人脐带间充质干细胞条件培养基联合白藜芦醇对人滋养层细胞的作用

目的:探讨人脐带间充质干细胞条件培养基联合白藜芦醇对人绒毛膜外滋养层细胞凋亡的影响。方法:通过CCK8细胞活力检测试剂盒测定白藜芦醇及其与人脐带间充质干细胞条件培养基共同处理人绒毛膜外滋养层细胞HTR8后对细胞增殖及活性的影响;细胞迁移试验检测白藜芦醇和人脐带间充质干细胞条件培养基对细胞迁移能力的影响;显微镜观察细胞形态,并用流式细胞仪检测细胞凋亡率的变化;Western blot检测白藜芦醇和人脐带间充质干细胞条件培养基对细胞凋亡相关蛋白Bax、Bcl-2以及迁移相关蛋白MMP-9表达的影响。结果:白藜芦醇能够抑制HTR8细胞增殖,抑制细胞迁移及MMP-9蛋白的表达,改变Bax和Bcl-2蛋白表达诱导细胞凋亡的作用。而人脐带间充质干细胞条件培养基能够逆转白藜芦醇对细胞的抑制作用。结论:人脐带间充质干细胞条件培养基能够通过调控Bax、Bcl-2、MMP-9的蛋白表达逆转白藜芦醇对人绒毛膜外滋养层细胞的抑制作用。人脐带间充质干细胞条件培养基可作为潜在的治疗人绒毛膜外滋养层细胞功能障碍的临床手段,孕妇需要小心使用白藜芦醇。     

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.     

基质衍生因子(SDF-1)对骨髓间充质干细胞定向迁移的影响

目的:研究基质细胞衍生因子-1(SDF-1)/CXCR4轴在骨髓间充质干细胞迁徙到受损胰腺中的作用。方法:密度梯度离心、贴壁培养骨髓间充质干细胞,建立STZ诱导糖尿病模型并制备正常和受损胰腺组织提取液,利用Transwell小室体外迁移体系观察不同浓度SDF-1和不同组织提取液对骨髓间充质干细胞的趋化作用,及SDF-1/CXCR4特异抑制剂AMD3100对骨髓间充质干细胞迁移的影响。结果:成功培养了骨髓间充质干细胞并建立了糖尿病大鼠模型。SDF-l对骨髓间充质干细胞有剂量依赖性的趋化作用,造模1周的胰腺组织提取液对骨髓间充质干细胞有明显的趋化作用,而这种作用可部分被SDF-1受体CXCR4的抑制剂AMD3100抑制。结论:受损胰腺组织提取液对骨髓间充质干细胞有明显的趋化作用,SDF-1/CXCR4轴可能在组织提取液趋化骨髓间充质干细胞迁移中起主要的作用。     

黄酒中抑制同型半胱氨酸诱导的大鼠血管平滑肌细胞增殖和迁移成分探讨

目的:探索黄酒中具有抑制同型半胱氨酸(Hcy)诱导的大鼠血管平滑肌细胞(VSMCs)增殖和迁移作用的活性成分。方法:SD大鼠主动脉VSMCs原代细胞培养鉴定,取4~7代细胞用于实验。VSMCs分为control组、Hcy(浓度为1 mmol/L)组、低聚糖组(Hcy+黄酒低聚糖)、多肽组(Hcy+黄酒多肽)、多酚组(Hcy+黄酒多酚)、酒精组(Hcy+酒精)、黄酒组(Hcy+黄酒)共7组。MTT法检测各组VSMCs的增殖情况;划痕法和Transwell法检测各组VSMCs的迁移情况;Western blot法检测各组VSMCs中基质金属蛋白酶-2/9(MMP-2/9)、基质金属蛋白酶的组织抑制剂-2(TIMP-2)的表达情况;明胶酶谱检测各组VSMCs中MMP-2/9的活性。结果:与control组相比,Hcy组VSMCs增殖迁移增加、MMP-2/9表达和活性增加(P0.01)。与Hcy组相比,多肽组、多酚组、黄酒组VSMCs增殖迁移减少、MMP-2/9表达和活性减少(P0.05)。各组之间TIMP-2的表达没有显著差异。结论:黄酒中的多肽类和多酚类成分具有抑制Hcy诱导的VSMCs增殖和迁移的作用。     

