软骨细胞上清液诱导冻存复苏后人骨髓间充质干细胞的研究

目的:探讨人软骨细胞培养上清诱导冻存人骨髓充质干细胞向软骨细胞分化的可行性.方法:取进行全髋关节置换术老年患者的骨髓和软骨组织,利用密度梯度离心法、全骨髓培养法分别培养骨髓间充质干细胞,冻存备用.培养软骨细胞,观察细胞生长,收集软骨细胞培养上清.复苏冻存的人骨髓间充质干细胞,观察复苏后细胞生长状态.利用收集的软骨细胞培养上清对复苏间充质干细胞进行定向诱导,诱导培养2周,观察细胞外观表型变化,Ⅱ型胶原免疫组化检测诱导后人骨髓间充质干细胞Ⅱ型胶原的表达.结果:密度梯度离心法与全骨髓培养法均可分离获得人骨髓间充质干细胞,原代生长前者优于后者.复苏细胞仍进行可传代,与正常生长骨髓间充质细胞无明显差异,均可传至第8代.软骨细胞培养上清诱导2周后,细胞形状向圆形,多角形转变,冻存骨髓间充质干细胞Ⅱ型胶原免疫组化检测Ⅱ型胶原表达阳性.结论:老年人骨髓间充质干细胞仍具有向软骨细胞转化的能力,冻存不影响其转化能力.     

骨髓间充质干细胞对器官移植中记忆性T细胞的影响

探讨骨髓间充质干细胞在器官移植中记忆性T淋巴细胞功能的影响。通过同种异基因皮肤移植的方法诱导CD8~+记忆性T淋巴细胞的产生,在体外应用混合淋巴增殖实验观察骨髓间充质干细胞对经过刺激后的T细胞增殖情况的影响;另一方面,通过同种异基因小鼠心脏移植模型的建立,在体内验证和观察骨髓间充质干细胞对小鼠移植器官生存寿命的影响。骨髓间充质干细胞在混合淋巴增殖实验中,可以有效抑制CD8~+记忆性T淋巴细胞的增殖能力,实验组淋巴细胞增殖指数明显低于对照组(t=4.575,p0.05);在小鼠心脏移植模型中,输注骨髓间充质干细胞后移植心脏的生存寿命明显增加,差异有统计学意义(p0.05)。骨髓间充质干细胞能够有效抑制器官移植中CD8~+记忆性T淋巴细胞的增殖,诱导免疫耐受,延长异体器官存活时间。     

黄芪多糖对多发性骨髓瘤患者骨髓间充质干细胞增殖与细胞因子表达的影响

目的:探讨黄芪多糖对多发性骨髓瘤(multiple myeloma,MM)患者骨髓间充质干细胞(bone mesenchymal stem cells,BMSC)增殖与细胞因子表达的影响.方法:采用密度梯度离心法分离MM患者骨髓间充质干细胞(MM-BMSC)及正常人骨髓间充质干细胞(ND-BMSC).取第3代BMSC,测定细胞的生长曲线及细胞因子的表达.分别采用MTT法、细胞流式术检测不同浓度APS对MM-BMSC的细胞增殖、细胞周期的影响,ELISA法测定IL-1β与IL-6的表达水平.结果:成功分离得到MM-BMSC和ND-BM-SC,细胞生长曲线显示MM-BMSC增殖缓慢,倍增时间为86h,而ND-BMSC的倍增时间为60h,两者相比差异具有统计学差异(P＜0.05).基础正常情况下,MM-BMSC与ND-MSC分泌的IL-1β、IL-6的水平有明显差异(P＜0.05).APS可促进MM-BMSC增殖,其中1 mg/mL APS的作用最为明显(P＜0.01).MM-BMSC基本都处于静止期,1 mg/mL APS处理的MM-BMSC处于G0/G1期细胞比例较其明显降低(P＜0.05),S,G2/M期细胞比例较其明显增高(P＜0.05),且1 mg/mL APS可显著下调MM-BMSC分泌IL-1β及IL-6的水平(P＜0.05).结论:多发性骨髓瘤患者骨髓间充质干细胞IL-1β、IL-6分泌水平异常,1 mg/mL黄芪多糖可促进骨髓间充质干细胞的增殖并下调其IL-1β,IL-6的分泌.     

