碱性成纤维细胞生长因子诱导非贴壁骨髓基质细胞向成骨细胞分化

目的：探讨碱性成纤维细胞生长因子(bFGF)对非贴壁骨髓前体细胞(NASP)的增殖分化作用。方法：分离骨髓前体细胞体外培养，用碱性磷酸酶化学染色及图象分析测定细胞克隆数；采用免疫组化(ABC法)进行骨钙素及碱性磷酸酶(ALP)测定，研究bFGF对NASP的促增殖作用。结果：bFGF能促进NASP细胞增殖，诱导NASP细胞产生碱性磷酸酶及骨钙素。结论：bFGF主要作用于非贴壁生长的基质前体细胞，bFGF能增加骨的数量，促进骨样组织形成。     

脂肪来源干细胞向成骨分化及褪黑素干预细胞生物学活性的变化

背景：研究表明褪黑素能促进脂肪来源干细胞向神经元转化,但对脂肪来源干细胞成骨分化后细胞的作用报道较少。 目的：观察脂肪来源干细胞向成骨分化及褪黑素对成骨分化后细胞生物学活性的影响。 方法：①取昆明种小鼠附睾处脂肪利用Ⅰ型胶原酶消化法和差速贴壁法获取、纯化脂肪来源干细胞,应用CD44免疫组化染色对细胞进行鉴定。②取第2代脂肪来源干细胞,加入成骨诱导培养基培养,行碱性磷酸酶染色和von Kossa法检测细胞分化情况。③取成骨诱导后的细胞,加入不同浓度的褪黑素,利用MTT法检测24,48 h时细胞增殖情况。④取成骨诱导后的细胞,加入最佳浓度的褪黑素,分别检测加入药物3,6 d时的碱性磷酸酶活性。 结果与结论：①接种48 h后大多数细胞贴壁,接种后4 d细胞呈现多种细胞形态,并形成大小不一的细胞集落,传代后的细胞形态趋于稳定,均称长梭型,培养细胞CD44呈阳性表达,阳性颗粒位于胞膜,呈棕黄色。②成骨诱导后的细胞应用von Kossa法染色呈阳性,黑色沉积物在细胞外的基质中呈网格状分布;碱性磷酸酶染色呈阳性,细胞内可见棕黑色颗粒。③在24 h和48 h时间点,1,10,100 μmol/L组的吸光度值明显大于空白组(P < 0.05),选择这3个浓度作为最佳的褪黑素浓度。④各组成骨诱导后细胞内碱性磷酸酶活性随时间延长明显增加,差异有显著性意义(P < 0.05)。褪黑素组(1,10,100 μmol/L)在3 d时的碱性磷酸酶活性与空白组相比无明显差异(P > 0.05),在6 d时较空白组明显增加(P < 0.05)。提示褪黑素可以增强脂肪来源干细胞成骨分化后细胞增殖,并提高细胞内碱性磷酸酶活性。 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

小分子化合物诱导大鼠脂肪基质干细胞向神经元的分化

目的:探讨小分子化合物诱导大鼠脂肪基质干细胞(ADSCs)向神经元的分化。方法:用Trichostatin A(TSA)、RG-108、8-BrcAMP和Rolipram 4种小分子物质对SD大鼠的ADSCs进行诱导分化,用免疫荧光形态学、免疫印迹和qRT-PCR等方法分别检测诱导前、后ADSCs神经元标志的表达。结果:诱导7 d时,ADSCs呈明显的神经元样结构,胞体呈圆形,具有多个突起,神经元样形态变化率高达95.12%±2.65%;诱导后的ADSCs nestin、β-tubulinⅢ、MAP2和ChAT在mRNA和蛋白水平的表达均有升高。结论:小分子化合物可将ADSCs诱导分化为神经元样细胞,其可作为治疗神经系统疾病及损伤的干细胞移植的种子细胞。     

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.     

生物陶瓷与细胞外基质对成骨细胞生长及代谢影响

本研究采用了体外细胞培养技术,将人胚成骨细胞分别与不同生物陶瓷(HA,TCP,FHA,BGC)及复合生物陶瓷(BMP,TGF-β1)共同培养.通过相差显微镜及扫描电镜观察材料表面及周围细胞形态及附着情况,分子生物学方法测定细胞增殖率,碱性磷酸酶(ALP)、骨钙素(OC)、白细胞介素6(IL-6)及转化生长因子β1(TGF-β1)分泌水平,探讨其作用机理.结果发现复合生物陶瓷细胞增殖率,ALP、OC、IL-6及TGF-β1水平明显高于相应单一材料及空白对照.而不同的复合材料细胞增殖率及细胞基质蛋白分泌有所差异.FHA BMP,TCP BMP及HA BGC TGF-β1显示了较高水平.不同材料培养不同时间对细胞生长及代谢影响有所不同,反映了成骨细胞培养方法评价人工骨替代材料成骨活性规律是切实可行的.     

