Lin28调节小鼠胚胎干细胞向原始生殖细胞分化

目的 探讨小鼠胚胎干细胞(mouse embryonic stem cells,mESCs)向原始生殖细胞(primordial germ cells,PGCs)分化过程中特异基因表达变化及可能机制.方法 mESCs分化形成拟胚体(embryoid bodies,EBs),不同浓度atRA(1μM,2 μ M,5 μ M)持续诱导EBs 16h、2d和5d,RT-PCR、和免疫荧光分别检测Lin28和Blimp1以及相应蛋白的表达变化.结果 atRA诱导16h的EBs其Lin28 mRNA表达量较高,随着诱导时间延长而逐渐降低,Blimp1则无明显变化.WB结果显示EBs的Lin28蛋白表达随诱导时间延长逐渐减弱,而Blimp1蛋白表达逐渐增强.免疫荧光显示Lin28和Blimp1阳性信号均定位于细胞胞浆,其变化特点与WB结果相一致.结论 EBs经atRA诱导后可影响Lin28的表达,Lin28和Blimp1的变化并不协调,其蛋白表达随着诱导会随之发生变化.atRA诱导可能使PGCs分化的Lin28低水平表达时期提前出现.     

全反式视黄酸影响小鼠胚胎干细胞向原始生殖细胞分化的相关基因表达

目的 探讨全反式视黄酸(all-trans retinoic acid,atRA)诱导小鼠胚胎干细胞(mouse embryonic stem cells,mESCs)向原始生殖细胞(Primordial germ cellS,PGCs)分化的相关基因表达改变及其机制.方法 mESCs分化形成拟胚体(embryoid bodies,Ebs),不同浓度atRA(1 μM,2 μM,5 μM)持续诱导Ebs 16h、2d和5d,观察Ebs形态变化,实时PCR和Western blot检测PGCs分化相关基因和蛋白表达变化.分析基因肩动子中RA反应元件,以Stra8(Stimulated by Retinoic Acid Gene 8,Stra8)基因为阳性对照,确定各基因对atRA刺激的原发性反应和继发性反应.结果 不同浓度atRA诱导Ebs 16h和2d后形态无明显变化.诱导后Ebs的PGCs分化相天基因mRNA表达分4种情况:16h内(Stra8)表达量达最大,然后下降;2d内(Scp3)表达量达最大,然后下降;5d达到最大量(Mvh);表达量无明显变化(Fragilis,Blimp-1和Prm1).Mvh表达符合继发反应特点;Stra8和Scp3表达符合原发反应特点;Blimp1,Prm1和Fragilis表达可能与atRA调节无关.Stra8蛋白变化与mRNA变化相一致,而Mvh则不一致.结论 atRA诱导mESCs向PGCs分化,各阶段标志基因表达变化均在相对短的时间内完成(2～5d),atRA诱导浓度可选用1 μM.根据基因表达模式初步得出,atRA调控的靶基因为Mvh,具备继发反应特点,与特异性分化有关,而Blimp-1、Fragilis和Prm1调控PGCs分化可能与atRA无关.     

β1转化生长因子对瘢痕成纤维细胞纤维粘连蛋白及其受体α5β1整合素表达的影响

目的　研究 β1转化生长因子 (TGF β1)对瘢痕成纤维细胞纤维粘连蛋白 (FN)及其受体α5β1整合素表达的影响 ,探讨TGF β1、FN及其受体α5β1整合素在增生性瘢痕形成发展中的作用。方法　采用细胞培养、ELISA法、免疫组织化学、图像分析技术 ,观察浓度分别为 0、5、10、2 5、5 0、10 0 μg/L的TGF β1作用下 ,瘢痕成纤维细胞FN及其受体α5β1整合素表达的变化。结果　TGF β1浓度在 10～ 5 0μg/L之间时 ,可明显刺激瘢痕成纤维细胞FN及其受体α5β1整合素的表达 ,与对照组相比较 ,两者差异有显著性意义 (P <0 .0 5 ) ,其作用呈一定的剂量效应关系 ,随着TGF β1浓度增加 ,促合成作用也随之增加 ,浓度在 10 0 μg/L时 ,作用达到饱和。 结论　TGF β1作用下FN及其受体α5β1整合素在成纤维细胞中的过度表达可能与增生性瘢痕形成有关。     

