菲立磁胞内标记食蟹猴骨髓基质细胞(英文)

背景:有关利用菲立磁体外细胞标记研究选择的动物主要是啮齿类动物,而研究灵长类动物食蟹猴的报道目前仍是空白。目的:观察利用菲立磁和转染试剂体外磁性标记食蟹猴骨髓基质细胞方法的可行性。方法:无菌条件下取食蟹猴骨髓,梯度密度离心法分离获取骨髓基质细胞,使用菲立磁-多聚赖氨酸复合物标记骨髓基质细胞,普鲁士蓝染色、电镜和锥虫蓝排除实验等方法鉴定菲立磁-多聚赖氨酸复合物标记食蟹猴骨髓基质细胞的效率和细胞的活力,倒置相差显微镜和免疫细胞化学方法检测骨髓基质细胞的增殖和分化能力。结果与结论:菲立磁可以高效率地标记骨髓基质细胞,标记效率在99%左右。光镜和电镜下骨髓基质细胞胞质内分别可见细小的蓝色铁颗粒和许多包裹铁颗粒的囊泡。菲立磁-多聚赖氨酸复合物标记对骨髓基质细胞的活力、增殖和分化等能力没有明显影响。提示菲立磁可以用来体外标记食蟹猴骨髓基质细胞。     

绿色荧光蛋白、菲立磁体外双标记神经干细胞及其活性的研究

目的探索用绿色荧光蛋白(green fluorescent protein,GFP)重组逆转录病毒和菲立磁(超顺磁性氧化铁,,Superparamagnetic Iron Oxide,SPIO)体外双标记神经干细胞的可行性及其对细胞活力的影响。方法用病毒介导的GFP基因转导大鼠胚胎神经干细胞。用SPIO和Lipofectamine体外磁化标记经GFP标记的胚胎神经干细胞。用荧光显微镜观察双标记神经干细胞,并行普鲁士蓝染色,了解转染成功率。MTT(四甲基偶氮唑蓝)法检测标记神经干细胞的细胞增殖活力。结果双标记神经干细胞分化的细胞荧光显微镜下观察见发出绿色荧光,普鲁士蓝染色呈阳性。而未标记神经干细胞荧光显微镜下观察未见发出绿色荧光,普鲁士蓝染色呈阴性。MTT法检测发现GFP和SPIO双标记神经干细胞的增殖活力与未标记神经干细胞相比无改变。结论绿色荧光蛋白重组逆转录病毒和菲立磁可以有效地标记体外分离培养的大鼠胚胎神经干细胞。双标记神经干细胞的增殖、分化活力与未标记神经干细胞相比无改变。     

超顺磁性氧化铁微粒磁标记骨髓间充质干细胞效率实验研究

目的探讨不同浓度超微超顺磁性氧化铁微粒（USPIO）与多聚左旋赖氨酸（PLL）标记大鼠骨髓问充质十细胞（BMSCs）的磁标记效率及对细胞生长活力的影响,寻找最佳配比浓度。方法实验采用6周龄Wister近交系大鼠,150g芹右．雄性。用贴壁法分离培养BMSCs。使用6nmUSPIO—PLL复合物标记BMSCs。实验分6组,对照组为未标记的BMSCs（A组）。按照PLL的有无及PLL质量浓度梯度（0、0．25、0．50、0．75、1．00μg／mL）分为5个实验组（B～F组）;其中每个实验组再根据不同浓度铁离子与PLL结合（USPIO的终质量浓度分别为25、50、100、150μg/mL）。使用电子显微镜及光学显微镜观察标记后的BMSCs及BMSCs内的USPIO微粒。BMSCs活力测定采用台盼蓝排除实验。BMSCs生长曲线绘制采用MTT法。MRI观察体外标记后BMSCs的显影。火焰法测量BMSCs内铁含量,验证铁含量与MRI信号的关系。统计学分析采用方差分析。,结果台盼蓝染色证实90％以上标记后BMSCs均拒染台盼蓝。根据BMSCs生长曲线判断单纯使用铁离子质量浓度达到200μg／mL时或PLL用量达1．00μg／mL会对BMSCs的生长产生一定的抑制作用。火焰法测得单独USPIO标记BMSCs与USPIO—PLL标记BMSCs铁含量差异有统计学意义（P〈0．05）。T2WI及SWI序列图像从B组至F组信号强度逐级下降,反映出铁离子的变化情况。结论使用USPIO（100μg／mL）结合PLL（0．75μg／mL）标记BMSCs即对细胞活性和生长无不利影响．且标记BMSCs的信号强度较强。     

