Snail基因修饰对骨髓基质干细胞骨架结构稳定作用及促迁移和对无血清培养诱导细胞凋亡的保护作用研究

转录因子Snail是调控肿瘤细胞迁徙转移的重要调控分子,基于干细胞与肿瘤细胞分子机制的重叠性,提出通过借鉴肿瘤细胞迁移的相关机制以用于提高骨髓基质干细胞向缺氧受损组织迁移能力的假设和研究思路,探讨Snail基因在人骨髓基质干细胞(MSCs)中的转染和表达情况,及转染后对基质干细胞促迁移作用、骨架结构的稳定作用及对无血清培养诱导细胞凋亡的保护作用。密度梯度离心法及细胞体外培养分离纯化人骨髓MSCs,脂质体法将重组表达载体pCAGGSneo-Snail-HA转染MSCs,G418筛选稳定表达,流式细胞仪检测MSCs表面抗原,采用免疫荧光染色技术检测转染后MSCs报告基因HA及目的基因Snail表达,Transwell细胞迁移实验和Western-blot评估细胞迁移能力和检测有关细胞信号转导通路分子水平变化,荧光染色分析细胞骨架,Sub-G1凋亡峰流式细胞仪检测细胞凋亡率并评估细胞抗凋亡能力。经流式细胞仪选择检测分离纯化扩增MSCs表面分子特点为CD34(-)/CD29( ),Snail及报告基因在转染后MSCs呈阳性表达,Snail质粒转染MSCs(MSCs-Sna)较对照空质粒转染MSCs(MSCs-neo)细胞迁移率增加(P<0.05),PI-3K信号通路特异性抑制剂Wortmannin能显著抑制此迁移率的增加,无血清培养72h后,MSCs-Sna凋亡率较MSCs-neo低(P<0.05)。经Snail基因转染,MSCs迁移能力、骨架结构的稳定性及在无血清培养环境中抗凋亡能力增加。     

TGF-b1对骨髓间充质干细胞迁移力及 Snail表达的影响

采用密度梯度离心结合贴壁法分离、培养大鼠骨髓间充质干细胞(BMMSCs),用免疫荧光法对培养第3代的细胞进行鉴定.用改良的Transwell小室及MTT法检测不同浓度TGF- b1对细胞迁移力及细胞增殖的影响;流式细胞仪检测细胞凋亡;RT-PCR检测TGF-b1作用不同时间细胞snail、基质金属蛋白酶2(MMP-2)mRNA表达;免疫荧光和Western印迹检测snail表达情况.结果表明密度梯度离心结合贴壁法能有效分离、纯化大鼠BMMSCs,免疫荧光检测显示培养的细胞CD29、CD44表达阳性,而CD34、CD45表达阴性;外源性TGF-b1对BMMSCs迁移力的促进作用具有剂量依赖性,在2 ng/ml时达到最高.高浓度却抑制BMMSCs的迁移.在2 ng/ml TGF-b1刺激下,细胞凋亡明显降低,snail、MMP-2 mRNA及snail表达明显增高,但对细胞增殖无明显影响.通过研究TGF-b1对BMMSCs的迁移力的影响及作用机制,为体外调控BMMSCs高效迁移入脑从而发挥其修复神经损伤作用提供实验依据和理论基础.     

骨髓基质干细胞向心肌细胞诱导分化的实验研究

目的探讨大鼠骨髓基质干细胞在体外和体内向心肌细胞诱导分化的能力,为下一步的细胞移植治疗心肌梗死提供实验基础.方法体外诱导实验中,将不同浓度的5-氮胞苷作用于不同培养时间的骨髓基质干细胞,摸索5-氮胞苷的最佳诱导时机和浓度,观察诱导后细胞形态变化,并用免疫细胞化学染色检测心肌特异性肌钙蛋白T的表达;在体内实验中,培养扩增的骨髓基质干细胞经BrdU标记后,自体移植于正常心肌内,分别通过BrdU和心肌特异性肌钙蛋白T免疫组织化学染色检测移植细胞的存活和分化情况.结果体外诱导实验中,5-氮胞苷的诱导作用以10μmol/L的浓度对传代细胞进行两次诱导,效果最好,不仅能诱导出表达心肌特异蛋白的心肌样细胞,而且这些细胞在体外能够自发搏动.体内诱导实验中,移植的细胞在正常心肌微环境中能够存活并分化为心肌细胞.结论骨髓基质干细胞在体外化学诱导和体内心肌微环境诱导时均能分化为心肌细胞,可用于细胞移植治疗心肌梗死的实验.     

