软骨损伤后基质细胞衍生因子1的表达

目的：研究骨髓间充质干细胞趋化聚集软骨损伤病灶的可能机制，为调控和干预软骨损伤时干细胞定向迁移和聚集提供理论和实验依据。 方法：制作兔软骨损伤模型，分别于制模后2、5、7、10、14、28天处死兔子，取损伤灶及边缘区组织，利用组织裂解液提取总蛋白，分别行酶联免疫法夹心法检测SDF-1表达和体外细胞迁移实验，部分兔软骨损伤制模成功后，行细胞体内移植，观察SDF-1对MSCs、软骨细胞的迁移影响。 结果：SDF-1的表达呈现出时间变化的趋势,制模后7天达到高峰水平。制模后的软骨组织对MSC迁移的影响与SDF-1的表达呈相同的变化程序,制模后7天对MSC的趋化作用最强。SDF-1 存在时，MSCs 迁移活跃，应用抗体封闭CXCR4 后，这种迁移显著减弱。体内移植的MSCs主要聚集在损伤灶周围，但在封闭CXCR4 后，这种聚集现象大大减弱。 结论：软骨损伤的早期,损伤灶内修复组织中SDF-1的表达明显升高,并可在损伤后2周内维持在一个相对高的水平,软骨损伤灶内SDF-1表达量上调是吸引骨髓干细胞归巢的可能机制之一，通过调节基质细胞源性因子1及其受体CXCR4对MSCs的趋化作用，可望调控干细胞向靶组织的趋化聚集量，达到治疗目的。     

大鼠骨髓间质干细胞静脉移植在脊髓损伤中的定向迁移

目的观察大鼠骨髓间质干细胞(ratbonemarrowstromalcells,rMSCs)静脉移植在体内的定向迁移。方法分离培养rMSCs,流式细胞术检测其表面标志,运用改良Allen法制备大鼠T10脊髓损伤模型,随机分为假手术组、对照组、rMSCs静脉移植组。假手术组、rMSCs静脉移植组同时于大鼠损伤后72小时经尾静脉移植溴脱氧尿苷(BrdU)标记MSCs。免疫组化技术检测rMSCs在体内迁移、存活以及分化情况。结果rMSCs体外分离培养扩增5代,细胞数可达1～2×1011个,具有多态性和贴壁生长特性,流式细胞术检测CD34、CD45阴性,CD29、CD90表达阳性。移植的rMSCs在宿主损伤脊髓中聚集并存活,3～5W后即有部分移植细胞表达神经元特异性烯醇化酶(neuronspecificenolase,NSE)、神经丝蛋白(neurofilament,NF)、微管相关蛋白(microtubuleassociatedprotein2,MAP2)。结论rMSCs体外扩增迅速,具有干细胞特性,经静脉移植在宿主体内可向损伤区脊髓聚集存活分化。     

趋化因子SDF-1体外趋化骨髓基质细胞迁移的实验研究

目的探讨趋化因子SDF-1在体外对骨髓基质细胞的迁移作用。方法采用全骨髓法培养成年Wistar大鼠骨髓基质细胞。取第五代骨髓基质细胞行免疫荧光鉴定；后通过细胞免疫荧光及RT-PCR的方法检测骨髓基质细胞表达趋化因子受体CXCR4的情况．利用Boyden小室法探讨趋化因子SDF-1对骨髓基质细胞的体外趋化作用及其特异性。结果第五代骨髓基质细胞都表达间充质干细胞标记物Vimentin、Laminin及Fibronectin；细胞免疫荧光及RT-PCR结果证实骨髓基质细胞表达趋化因子受体CXCR4；趋化因子SDF-1（5、50、500ng／mL）体外可趋化骨髓基质细胞迁移，抗SDF-1多克隆抗体可对抗其趋化迁移作用。结论SDF-1／CXCR4通路参与骨髓基质细胞体外迁移，为进一步研究骨髓基质细胞的迁移机制提供了理论依据。     