超顺磁性氧化铁标记对骨髓间充质干细胞多向分化诱导的影响

研究兔骨髓间充质干细胞(BMSCs)经超顺磁性氧化铁(SPIO)标记后,体外成骨、成脂及成软骨诱导能力的变化。体外贴壁培养和扩增兔BMSCs,采用SPIO(25μg/ml)联合硫酸鱼精蛋白转染剂标记兔BMSCs,分别采用适宜的成骨、成脂及成软骨诱导培养液对磁标记BMSCs进行体外定向诱导培养3周,诱导过程中观察细胞形态学变化。3周后对成骨定向诱导组进行钙结节茜素红染色和碱性磷酸酶(ALP)组化染色;对成脂肪定向诱导组行油红-O染色;对成软骨定向诱导组行番红O染色、II型胶原免疫组化染色检测观察胞外基质糖胺多糖、II型胶原的分泌和表达。利用Image-Pro Plus图像分析系统对免疫细胞化学染色进行光密度半定量分析。结果表明:超顺磁性氧化铁粒子标记BMSCs后普鲁士染色和电镜检查显示细胞胞浆内含致密铁颗粒;磁标记BMSCs在成骨、成脂及成软骨潜在多向分化诱导能力上与对照组未标记细胞相比无统计学差异。提示,SPIO联合硫酸鱼精蛋白转染剂能成功标记BMSCs,超顺磁性氧化铁标记对兔BMSCs体外多向分化诱导能力无明显影响,合理应用这种新型细胞标记技术将促进对组织工程种子细胞的研究。     

超顺磁性氧化铁标记骨髓间充质干细胞的实验研究

目的探讨超顺磁性氧化铁纳米颗粒（SPIO）标记青山羊骨髓间充质干细胞的方法,并确定标记颗粒在细胞内的具体位置,评价标记效果。方法抽取骨髓离心,分离间充质干细胞制成单细胞悬液传代培养及扩增,用SPIO标记第三代骨髓间充质干细胞,显微镜下观察发现细胞内胞吞入大量SPIO颗粒后消化培养细胞,离心成团,于2.5%戊二醛固定,透射电镜常规制样,镜下观察并采集图像。结果电镜下骨髓间充质干细胞的超微结构保存良好,胞质内出现大量电子密度高的SPIO颗粒,呈小团状或点状分布,主要位于溶酶体内及滑面内质网、线粒体及胞浆中的膜性结构上,细胞膜表面和核膜上也可见少量的散在颗粒。结论透射电镜下显示用SPIO标记骨髓间充质干细胞阳性率高,超微结构保存良好,准确定位了SPIO在胞内的位置,此法可为干细胞移植的示踪提供依据。     

骨髓基质细胞对脑神经干细胞分化为神经元的诱导作用

目的：观察成年大鼠骨髓基质细胞诱导的新生大鼠中脑神经干细胞分化为神经元的机制。方法：采用骨髓基质细胞和神经干细胞共培养方法，通过显微镜观察神经干细胞的分化状态，使用免疫组织化学技术，分析神经元在神经细胞后代中所占的比例。结果：（1）骨髓基质细胞可诱导神经干细胞分化为高比例神经元；（2）骨髓基质细胞可促进神经元的存活。结论：骨髓基质细胞可提供神经干细胞分化为神经元和促进神经元存活的信号物质。     

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。     

骨髓基质细胞促进神经干细胞增殖分化

目的：探讨骨髓基质细胞（BMSCs）对神经干细胞（NSCs）增殖分化的影响。 方法:在体外比较NSCs在单独培养和在BMSCs条件培养液中培养下的分化和增殖情况。 结果:应用BMSCs条件培养液培养NSCs，分化的神经元比例较显著高于NSCs单独培养（41.1%±3.2% vs 23.3%±16.5%，P<0.05），而分化的星形胶质细胞所占比例显著降低（33.8%±4.9% vs 65.0%±10.4%，P<0.01），同时增殖细胞所占比例也显著增高（74.7％±4.7％ vs 51.4％±12.3％，P<0.01）。 结论:BMSCs对NSCs有促进其增殖和向神经元分化的作用。NSCs与BMSCs联合移植可能会增强NSCs移植的抗脑损伤作用。     

