壳聚糖-明胶-果胶膜对骨髓基质干细胞的细胞行为研究

目的 考察壳聚糖-明胶-果胶膜对骨髓基质干细胞（MSC）的作用,为组织工程提供一种新的可望用于组织工程的生物材料. 方法 将MSC接种到壳聚糖-明胶-果胶（CS-Gel-P）膜和壳聚糖-明胶（CS-Gel）膜上,观察其生长形态,用MTT法测定其细胞活性,并考察其碱性磷酸酶活性表达. 结果 在CS-Gel-P膜上接种的MSC表现出比在CS-Gel膜和组织培养板（TCPS）上更好黏附性能,同时在不同时间点的碱性磷酸酶表达也均高于CS-Gel膜,在两种不同膜上的MSC的细胞活性差异无统计学意义. 结论 果胶的加入可以促进MSC的黏附和生长,同时更能促进MSC诱导分化为成骨细胞.     

工程化的神经干细胞的研究进展

干细胞是目前生物医学领域研究的热点之一.神经干细胞(NSCs)的研究和应用尤为引人注目.神经干细胞是一类广泛存在于中枢神经系统内,能自我复制、自我更新,具有多分化潜能的细胞.所谓工程化的神经干细胞是指利用基因工程等技术,使神经干细胞能高效稳定地表达各种神经营养因子,促进各种中枢神经系统疾病尤其是脊髓损伤的修复.就工程化的神经干细胞的研究现状进行综述,并提出面临的问题和今后的研究方向.     

壳聚糖支架与神经干细胞生物相容性的研究

为探讨壳聚糖多孔支架与神经干细胞(NSCs)的生物相容性,应用冷冻干燥技术制备壳聚糖多孔支架,将神经干细胞克隆球接种于壳聚糖支架载体上,分为NSCs+支架组和NSCs+支架+NGF组。培养2周后冰冻切片,行Nissl染色及微管相关蛋白-2(MAP-2)、胶质纤维酸性蛋白(GFAP)和2,3-环核苷酸磷酸二酯酶(CNP)免疫组化染色来检测NSCs的分化,并进行MAP-2阳性细胞计数、胞体面积、细胞周长的图像处理和统计分析。结果显示:壳聚糖支架孔隙率为90%,支架孔径为50～350μm。NSCs可以在壳聚糖支架上存活、迁移并分化成神经元、星形胶质细胞和少突胶质细胞。NSCs+支架+NGF组的MAP-2阳性细胞数明显多于NSCs+支架组,且胞体较大,突起较多且长。上述结果提示,壳聚糖支架与NSCs具有良好的生物相容性,外源性的NGF能够促进壳聚糖支架中的NSCs向神经元分化。     

壳聚糖支架上骨髓间充质干细胞的浓度及黏附生长

背景：组织工程的研究重点是利用少量的细胞经体外培养、扩增后, 在一定环境下附着在三维多孔支架上并良好生长为后期的组织器官重建修复做好基础。 目的：对不同浓度兔骨髓间充质干细胞复合至壳聚糖支架用于组织工程再生修复进行评价。 方法：取5×10⁵脱乙酰度为95%的壳聚糖粉末通过冷冻干燥法制备壳聚糖支架,取1×10⁶,1×10⁷,1×10⁸,1×10⁹ L^-1细胞体积各100 μL复合至壳聚糖支架后1,3,5,7,9 d以光镜,扫描电镜,MTT法观察骨髓间充质干细胞的生长与分裂增殖情况。 结果与结论：壳聚糖海绵状多孔支架为5 mm×5 mm×3 mm,孔径190~380 μm,平均孔径290 μm,孔相通性较好,空隙率为(84.00±4.62)%。细胞/支架共培养72 h后各浓度细胞组均可渗入壳聚糖支架多孔结构内黏附生长。1×10⁷,1×10⁸,1× 10⁹ L^-1浓度细胞组在支架上成蔟生长,部分细胞与支架融合。结果提示,1×10⁷,1×10⁸L^-1组细胞更利于骨髓间充质干细胞在壳聚糖支架的黏附生长,用于组织再生修复。     