TNF-α刺激大鼠骨髓间充质干细胞的机制研究

目的:研究肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)刺激大鼠骨髓间充质干细胞(marrow-derived mesenchymalstem cells,MSCs)的作用机制。方法:采取大鼠骨髓,以密度梯度离心分离出单个核细胞(MNCs),于体外培养并由牛垂体提取物(PEX)诱导扩增传代培养出骨髓间充质干细胞(MSCs)。经形态学和流式细胞仪检测MSCs表面标志物鉴定后,用TNF-α刺激骨髓间充质干细胞(MSCs),通过酶联免疫吸附剂测定法(enzyme linked immunosorbent assay,ELISA)观察比较不同组别细胞的生长因子分泌和蛋白印迹法(western blot)来观察细胞中蛋白的变化。结果:①经形态学观察和流式细胞仪检测MSCs表面标志物鉴定,提示骨髓间充质干细胞的培养成功。②无TNF-α刺激组与TNF-α刺激组比较,TNF-α刺激组的生长因子分泌显著性增加,而通过磷酸化IκB的表达量显著性增加提示NF-κB被激活(P<0.05);同时TNF-α刺激组与TNF-α+NF-κB抑制剂组比较,TNF-α+NF-κB抑制剂组的生长因子分泌显著降低,而通过磷酸化IκB的表达量显著减少提示NF-κB的活性被抑制(P<0.05)。结论:NF-κB对TNF-α刺激下的骨髓间充质干细胞分泌生长因子有关键性作用。     

诱导体外培养的骨髓间充质干细胞向心肌样细胞的分化

选用Wistar大鼠分离骨髓间充质干细胞作体外培养及鉴定其表达抗原CD44、CDw90;采用10μmol／L 5-氮胞苷诱导第1代的骨髓间充质干细胞,于诱导后2、4周进行免疫细胞化学反应检测α-横纹肌肌动蛋白、肌钙蛋白T。证实体外培养的第1代骨髓间充质干细胞经5-氮胞苷诱导可分化为心肌样细胞,为指导体外诱导的心肌细胞应用于。临床提供一定的理论依据和技术手段。     

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.     

TNF-α刺激大鼠骨髓间充质干细胞的机制研究

目的：研究肿瘤坏死因子-α（Tumor necrosis factor-α,TNF-α）刺激大鼠骨髓间充质干细胞（marrow-derived mesenchymalstem cells,MSCs）的作用机制。方法：采取大鼠骨髓,以密度梯度离心分离出单个核细胞（MNCs）,于体外培养并由牛垂体提取物（PEX）诱导扩增传代培养出骨髓间充质干细胞（MSCs）。经形态学和流式细胞仪检测MSCs表面标志物鉴定后,用TNF-α刺激骨髓间充质干细胞（MSCs）,通过酶联免疫吸附剂测定法（enzyme linked immunosorbent assay,ELISA）观察比较不同组别细胞的生长因子分泌和蛋白印迹法（western blot）来观察细胞中蛋白的变化。结果：①经形态学观察和流式细胞仪检测MSCs表面标志物鉴定,提示骨髓间充质干细胞的培养成功。②无TNF-α刺激组与TNF-α刺激组比较,TNF-α刺激组的生长因子分泌显著性增加,而通过磷酸化IκB的表达量显著性增加提示NF-κB被激活（P〈0.05）;同时TNF-α刺激组与TNF-α＋NF-κB抑制剂组比较,TNF-α＋NF-κB抑制剂组的生长因子分泌显著降低,而通过磷酸化IκB的表达量显著减少提示NF-κB的活性被抑制（P〈0.05）。结论：NF-κB对TNF-α刺激下的骨髓间充质干细胞分泌生长因子有关键性作用。     

Effect of the cyclic stretch on the expression of osteogenesis genes in human periodontal ligament cells

