乳鼠雪旺氏细胞的纯化培养研究

目的：获得高纯度的乳鼠雪旺氏细胞。方法：采用出生后３～５天乳鼠的坐骨、臂丛神经，植块法培养乳鼠雪旺氏细胞；并通过精细剥除神经外膜、组织块反复再植、低浓度胰酶快速消化、差速贴壁法的有机结合对雪旺氏细胞进行纯化；继用抗Ｓ－１００蛋白单抗通过间接免疫荧光法及ＡＢＣ法进行细胞鉴定。结果：首次植块获得的细胞经纯化后雪旺氏细胞可高达８５％以上，反复植块者可达９５％，甚至高达９８％。结论：本方法简单、稳定，所获得的雪旺氏细胞纯度高、数量大、受非实验因素影响小     

Co-culture with Schwann cells is an effective way for adipose-derived stem cells neural transdifferentiation

Introduction

Adipose-derived stem cells (ADSCs) could accomplish neural transdifferentiation with the presence of certain growth factors in vitro. It has been proved that bone marrow stromal cells (BMSCs) can realize neural transdifferentiation only by being co-cultured with Schwann cells (SCs), and in our former studies we have confirmed that ADSCs could do so too. This paper aims to investigate whether the neural induction efficiency of co-culture is as high as that of other strategies using chemicals or chemicals combined with some growth factors.

Material and methods

We isolated and multiplied ADSCs from adult Sprague-Dawley rats, and SCs from sciatic nerves of 1-to-2-day-old Sprague-Dawley rat pups, then induced ADSCs neural transdifferentiation through 2% dimethyl sulphoxide (DMSO) and DMSO combined with growth factors. Meanwhile we co-cultured ADSCs and SCs in Transwell culture dishes without intercellular contacts. Immunostaining and RT-PCR were adopted to investigate the neural transdifferentiation of ADSCs. Then we compared the expression differences for genes S100, nestin and GFAP of the above three protocols by real-time PCR.

Results

Both immunostaining and RT-PCR proved that ADSCs could accomplish neural transdifferentiation through each of the above three protocols. And real-time PCR further shows that the gene expression relative quantities for the above three genes are not statistically different between co-culture and induction through DMSO combined with growth factors (p > 0.05), but both of them are statistically different from induction only by DMSO (p < 0.05).

Conclusions

Co-culturing ADSCs and SCs may be a simple, effective and practical way for ADSCs neural transdifferentiation.     

体外培养Schwann细胞缺氧模型的建立

目的:建立体外培养Schwann细胞的缺氧模型。方法:取体外培养Schwann细胞,置于通入体积分数为5%CO2和95%N2的有机玻璃盒中继续缺氧培养10、15和20min,用H-E染色观察细胞形态,MTT比色试验检测细胞的活力。结果:缺氧10min组细胞形态及活力与正常组无明显差异;而缺氧15min组的细胞突起回缩,活力为缺氧前的66·3%;缺氧20min组的细胞多数死亡,活力仅为缺氧前的20·6%。结论:本方法可以成功建立有效、简便、易行的Schwann细胞缺氧模型。     

In vitro evaluation of random and aligned polycaprolactone/gelatin fibers via electrospinning for bone tissue engineering

Scaffold, as an essential element of tissue engineering, should provide proper chemical and structural cues to direct tissue regeneration. In this study, aligned and random polycaprolactone (PCL)/gelatin fibrous scaffolds with different mass ratio were electrospun. Chemical, structural, and mechanical properties of PCL/gelatin fibrous scaffolds were characterized by FTIR and tensile measurements. The average diameters of different groups were between 334.96?±?41.43?nm and 363.78?±?50.49?nm. Blending PCL with gelatin increased the mechanical properties of the scaffolds. The cell culture results demonstrated that the mass ratio of PCL and gelatin showed no obvious effects on cell behavior, whereas the cell growth behavior was affected by the fibers orientation. Higher elongation ratio, enhanced cell proliferation and elevated alkaline phosphatase activity were observed for cells cultured on aligned fibers. The findings in our research provide insightful information for the design and fabrication of scaffolds for bone tissue engineering.     