MRI活体示踪超顺磁性氧化铁标记兔骨髓间充质干细胞的自体皮下移植

背景：高磁场MRI已被成功应用于示踪移植超顺磁性氧化铁标记骨髓基质干细胞的研究,但目前还有应用低磁场MRI示踪移植干细胞的报道。 目的：探讨用0.2 T MRI活体示踪自体皮下移植磁化标记骨髓基质干细胞的分布和迁移的可行性。 方法：从兔骨髓中分离培养骨髓基质干细胞,采用超顺磁性氧化铁和BrdU双重标记后,与壳聚糖复合植入兔自体大腿皮下。自体皮下移植未标记骨髓基质干细胞和皮下单纯注射超顺磁性氧化铁为对照。 结果与结论：超顺磁性氧化铁标记骨髓基质干细胞经普鲁士蓝染色和电镜检查证实细胞胞浆含致密铁颗粒。超顺磁性氧化铁标记后自体皮下移植的兔骨髓基质干细胞在GRET2*WI序列成像时产生特征性的低信号改变至少维持8周,且信号逐渐从移植部位进入组织深处。但普鲁士蓝染色和BrdU免疫组化显示大部分的移植细胞仍停留在原移植部位。提示体外超顺磁性氧化铁能有效地标记骨髓基质干细胞,利用0.2 T MRI活体示踪自体皮下移植的超顺磁性氧化铁标记兔骨髓基质干细胞分布和迁移是可行的。     

体外诱导恒河猴骨髓基质细胞分化为神经细胞的分化条件

目的比较血清维甲酸(retinoic acid,RA)、胶质细胞源神经营养因子(glial cell line-derived neurotrophic factor,GDNF)及脑源性神经营养因子(brain derived neurotrophic factor,BDNF)等在不同浓度诱导条件下使恒河猴骨髓基质细胞(bone marrow stromal cells)诱导分化为神经干细胞(NSCs)及成熟神经细胞的分化条件。方法用Nestin、CD133抗体免疫细胞化学染色,鉴定NSCs;用NSE、β-tublin鉴定神经元;用GFAP鉴定神经胶质细胞,膜片钳检测分化成熟细胞的电生理特性。结果培养第8天多数细胞表现出Nestin及CD133抗原阳性,即为NSCs细胞;诱导后3天即有神经元样细胞出现,此后神经元样细胞逐渐增多,膜片钳检测发现这些细胞具有类似神经细胞的电生理特性。同时,与其他培养条件相比较,低浓度血清(2．5％) RA GDNF组诱导分化效能最高。结论应用RA GDNF及配合使用低浓度血清能够高效诱导骨髓源NSCs向成熟神经细胞分化。     

骨髓基质干细胞复合煅烧骨的皮下成骨

背景：研究证明骨髓基质干细胞与煅烧骨支架材料结合后可形成组织工程化骨,但在动物体内的生物相容性及皮下诱导成骨的能力国内报道较少。 目的：观察骨髓基质细胞复合异种煅烧骨植入BALB/c裸鼠背部皮下的成骨性能及煅烧骨材料作为组织工程骨支架材料的可行性。 方法：选用经脱脂及脱蛋白处理后高温煅烧形成的骨支架材料与梯度密度离心法分离培养至第3代的羊骨髓基质干细胞构建细胞-煅烧骨复合物植入BALB/c裸鼠背部皮下,选同期对侧背部皮下植入单纯煅烧骨为对照组。 结果与结论：煅烧后的松质骨块为白垩色,表面呈蜂窝状多孔结构,保留了天然松质骨的多孔状空间结构。骨小梁结构完整,孔隙相互连通。骨髓基质干细胞接种到煅烧骨后24 h可见大量细胞黏附于支架上,7 d后细胞分泌大量细胞外基质,细胞与基质分界不清,细胞能在材料上良好地黏附、增殖与生长,细胞活性未受到支架材料的影响。植入4周后,两组均可见煅烧骨边缘出现少量残片,细胞-煅烧骨复合物组煅烧骨孔隙周边可发现骨细胞,对照组煅烧骨表面可见纤维结缔组织包绕。植入后8周,两组均可见到煅烧骨部分降解为片状类骨质,周围有成纤维细胞包绕,排列紧密,形态多样,细胞-煅烧骨复合物组煅烧骨孔隙内可见煅烧骨表面有排列成行的成骨细胞,孔隙间有散在淋巴细胞浸润。对照组标本可见孔隙内有大量结缔组织长入,未见明显成骨迹象。结果说明,经高温煅烧后的松质骨材料,具有良好的生物相容性和生物安全性,可作为骨髓基质干细胞的良好载体,复合后植入体内能够诱导新生骨组织形成,可作为骨缺损组织工程修复的支架材料。     