细胞基质衍生因子-1α在脑梗死中作用的研究进展

趋化因子细胞基质衍生因子-1α(stromal derived factor-1α,SDF-1α)及其受体CXCR4、CXCR7在多种细胞及组织中广泛表达,对中枢神经的发育起着重要作用。近年来研究表明,SDF-1α-CXCR4/CXCR7趋化轴在脑梗死后新生血管的形成及内源性神经干细胞的增殖并迁移至梗死区进行修复的过程中发挥着重要作用,此外,还有影响炎症反应的作用,有可能成为脑梗死治疗的新的靶点。     

基质细胞衍生因子-1诱导神经干细胞靶向性迁移的实验研究

目的 观察基质细胞衍生因子-1(SDF-1)对神经干细胞(NSCs)迁移的影响.方法 由胚胎大鼠脑组织获取NSCs并进行传代培养和分化鉴定,使用流式细胞术检测NSCs纯度及SDF-1特异性受体CXCR4的表达情况,之后利用Blind-Well小室体外迁移体系观察不同浓度的SDF-1(0 ng/L、1 ng/mL、10 ng/mL、50 ng/mL、100 ng/mL、500 ng/mL和1000 ng/mL)对NSCs迁移数量的影响,并使用μ-载片观察SDF-1对NSCs迁移距离的影响.结果成功分离培养得到了能够在体外不断分裂增殖、具有多向分化潜能的NSCs,连续传3代后绝大部分细胞nestin表达阳性,nestin和CXCR4的共表达率达到80%左右.细胞趋化实验结果表明,SDF-1对NSCs有较强的趋化作用,随着SDF-1浓度的升高,发生迁移的细胞数量也随之增加,并于SDF-1浓度为500ng/mL时达到最高峰[(256.79±38.27)个细胞/每高倍视野].在μ-载片细胞生长通道两侧的SDF-1浓度梯度(500ng/mL→0ng/mL)作用下,由细胞克隆球迁出的细胞呈不对称分布,通常有更多的迁出细胞分布于趋化因子浓度较高的一侧,且该侧细胞的最大迁移距离也比对侧远.结论 SDF-1与其特异性受体CXCR4相巨作用能够诱导NSCs发生靶向性迁移.     

基质细胞衍生因子1趋化神经干细胞迁移的体外效应

背景：神经干细胞的迁移在神经系统的发育和损伤修复中起着至关重要的作用,近来研究表明趋化因子参与神经干细胞的迁移,但关于其迁移机制目前尚不清楚。 目的：观察体外条件下基质细胞衍生因子1对胎鼠海马神经干细胞的趋化迁移作用。 方法：通过无血清法体外分离、培养及鉴定胎鼠海马神经干细胞;细胞免疫荧光及RT-PCR检测其CXCR4是否表达;观察不同浓度基质细胞衍生因子1对神经干细胞的趋化迁移作用,中和CXCR4受体以验证基质细胞衍生因子1趋化迁移作用的特异性。 结果与结论：胎鼠海马来源神经干细胞表达趋化因子受体CXCR4,呈阳性。RT-PCR琼脂糖凝胶电泳后出现643 bp特异性扩增条带。体外条件下基质细胞衍生因子1趋化迁移随浓度而增强,500 μg/L为最佳趋化浓度。加入抗CXCR4多克隆抗体中和后,神经干细胞迁移较基质细胞衍生因子1组明显减少,与对照组比较,差异无显著性意义(P > 0.05),提示抗CXCR4多克隆抗体可阻断趋化迁移作用。     