肌糖蛋白TN-C促进人骨髓基质干细胞向成骨细胞的分化

目的 研究肌糖蛋白(Tenascin-c,TN-C)对人骨髓基质干细胞向成骨细胞分化的诱导作用,为TN-C在骨折愈合中的应用提供理论依据.方法 用全骨髓培养法获取人骨髓基质干细胞(hBMSCs),第6～10代的细胞用于本研究.体外培养的hBMSCs用自行制备的不同浓度重组TN-C (rTN-C)蛋白处理(0、1、10、50和100 nmol/L),于处理后不同时间进行细胞茜素红染色,检测细胞的矿化;或于不同时间收集细胞总RNA进行RT-PCR检测碱性磷酸酶(ALP)和骨钙素(OCN) mRNA水平的变化;同时利用ALP检测试剂盒和骨钙素放射性免疫试剂盒分别检测ALP活性和细胞培养基中OCN含量.结果 1)rTN-C处理hBMSCs 10 d后,rTN-C呈剂量依赖性促进成骨细胞的矿化(P＜0.05);2) rTN-C处理hBMSCs后,细胞ALP活性及培养基上清中OCN含量显著高于未处理对照组(P＜0.05).结论 肌糖蛋白对人BMSC向成骨细胞的分化有一定的促进作用.     

miR-3960对成骨细胞分化的影响**☆

背景：成骨细胞的分化成熟过程涉及多种激素和细胞因子对成骨细胞分化相关基因表达的调控,近来发现多个微小RNAs也参与了这一调控过程。 目的：观察miR-3960在小鼠骨髓基质细胞向成骨细胞分化过程中的作用。 方法：将miR-3960表达载体pSilencer4.1-miR-3960转染骨髓基质细胞,构建miR-3960过表达细胞模型,随后予300 μg/L骨形态发生蛋白2诱导分化,观察成骨细胞分化指标变化。 结果与结论：转染pSilencer4.1-miR-3960能够在细胞中稳定地高表达miR-3960。miR-3960过表达促进骨髓基质细胞向成骨细胞分化过程中的碱性磷酸酶活性增高和骨钙素分泌,增加细胞中的钙沉积量。抑制miR-3960降低骨髓基质细胞向成骨细胞分化过程中的碱性磷酸酶活性,减少骨钙素分泌,降低钙沉积量。表明miR-3960可以促进成骨细胞分化。     

鼠骨髓基质干细胞的特性及向成骨细胞的分化

目的分离纯化成年小鼠骨髓基质干细胞并进行定向诱导。方法利用Percoll液分离成年小鼠骨髓基质干细胞,Ter119、CD45磁珠纯化细胞,培养2-3代的细胞用矿化液进行定向诱导。对诱导前后的细胞进行免疫组化染色。结果 镜下见原代细胞呈多种形态。Ter119、CD45磁珠筛选可获得纯度达57.95％以上骨髓基质干细胞。碱性磷酸酶、Von Kossa染色阳性,油红染色弱阳性,矿化液诱导2周后,可形成钙结节。结论利用Percoll液结合磁珠分离法是离体条件下获得骨髓基质干细胞的一个简便有效的方法,纯化后定向诱导可得到较纯的成骨细胞。     