Smooth muscle-like tissues engineered with bone marrow stromal cells

Bone marrow-derived cells have demonstrated the ability to differentiate into multiple mesenchymal cell lineages. Here we tested whether smooth muscle (SM)-like tissues can be created in vivo with bone marrow stromal cells (BMSCs). Cultured canine BMSCs, which expressed SM cell-specific markers including SM alpha-actin and SM myosin heavy chain, were seeded on three-dimensional, biodegradable polymer scaffolds and implanted into peritoneal cavity of athymic mice. The cell-scaffold constructs retrieved 4 weeks after implantation formed three-dimensional tissues. Immunohistochemical analyses showed that the tissue reconstructs expressed SM alpha-actin and SM myosin heavy chain. Masson's trichrome staining showed the presence of significant amounts of collagen in the tissue reconstructs. Cells labeled with a fluorescent tracer prior to implantation were still present in the tissue reconstructs 4 weeks after implantation. Non-seeded scaffolds (control groups) retrieved 4 weeks after implantation did not exhibit extensive tissue formation. This study demonstrates the potential of BMSCs as an alternative cell source for tissue engineering of SM.     

Osteogenic differentiation and angiogenesis with cocultured adipose-derived stromal cells and bone marrow stromal cells

The purpose of this study was to determine the influence of cocultured adipose-derived stromal cells (ASCs) in enhancing the osteogenic differentiation and angiogenesis of bone marrow stromal cells (BMSCs) as well as the underlying mechanism and the optimal ratio. Two in vitro coculture models, segregated cocultures using transwell and mixed cocultures, were employed to assess the indirect and direct effects of coculture respectively. Coculture was carried out for 14 days using 1 × 10⁵ BMSCs and ASCs of variable number. BMSCs, ASCs, or both were seeded in PLGA scaffold and implanted in the subcutaneous tissue of 25 nude mice for in vivo analysis of angiogenesis. To evaluate the orthotopic bone formation, critical size calvarial defects were created on 20 mice, and implanted with hydroxyapatite/β-tricalcium phosphate granules plus BMSCs, ASCs, or both. From both transwell and mixed coculture model, 1 × 10⁵ BMSCs cocultured with 0.5 × 10⁵ ASCs showed significantly greater osteogenic differentiation and mineralization than BMSCs alone. The mixed ASC/BMSC coculture at or above a ratio of 0.5/1 showed increased secretion of vascular endothelial growth factor (VEGF), and induced effective tube formation from human umbilical vein endothelial cells, which were comparable to ASCs. Cytokine profiling assay and gene expression study showed elevated levels of angiogenic factors VEGF and CXCL1, osteogenic factor Wnt5a as well as transforming growth factor (TGF)-βR1 and SMAD3 from BMSCs when cocultured with ASCs. After 5 weeks of implantation, polylactic-co-glycolic acid (PLGA)-ASCs-BMSCs had a number of vascular structures comparable to PLGA-ASCs and significantly greater than PLGA-BMSCs. Calvarial defects treated with ceramic/BMSCs/ASCs had greater area of repair and better reconstitution of osseous structure than the defects treated with ceramic/ASCs or ceramic/BMSCs after 10 weeks. In conclusion, ASCs added to BMSCs promoted osteogenesis and angiogenesis at the optimal ASC/BMSC ratio of 0.5/1.     

Degradation of polysaccharide hydrogels seeded with bone marrow stromal cells

In order to produce hydrogel cell culture substrates that are fit for the purpose, it is important that the mechanical properties are well understood not only at the point of cell seeding but throughout the culture period. In this study the change in the mechanical properties of three biopolymer hydrogels alginate, low methoxy pectin and gellan gum have been assessed in cell culture conditions. Samples of the gels were prepared encapsulating rat bone marrow stromal cells which were then cultured in osteogenic media. Acellular samples were also prepared and incubated in standard cell culture media. The rheological properties of the gels were measured over a culture period of 28 days and it was found that the gels degraded at very different rates. The degradation occurred most rapidly in the order alginate > Low methoxy pectin > gellan gum. The ability of each hydrogel to support differentiation of bone marrow stromal cells to osteoblasts was also verified by evidence of mineral deposits in all three of the materials. These results highlight that the mechanical properties of biopolymer hydrogels can vary greatly during in vitro culture, and provide the potential of selecting hydrogel cell culture substrates with mechanical properties that are tissue specific.     