骨髓基质干细胞对MPP+制备的PC12细胞凋亡的影响

目的探讨BMSCs分泌物对MPP^＋诱导的PC12细胞凋亡的保护作用。方法在体外培养、纯化BMSCs并收集其培养上清,流式细胞术分析PC12细胞的凋亡率;通过免疫细胞化学法和RT-PCR法检测PC12细胞bcl-2和TH在蛋白和mRNA水平的表达。结果BMSCs可在体外分离扩增,其表面抗原CD44阳性而CD45阴性,流式细胞术检测显示BMSCs上清液组加MPP^＋处理组（联合组）细胞凋亡率低于MPP^＋损害组;免疫细胞化学法和RT-PCR法显示联合组的bcl-2和TH蛋白和mRNA水平与MPP^＋损害组相比明显增加。结论BMSCs能分泌多种对退变多巴胺能神经元有保护作用的神经营养因子,因此BMSCs对MPP^＋诱导的PC12细胞凋亡有保护作用,其保护作用的强弱与BMSCs上清液浓度有关,其作用机制可能是通过上调bcl-2来实现的。     

Sirt3对过氧化氢诱导的骨髓间充质干细胞凋亡的保护作用

目的:通过调控骨髓间充质干细胞(mesenchymal stem cells,MSCs)中的Sirtuin-3(Sirt3)蛋白的表达水平,阐明Sirt3对过氧化氢(H2O2)诱导MSCs凋亡的保护作用及其机制。方法:将大鼠MSCs分为正常对照组、H2O2刺激组、H2O2+Sirt3转染组、H2O2+Sirt3 si RNA转染组。利用Western blot法检测Sirt3及cyclophilin d蛋白的表达水平、利用免疫沉淀法检测cyclophilin d乙酰化水平、利用流式细胞仪检测细胞凋亡。结果:1H2O2刺激使MSCs中Sirt3表达水平降低。2转染Sirt3可减少H2O2诱导的MSCs的凋亡,而转染Sirt3 si RNA可增加H2O2诱导的MSCs的凋亡。3Sirt3蛋白的含量并不影响cyclophilin d的表达,但影响cyclophilin d的乙酰化水平。结论:Sirt3可能通过减少线粒体通透性转换孔(mitochondrial permeablity transition pore,m PTP)中的关键蛋白cyclophilin d的乙酰化水平,进一步抑制m PTP的开放,从而减少H2O2诱导的MSCs的凋亡。     

大鼠骨髓基质干细胞的分离培养与向神经元样细胞诱导分化的实验研究

目的 探讨大鼠骨髓基质干细胞的提取、分离培养和体外扩增的最佳条件,研究其在体外培养中定向诱导分化为神经元样细胞的可能。方法 通过密度梯度离心和贴壁培养法从成年大鼠骨髓中分离骨髓基质干细胞,进行培养扩增,观察其生长特性;用2-巯基乙醇(β-mercaptoethanol,β-ME)对传代细胞诱导分化,并通过免疫细胞化学染色鉴定分化细胞的类型。结果 原代培养时形成由基质干细胞组成的细胞集落,细胞集落14d时接近融合,传代后,细胞体积变大,约5～7d传代一次。β-ME诱导后,70％以上的细胞在形态上呈神经元样,免疫细胞化学染色呈NSE阳性,GFAP阴性,说明诱导分化的细胞为神经元,而不是星形胶质细胞。结论 骨髓基质干细胞在体外培养条件下生长良好,并可连续传代;在β-ME作用下可被诱导分化为神经元样细胞。     

溶血磷脂酸抗缺氧无血清诱导的大鼠骨髓间充质干细胞凋亡作用

在体外缺氧无血清条件下模拟心肌缺血微环境,研究溶血磷脂酸(LPA)对骨髓间充质干细胞(BMMSCs)的抗凋亡作用.应用Hoechst33342染色和膜联蛋白V/PI双染流式细胞术观察细胞凋亡,并利用Western印迹方法检测ERK1/2和Akt的磷酸化水平变化.结果表明缺氧无血清条件引起明显的细胞凋亡,LPA处理6 h和1 6 h 组细胞凋亡率较缺氧无血清组显著降低(P＜0.05),而1 h组细胞凋亡率与缺氧无血清组无显著性差异.LPA处理组(1 h,6h,1 6 h组)ERK1/2磷酸化水平较缺氧无血清组降低,Akt磷酸化水平较缺氧无血清组增高.在缺氧无血清环境中,LPA有利于增强BMMSCs的抗凋亡能力,提高BMMSCs存活率,为临床提高BMMSCs治疗缺血性心肌病的疗效提供理论依据.     