趋化因子CXCL-8趋化骨髓间充质干细胞迁移的体外效应

背景：早期研究表明趋化因子参与骨髓间充质干细胞的体外趋化,确定骨髓间充质干细胞体内迁移的分子机制,对其介导的细胞治疗中枢神经系统损伤和疾病有重要作用。 目的：观察体外条件下趋化因子CXCL-8对大鼠骨髓间充质干细胞迁移的趋化作用。 设计、时间及地点：细胞学体外观察,于2007-10/2008-06在青岛大学医学院附属医院脑血管病研究所完成。 材料：纯种SPF级成年Wistar大鼠8只,由青岛市实验动物和动物实验中心提供。鼠重组趋化因子CXCL-8为Santa cruz公司产品。 方法：采用全骨髓法分离培养大鼠骨髓间充质干细胞,胰蛋白酶消化传代。取500 μL趋化因子CXCL-8,加入含体积分数为0.5%胎牛血清的DMEM条件培养基,分别将质量浓度调整为5,50,250,500 μg/L加到趋化板下层,对照组单纯添加DMEM条件培养基,以8 μm孔径的聚碳酸酯膜覆盖。调整骨髓间充质干细胞浓度至1.5×109 L-1,取100 μL细胞悬液加到趋化板上层。为验证趋化因子CXCL-8作用的特异性,将经抗CXCR6多克隆抗体孵育12 h后的骨髓间充质干细胞加到趋化板上层,趋化因子CXCL-8加到趋化板下层。 主要观察指标：骨髓间充质干细胞趋化因子受体CXCR1的表达,趋化因子CXCL-8对骨髓间充质干细胞体外趋化迁移作用及其特异性检测。 结果：骨髓间充质干细胞趋化因子受体CXCR1呈阳性表达,位于细胞膜和细胞浆中,RT-PCR琼脂糖凝胶电泳后出现215 bp特异性扩增条带。与对照组比较,加入5~500 μg/L趋化因子CXCL-8后,骨髓间充质干细胞迁移数均明显增加（P < 0.05）。加入抗CXCR1多克隆抗体后,骨髓间充质干细胞迁移数较250 μg/L趋化因子CXCL-8组明显减少（P < 0.05）。 结论：体外条件下趋化因子CXCL-8在5~500 μg/L质量浓度范围可趋化骨髓间充质干细胞迁移,封闭趋化因子受体CXCR1后其趋化迁移作用明显减弱。     

趋化因子CXCL-16体外趋化骨髓基质细胞迁移的实验研究

目的 探讨趋化因子CXCL-16对骨髓基质细胞的体外迁移作用.方法 采用全骨髓法培养成年Wistar-大鼠骨髓基质细胞,取第5代骨髓基质细胞行流式细胞仪鉴定;然后通过细胞免疫荧光及RT-PCR的方法检测骨髓基质细胞表达趋化因子受体CXCR6的情况,利用Transwell小室法探讨趋化因子CXCL-16对骨髓基质细胞的体外趋化作用及其特异性.结果 第5代骨髓基质细胞的间充质干细胞的标志CD29的表达呈阳性,而造血干细胞表面标志CD45呈阴性;细胞免疫荧光及RT-PCR结果从基因和蛋白两方面证实骨髓基质细胞表达趋化因子受体CXCR6,趋化凶子CXCL-16(5～500 ng/ml)体外可趋化骨髓基质细胞迁移,抗CXCR6多克隆抗体可对抗其趋化迁移作用.结论 CXCL-16/CXCR6通路参与骨髓基质细胞体外迁移,为进一步研究骨髓基质细胞的迁移机制提供了理论依据.     

骨髓间质干细胞经静脉移植后在脑缺血大鼠的体内分布与迁移

目的 观察骨髓间质干细胞经静脉移植后在脑缺血大鼠体内的存活、分布及迁移情况,为细胞静脉移植奠定基础。方法密度梯度离心法分离大鼠骨髓间质干细胞(rat bone mescnchymal stem cels,rMSCs),经体外培养并扩增,线拴法制作大鼠大脑中动脉闭塞(MCAO)脑局灶缺血模型,rMSCs Hoechst33342标记后,通过静脉移植,经过1周、3周、5周后处死大鼠,取脑、心、肺、肾、肝、脾组织切片,在倒置荧光显微镜下观察细胞在体内的存活、分布及迁移情况。结果大鼠脑缺血后,经静脉移植骨髓间质干细胞在脑内能较长时间存活,静脉移植的细胞集中于脑缺血灶周围,而脑缺血对侧及心、肺、肾、肝、脾等组织少有细胞发现。结论rMSCs通过静脉途径进行移植后,能够向损伤部位迁移,与宿主脑组织整合在一起。骨髓间质干细胞经静脉移植用于治疗脑缺血具有可行性。     