整合素在着床中的作用

近年来 ,细胞外基质成分 ( extracellularmatrix,ECM)与细胞表面受体相互作用的机制 ,随着研究的深入已经越来越清楚。这种受体命名为整合素 ( integrin) ,是一种完整的跨膜糖蛋白 ,由两种不同亚单位组成的异二聚体 ,包括纤粘连蛋白 ( fibronectin,FN)、层粘连蛋白( lamimin,L N)、胶原 ( collagen,COL )及玻璃粘连蛋白 ( vitronectin,VN)等受体。整合素和ECM相互作用促进细胞的粘附、生长、迁徙、增殖及分化 [1] 。子宫、输卵管及早期胚胎中广泛存在整合素和 ECM。在精卵识别过程中 ,整合素起调节作用 ,促使胚泡粘附于子宫内膜上皮…     

二十碳五烯酸对白蛋白诱导肾小管上皮细胞转分化的影响

目的 探讨二十碳五烯酸(eicosapentaenoic acid,EPA)对人白蛋白诱导的人近端肾小管上皮细胞(HK-2)发生转分化的干预作用.方法 体外培养人HK-2细胞,随机分为6组:A组,空白对照组(未加任何刺激物);B组,单纯EPA组(加入30 μmol/L EPA);C组,血白蛋白(albumin,Alb)诱导组(加入5 mg/ml Alb);D组,低剂量EPA干预组(5mg/ml Alb+ 10 μmol/L EPA);E组,中剂量EPA干预组(5mg/ml Alb+ 30 μmol/L EPA);F组,高剂量EPA干预组(5 mg/ml Alb+ 50μmol/L EPA).细胞免疫荧光检测E钙黏蛋白(E-cadherin)、α平滑肌肌动蛋白(α-smooth muscle ac-tin,α-SMA)的表达;ELISA法检测纤维连接蛋白(fibronectin,FN)的水平;采用RT-PCR检测转化生长因子β1(transforming growth factor β1,TGF-β1)的mRNA表达.结果 与A组比较,B组TGF-β1mRNA、α-SMA、FN、E-cadherin表达均无统计学差异(P＞0.05);C组TGF-β1 mRNA、α-SMA、FN较其他各组表达明显增高(P＜0.05),而E-cadherin表达明显下降(P＜0.05).与C组比较,D组、E组、F组TGF-β1 mRNA、α-SMA、FN表达逐渐下降,两组间比较差异有统计学意义(P＜0.05);E-cadherin的表达逐渐增高,各组间比较差异有统计学意义(P＜0.05).提示EPA可显著抑制Alb刺激人肾小管上皮细胞表达E-cadherin、α-SMA,同时下调上清液中FN水平,且其抑制作用的强弱与EPA浓度密切相关.EPA还明显下调了Alb刺激后TGF-β1的表达.结论 一定浓度的人白蛋白可以诱导体外培养的HK-2细胞发生转分化;EPA可一定程度上抑制白蛋白诱导HK-2发生转分化过程,且呈剂量依赖性.     