不同氧浓度对骨髓基质细胞向成骨细胞分化的影响

目的:观察不同低氧环境对在向成骨细胞分化培养系中的骨髓基质细胞核心结合因子α1(Cbfα1/Runx2)、骨形态发生蛋白2(BMP2)和过氧化物酶体增殖物激活受体γ2(PPAR-γ2)表达的影响,探讨低氧环境对成骨细胞分化的影响。方法:取4月龄雌性SD大鼠骨髓基质细胞在生长培养基中培养传代后,随机分成4组,每组样本数为8,加入分化培养基,分别将细胞放入4个不同氧浓度中继续培养,3d后进行下面的实验:用Trizol提取细胞总RNA,用半定量逆转录PCR(RT-PCR)检测(Cbfα1/Runx2)、BMP2和PPARγ2mRNA的表达;用Western blotting检测(Cbfα1/Runx2)、BMP2蛋白的表达。结果:1.与常氧组(20%)相比,各低氧组Runx2mRNA和Runx2蛋白的表达明显增加。2.与常氧组(20%)相比,各低氧组BMP2mRNA的表达明显增加,低氧可促进BMP2蛋白的表达。3.与常氧组(20%)相比,各低氧组PPARγ2mRNA的表达明显降低。结论:低氧能明显促进Runx2mRNA、BMP2mRNA和Runx2蛋白的表达,且氧浓度越低,Runx2mRNA、BMP2mRNA和Runx2、BMP2蛋白表达越多,相反,低氧能明显抑制骨髓基质细胞PPARγ2mRNA的表达,且氧浓度越低,PPARγ2mRNA的表达越低,表明低氧明显抑制骨髓基质细胞向脂肪细胞分化而促进其向成骨细胞分化。     

脂肪基质细胞诱导分化为星形胶质细胞过程中Bax/Bcl-2蛋白与细胞凋亡

背景:神经退行性疾病是一大类能够导致患者工作和生活能力丧失的重大神经系统疑难疾病,目前对于该病尚无有效的治疗方法,脂肪基质细胞能够在体外诱导分化为神经细胞,有望为该类疾病的治疗提供新的方法.目的:探究脂肪基质细胞诱导分化为星形胶质细胞过程中Bax/Bcl-2蛋白的表达变化,进一步研究诱导过程中是否存在线粒体途径介导的细...     

改构型酸性成纤维细胞生长因子对大鼠动脉血栓形成的影响

目的探讨改构型酸性成纤维细胞生长因子(modified acidic fibroblast growth factor,MaFGF)对大鼠颈总动脉血栓形成的影响。方法Wistar大鼠50只,随机分成生理盐水组、肝素组、MaFGF组(又分MaFGF1组、MaFGF组、MaFGF组),每组10只。于大鼠的股静脉分别注射生理盐水(0.5ml/100g)、肝素钠(0.1U/10023g),MaFGF1组(10U/100g)、MaFGF2组(100U/100g)、MaFGF3组(1000U/100g),给药后5min,用保护电极刺激颈总动脉,直至血压曲线逐渐消失,记录刺激开始至压力曲线消失的时间,即血栓堵塞时间。结果肝素组、MaFGF2组、MaFGF3组与生理盐水组比较,平均堵塞时间延长(P<0.05或P<0.01);肝素组与各MaFGF组之间比较,肝素的平均堵塞时间明显较长(P<0.001)。结论MaFGF可以延长大鼠动脉血栓形成的时间,并呈现一定的量效关系。     

血小板源性生长因子对体外培养内皮细胞碱性成纤维细胞生长因子水平的影响

目的 :观察血小板源性生长因子 (PDGF)对于培养脐静脉内皮细胞碱性成纤维细胞生长因子 (bFGF)水平的影响 ,探讨PDGF促进新生血管形成的机制。方法 :通过内皮细胞的培养和免疫组织化学等方法检测PDGF对缺氧和常规培养内皮细胞bFGF水平的影响以及不同浓度PDGF促bFGF表达的作用。结果 :缺氧培养与常规培养均能增加内皮细胞胞浆bFGF水平 ,且呈剂量依赖性。结论 :PDGF可能通过直接促血管形成生长因子bFGF的介导作用间接促进新生血管的形成。     

Human Urine-derived Stem Cells Seeded Surface Modified Composite Scaffold Grafts for Bladder Reconstruction in a Rat Model

We conducted this study to investigate the synergistic effect of human urine-derived stem cells (USCs) and surface modified composite scaffold for bladder reconstruction in a rat model. The composite scaffold (Polycaprolactone/Pluronic F127/3 wt% bladder submucosa matrix) was fabricated using an immersion precipitation method, and heparin was immobilized on the surface via covalent conjugation. Basic fibroblast growth factor (bFGF) was loaded onto the heparin-immobilized scaffold by a simple dipping method. In maximal bladder capacity and compliance analysis at 8 weeks post operation, the USCs-scaffold^heparin-bFGF group showed significant functional improvement (2.34 ± 0.25 mL and 55.09 ± 11.81 µL/cm H₂O) compared to the other groups (2.60 ± 0.23 mL and 56.14 ± 9.00 µL/cm H₂O for the control group, 1.46 ± 0.18 mL and 34.27 ± 4.42 µL/cm H₂O for the partial cystectomy group, 1.76 ± 0.22 mL and 35.62 ± 6.69 µL/cm H₂O for the scaffold group, and 1.92 ± 0.29 mL and 40.74 ± 7.88 µL/cm H₂O for the scaffold^heparin-bFGF group, respectively). In histological and immunohistochemical analysis, the USC-scaffold^heparin-bFGF group showed pronounced, well-differentiated, and organized smooth muscle bundle formation, a multi-layered and pan-cytokeratin-positive urothelium, and high condensation of submucosal area. The USCs seeded scaffold^heparin-bFGF exhibits significantly increased bladder capacity, compliance, regeneration of smooth muscle tissue, multi-layered urothelium, and condensed submucosa layers at the in vivo study.     