细胞趋化因子SDF-1α对小胶质细胞趋化作用的研究

目的观察细胞趋化因子SDF-1对小胶质细胞的趋化作用以及机制探讨。方法体外培养BV2细胞系,给予不同浓度SDF-1α,进行迁移实验以确定SDF-1α对小胶质细胞趋化作用的浓度;将C6小鼠分为对照组和SDF-1α干预组,分别予以侧脑室注射PBS和SDF-1α各8周,采用免疫荧光组织化学方法观察注射后小鼠脑内小胶质细胞的数量,进一步采用Western Blot检测小胶质细胞标志物Iba-1的表达水平。结果予以SDF-1干预6 h后BV2细胞的迁移指数增加,SDF-1的受体CXCR4拮抗剂AMD3100能抑制SDF-1所致的小胶质细胞迁移;长程的SDF-1α侧脑室注射能够增加C6小鼠皮层和海马小胶质细胞的数量;Western Blot检测发现SDF-1α干预组脑组织中Iba-1表达量也明显增加。结论 SDF-1在离体和在体实验中均能趋化小胶质细胞的迁移。     

Stromal cell-derived factor 1-mediated CXCR4 signaling in rat and human cortical neural progenitor cells

Stromal cell-derived factor 1 (SDF-1) and the chemokine receptor CXCR4 are highly expressed in the nervous system. Knockout studies have suggested that both SDF-1 and CXCR4 play essential roles in cerebellar, hippocampal, and neocortical neural cell migration during embryogenesis. To extend these observations, CXCR4 signaling events in rat and human neural progenitor cells (NPCs) were examined. Our results show that CXCR4 is expressed in abundance on rat and human NPCs. Moreover, SDF-1alpha induced increased NPCs levels of inositol 1,4,5-triphosphate, extracellular signal-regulated kinases 1/2, Akt, c-Jun N-terminal kinase, and intracellular calcium whereas it diminished cyclic adenosine monophosphate. Finally, SDF-1alpha can induce human NPC chemotaxis in vitro, suggesting that CXCR4 plays a functional role in NPC migration. Both T140, a CXCR4 antagonist, and pertussis toxin (PTX), an inactivator of G protein-coupled receptors, abrogated these events. Ultimately, this study suggested that SDF-1alpha can influence NPC function through CXCR4 and that CXCR4 is functional on NPC.     

The relationship between SDF-1α/CXCR4 and neural stem cells appearing in damaged area after traumatic brain injury in rats

Abstract

Objective: The actual relationship between neural stem cells and SDF-1α/CXCR4 after brain injury has not yet been elucidated, although recent studies have speculated that stromal cell-derived factor-1α (SDF-1α) and its receptor, CXCR4, could contribute to neural stem cells migration after brain injury. In the present study, the temporal relationship between neural stem cells (NSCs) and SDF-1α/CXCR4 around a damaged area was investigated using a rat traumatic brain injury (TBI) model.

Methods: We used molecular biology techniques and immunohistochemistry to investigate the relationship between SDF-1α/CXCR4 expression and NSCs existence around a damaged area after TBI in the rat brain.

Results: SDF-1α mRNA expression and SDF-1α protein synthesis did not increase after TBI. However, SDF-1α leaked from the injured area and diffused into the cortex 1–3 days after TBI. Subsequently, the levels of CXCR4 mRNA expression and CXCR4 protein synthesis increased significantly. Many small cells with a nestin-positive cytoplasm and fibers also showed immunopositivity for both CXCR4 and SOX-2, but not for GFAP, 3–7 days after TBI. Moreover, a proportion of the CXCR4-positive cells and fibers also showed immunostaining for neurofilaments.

Discussion: These results suggest that the leaked SDF-1α attracted CXCR4-positive NSCs as well as elongated nerve fibers. It is considered that the SDF-1α/CXCR4 system in the brain contributes to neural stem cells appearance and maturation after TBI. Therefore, exploitation of the SDF-1α/CXCR4 system around a damaged area may improve the brain dysfunction after TBI.     