机械拉伸对骨髓间充质干细胞迁移行为的影响

目的:研究机械拉伸刺激对大鼠骨髓间充质干细胞迁移行为的影响并探讨其相关分子机制。方法:应用单轴机械拉伸加载装置考察不同条件的周期拉伸刺激对大鼠骨髓间充质干细胞迁移行为的影响,采用Transwell和划痕法评价细胞迁移能力,采用明胶酶谱法检测基质金属蛋白酶-2,-9(MMP-2,-9)表达的变化。结果:适宜的拉伸刺激可以明显促进大鼠骨髓间充质干细胞的迁移能力,1 Hz、10%应变拉伸8 h后可以使细胞迁移数量增加到对照组的1.58倍。拉伸刺激诱导骨髓间充质干细胞基质金属蛋白酶-2,-9(MMP-2,-9)表达。抑制剂GM6001抑制了拉伸诱导的MMP-2,-9分泌增加,同时抑制了拉伸刺激对细胞迁移的促进作用。结论:机械拉伸刺激影响大鼠骨髓间充质干细胞的迁移行为,MMP-2,-9在此过程中可能起着重要介导作用。     

骨髓间充质干细胞分化为心肌细胞过程中Notch表达的研究

目的研究骨髓间充质干细胞分化为心肌细胞过程中Notch表达的研究。方法用密度梯度离心法分离培养犬骨髓间充质干细胞,按照酶法及差速贴壁法分离培养心肌细胞。观察干细胞增殖及传代情况。单独培养的干细胞为对照组,实验组将骨髓间充质干细胞与心肌细胞共培养,用RT-PCR、免疫细胞化学、MTT等方法检测干细胞分化为心肌细胞的情况,及干细胞在增殖与分化为心肌细胞过程中Notch信号系统的表达情况。结果骨髓间充质干细胞呈梭形、旋涡样生长,增殖及传代能力强,并可诱导分化为心肌样细胞,免疫荧光示心肌细胞标志物的表达。RT-PCR及免疫细胞化学显示实验组有Notch信号通路受体及配体的表达,而对照组表达微弱。结论骨髓间充质干细胞在增殖及分化过程中存在Notch信号通路,在干细胞分化为心肌细胞过程中Notch信号系统的表达上调。     

阿司匹林对骨髓间充质干细胞移植治疗缺血性脑卒中的影响研究进展

阿司匹林是缺血性脑卒中患者急性期治疗药物及卒中再发的二级预防常用药物,骨髓间充质干细胞(BMSCs)移植是治疗缺血性脑血管疾病的新的新兴技术。已证实阿司匹林可抑制骨髓间充质干细胞的增殖及影响骨髓间充质干细胞的分化。本文就阿司匹林对骨髓间充质干细胞移植治疗缺血性脑卒中的影响等进行综述。     

猪骨髓间充质干细胞的分离培养及核移植后重构胚胎发育能力的研究

不同类型的细胞核移植效率不同,原因之一可能是不同类型细胞核移植后进行重编程的潜力不同.本实验对猪骨髓间充质干细胞(porcine bone marrow mesenchymal stem cells,pMSCs)体外分离培养的方法进行了优化.对猪骨髓间充质干细胞的增殖及生长特性进行了观察分析,并以其作为供体细胞进行核移植,对此类型细胞进行重编程的潜力进行了评估.结果表明用密度梯度离心法分离猪骨髓间充质干细胞优于全骨髓贴壁法:猪骨髓间充质干细胞数目在培养第6天达到峰值,传代培养10 h时,贴壁率达到78.50%;传代培养后第4天分裂指数最高,为24.00‰;以猪骨髓间充质干细胞(pMSCs)和猪胎儿成纤维细胞(PF)分别作为供核细胞构建核移植胚胎,其体外囊胚发育率分别为14.63%与15.07%(P＞0.05),孤雌对照组囊胚发育率为30.91%(P＜0.05);而三组囊胚细胞数分别为30.67±17.7、24.1±6.5和25.8±11.4(P＞0.05).实验表明,体外培养的猪骨髓间充质干细胞生长增殖旺盛,生物学性状稳定.并适合作为核移植供体细胞.     

Nucleostemin在大鼠骨髓基质干细胞向成骨细胞诱导分化中的研究

目的探讨Nucleostemin（NS）在大鼠骨髓基质干细胞向成骨细胞诱导分化中的机制及作用。方法取4～6周龄雄性SD大鼠两侧股骨、胫骨骨髓基质细胞,原代及传代培养。取第3代骨髓间充质干细胞分别用普通和矿化培养基培养,通过相差倒置显微镜观察细胞生长、MTT法检测细胞增殖、碱性磷酸酶和茜素红钙染色了解成骨活性。免疫组化SABC法及免疫荧光法检测NS在细胞中的表达情况,比较NS分别在普通培养基和成骨细胞诱导培养基作用下的表达情况。结果大鼠骨髓间充质干细胞在矿化培养基诱导下其NS的表达较普通培养基培养下的表达明显减弱,且呈时间依赖性衰减。成骨细胞的NS表达则为阴性。在矿化液作用下,骨髓间充质干细胞可诱导为成骨细胞。MTT显示矿化液培养细胞生长潜伏期长,对数生长期较对照组延长。结论NS作为核干细胞因子,在干细胞中和某些肿瘤细胞中表达丰富。细胞分化后,其表达明显减少。因此,NS很可能作为大鼠基质干细胞是否具有潜在分化能力的标志性因子。     