烧伤大鼠创面再上皮化过程中表皮角质形成细胞基底膜相关基因的表达

目的　检测烧伤大鼠创面再上皮化过程中表皮角质形成细胞(KCs)基底膜(BM)相关基因的表达。　方法　24只SD大鼠造成背部45cm2 深Ⅱ度烫伤, 按照基因表达检测时间随机分为A组(伤后3d)、B组(伤后10d)、C组(伤后14d)、D组(创面完成再上皮化后),每组6只。伤后3、10、14d取距创缘1cm处皮肤标本,创面完成再上皮化后取创面中心皮肤标本,分别制备KCs悬液。另取6只SD大鼠背部皮肤作为正常对照组。利用基因芯片技术检测各组大鼠创面再上皮化不同阶段KCsBM相关基因的差异性表达。　结果　伤后3d,KCs层粘连蛋白γ1、整合素β8基因表达上调,基因表达数据与正常对照比值分别为2. 068和2. 200。再上皮化过程中(伤后10、14d)层粘连蛋白受体1、整合素β1基因表达上调,β2、β7表达下调,基因表达数据与正常对照的比值分别为2. 472、2. 658、0. 419和0. 462。Ⅳ胶型原α1、α3基因各组均上调,基因表达数据与正常对照的比值为2. 547和2 036。　结论　创面再上皮化过程中整合素β1、层粘连蛋白γ1、层粘连蛋白受体1、Ⅳ型胶原α1、α3基因表达上调,有利于新生皮肤BM的构建和KCs与BM之间形成稳定的连接。     

小鼠iPS细胞具备诱导性原始生殖细胞分化潜能的研究

目的:探讨小鼠诱导性多能干细胞IP14D-1是否具备诱导性原始生殖细胞(induced primordial germcells,iPGCs)分化潜能,以及特异基因表达变化及可能机制。方法:未分化IP14D-1培养扩增,分化形成诱导性拟胚体(induced embryoid bodies,iEBs)。RT-PCR和免疫荧光分别检测4、7、9 d的iEBs中Lin28、Blimp1、Stra8和Mvh的表达变化和蛋白定位情况。结果:未分化IP14D-1同小鼠胚胎干细胞(mouse embryonic stem cells,mESCs)相同,Lin28表达较弱,Blimp1表达相对较强,Mvh和Stra8也在这两种细胞及其相应iEBs和EBs中表达,但均无明显差别。IP14D-1分化形成的iEBs从4 d生长到7 d时,Lin28表达逐渐增强,到9 d时表现为下降,Blimp1表达则随着iEB生长时间延长而逐渐降低。结论:建立了IP14D-1和相应拟胚体(iEBs)的完善、稳定的培养及分化体系;未分化IP14D-1与mESCs在Lin28、Blimp1、Mvh和Stra8表达方面无明显差别;iEB和EBs的Mvh和Stra8表达也无明显差别。IP14D-1及iEBs具有iPGCs分化潜能,且可能是7 d的iEBs内iPGCs分化数量较多,之后进入iPGCs分化的Lin28低表达时期。     

整合素连锁激酶介导转化生长因子β1上调肾小管上皮细胞表达纤连蛋白的研究

目的 研究转化生长因子β1（TGF-β1）诱导肾小管上皮细胞合成纤连蛋白（FN）与整合素连锁激酶（ILK）表达的关系.方法 培养人肾小管上皮细胞（HKC）,用蛋白印迹方法检测TGF-β1诱导HKC合成FN和表达ILK的作用.通过基因转染的方法,将含人野生型ILK基因的表达质粒pCMV-wtILK和无激酶活力的人ILK基因表达质粒pCMV-kdILK转染HKC细胞,观察ILK过度表达和抑制ILK活力对TGF-β1诱导的HKC合成FN的影响.结果 TGF-β1能够刺激HKC合成FN,其作用呈剂量依赖性.TGF-β1刺激8 h即可上调HKC细胞ILK的表达,与TGF-β1所致的FN合成的增加相一致.转染pCMV-wtILK质粒的HKC细胞,其FN的表达呈增加趋势.转染pCMV-kdILK抑制ILK的活力能够显著拮抗TGF-β1刺激HKC表达FN的作用.结论 TGF-β1刺激肾小管上皮细胞FN合成与其所致的ILK表达密切相关.抑制ILK的活力能够拮抗TGF-β1导致的FN合成.     