Periodontal ligament cells can potentially differentiate into osteoblast-like cells and influence the remodeling of periodontal tissues under mechanical strain conditions. In the present study, Gene chip technology was adopted to investigate the effect of the cyclic stretch on the expression of osteogenic-related genes in human periodontal ligament cells (HPDLCs). Cultured HPDLCs were subjected to 12% elongation cyclic stretch for 24 h using a Flexercell Strain Unit, and then GEArray Q series human osteogenesis gene expression profile chip with 96 spot array numbers was used to conduct parallel analyses on the change of the related gene expression in the osteogenic differentiation of HPDLCs stimulated by cyclic stretch. The results show that after the HPDLCs were stimulated by the cyclic stretch, the expression of 21 osteogenic-related genes was significantly upregulated, including 10 growth factor genes and their associated molecules, 10 extracellular matrix genes and their associated proteins, and 1 cell adhesion molecule. Two genes were significantly downregulated, including one growth factor gene and one cell adhesion molecule. Then the expressions of 10 candidate genes were validated using Real-time RT-PCR. These results indicate that cyclic stretch with 12% deformation can stimulate or inhibit some gene expression which was associated with the process of HPDLCs differentiation.     

Effects and mechanisms of substance P on the proliferation and angiogenic differentiation of bone marrow mesenchymal stem cells: Bioinformatics and in vitro experiments

The slight release of substance P (SP) from the end of peripheral nerve fibers causes a neurogenic inflammatory reaction, promotes vascular dilation and increases vascular permeability. However, whether SP can promote the angiogenesis of bone marrow mesenchymal stem cells (BMSCs) under high glucose conditions has not been reported. This study analyzed the targets, biological processes and molecular mechanisms underlying the effects of SP on BMSCs. BMSCs cultured in vitro were divided into a normal control group, high glucose control group, high glucose SP group and high glucose Akt inhibitor group to verify the effects of SP on BMSCs proliferation, migration and angiogenic differentiation. SP was found to act on 28 targets of BMSCs and participate in angiogenesis. Thirty-six core proteins, including AKT1, APP, BRCA1, CREBBP and EGFR, were identified. In a high glucose environment, SP increased the BMSCs proliferation optical density value and cell migration number and reduced the BMSCs apoptosis rate. In addition, SP induced BMSCs to highly express the CD31 protein, maintain the wall structure integrity of the matrix glue mesh and promote increases in the number of matrix glue meshes. These experiments showed that in a high glucose environment, SP acts on 28 targets of BMSCs that encode core proteins, such as AKT1, APP and BRCA1, and improves BMSCs proliferation, migration and angiogenic differentiation through the Akt signaling pathway.     

周期加载对VEC-304细胞株迁移、增殖分布的影响

在弹性膜上接种VEC－304,融合生长后擦去周边细胞形成一规则圆斑,使用弹性膜周期加载装置,对生长在弹性膜上的VEC－304圆斑施以15％拉伸应变,经显微镜观察,计算机图像处理了解细胞增殖、迁移时间过程及增殖细胞在应变场中的分布,细胞银染了解细胞形态及细胞连接情况,免疫荧光染色观察增殖细胞的分裂极。结果发现：（1）随加载时间延长圆斑沿与应变垂直向伸展成椭圆形,24h两径出现明显差异;（2）随时间增加圆斑面积扩大,但实验组沿与加载垂直方向的增大较沿加载方向为大;（3）细胞有丝分裂器（中心粒）位于细胞短轴即加载方向;（4）加载后细胞较对照组排列紧密,细胞间距更小,且出现较为明显的重叠生长现象。VEC－304在应变场中的运动迁移、增殖具有方向依赖性,周期加载可以促进细胞间连接。     

Cyclic stretch attenuates effects of hyperoxia on cell proliferation and viability in human alveolar epithelial cells

The treatment of severe lung disease often requires the use of high concentrations of oxygen coupled with the need for assisted ventilation, potentially exposing the pulmonary epithelium to both reactive oxygen species and nonphysiological cyclic stretch. Whereas prolonged hyperoxia is known to cause increased cell injury, cyclic stretch may result in either cell proliferation or injury depending on the pattern and degree of exposure to mechanical deformation. How hyperoxia and cyclic stretch interact to affect the pulmonary epithelium in vitro has not been previously investigated. This study was performed using human alveolar epithelial A549 cells to explore the combined effects of cyclic stretch and hyperoxia on cell proliferation and viability. Under room air conditions, cyclic stretch did not alter cell viability at any time point and increased cell number after 48 h compared with unstretched controls. After exposure to prolonged hyperoxia, cell number and [(3)H]thymidine incorporation markedly decreased, whereas evidence of oxidative stress and nonapoptotic cell death increased. The combination of cyclic stretch with hyperoxia significantly mitigated the negative effects of prolonged hyperoxia alone on measures of cell proliferation and viability. In addition, cyclic stretch resulted in decreased levels of oxidative stress over time in hyperoxia-exposed cells. Our results suggest that cyclic stretch, as applied in this study, can minimize the detrimental effects of hyperoxia on alveolar epithelial A549 cells.     