转染腺相关病毒对骨髓间充质干细胞分化潜能的影响

目的:探讨转染腺相关病毒对骨髓间充质干细胞分化潜能的影响.方法:运用密度梯度离心法分离骨髓间充质干细胞;将pAAV-GFP、pAAV-RC、pHelper用磷酸钙法共转染HEK-293细胞,得到rAAV-GFP,转染骨髓间充质干细胞.进行成骨诱导分化,观察rAAV-GFP对骨髓间充质干细胞分化潜能的影响.结果:与未转染rAAV-GFP的间充质干细胞相比较,转染rAAV-GFP后,骨髓间充质干细胞分化潜能未见改变,均表现为细胞浆蓝紫色,核周明显.结论:转染腺相关病毒对骨髓间充质干细胞分化潜能未见明显影响,为基因修饰腺相关病毒转染骨髓间充质干细胞进行体内移植提供了实验基础.     

诱导MSCs向成骨细胞分化相关因素研究现状

骨髓间充质干细胞(mesenchymal steml cells,MSCs)是干细胞家族的重要成员,是一类具有自我增殖和多项分化潜能的多能干细胞.MSCs定向分化为成骨细胞将可能为包括骨缺损、骨折不愈合、股骨头坏死等多种骨科难治性疾病的治疗带来一场革新.本文主要介绍了化学因素、生物因素、他汀类药物、物理因素、转基因技术以及中医药等相关因素诱导MSCs向成骨细胞分化的研究现状.     

Mechanical regulation of HB-GAM expression in bone cells

Bone adaption upon mechanical stimulation is accompanied by changes in gene expression. In this context we investigated the influence of mechanical loading on heparin binding growth associated molecule (HB-GAM) expression, an extracellular matrix molecule which in cell culture has been shown to stimulate the differentiation of osteoblasts. We obtained information on the participating signal transduction pathways using a mitogenic loading regimen. Specific inhibitors of various signal transduction pathways were added to loaded cells and to unloaded controls. By semi-quantitative PCR studies we demonstrated a rapid decrease of HB-GAM expression in primary osteoblasts and SaOs-2 cells by 20-30% upon mechanical loading within 30min. We showed that the RGD-integrin interaction is involved in the regulation of HB-GAM expression. Furthermore, integrity of the cytoskeleton, stretch-activated, and voltage-sensitive Ca(2+) channels as well as gap junctional communication are necessary for the downregulation of HB-GAM expression by mechanical loading.     

Matrix nanotopography as a regulator of cell function

The architecture of the extracellular matrix (ECM) directs cell behavior by providing spatial and mechanical cues to which cells respond. In addition to soluble chemical factors, physical interactions between the cell and ECM regulate primary cell processes, including differentiation, migration, and proliferation. Advances in microtechnology and, more recently, nanotechnology provide a powerful means to study the influence of the ECM on cell behavior. By recapitulating local architectures that cells encounter in vivo, we can elucidate and dissect the fundamental signal transduction pathways that control cell behavior in critical developmental, physiological, and pathological processes.     

Mechanoregulation of chondrocyte proliferation, maturation, and hypertrophy: ion-channel dependent transduction of matrix deformation signals

Mechanical stress-induced matrix deformation plays a fundamental role in regulating cellular activities; however, little is known about its underlying mechanisms. To understand the effects of matrix deformation on chondrocytes, we characterized primary chondrocytes cultured on three-dimensional collagen scaffoldings, which can be loaded mechanically with a computer-controlled "Bio-Stretch" device. Cyclic matrix deformation greatly stimulated proliferation of immature chondrocytes, but not that of hypertrophic chondrocytes. This indicates that mechanical stimulation of chondrocyte proliferation is developmental stage specific. Synthesis of cartilage matrix protein (CMP/matrilin-1), a mature chondrocyte marker, and type X collagen, a hypertrophic chondrocyte marker, was up-regulated by stretch-induced matrix deformation. Therefore, genes of CMP and type X collagen are responsive to mechanical stress. Mechanical stimulation of the mRNA levels of CMP and type X collagen occurred exactly at the same time points when these markers were synthesized by nonloading cells. This indicates that cyclic matrix deformation does not alter the speed of differentiation, but affects the extent of differentiation. The addition of the stretch-activated channel blocker gadolinium during loading abolished mechanical stimulation of chondrocyte proliferation, but did not affect the up-regulation of CMP mRNA by mechanical stretch. In contrast, the calcium channel blocker nifedipine inhibited both the stretch-induced proliferation and the increase of CMP mRNA. This suggests that stretch-induced matrix deformation regulates chondrocyte proliferation and differentiation via two signal transduction pathways, with stretch-activated channels involved in transducing the proliferative signals and calcium channels involved in transducing the signals for both proliferation and differentiation.     