骨髓基质细胞体外诱导分化为神经元样细胞的研究

本实验观察了骨髓基质细胞(BMSCs)经化学诱导向神经细胞分化的过程,并初步探索其分化机制。首先分离培养扩增纯化SD大鼠BMSCs,应用β-巯基乙醇(BME)对第4代BMSCs进行诱导分化,分化稳定后撤除诱导液继续常规培养2周。结果显示:诱导分化前,细胞呈现梭形,平行排列生长。诱导分化后,多数BMSCs伸出突起,并发出初级和次级分支,相互交织成网状结构,成典型的神经元样细胞形态,分化过程中有部分细胞漂浮死亡。撤除诱导液常规培养2周后,分化的细胞逐渐缓慢恢复到原来成纤维细胞样长梭状形态。透射电镜观察到分化后细胞具有发育早期神经元的超微结构特点,免疫组织化学方法证实,神经干细胞标志蛋白(Nestin)在诱导分化后即开始表达,1h达到高峰,此时Nestin阳性细胞率为(29.35±1.45)%,之后随时间逐渐下降,5h时降至很低;神经元细胞标志蛋白-神经元特异性烯醇化酶(NSE)在诱导分化前不表达,在诱导分化后随时间表达逐渐增强,5h时阳性细胞率最高为(57.53±2.63)%;撤除诱导液常规培养2周时,Nestin、NSE表达均为阴性。星形胶质细胞标志蛋白-胶质纤维酸性蛋白(Glial fibrillary acidic protein GFAP)测定在不同组、不同时间点均为阴性结果。提示,BMSCs体外经BME化学诱导可向神经元样细胞分化,不能分化为胶质细胞;分化机制可能是先分化为神经干细胞,再转分化为神经元样细胞,这一化学分化过程是迅速、短暂、可逆的,且对细胞有损伤,因此,化学诱导后的BMSCs不适宜做细胞疗法的种子细胞。     

骨髓间充质干细胞在肝部分切除模型小鼠体内向肝细胞分化

以含20%胎牛血清的DM EM低糖培养基,原代细胞直接贴壁培养,48 h后换液继续培养,分离BALB/C小鼠的骨髓间充质干细胞,该细胞分为CD44 CD29 与CD44-CD29 两群。将第5代骨髓间充质干细胞注入部分肝切除小鼠肝右叶,术后5 d和14 d分别处死动物,测体重、肝重与肝功,制备肝组织芯片,原位杂交和免疫荧光同步检测Y染色体与肝细胞标记(白蛋白或CK 18),并作图像叠加。发现移植组小鼠在移植后5 d与14 d在注射肝叶与非注射肝叶内皆可检出大量同时表达Y染色体与白蛋白及Y染色体与CK 18的细胞。术后14 d,移植组肝重与肝脏器指数高于模型组。表明骨髓间充质干细胞能在有再生需求的小鼠体内向肝细胞分化并参与再生。     

Advanced Properties of Urine Derived Stem Cells Compared to Adipose Tissue Derived Stem Cells in Terms of Cell Proliferation,Immune Modulation and Multi Differentiation

Adipose tissue stem cells (ADSCs) would be an attractive autologous cell source. However, ADSCs require invasive procedures, and has potential complications. Recently, urine stem cells (USCs) have been proposed as an alternative stem cell source. In this study, we compared USCs and ADSCs collected from the same patients on stem cell characteristics and capacity to differentiate into various cell lineages to provide a useful guideline for selecting the appropriate type of cell source for use in clinical application. The urine samples were collected via urethral catheterization, and adipose tissue was obtained from subcutaneous fat tissue during elective laparoscopic kidney surgery from the same patient (n = 10). Both cells were plated for primary culture. Cell proliferation, colony formation, cell surface markers, immune modulation, chromosome stability and multi-lineage differentiation were analyzed for each USCs and ADSCs at cell passage 3, 5, and 7. USCs showed high cell proliferation rate, enhanced colony forming ability, strong positive for stem cell markers expression, high efficiency for inhibition of immune cell activation compared to ADSCs at cell passage 3, 5, and 7. In chromosome stability analysis, both cells showed normal karyotype through all passages. In analysis of multi-lineage capability, USCs showed higher myogenic, neurogenic, and endogenic differentiation rate, and lower osteogenic, adipogenic, and chondrogenic differentiation rate compared to ADSCs. Therefore, we expect that USC can be an alternative autologous stem cell source for muscle, neuron and endothelial tissue reconstruction instead of ADSCs.     