神经再生素促进神经干细胞的生长和分化

目的:研究神经再生素对体外培养神经干细胞分化的促进作用及对其生长相关蛋白(GAP43)、神经丝蛋白(NF-H)表达的影响.方法:取出生3～5d的新生SD大鼠大脑皮层进行神经干细胞的体外培养、鉴定,神经干细胞与0、 1、 2mg/L的神经再生素(NRF)共培养8d,相差显微镜观察分析,应用Real-time PCR对与不同浓度的NRF(0、 1、 2、 4、 8mg/L)共培养8d的神经干细胞进行GAP43、 NF-H的表达量检测.结果:成功培养出具有多向分化潜能的神经干细胞;神经再生素可明显促进神经干细胞的分化,并能在一定范围内随浓度递增而有效促进GAP43、 NF-H的表达,且最佳作用浓度为4mg/L.结论:神经再生素可以促进神经干细胞的生长和分化.     

干细胞及神经导管支架修复神经缺损

背景：组织工程的发展为神经缺损的修复提供了可能,种子细胞与导管支架制成的复合体是构建组织工程神经的核心。 目的：从干细胞的组织工程应用及构建具有良好生物相容性的导管支架材料角度,探索如何更好的修复神经损伤。 方法：以“干细胞,神经损伤,修复,神经导管,神经支架材料”为中文关键词,以“stem cells,nerve damage,repair,nerve guide conduit material,scaffold materials,nerve tissue engineering”为英文关键词,采用计算机检索CNKI和Medline数据库1996-01/2011-01有关不同来源干细胞和导管支架材料修复神经缺损的相关文章,排除重复研究或Meta分析类文章,筛选纳入30篇文献进行评价。 结果与结论：移植神经干细胞可以在神经系统存活、增殖、迁移,在不同部位分化为相应的细胞,因此给神经修复领域带来新的希望。另外,随着生物材料的发展,神经导管材料修复神经缺损也取得了优良的效果,具有良好的应用前景。将神经干细胞复合导管可降解生物材料有望能更好的满足神经支架的要求,达到修复和重建的目的。     

神经干细胞在新型复合支架中的生长和分化

BACKGROUND:Neural stem cells with self-proliferation and differentiation potential are the ideal seed cells for central nervous tissue engineering. Although collagen and silk fibroin as biological scaffold materials have been widely used, both of them used alone have certain shortcomings. Is it possible to combine the two materials to build a novel neural tissue-engineered scaffold? What is the effect of this novel scaffold on the growth and differentiation of neural stem cells? OBJECTIVE:To observe the growth and differentiation of neural stem cells seeded onto the novel composite scaffold. METHODS:The rat embryonic neural stem cells were inoculated onto new composite scaffolds, and then, their growth and differentiation were observed by light microscopy and scanning electron microscopy. Neural stem cells were cultured in conventional suspension culture as control group. Cell counting kit-8 assay was used to detect viability of neural stem cells in the two groups. Three-dimensional composite scaffolds carrying neural stem cells were sliced into paraffin sections to observe the growth and differentiation of neural stem cells by hematoxylin-eosin staining and immunofluorescence staining. RESULTS AND CONCLUSION: Neural stem cells cultured on the new composite scaffold grew and differentiated well, and interconnected synapses were observed. Cell counting kit-8 assay showed that neural stem cells on the scaffold grew well, and the cell viability was significantly higher in the composite scaffold group than that in the control group (P < 0.05). Hematoxylin-eosin staining and immunofluorescence staining of paraffin sections further provided evidence for good growth and differentiation of neural stem cells on the scaffold. These results indicate that the novel composite scaffold with good biocompatibility benefits the growth and differentiation of neural stem cells, promising a favorable application prospect.     