整合素及相关信号转导因子在结直肠癌转移中的作用机制

目的研究整合素αvβ3、黏着斑激酶(FAK)、纤连蛋白(FN)和基质金属蛋白酶-9(MMP-9)在结直肠癌转移中的作用机制。方法应用半定量逆转录聚合酶链反应(SQRT-PCR)和免疫组织化学方法,检测30例结直肠癌标本中整合素αvβ3、FAK、FN和MMP-9mRNA转录水平和蛋白表达变化。结果结直肠癌组织中整合素αvβ3亚基转录水平明显增高(P<0.05),FAK、FN和MMP-9mRNA转录水平随肿瘤浸润深度增加和Dukes分期进展而升高(P<0.05),且在淋巴结转移组升高明显(P<0.05);FAK和细胞外基质中FN、MMP-9蛋白表达水平与其基因mRNA转录水平改变相一致,但基底膜上的FN蛋白表达改变则完全相反。结论结直肠癌细胞转移初始阶段,FN-整合素αvβ3-细胞骨架-FAK系统由细胞外向细胞内传递信号,产生MMP-9等因子,完成癌细胞脱落、黏附、降解、转移的过程,且上述基因的转录、表达水平越高,结直肠癌的侵袭转移潜能越强。     

TGF—β1基因修饰诱导脂肪干细胞成软骨分化的实验研究

[目的]本研究通过选择对软骨细胞ECM合成具有促进作用的TGF-β1转染脂肪干细胞(ADSCs),观察转染后目的基因的稳定表达和对软骨细胞外基质(ECM)的两种重要组分:Ⅱ型胶原(type Ⅱ collage,ColⅡ)和aggrecan的合成,为构建新型的组织工程软骨提供实验依据.[方法]TGF-β1基因转染脂肪干细胞及阳性细胞克隆株的筛选;RT-PCR检测TGF-β1、纤连蛋白(fibronectin,FiN)、ColⅡ、Aggrecan的表达;Western-blot检测TGF-β1.[结果]RT-PCR结果显示:实验组(TGF-β1稳定转染组)的TGF-β1、FN、Col Ⅱ、Aggrecan的表达较空染组和正常对照组均明显增多(P＜0.01);Western blot检测实验组(TGF-β1稳定转染组)TGF-β1蛋白的表达较空染组和正常对照组均明显增多(P＜0.01).[结论]成功分离培养脂肪干细胞;以脂质体介导法将TGF-β1目的基因成功转染ADSCs,转染后成软骨分化的特异性细胞外基质增多,表明转染TGF-β1基因可以促使ADSCs向软骨细胞方向分化,从而为软骨组织工程提供新的思路和方法.     

诱导多能干细胞体外向生精细胞自发分化潜能的研究

目的:探讨诱导多能干细胞(iPS)体外培养自发分化过程中生精细胞相关基因的表达,评估iPS体外向生精细胞自发分化的潜能。方法:经类胚体(EB)形成,体外诱导iPS向生精细胞分化,实时定量PCR和PCR检测生精细胞相关基因的表达。结果:实时定量PCR和PCR结果显示iPS经EB形成诱导分化后生精细胞不同时期的相关基因均有不同程度表达。iPS体外培养自发分化后生精细胞相关基因出现4种时间表达特征:Oct4基因表达量呈波浪状上升;Dppa3和Stra8基因表达量随诱导时间延长而下降;Dazl基因表达量呈波浪状下降;减数分裂前期基因Tex14、Msy2,减数分裂期基因Scp1、Scp3以及单倍体基因Akap3随着诱导时间延长先表达增加,而后表达下降。结论:iPS在经EB自发分化过程中表达生精细胞不同时期的相关基因,并且表达雄性配子单倍体基因,具有向雄性配子的分化潜能。     

Why human extragonadal germ cell tumours occur in the midline of the body: old concepts, new perspectives

