞,取第3代细胞随机分为4组：成骨诱导组、成骨诱导＋电磁照射组、电磁场照射组、空白对照组。观察各组骨髓间充质干细胞培养过程中细胞形态、数量、总蛋白量等方面变化。 结果与结论：与未经高频脉冲电磁场照射组相比,经高频脉冲电磁场照射后,骨髓间充质干细胞胞体稍有增多,但分化方面区别微弱。电磁场照射组细胞增殖速度、总蛋白含量均低于较空白对照组(P < 0.05)。但电磁照射组细胞凋亡率较空白对照组增加(P < 0.05)。说明高频脉冲电磁场促进骨髓间充质干细胞的成骨分化趋势不明显,可抑制其增殖,促进其凋亡。 关键词：脉冲电磁场;骨髓间充质干细胞;高频;成骨分化;凋亡     

人胎盘间充质干细胞体外向胆碱能样神经元的分化*

背景：胎盘中含有与骨髓中类似的间充质干细胞,也可能具有多向分化能力。 目的：探讨人胎盘间充质干细胞体外能否定向诱导分化为胆碱能样神经元。 方法：采用酶消化法分离培养人胎盘间充质干细胞,传至第4代,先用胎牛血清+碱性成纤维细胞生长因子诱导培养2 d,然后分为3组：方案Ⅰ加入含碱性成纤维细胞生长因子、2-巯基乙醇、维甲酸、神经生长因子的DMEM/F12继续诱导19 d;方案Ⅱ在其基础上,诱导液中另加入表皮生长因子、Heparin;阴性对照组仅加入含胎牛血清的DMEM/F12培养液,不添加任何诱导剂。 结果与结论：胎盘组织消化后获得的贴壁细胞,经2周培养后逐渐形成扁平单层细胞,呈平行排列或漩涡样成簇生长,为梭形,传至第3代细胞形态较均一。第4代人胎盘间充质干细胞强表达CD44,CD29,不表达CD34,CD45,CD106及HLA-DR。人胎盘间充质干细胞经方案Ⅰ和方案Ⅱ诱导2周后,均可检测到nestin、chat mRNA的表达,且chat,Ache,nestin阳性率显著高于阴性对照组细胞(P < 0.05)。与阴性对照组比较,经两种方案诱导的人胎盘间充质干细胞培养上清液中乙酰胆碱浓度均明显升高(P < 0.05),且方案Ⅰ诱导组升高幅度明显大于方案Ⅱ诱导组(P < 0.05)。提示联合多种生长因子分阶段进行诱导,人胎盘间充质干细胞可在体外分化为具有合成及释放降解乙酰胆碱能力的胆碱能样神经元细胞。     

大鼠骨髓间充质干细胞体外增殖与多向分化的能力

背景：生理条件下机体内多数骨髓间充质干细胞增殖并不明显,然而在一定刺激下可表现出旺盛的有丝分裂活动,具有很强的增殖倍增能力,且体外实验表明其具有多向分化潜能。 目的：进一步验证体外分离培养的大鼠骨髓间充质干细胞生长增殖与多向分化潜能。 方法：全骨髓法体外分离培养大鼠骨髓间充质干细胞,贴壁筛选法进行纯化,倒置相差显微镜下观察细胞形态及生长特征,MTT法绘制细胞生长曲线,免疫组化法对细胞表面干细胞标志CD44进行鉴定。取传至第4代细胞,分别用成骨、成软骨、成脂肪和成神经诱导剂予以培养,通过碱性磷酸酶染色、Ⅱ型胶原免疫组化染色、油红O染色、NeuN抗体免疫组化染色进行分化能力鉴定。 结果与结论：分离培养的细胞呈长梭形或多边形,细胞生长曲线呈S形,群体倍增时间约为31 h,免疫细胞化学染色后CD44呈阳性表达,CD34呈阴性。第4代骨髓间充质干细胞成骨诱导2周后出现钙盐沉积,成软骨诱导培养2周后Ⅱ型胶原检测呈阳性,成脂肪诱导培养2周后在细胞的胞浆内充满大量红色脂肪滴,成神经诱导6 h后细胞出现突起,类似神经元的轴突和树突纤维,NeuN免疫组化染色呈阳性。表明体外培养的大鼠骨髓间充质干细胞生长增殖能力旺盛,可向成骨细胞、软骨细胞、脂肪细胞、神经元样细胞方向分化。