干细胞移植与神经修复

背景：随着生命科学发展,利用干细胞来源的组织工程技术,有可能使受损的神经组织恢复再生能力。 目的：针对较为重要的几种干细胞在神经系统修复中的临床应用做一综述,以期阐明各自适用范围和临床效度,为临床医师按需选择提供参考。 方法：应用计算机检索PubMed 数据库中1998至2011年期间的关于干细胞移植在神经修复方面研究的文章,检索词为“Stem cells, Transplantation, Nerve repair”,限定语种为“English”。同时,检索万方数据库中2006至2011年期间的相关文章,检索词为“干细胞,移植,神经修复”,限定语种为中文。 结果与结论：初次检索得到288篇文章,选择与移植干细胞的来源、分化、特征及其在神经修复方面相关的文章,包括基础研究和临床研究,观察对象无论为人或动物,均进行审校。根据纳入标准,在排除重复和个案报道类文章之后,重点选择32篇文章进行综述。说明干细胞移植为神经系统疾病的治疗提供了一条新途径,使传统认为的不可修复、不可再生的神经组织的结构修复和功能重建成为可能。干细胞移植是一个全新的领域,还有许多问题亟待解决。     

骨髓基质细胞和壳聚糖/明胶共混材料生物相容性研究

目的:研究壳聚糖/明胶共混材料对离体培养的骨髓基质细胞粘附及增殖的影响,寻找骨髓基质细胞新的载体材料。方法:取2周龄幼兔的长骨采集骨髓,培养骨髓基质细胞,体外扩增1周后,种植于纯壳聚糖和壳聚糖/明胶共混材料的表面。在倒置光学显微镜、扫描电镜的辅助下,观察细胞的粘附和生长情况,种植7d后用透射电镜观察细胞功能状况,用MTT方法检测种植后2d、4d、6d、8d细胞的增殖情况。结果:壳聚糖/明胶共混材料和纯壳聚糖能促进骨髓基质细胞在材料表面粘附并保持其在机体内的形态。壳聚糖/明胶共混材料表面的骨髓基质细胞功能活跃。在材料表面和培养板表面培养的骨髓基质细胞均能持续增殖,而壳聚糖/明胶共混材料能显著促进骨髓基质细胞的增殖(P<0.01)。结论:壳聚糖/明胶共混材料保持了壳聚糖的某些生物活性,同时由于加入明胶,能促进骨髓基质细胞的增殖,可作为骨髓基质细胞的载体应用于组织工程。     

干细胞、组织工程支架与神经营养因子在脊髓损伤治疗中的应用

文题释义：组织工程支架：目前干细胞移植已被证明可以促进脊髓损伤后的神经元再生,但是存在移植细胞存活率、定植率较低等局限性。组织工程支架可以提供一个三维空间,模拟更有利的内源性微环境,为移植干细胞提供有效支持,从而促进干细胞在体内黏附、迁移和分化。间充质干细胞：是一种多能干细胞,它具有干细胞的所有共性,即自我更新和多向分化能力。它又具有其他干细胞所没有的优势如易于分离提取、不存在伦理问题、不存在免疫排斥风险等,目前已成为组织工程技术中较为常用的种子细胞。  摘要背景：干细胞具有多向分化潜能,组织工程支架可以为其提供支持,神经营养因子则可以促进干细胞的分化,3者联合移植治疗方案促进脊髓损伤功能恢复是当前研究热点之一。目的：综述近年来干细胞、组织工程支架、神经营养因子及其联合移植治疗脊髓损伤的研究进展。方法：作者检索PubMed、Sciencedirect、Medline、中国知网等数据库2000年1月至2019年10月的相关文章。英文检索词为“spinal cord injury,mesenchymal stem cells,issue engineering,neurotrophic factor”,中文检索词为“脊髓损伤,间充质干细胞,组织工程,神经营养因子”,检索文献类型为研究原著,初检文章712篇,再经过严格筛选后,对符合要求的79篇文献进行分类综述。结果与结论：单纯的干细胞移植并不能重建脊髓的复杂结构和稳定性,而单纯的生物材料、神经营养因子也不能替代脊髓损伤过程中的神经元丢失,因此联合移植是脊髓损伤治疗的研究方向。但是由于脊髓损伤的部位、损伤的组织及其修复时的营养需求各异,如何将干细胞、组织工程支架、神经营养因子联合的方案最优化仍是一个巨大的挑战。ORCID: 0000-0001-8505-0231(季航宇) 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     