Uniaxial strain upregulates matrix-degrading enzymes produced by human vascular smooth muscle cells

Arteries remodel in response to environmental changes. We investigated whether mechanical strain modulates production of matrix metalloproteinase (MMP)-2 and -9 by cultured vascular smooth muscle cells (SMC). MMP-2 and MMP-9 expression were tested using human saphenous vein SMC cultured on silicone membranes at rest or subjected to physiological levels (5%) of stationary or cyclical (1 Hz) uniaxial strain. Compared with control, stationary strain significantly increased MMP-2 mRNA levels at all time points, whereas cyclic strain decreased it after 48 h. Both secreted and cell-associated pro-MMP-2 levels were increased by stationary strain at all times (P < 0.01), whereas cyclic strain decreased secreted levels after 48 h (P < 0.02). MMP-9 mRNA levels and pro-MMP-9 protein were increased after 48 h of stationary stretch (P < 0.01) compared with both no strain and cyclic strain. Our study indicates that vascular SMC show a selective response to different types of strain. We suggest that local increases in stationary mechanical strain resulting from stenting, hypertension, or atherosclerosis may lead to enhanced matrix degradation by SMC.     

Balance of life and death in alveolar epithelial type II cells: proliferation, apoptosis, and the effects of cyclic stretch on wound healing

After acute lung injury, repair of the alveolar epithelium occurs on a substrate undergoing cyclic mechanical deformation. While previous studies showed that mechanical stretch increased alveolar epithelial cell necrosis and apoptosis, the impact of cell death during repair was not determined. We examined epithelial repair during cyclic stretch (CS) in a scratch-wound model of primary rat alveolar type II (ATII) cells and found that CS altered the balance between proliferation and cell death. We measured cell migration, size, and density; intercellular gap formation; cell number, proliferation, and apoptosis; cytoskeletal organization; and focal adhesions in response to scratch wounding followed by CS for up to 24 h. Under static conditions, wounds were closed by 24 h, but repair was inhibited by CS. Wounding stimulated cell motility and proliferation, actin and vinculin redistribution, and focal adhesion formation at the wound edge, while CS impeded cell spreading, initiated apoptosis, stimulated cytoskeletal reorganization, and attenuated focal adhesion formation. CS also caused significant intercellular gap formation compared with static cells. Our results suggest that CS alters several mechanisms of epithelial repair and that an imbalance occurs between cell death and proliferation that must be overcome to restore the epithelial barrier.     

Physiological cyclic stretch directs L-arginine transport and metabolism to collagen synthesis in vascular smooth muscle.

Application of cyclic stretch (10% at 1 hertz) to vascular smooth muscle cells (SMC) increased L-arginine uptake and this was associated with a specific increase in cationic amino acid transporter-2 (CAT-2) mRNA. In addition, cyclic stretch stimulated L-arginine metabolism by inducing arginase I mRNA and arginase activity. In contrast, cyclic stretch inhibited the catabolism of L-arginine to nitric oxide (NO) by blocking inducible NO synthase expression. Exposure of SMC to cyclic stretch markedly increased the capacity of SMC to generate L-proline from L-arginine while inhibiting the formation of polyamines. The stretch-mediated increase in L-proline production was reversed by methyl-L-arginine, a competitive inhibitor of L-arginine transport, by hydroxy-L-arginine, an arginase inhibitor, or by the ornithine aminotransferase inhibitor L-canaline. Finally, cyclic stretch stimulated collagen synthesis and the accumulation of type I collagen, which was inhibited by L-canaline. These results demonstrate that cyclic stretch coordinately stimulates L-proline synthesis by regulating the genes that modulate the transport and metabolism of L-arginine. In addition, they show that stretch-stimulated collagen production is dependent on L-proline formation. The ability of hemodynamic forces to up-regulate L-arginine transport and direct its metabolism to L-proline may play an important role in stabilizing vascular lesions by promoting SMC collagen synthesis.     