丙二醛对体外培养下小鼠骨髓间充质干细胞凋亡的诱导作用

为了探索丙二醛对小鼠骨髓间充质干细胞(MSCs)凋亡的诱导作用及其机制,在不同浓度的丙二醛培养体系中孵育MSCs 24 h,用TUNEL法、流式细胞术检测MSC凋亡率,并用实时定量RT-PCR、Western印迹检测Bcl-2、Bax及Caspase-3基因的表达水平。结果发现,MDA能浓度依赖性地增加TUNEL阳性细胞百分率、亚G1峰细胞百分率,同时下调Bcl-2 mRNA及蛋白的表达,上调Bax mRNA和Caspase-3 mRNA及蛋白的表达.这些结果表明:在体外培养条件下,丙二醛可诱导小鼠骨髓间充质干细胞的凋亡,其作用机制与Bcl-2、Bax和Caspase-3基因表达水平的变化有关。     

Human Bone Marrow Mesenchymal Stem Cells Induce Collagen Production and Tongue Cancer Invasion

Tumor microenvironment (TME) is an active player in carcinogenesis and changes in its composition modify cancer growth. Carcinoma-associated fibroblasts, bone marrow-derived multipotent mesenchymal stem cells (BMMSCs), and inflammatory cells can all affect the composition of TME leading to changes in proliferation, invasion and metastasis formation of carcinoma cells. In this study, we confirmed an interaction between BMMSCs and oral tongue squamous cell carcinoma (OTSCC) cells by analyzing the invasion progression and gene expression pattern. In a 3-dimensional myoma organotypic invasion model the presence of BMMSCs inhibited the proliferation but increased the invasion of OTSCC cells. Furthermore, the signals originating from OTSCC cells up-regulated the expression of inflammatory chemokines by BMMSCs, whereas BMMSC products induced the expression of known invasion linked molecules by carcinoma cells. Particularly, after the cell-cell interactions, the chemokine CCL5 was abundantly secreted from BMMSCs and a function blocking antibody against CCL5 inhibited BMMSC enhanced cancer invasion area. However, CCL5 blocking antibody did not inhibit the depth of invasion. Additionally, after exposure to BMMSCs, the expression of type I collagen mRNA in OTSCC cells was markedly up-regulated. Interestingly, also high expression of type I collagen N-terminal propeptide (PINP) in vivo correlated with the cancer-specific mortality of OTSCC patients, whereas there was no association between cancer tissue CCL5 levels and the clinical parameters. In conclusion, our results suggest that the interaction between BMMSC and carcinoma cells induce cytokine and matrix molecule expression, of which high level of type I collagen production correlates with the prognosis of OTSCC patients.     

成纤维生长因子-2与肝细胞生长因子诱导骨髓间充质干细胞分化为肝细胞样细胞的作用比较

比较成纤维生长因子-2(FGF-2)与肝细胞生长因子(HGF)诱导大鼠骨髓间充质干细胞(BM-MSCs)向肝细胞分化的能力,并进一步的研究BM-MSCs诱导成肝细胞所需的最佳诱导因子以及其用量。体外获取、培养大鼠BM-MSCs,将第3代BM-MSCs采用不同剂量的FGF-2和HGF诱导。诱导后,在显微镜下观察细胞形态学的改变;Shiff染色法检测糖原的分泌;ELISA法检测AFP的分泌;免疫细胞化学法检测白蛋白和CK19的分泌。诱导后BM-MSCs由梭形向多角形、卵圆形方向变化,AFP第3天就有分泌,第12天达到高峰,以后逐渐减少;白蛋白、CK19和糖原第12天即有阳性表达,以后随着诱导时间的延长阳性率逐渐升高。第2组和第4组比其他组分泌的白蛋白、CK19和糖原均多。FGF-2比HGF具有更强的诱导BM-MSCs向肝细胞分化的能力。     

Circadian Clock Genes Modulate Human Bone Marrow Mesenchymal Stem Cell Differentiation,Migration and Cell Cycle

Many of the components that regulate the circadian clock have been identified in organisms and humans. The influence of circadian rhythm (CR) on the regulation of stem cells biology began to be evaluated. However, little is known on the role of CR on human mesenchymal stem cell (hMSCs) properties. The objective of this study was to investigate the influence of CR on the differentiation capacities of bone marrow hMSCs, as well as the regulation of cell cycle and migration capabilities. To that, we used both a chemical approach with a GSK-3β specific inhibitor (2’E,3’Z-6-bromoindirubin-3’-oxime, BIO) and a knockdown of CLOCK and PER2, two of the main genes involved in CR regulation. In these experimental conditions, a dramatic inhibition of adipocyte differentiation was observed, while osteoblastic differentiation capacities were not modified. In addition, cell migration was decreased in PER2^-/- cells. Lastly, downregulation of circadian clock genes induced a modification of the hMSCs cell cycle phase distribution, which was shown to be related to a change of the cyclin expression profile. Taken together, these data showed that CR plays a role in the regulation of hMSCs differentiation and division, and likely represent key factor in maintaining hMSCs properties.     