Chitosan,Gelatin and Poly(L-Lysine) Polyelectrolyte-Based Scaffolds and Films for Neural Tissue Engineering

Biomaterial implants are a promising strategy to replace neural tissue that is lost after traumatic nerve damage. Chitosan (Ch) is a suitable material for nerve implantation when it is used at a minimum amount of 2% (w/v). The goal of this study was to determine the best mixture of 2% Ch with gelatin (G) and poly(L-lysine) (PLL) for use in neural tissue engineering. Using different physicochemical approaches we showed that all mixtures formed polyelectrolyte complexes with distinct electrostatic interactions between their compounds. This gave rise to different gel morphologies, among which Ch + G exhibited a significantly smaller pore size, unlike Ch + G + PLL. However, thermal resistance to degradation and the wettability of the Ch-based films were not affected. Additionally, these differences affected glial cells growth in long-term (14 days) cultures performed on Ch-based films. Astrocytes and olfactory ensheathing cells proliferated on G and Ch + G films which induced both flattened and spindle cell morphologies. Meanwhile, cortical and hippocampal neurons were similarly viable in all studied films and significantly lower than those observed in controls. Lastly, neurites from dorsal root ganglia extended the most on Ch + G films. These results show that a Ch + G mixture is a promising candidate for use in neural tissue engineering.     

Plasma Enhancement of In Vitro Attachment of Rat Bone-Marrow-Derived Stem Cells on Cross-Linked Gelatin Films

Abstract

In this work, nitrogen, oxygen and air glow discharges powered by 50 Hz AC power supply are used for the treatment of type-A gelatin film cross-linked by a dehydrothermal (DHT) process. The properties of cross-linked gelatin were characterized by contact angle measurement, atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS) analysis. The results showed that the water contact angle of gelatin films decrease with increasing plasma treatment time. The treatment of nitrogen, oxygen and air plasma up to 30 s had no effects on the surface roughness of the gelatin film as revealed by AFM results. The XPS analysis showed that the N-containing functional groups generated by nitrogen and air plasma, and O-containing functional groups generated by oxygen and air plasmas were incorporated onto the film surface, the functional groups were found to increase with increasing treatment time. An in vitro test using rat bone-marrow-mesenchym-derived stem cells (MSCs) revealed that the number of cells attached on plasma-treated gelatin films was significantly increased compared to untreated samples. The best enhancement of cell attachment was noticed when the film was treated with nitrogen plasma for 15–30 s, oxygen plasma for 3 s, and air plasma for 9 s. In addition, among the three types of plasmas used, nitrogen plasma treatment gave the best MSCs attachment on the gelatin surface. The results suggest that a type-A gelatin film with water contact angle of 27–28° and an O/N ratio of 1.4 is most suitable for MSCs attachment.     