Tissue-engineered bone repair of goat-femur defects with osteogenically induced bone marrow stromal cells

Tissue engineering can generate bone tissue and has been shown to provide a better means of repairing weight-bearing bone defect. Previous studies, however, have heretofore been limited to the use of nonosteogenically induced bone marrow stromal cells (BMSCs) or the application of slow-degradation scaffolds. In this study, weight-bearing bone was engineered using osteogenically induced BMSCs. In addition, coral was used as a scaffold material, due to its proper degradation rate for the engineering and repair of a goat femur defect. A 25 mm long defect was created at the middle of the right femur in each of 10 goats. The rates of defect repair were compared in an experimental group of ten goats receiving implants containing osteogenically induced BMSCs and in the control group of goats (n = 10) receiving just coral cylinders. In the experimental group, bony union was observed by radiographic and gross view at 4 months, and engineered bone was further remodeled into newly formed cortexed bone at 8 months. There was increased gray density of radiographic rays in the repaired area, which was significantly different (p < 0.05) from that of the control group. H&E staining demonstrated that trabecular bone was formed at 4 months. Moreover, irregular osteon was observed at 8 months. Most importantly, the tissue-engineered bone segment revealed a similarity to the left-side normal femur in terms of bend load strength and bend rigidity, showing no significant difference (p > 0.05). In contrast, the coral cylinders of the control group showed no bone formation. Furthermore, almost complete resorption of the carrier had occurred, being evident at 2 months in the control group. H&E staining demonstrated that a small amount of residual coral particle was surrounded by fibrous tissue at 4 months whereas the residues disappeared at 8 months. Based on these results, we conclude that engineered bone from osteogenically induced BMSCs and coral can ideally heal critical-sized segmental bone defects in the weight-bearing area of goats.     

脱基质小牛骨粉复合骨髓基质干细胞的成骨作用

背景：利用骨组织工程技术在上颌窦提升中的成骨研究是目前口腔种植学到研究热点。 目的：探讨骨组织工程在上颌窦底提升同期牙种植中的成骨效果。 方法：体外分离培养犬骨髓基质干细胞,将细胞与脱基质小牛骨粉复合培养并向成骨细胞定向诱导分化。健康成年犬12只行双侧上颌窦底提升同期牙种植,一侧植入复合物,另一侧植入脱基质小牛骨粉做对照。 结果与结论：种植体稳固无松动,上颌窦黏膜完整。实验侧可见新生骨形成较早、骨量较多;对照侧新生骨形成较慢。X射线显示实验侧新生骨质致密,与种植体结合紧密。随时间的增加,牵出力增大,12,24周时两侧差异有显著性意义(P < 0.05)。组织形态学检测显示新生骨面积逐渐增加,12,24周时差异有显著性意义(P < 0.05)。提示组织工程化骨在上颌窦提升同期牙种植中可获得良好的成骨效果。     

骨组织工程的种子细胞--骨髓基质细胞的研究进展

骨髓基质细胞作为骨组织工程的种子细胞具有广阔前景.许多实验证实骨髓基质细胞具有间充质干细胞特性,表现为较强的增殖能力和向多种间充质细胞分化的潜能.目前已建立了体外培养骨髓基质干细胞的方法,而且正在摸索进一步纯化的方法和诱导分化的条件.已有利用其成骨特性体内移植实验,表明在适当的条件下,接种在组织工程材料上的骨髓基质细胞可以形成新骨.     

骨髓基质干细胞治疗骨不连的新进展

背景：骨髓基质干细胞体外培养增殖力强、易于向成骨细胞及软骨细胞方向分化且成骨性能稳定等特点,成为骨组织工程中合适的种子细胞。 目的：总结分析采用骨髓基质干细胞作为种子细胞,分析其直接移植于骨不连部位或复合支架或转基因治疗骨不连所具有的优劣势。 方法：检索1992/2011西文生物医学期刊文献数据及CNKI 数据库有关骨不连研究,骨髓基质干细胞分离、培养,在骨不连方面的应用,骨组织工程细胞支架方面的文献,英文检索词为“bone marrow stromal stem cells,nonunions, repairing,tissue engineering”,中文检索词为“骨髓基质干细胞,骨修复,骨不连,组织工程”。排除重复性研究,保留23篇进一步归纳总结。 结果与结论：利用骨髓基质干细胞作为种子细胞,直接植入骨不连部位,或与适当的支架材料结合,或用骨髓基质干细胞作为靶细胞,导入外源目的基因诱导成骨的基因治疗来修复骨缺损的方法,给骨缺损的治疗带来光明的前景。但同时也存在骨髓基质干细胞增殖、分化合适条件难以准确确定,经皮移植自体骨髓基质干细胞植入体内后容易流失,不能在植入部位形成有效的细胞浓度,支架材料尚不能完全符合临床要求,以及如何将骨组织工程与基因治疗的方法结合起来等问题,需要进一步的研究。