Physiological cyclic stretch causes cell cycle arrest in cultured vascular smooth muscle cells

Smooth muscle cells (SMC) are the major cellular component of the blood vessel wall and are continuously exposed to cyclic stretch due to pulsatile blood flow. This study examined the effects of a physiologically relevant level of cyclic stretch on rat aortic vascular SMC proliferation. Treatment of static SMC with serum, platelet-derived growth factor, or thrombin stimulated SMC proliferation, whereas exposure of SMC to cyclic stretch blocked the proliferative effect of these growth factors. The stretch-mediated inhibition in SMC growth was not due to cell detachment or increased cell death. Flow cytometry analysis revealed that cyclic stretch increased the fraction of SMC in the G(0)/G(1) phase of the cell cycle. Stretch-inhibited G(1)/S phase transition was associated with a decrease in retinoblastoma protein phosphorylation and with a selective increase in the cyclin-dependent kinase inhibitor p21, but not p27. These results demonstrate that cyclic stretch inhibits SMC growth by blocking cell cycle progression and suggest that physiological levels of cyclic stretch contribute to vascular homeostasis by inhibiting the proliferative pathway of SMC.     

Matrix metalloproteinase 2 (gelatinase A) is related to migration of keratinocytes.

The role of matrix metalloproteinases (MMPs) in cell migration was studied by measuring cell growth, migration, and production of MMP-2 and -9 in oral mucosal and skin keratinocytes cultured in the presence of synthetic MMP inhibitors. MMP-2 was the major gelatinolytic MMP produced by these cells while MMP-9 was produced at a low basal level. Inhibitor effects on MMP-9 production were therefore studied in keratinocytes stimulated by tumor necrosis factor alpha (TNFalpha). Tetracycline analogues at concentrations that inhibited the production of MMP-2 but not MMP-9 were able to drastically inhibit migration of both mucosal and skin keratinocytes. Tetracycline analogues also inhibited keratinocyte growth, an effect not found for the other inhibitors tested. Heterocyclic carbonate-derived compounds (LWs) that inhibited MMP-9 but not MMP-2 production had no effect on cell migration. Batimastat, a potent MMP inhibitor, did not have any effect on MMP production or cell growth but did inhibit keratinocyte migration. Tumor growth factor beta (TGFbeta) increased keratinocyte migration as well as both cell-associated and secreted MMP-2 production in wounded cell cultures. The secreted enzyme was partially converted into an active form. In this model batimastat totally blocked TGFbeta-promoted keratinocyte migration. Immunostaining of keratinocytes advancing into the wound revealed that MMP-2 was localized in extracellular matrix contactlike structures against the endogenously produced laminin-5-rich matrix. MMP-9 was localized diffusely along the cell membranes. Using in situ hybridization we observed that in chronically inflamed human gingiva MMP-2 is expressed in epithelium extending into subepithelial connective tissue. These results suggest that MMP-2 plays a specific role in epithelial migration, possibly by detaching the advancing cells from the pericellular matrix or by activating other MMPs.     

Mechanical stretch induces MMP-2 release and activation in lung endothelium: role of EMMPRIN

High-volume mechanical ventilation leads to ventilator-induced lung injury. This type of lung injury is accompanied by an increased release and activation of matrix metalloproteinases (MMPs). To investigate the mechanism leading to the increased MMP release, we systematically studied the effect of mechanical stretch on human microvascular endothelial cells isolated from the lung. We exposed cells grown on collagen 1 BioFlex plates to sinusoidal cyclic stretch at 0.5 Hz using the Flexercell system with 17-18% elongation of cells. After 4 days of cell stretching, conditioned media and cell lysate were collected and analyzed by gelatin, casein, and reverse zymograms as well as Western blotting. RT-PCR of mRNA extracted from stretched cells was performed. Our results show that 1) cyclic stretch led to increased release and activation of MMP-2 and MMP-1; 2) the activation of MMP-2 was accompanied by an increase in membrane type-1 MMP (MT1-MMP) and inhibited by a hydroxamic acid-derived inhibitor of MMPs (Prinomastat, AG3340); and 3) the MMP-2 release and activation were preceded by an increase in production of extracellular MMP inducer (EMMPRIN). These results suggest that cyclic mechanical stretch leads to MMP-2 activation through an MT1-MMP mechanism. EMMPRIN may play an important role in the release and activation of MMPs during lung injury.