PLGA微囊促进树突细胞抗原提呈作用的体外观察

本研究在体外将含卵清蛋白(OVA)的PLGA微囊与小鼠骨髓诱导的树突细胞(BM-DC)相互作用，然后用卵清蛋白特异性杂交瘤T细胞检测OVA抗原在BM-DC表面的表达，结果显示：PLGA微囊可以在体外显著促进BM-DC对OVA抗原的呈递。     

RGD多肽修饰的改性PLGA仿生支架材料对骨髓间充质干细胞粘附、增殖及分化影响的研究

目的：探索RGD多肽修饰的改性PLGA支架材料上骨髓基质细胞的增殖、粘附及分化情况。方法用异型双功能交联剂Sulfo-LC-SPDP将GRGDSPC多肽共价结合到改性PLGA支架材料上，以未接多肽的改性PLGA材料做对照，取第三代MSC接种到材料上，培养1d、2d、3d、4d后比较材料上的细胞密度来反映细胞的增殖程度；取第三代MSC接种到材料上，培养4h、12h后沉淀法定量检测粘附的细胞数，培养24h后摄光镜图像比较粘附细胞的数量和形态，并用FITC连接的鬼笔环肽对细胞骨架染色，在荧光显微镜下观察细胞骨架的组织情况；取第三代MSC接种到材料上，用成骨性培养基培养7d、14d、21d，检测细胞中ALP活性来了解MSC分化情况。结果：培养1d、2d、3d、4d后细胞的增殖程度无显著性差异；培养4h、12h后实验组细胞粘附率均显著高于对照组，且24h后细胞的粘附质量、细胞骨架的组织情况也较对照组为好；培养14d后实验组细胞表达显著高的ALP活性。结论：RGD多肽修饰对细胞增殖无明显促进作用，但能提高改性PLGA支架材料对骨髓基质细胞的粘附性，对MSC向成骨细胞分化有显著促进作用。     

Neural Progenitor and Hemopoietic Stem Cells Inhibit the Growth of Low-Differentiated Glioma

The effects of neural progenitor and hemopoietic stem cells on C6 glioma cells were studied in in vivo and in vitro experiments. Considerable inhibition of proliferation during co-culturing of glioma cells with neural progenitor cells was revealed by quantitative MTT test and bromodeoxyuridine incorporation test. Labeled neural progenitor and hemopoietic stem cells implanted into the focus of experimental cerebral glioma C6 survive in the brain of experimental animals for at least 7 days, migrate with glioma cells, and accumulate in the peritumoral space. Under these conditions, neural progenitor cells differentiate with the formation of long processes. Morphometric analysis of glioma cells showed that implantation of neural progenitor and hemopoietic stem cells is accompanied by considerable inhibition of the growth of experimental glioma C6 in comparison with the control. The mechanisms of tumor-suppressive effects of neural and hemopoietic stem cells require further investigation.     

Effects of the Surface Characteristics of Nano-Crystalline and Micro-Particle Calcium Phosphate/Chitosan Composite Films on the Behavior of Human Mesenchymal Stem Cells In Vitro

Calcium phosphate (CaP) compounds, the main inorganic constituent of mammalian bone tissues, are believed to support bone precursor cell growth and osteogenic differentiation. Chitosan, a deacetylated derivative of chitin, is a versatile biopolymer to offer broad possibilities for cell-based tissue engineering. In the present study, different scales of CaP crystals on chitosan membranes were prepared for culture of human mesenchymal stem cells (hMSCs) in vitro. A series of aqueous CaP suspensions with different concentrations were mixed with chitosan solution and chitosan/calcium phosphate (C/CaP) films were fabricated by the solvent-casting method. With different weight ratios of CaP in chitosan solution, the various surface characteristics of nano-amorphous (C/CaP 0.1), nano-crystalline (C/CaP 0.5) and micro-particle (C/CaP 2) CaP compounds were examined by scanning electron microscopy and electron dispersion spectroscopy. X-ray diffraction on micro-particles of CaP indicated the formation of crystalline hydroxyapatite. The behavior of hMSCs, including proliferation, cell spreading and osteogenic differentiation, was studied on the C/CaP films. In basal culture medium, the incorporation of CaP into chitosan films could promote the proliferation of hMSCs. The C/CaP 0.5 film with connected CaP nano-crystals had better cellular viability. The fluorescence microscope images at 14 days of culture revealed extensive networks of F-actin filaments of hMSCs on chitosan, C/CaP 0.1 and C/CaP 0.5 films. The cellular morphology on C/CaP 2 film with discrete CaP micro-particles was partly restrained. In osteogenic medium, the alkaline phosphatase (ALP) activity of hMSCs increased and showed the process of osteogenic differentiation. The ALP levels on C/CaP 2 film were higher than those on C/CaP 0.1 and C/CaP 0.5 films. These results demonstrated that the crystallinity and topography of CaP on chitosan membranes could modulate the behaviors of cultured hMSCs in vitro.     