Hypotheses on the origin and distribution of extragonadal germ cell tumours (GCTs) and teratomas are briefly reviewed and revisited in the light of (i) new developments in the classification of GCTs, (ii) data on genomic imprinting of these neoplasms and (iii) the recent finding that germ cells can be derived from mouse and human embryonal stem (ES) cells. Only the Type I (infantile teratomas/yolk sac tumours) and Type II GCTs (seminomatous tumours and non-seminomas) occur in the gonads and extragonadal localizations. The data on genomic imprinting lend support to the hypothesis that they are derived from germ cells. These precursor cells could have differentiated from ES cells in extragonadal localizations. Their distribution along the midline of the body is still best explained by the migration of primitive germ cells during development. The narrower distribution of the Type II than the Type I GCTs is probably due to the more strict conditions for survival and proliferation of primordial germ cells (PGCs)/gonocytes from which the Type II tumours originate, when compared with the precursor cells of Type I tumours, probably primitive germ cells closer to the ES cell. The known niches in which the Type II tumours develop have in common that they contain feeder cells expressing stem cell factor (SCF) - the ligand for the SCF receptor c-KIT, involved in proliferation and survival of PGCs/gonocytes - and contain GBY including the gene TSPY.     

Generation of male germ cells from induced pluripotent stem cells (iPS cells): an in vitro and in vivo study

Recent studies have reported that induced pluripotent stem (iPS) cells from mice and humans can differentiate into primordial germ cells. However, whether iPS cells are capable of producing male germ cells is not known. The objective of this study was to investigate the differentiation potential of mouse iPS cells into spermatogonial stem cells and late-stage male germ cells. We used an approach that combines in vitro differentiation and in vivo transplantation. Embryoid bodies (EBs) were obtained from iPS cells using leukaemia inhibitor factor (LIF)-free medium. Quantitative PCR revealed a decrease in Oct4 expression and an increase in Stra8 and Vasa mRNA in the EBs derived from iPS cells. iPS cell-derived EBs were induced by retinoic acid to differentiate into spermatogonial stem cells (SSCs), as evidenced by their expression of VASA, as well as CDH1 and GFRα1, which are markers of SSCs. Furthermore, these germ cells derived from iPS cells were transplanted into recipient testes of mice that had been pre-treated with busulfan. Notably, iPS cell-derived SSCs were able to differentiate into male germ cells ranging from spermatogonia to round spermatids, as shown by VASA and SCP3 expression. This study demonstrates that iPS cells have the potential to differentiate into late-stage male germ cells. The derivation of male germ cells from iPS cells has potential applications in the treatment of male infertility and provides a model for uncovering the molecular mechanisms underlying male germ cell development.     

Collagen IV significantly enhances migration and transplantation of embryonic stem cells: involvement of α2β1 integrin-mediated actin remodeling

Embryonic stem (ES) cell transplantation represents a potential means for the treatment of degenerative diseases and injuries. As appropriate distribution of transplanted ES cells in the host tissue is critical for successful transplantation, the exploration of efficient strategies to enhance ES cell migration is warranted. In this study we investigated ES cell migration under the influence of various extracellular matrix (ECM) proteins, which have been shown to stimulate cell migration in various cell models with unclear effects on ES cells. Using two mouse ES (mES) cell lines, ESC 26GJ9012-8-2 and ES-D3 GL, to generate embryoid bodies (EBs), we examined the migration of differentiating cells from EBs that were delivered onto culture surfaces coated with or without collagen I, collagen IV, Matrigel, fibronectin, and laminin. Among these ECM proteins, collagen IV exhibited maximal migration enhancing effect. mES cells expressed α2 and β1 integrin subunits and the migration enhancing effect of collagen IV was prevented by RGD peptides as well as antibodies against α2 and β1 integrins, indicating that the enhancing effect of collagen IV on cell migration was mediated by α2β1 integrin. Furthermore, staining of actin cytoskeleton that links to integrins revealed well-developed stress fibers and long filopodia in mES cells cultured on collagen IV, and the actin-disrupting cytochalasin D abolished the collagen IV-enhanced cell migration. In addition, pretreatment of undifferentiated or differentiated mES cells with collagen IV resulted in improved engraftment and growth after transplantation into the subcutaneous tissue of nude mice. Finally, collagen IV pretreatment of osteogenically differentiated mES cells increased osteogenic differentiation-like tissue and decreased undifferentiation-like tissue in the grafts grown after transplantation. Our results demonstrated that collagen IV significantly enhanced the migration of differentiating ES cells through α2β1 integrin-mediated actin remodeling and could promote ES cell transplantation efficiency, which may be imperative to stem cell therapy.     