人骨髓间充质干细胞培养方法

骨髓间充质干细胞(BMMSCs)是一种多潜能的成体干细胞,在细胞治疗和组织工程上具有广阔的应用前景。对供体年龄、分离方法、培养密度、培养基和培养基质表面性质对细胞增殖的影响进行了比较,重点阐述了用人自体血清结合多种细胞因子,替代胎牛血清培养BMMSCs的效果,转染端粒酶基因的BMMSCs的增殖能力和分化潜能,以及灌注培养反应器用于大规模培养的技术进展。     

Osteoblasts and Bone Marrow Mesenchymal Stromal Cells Control Hematopoietic Stem Cell Migration and Proliferation in 3D In Vitro Model

Background

Migration, proliferation, and differentiation of hematopoietic stem cells (HSCs) are dependent upon a complex three-dimensional (3D) bone marrow microenvironment. Although osteoblasts control the HSC pool, the subendosteal niche is complex and its cellular composition and the role of each cell population in HSC fate have not been established. In vivo models are complex and involve subtle species-specific differences, while bidimensional cultures do not reflect the 3D tissue organization. The aim of this study was to investigate in vitro the role of human bone marrow–derived mesenchymal stromal cells (BMSC) and active osteoblasts in control of migration, lodgment, and proliferation of HSCs.

Methodology/Principal Findings

A complex mixed multicellular spheroid in vitro model was developed with human BMSC, undifferentiated or induced for one week into osteoblasts. A clear limit between the two stromal cells was established, and deposition of extracellular matrix proteins fibronectin, collagens I and IV, laminin, and osteopontin was similar to the observed in vivo. Noninduced BMSC cultured as spheroid expressed higher levels of mRNA for the chemokine CXCL12, and the growth factors Wnt5a and Kit ligand. Cord blood and bone marrow CD34⁺ cells moved in and out the spheroids, and some lodged at the interface of the two stromal cells. Myeloid colony-forming cells were maintained after seven days of coculture with mixed spheroids, and the frequency of cycling CD34⁺ cells was decreased.

Conclusions/Significance

Undifferentiated and one-week osteo-induced BMSC self-assembled in a 3D spheroid and formed a microenvironment that is informative for hematopoietic progenitor cells, allowing their lodgment and controlling their proliferation.     

小鼠骨髓间充质干细胞的培养、鉴定和Survivin的表达研究

为培养及鉴定小鼠来源骨髓间充质干细胞,并测定细胞中Survivin的表达情况,采用全骨髓培养法获取骨髓间充质干细胞,绘制生长曲线,流式细胞仪检测细胞表面标志物,行成骨、成脂检测,RT-PCR测定Survivin表达情况.结果表明培养出的细胞呈长梭状成纤维细胞样,经流式细胞仪检测细胞表面高表达CD29、CD34、CD44、SCA-1,低表达CD117;细胞曲线显示传代细胞培养1～3d生长缓慢,第4d生长加快并于第7d达到高峰;成骨诱导20d经茜素红染色呈红色结节,成脂诱导14d油红O染色显示有大量脂质沉淀;RT-PCR结果显示Survivin mRNA阳性表达.经全骨髓培养法可以培养出大量骨髓间充质干细胞,同时Survivin在小鼠骨髓间充质干细胞中正常表达,提示可能参与骨髓间充质干细胞抗凋亡过程.     

Stimulating Factors and Cell Recruitment In Murine Bone Marrow Stem Cells and Emt6 Tumours

The role of a stimulating factor in cell recruitment and the kinetics of its secretion were investigated by in vivo and in vitro techniques. the association of these two methods made it possible to demonstrate that a non-cycling population liberates a factor which in turn stimulates quiescent bone marrow stem cells into DNA synthesis. Moreover, it seems that undamaged cells are capable of secreting this factor. A stimulating factor responsible for cell recruitment was also demonstrated in an experimental EMT6 tumour and the kinetics of its secretion reported.