The presumed atypical chemokine receptor CXCR7 signals through G(i/o) proteins in primary rodent astrocytes and human glioma cells

SDF-1/CXCL12 binds to the chemokine receptors, CXCR4 and CXCR7, and controls cell proliferation and migration during development, tumorigenesis, and inflammatory processes. It is currently assumed that CXCR7 would represent an atypical or scavenger chemokine receptor which modulates the function of CXCR4. Contrasting this view, we demonstrated recently that CXCR7 actively mediates SDF-1 signaling in primary astrocytes. Here, we provide evidence that CXCR7 affects astrocytic cell signaling and function through pertussis toxin-sensitive G(i/o) proteins. SDF-1-dependent activation of G(i/o) proteins and subsequent increases in intracellular Ca(2+) concentration persisted in primary rodent astrocytes with depleted expression of CXCR4, but were abolished in astrocytes with depleted expression of CXCR7. Moreover, CXCR7-mediated effects of SDF-1 on Erk and Akt signaling as well as on astrocytic proliferation and migration were all sensitive to pertussis toxin. Likewise, pertussis toxin abolished SDF-1-induced activation of Erk and Akt in CXCR7-only expressing human glioma cell lines. Finally, consistent with a ligand-biased function of CXCR7 in astrocytes, the alternate CXCR7 ligand, I-TAC/CXCL11, activated Erk and Akt through β-arrestin. The demonstration that SDF-1-bound CXCR7 activates G(i/o) proteins in astrocytes could help to explain some discrepancies previously observed for the function of CXCR4 and CXCR7 in other cell types.     

Critical role of SDF-1/CXCR4 signaling pathway in stem cell homing in the deafened rat cochlea after acoustic trauma

Previous animal studies have shown that stromal cell-derived factor-1(SDF-1)/CXC chemokine receptor-4(CXCR4) signaling pathway plays an important role in the targeted migration of bone marrow-derived mesenchymal stem cells(BMSCs) to the injured area. In the present study, we aimed to investigate the potential role of chemotactic SDF-1/CXCR4 signaling pathway in the homing of transplanted BMSCs to the injured cochlea after noise-induced hearing loss(NIHL) in a rat model. White noise exposure(110 d B) paradigm was used for hearing loss induction in male rats for 6 hours in 5 days. Distortion-product otoacoustic emission(DPOAE) responses were recorded before the experiment and post noise exposure. Hoechst 33342-labeled BMSCs and CXCR4 antagonist(AMD3100)-treated BMSCs were injected into the rat cochlea through the round window. SDF-1 protein expression in the cochlear tissue was assayed using western blot assay. The number of labeled BMSCs reaching the endolymph was determined after 24 hours. SDF-1 was significantly increased in the cochlear tissue of rats in the noise exposure group than in the control group. The number of Hoechst 33342-labeled BMSCs reaching the endolymph of the cochlea was significantly smaller in the AMD3100-treated BMSCs group than in the normal BMSCs group. Our present findings suggest that the SDF-1/CXCR4 signaling pathway has a critical role in BMSCs migration to the injured cochlea in a rat model of noise-induced hearing loss.     

The role of CXCR4 signaling in the migration of transplanted oligodendrocyte progenitors into the cerebral white matter

Enhancing the ability of either endogenous or transplanted oligodendrocyte progenitors (OPs) to engage in myelination may constitute a novel therapeutic approach to demyelinating diseases of the brain. It is known that in adults neural progenitors situated in the subventricular zone of the lateral ventricle (SVZ) are capable of generating OPs which can migrate into white matter tracts such as the corpus callosum (CC). We observed that progenitor cells in the SVZ of adult mice expressed CXCR4 chemokine receptors and that the chemokine SDF-1/CXCL12 was expressed in the CC. We therefore investigated the role of chemokine signaling in regulating the migration of OPs into the CC following their transplantation into the lateral ventricle. We established OP cell cultures from Olig2-EGFP mouse brains. These cells expressed a variety of chemokine receptors, including CXCR4 receptors. Olig2-EGFP OPs differentiated into CNPase-expressing oligodendrocytes in culture. To study the migratory capacity of Olig2-EGFP OPs in vivo, we transplanted them into the lateral ventricles of mice. Donor cells migrated into the CC and differentiated into mature oligodendrocytes. This migration was enhanced in animals with Experimental Autoimmune Encephalomyelitis (EAE). Inhibition of CXCR4 receptor expression in OPs using shRNA inhibited the migration of transplanted OPs into the white matter suggesting that their directed migration is regulated by CXCR4 signaling. These findings indicate that CXCR4 mediated signaling is important in guiding the migration of transplanted OPs in the context of inflammatory demyelinating brain disease.