Distribution of integrins and their matrix ligands in osteogenic sarcomas

We studies the expression of the members of the β1 integrin family and extracellular matrix ligands in osteosarcoma tissues. Immunostaining of primary osteosarcomas with use of specific antibodies for α1-α6 integrin subunits, fibronectin, type-I collagen, and laminin gave characteristic patterns: despite the diffuse expression of type-I collagen in all 16 osteosarcomas, collagen receptors were detected in only one. Laminin and laminin receptors were expressed poorly. In contrast, the α4 and α5 fibronectin receptors were found in 100 and 75%, respectively, which correlates very well with the strong expression of fibronectin in the stroma. The other ligand for α4, vascular cell adhesion molecule-1, was not expressed in the seven specimens tested. Therefore, primary osteosarcomas closely interact with fibronectin through α4 and possibly α5 subunits of the β1 integrin. We also examined the expression the expression was detected in all six specimens, however, the change varied. In contrast, osteosarcoma cells obtained from invasive portions of tumors exhibited new and augmented expression of certain integrins in two of the three cases in which their ligands were also expressed.     

Extracellular matrix components induce differentiation of the human osteoblastic cell line SV-HFO in a soluble factor-dependent manner

In this study, we examined the effect of extracellular matrix (ECM) components including type I collagen (Col I), type IV collagen (Col IV), laminin, and fibronectin on differentiation of the human osteoblastic cell line SV-HFO when the cells were treated with dexamethasone (Dex), which induces the cells' mineralization. All ECM proteins clearly increased the alkaline phosphatase (ALP) activity of SV-HFO cells in a dose-dependent fashion. Similarly, treatment with 1, 25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) induced the ECM components to enhance the osteocalcin (OC) synthesis of SV-HFO cells. Each ECM component had distinct effects depending on the soluble factor used; with Dex treatment, fibronectin more efficiently increased the ALP activity of Dex-treated SV-HFO cells than Col I, Col IV, or laminin, while with 1,25(OH)₂D₃ treatment, Col IV more efficiently increased on OC synthesis than the other ECMs. We also examined the expression of integrins, an ECM receptor family, using monoclonal antibodies. Flow cytometric analysis demonstrated that SV-HFO cells expressed all very late antigen (VLA) integrins including VLA-1 to VLA-6 as well as _v3 integrin, and that the expression of VLA integrins is regulated by the Dex-induced cell differentiation. These findings suggest that ECM components have soluble factor-dependent stimulatory effects on the regulation of osteoblastic differentiation of SV-HFO cells, possibly through the cell surface ECM receptors in the integrin family.     

Effect of cyclic strain and plating matrix on cell proliferation and integrin expression by ligament fibroblasts.