Invited review: mechanochemical signal transduction in the fetal lung.

Growth and maturation of fetal lungs are regulated by both humoral and physical factors. Mechanical stretch stimulates fetal lung cell proliferation and affects fetal lung maturation by influencing the production of extracellular matrix molecules and the expression of specific genes of fetal lung cells. These effects are mediated through special signal transduction pathways in fetal lung cells. Various in vivo and in vitro model systems have been developed to investigate the mechanotransduction process. The diversity and discrepancy of these studies have raised many questions. We will briefly summarize mechanical force-induced signals in fetal lung cell proliferation and differentiation and then discuss several important issues related to these studies.     

Conversion of mechanical force into biochemical signaling

Physical forces play important roles in regulating cell proliferation, differentiation, and death by activating intracellular signal transduction pathways. How cells sense mechanical stimulation, however, is largely unknown. Most studies focus on cellular membrane proteins such as ion channels, integrins, and receptors for growth factors as mechanosensory units. Here we show that mechanical stretch-induced c-Src protein tyrosine kinase activation is mediated through the actin filament-associated protein (AFAP). Distributed along the actin filaments, AFAP can directly active c-Src through binding to its Src homology 3 and/or 2 domains. Mutations at these specific binding sites on AFAP blocked mechanical stretch-induced c-Src activation. Therefore, mechanical force can be transmitted along the cytoskeleton, and interaction between cytoskeletal associated proteins and enzymes related to signal transduction may convert physical forces into biochemical reactions. Cytoskeleton deformation-induced protein-protein interaction via specific binding sites may represent a novel intracellular mechanism for cells to sense mechanical stimulation.     

Role of cytokines and extracellular matrix in the regulation of haemopoietic stem cells

The understanding of molecular mechanisms regulating the formation, growth and differentiation of haemopoietic stem cells has advanced considerably recently. Particular progress has been made in defining the cytokines, chemokines and extracellular matrix components which retain and maintain primitive haemopoietic cell populations in bone marrow. Furthermore, signal transduction pathways that are critical for haemopoiesis, both in vivo and in vitro, and that are activated by cytokines have also been identified and further characterised. The importance of these processes has, this year, been exemplified by the phenotypes of mice deficient in key signal transduction proteins and the discovery that mutations in the component proteins of some signalling pathways are linked to human diseases. Significant advances in understanding the molecular mechanisms for mobilisation of stem cells from bone marrow have also been made this year; this has potential importance for bone marrow transplantation.     

单细胞测序分析人类胚胎干细胞神经分化的分子机制

目的 探讨人类胚胎干细胞(ESCs)分化为神经细胞的关键性靶基因及分子机制,为临床靶向治疗神经康复患者提供分子理论依据.方法 基于GEO数据平台芯片,采用单细胞测序方法(scRNA-seq),利用R语言从多分子维度(单细胞差异基因、蛋白互作网络和基因通路等)分析人类ESCs分化过程中的关键Marker基因并利用质控和数...     

Integrin alphaIIbbeta3 induces the adhesion and activation of mast cells through interaction with fibrinogen

Integrin alphaIIb, a well-known marker of megakaryocyte-platelet lineage, has been recently recognized on hemopoietic progenitors. We now demonstrate that integrin alphaIIbbeta3 is highly expressed on mouse and human mast cells including mouse bone marrow-derived mast cells, peritoneal mast cells, and human cord blood-derived mast cells, and that its binding to extracellular matrix proteins leads to enhancement of biological functions of mast cells in concert with various stimuli. With exposure to various stimuli, including cross-linking of FcepsilonRI and stem cell factor, mast cells adhered to extracellular matrix proteins such as fibrinogen and von Willebrand factor in an integrin alphaIIbbeta3-dependent manner. In addition, the binding of mast cells to fibrinogen enhanced proliferation, cytokine production, and migration and induced uptake of soluble fibrinogen in response to stem cell factor stimulation, implicating integrin alphaIIbbeta3 in a variety of mast cell functions. In conclusion, mouse and human mast cells express functional integrin alphaIIbbeta3.