神经干细胞的生物学特性和体外培养鉴定

神经干细胞(NSCs)是具有自我更新和多向分化能力的细胞,存在于胚胎、胎儿和成人的脑室下区、齿状回、纹状体、室管膜下区等部位。通过机械分离或酶消化法能从其存在部位分离出NSCs。当培养基中表皮生长因子(EGF)和成纤维生长因子(bFGF)浓度均为20ng·mL-1时,NSCs能通过对称分裂和不对称分裂在体外增殖,当培养基中bFGF浓度为1～10ng·mL时,NSCs可向神经元和胶质细胞分化。目前,表达Nestin、自我增-1殖和多潜能分化能力是鉴定NSCs的三大条件,但还没有NSCs特异性的鉴定方法。     

Improved Survival and Regeneration of Irradiated Mouse Neural Stem Cells after Co-Culturing with Non-Irradiated Mouse Neural Stem Cells or Mesenchymal Stem Cells from the Adipose Tissue

Bulletin of Experimental Biology and Medicine - We studied the effect of neural stem cells (NSC) and mesenchymal stem cells (MSC) from mouse adipose tissue on survival, clonogenic activity, and...     

内皮细胞对神经干细胞增殖与分化的影响

已经确定神经干细胞在脑中不是随意分布,而是集中在血管周围的。尽管神经干细胞存在于血管周围,但是对于其与血管组成细胞之间的关系还不是很清楚。据报道,内皮细胞释放的可溶性因子可以刺激神经干细胞自我增殖,抑制其分化,并且提高神经元的比例。将内皮细胞与神经干细胞共同培养可以激活Notch途径来促进神经干细胞自我增殖。另外,血管内皮生长因子对神经细胞的生长也起着非常重要的作用,它促进了中枢神经系统星形胶质细胞的生长与分化。因此,内皮细胞不仅是传统意义上血管的组成部分,还是神经干细胞所在区域的重要成分,并且可以通过大脑产生的神经营养性分泌物来提高神经元的发生。     

Proliferation of Mesenchymal Stem Cell on Chitosan Films Associated with Convex Micro-topography

Topographical micro-structures of material surfaces are known to influence cell behavior. In this study, alignment and proliferation of mesenchymal stem cells (MSCs) on chitosan (CS) films with different microtopography was investigated. A modified solvent–non-solvent method was used to produce micro-well polystyrene films which were used as templates for preparation of micro-hilled CS films. The alignment and proliferation of MSCs on CS films were characterized by scanning electron microscopy and the MTT assay, respectively. Polystyrene films with different micro-well diameters were produced under different conditions. We successfully produced CS films with micro-hills of 10 μm (CS10) and 5–30 μm (CS5-30) by a molding approach. MSCs cultured on CS10 covered the tightly packed micro-hills and flattened polygonally. In contrast, MSCs on CS5-30 mostly adhered between loosely packed micro-hills and demonstrated elongated extensions. The proliferation rate of MSCs on CS5-30 was highest on day 4 (66% on CS5-30 versus 46% on CS10 and 24% on smooth CS) and day 8 (106% on CS5-30 versus 38% on CS10 and 72% on smooth CS). It is reasonable to suggest that spatially and dimensionally dispersed micro-hills on CS films facilitate the alignment and proliferation of MSCs. This may be used to physically modify CS for tissue-engineering applications.