The role of cell surface integrins in cell migration, proliferation, and attachment to matrix molecules is well known. Integrin-matrix interactions have been implicated in mechanotransduction and load transmission from the outside to the inside of the cell. In this study, the effect of cyclic strain on the cell proliferation, attachment, and expression of integrin subunits beta1, beta3, and alpha5 was determined in anterior cruciate ligament (ACL) and medial collateral ligament (MCL) fibroblasts grown on polystyrene, Type I collagen, laminin, elastin, and fibronectin. ACL fibroblast proliferation was not affected by growth substrate whereas MCL cells reached confluence more rapidly on fibronectin compared with collagen or polystyrene. Exposure to 5% cyclic strain resulted in a significant decrease in ACL and MCL fibroblast proliferation on fibronectin and Type I collagen. MCL cells showed a greater strain-dependent inhibition of cells grown on a fibronectin substrate than those grown on collagen. This matrix-dependent effect of strain on cell proliferation was not seen with ACL cells. Attachment of ACL and MCL fibroblasts was stronger to fibronectin compared with Type I collagen, laminin, and polystyrene. In the absence of applied load, the expression of beta1, beta3, and alpha5 subunits was not substrate dependent and the expression of beta1 and alpha5 integrin subunits was higher in MCL cells than ACL cells on all substrates. In contrast, the expression of beta3 integrin subunit was higher in ACL cells than MCL cells. In response to 5% strain, beta1, and alpha5 expression increased in all fibroblasts with MCL cells having a higher magnitude of expression. beta3 expression showed a 90% increase in response to load when grown on laminin for both MCL and ACL fibroblasts and demonstrated no change in expression on Type I collagen or fibronectin. The duration of applied strain from 2 versus 22 h had no effect on cell proliferation or integrin expression.     

Functional integrin subunits regulating cell-matrix interactions in the intervertebral disc.

Cellular interactions with the extracellular matrix are key factors regulating cell survival, differentiation, and response to environmental stimuli in cartilagenous tissues. Much is known about the extracellular matrix proteins in the intervertebral disc (IVD) and their variations with region, age, or degenerative state of the tissue. In contrast, little is known of the integrin cell surface receptors that directly bind to and interact with these matrix proteins in the IVD. In almost all tissues, these integrin-mediated cell-matrix interactions are important for transducing environmental cues arising from mechanical stimuli, matrix degradation fragments, and cytokines into intracellular signals. In this study, cells from the nucleus pulposus and anulus fibrosus regions of porcine IVDs were analyzed via flow cytometry to quantify integrin expression levels upon isolation and after monolayer culture. Assays of cell attachment to collagens, fibronectin, and laminin were performed after functional blocking of select integrin subunits to evaluate the role of specific integrins in cell attachment. In situ distribution and co-localization of integrins and laminin were also characterized. Results identify integrin receptors critical for IVD cell interactions with collagens (alpha1beta1) and fibronectin (alpha5beta1). Additionally, dramatic differences in cell-laminin interactions were observed between cells of the nucleus and anulus regions, including differences in alpha6 integrin expression, cell adhesion to laminin, and in situ pericellular environments. These findings suggest laminin-cell interactions may be important and unique to the nucleus pulposus region of the IVD. The results of this study provide new information on functional cell-matrix interactions in tissues of the IVD.     

诱导小鼠骨髓间充质干细胞向雄性生殖细胞的定向分化

目的探讨小鼠骨髓间充质干细胞是否能够在体外被诱导发生向雄性生殖细胞方向的分化。方法从雄性小鼠骨髓中分离能够长期贴壁生长的细胞,并鉴定其是否为间充质干细胞。对分离的细胞进行生殖细胞特异性报告基因标记(stra-8-GFP)。采用视黄酸诱导标记的细胞发生向生殖细胞方向的分化。通过观察报告基因表达和生殖细胞相关基因mRNA表达情况确定是否发生了分化。结果从小鼠骨髓中分离到的贴壁生长的细胞表达间充质干细胞的表面标志CD90、CD44、CD105和Sca-1;细胞在体外可以被诱导分化为成骨、成软骨及成脂肪细胞。报告基因标记的间充质干细胞在被视黄酸诱导2d后开始表达绿色荧光蛋白和生殖细胞相关基因Mvh、Fragilis和Stella的mRNA。未经视黄酸诱导的细胞不表达绿色荧光蛋白和生殖细胞相关基因。结论小鼠骨髓间充质干细胞在体外可以被视黄酸诱导发生向雄性生殖细胞方向的分化。