大鼠骨髓间充质干细胞向神经细胞和星形胶质细胞分化的研究

目的探讨体外诱导骨髓间充质干细胞(BMSCs)分化成神经细胞或星形胶质细胞。方法使用四种不同的诱导方式来处理BMSCs:-巯基乙醇(BME)、维甲酸(RA)、无血清培养基以及和新生鼠的脑星形胶质细胞共培养。结果四种方法处理后,BMSCs都能表达神经细胞标示物MAP2或星形胶质细胞标志物GFAP,并伴随着明显的细胞形态的改变。用RA诱导以及在无血清的培养基中培养,BMSCs主要分化成神经元样细胞,表达神经元的标志物MAP2,并且BME能促进这个过程。但在和星形胶质细胞共培养后,BMSCs主要分化成GFAP+细胞。结论BMSCs依赖于不同的诱导条件,具有分化成神经细胞或星形胶质细胞的可塑性。     

Upregulation of VEGF and FGF2 in normal rat brain after experimental intraoperative radiation therapy

The expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF)2 in the irradiated brain was examined to test how a single high dose radiation, similar to that used for intraoperative radiation therapy given to the normal cerebrum, can affect the vascular endothelium. After a burr hole trephination in the rat skull, the cerebral hemisphere was exposed to a single 10 Gy dose of gamma rays, and the radiation effect was assessed at 1, 2, 4, 6, and 8 weeks after irradiation. Histological changes, such as reactive gliosis, inflammation, vascular proliferation and necrosis, were correlated with the duration after irradiation. Significant VEGF and FGF2 expression in the 2- and 8-week were detected by enzyme-linked immunosorbent assay quantification in the radiation group. Immunohistochemical study for VEGF was done and the number of positive cells gradually increased over time, compared with the sham operation group. In conclusion, the radiation injuries consisted of radiation necrosis associated with the expression of VEGF and FGF2. These findings indicate that VEGF and FGF2 may play a role in the radiation injuries after intraoperative single high-dose irradiation.     

Effects of hepatocyte growth factor on collagen synthesis and matrix metalloproteinase production in keloids

Keloids are pathologic proliferations of the dermal layer of the skin resulting from excessive collagen production and deposition. Hepatocyte growth factor (HGF) increases the expression of matrix metalloproteinase (MMP)-1 and suppresses collagen synthesis to modulate extracellular matrix turnover. To investigate the anti-fibrotic effects of HGF, we examine the mRNA expression of collagen types I and III and matrix metalloproteinase (MMP-1, MMP-3) on human dermal fibroblast (HDF) cell lines and keloid fibroblasts (KFs, n = 5) after adding various amount of HGF protein. We also evaluated the enzymatic activity of MMP-2, MMP-9 by zymograghy. In HDFs treated with TGF-β1 and HGF protein simultaneously, both type I and III collagen mRNA expression significantly decreased (P < 0.05). Expression of MMP-1, MMP-3 mRNA also decreased. However, the mRNA expression of MMP-1, MMP-3 significantly increased in KFs with increasing amount of HGF in dose dependent manner (P < 0.05). The enzymatic activities of MMP-2 increased with increasing HGF protein in a dose-dependent manner. However, the enzymatic activity of MMP-9 did not change. These results suggest that the anti-fibrotic effects of HGF may have therapeutic effects on keloids by reversing pathologic fibrosis.     

Influence of Low Intensity Laser Irradiation on Isolated Human Adipose Derived Stem Cells Over 72 Hours and Their Differentiation Potential into Smooth Muscle Cells Using Retinoic Acid

Introduction  

Human adipose derived stem cells (hADSCs), with their impressive differentiation potential, may be used in autologous cell therapy or grafting to replace damaged tissues. Low intensity laser irradiation (LILI) has been shown to influence the behaviour of various cells, including stem cells.