Rat bone marrow stromal cells could be induced into Schwann cell precursor-like cells in vitro

Schwann cells (SC) which are myelin forming cells in peripheral nervous system can remyelinate demyelinated central nervous system (CNS) axons in vivo. However, potential drawbacks to the use of SC in the CNS are nevertheless apparent, and Schwann cell precursors (SCP) are favourable cells for myelin repair in the CNS. But for clinical use, it is difficult to obtain sufficient large number of SCP. In the present study, rat bone marrow stromal cells (MSCs) were cultured, identified and then converted into neurospheres. Then neurospheres were identified and induced into SCP-like cells. SCP-like cells were flattened in shape, p75⁺GFAP⁻S-100⁻nestin⁻, and could differentiate into SC-like cells, similar to genuine SCP. Our data suggested that MSCs could be induced into SCP-like cells.     

施万细胞诱导共培养骨髓基质细胞向多巴胺能神经元分化

观察施万细胞(SCs)对骨髓基质细胞(BMSCs)的诱导分化作用。分离和体外培养SD大鼠SCs和BMSCs,分为SCs+BMSCs共培养(实验组)和BMSCs单独培养(对照组)。用流式细胞仪(FCM),S100、Brdu/nestin、Brdu/TH免疫荧光法分别鉴定BMSCs、SCs和BMSCs的分化情况。第2代SCs呈S100阳性,第3代BMSCs呈CD29和CD90阳性,CD45阴性。二者共培养3d后,可见Brdu/nestin免疫荧光双标细胞,阳性率达28.3±1.3%,与对照组比较,P<0.05。7d后,Brdu/nestin双标细胞减少,出现Brdu/TH免疫荧光双标细胞,阳性率为19.2±1.6%,与对照组比较,P<0.05。而对照组始终只见Brdu单标细胞。上述结果表明SCs可诱导共培养的BMSCs分化为DA能神经元。     

Comparison in the biological characteristics between primary cultured sensory and motor Schwann cells

Schwann cells (SCs) express distinct sensory and motor phenotypes, which are associated with modality-specific promotion of axon growth. Here we compared cell proliferation and migration of primary cultured sensory and motor SCs and determined the mRNA expression of several genes, nap1l1, dok4, lpp, mmp-9 and l1cam, in two phenotypes of SCs. The results showed that the rate of cell proliferation or migration was higher in sensory SCs than in motor SCs, and the five proliferation or migration-related genes also had higher expression in sensory SCs than in motor SCs. These findings may provide a basis for deeply studying the biological differences between sensory and motor SCs.     

简易雪旺氏细胞培养方法及用于凋亡实验的研究

目的：寻找一种快速简便的雪旺氏细胞培养方法以期获得研究神经再生的细胞材料并用于细胞凋亡的实验研究。方法：采用两次差速贴壁结合Ara-c法获得雪旺氏细胞,加入过氧化氢并经1％的碘化吡啶（PI）染色观察雪旺氏细胞凋亡的情况。结果;采用该法能获得较纯的雪旺氏细胞,加入过氧化氢后经1％的碘化(PI)杂色雪旺氏细胞有不同程度的凋亡。结论：过氧化氮产生的氧自由基能诱导细胞的凋亡,提示在神经组织的创伤过程中神经胶质细胞的凋亡可能与氧自由基损伤有关。     

Enhanced differentiation of human neural crest stem cells towards the Schwann cell lineage by aligned electrospun fiber matrix

Human pluripotent stem cell-derived neural crest stem cells (NCSCs) provide a promising cell source for generating Schwann cells in the treatment of neurodegenerative diseases and traumatic injuries in the peripheral nervous system. Influencing cell behavior through a synthetic matrix topography has been shown to be an effective approach to directing stem cell proliferation and differentiation. Here we have investigated the effect of nanofiber topography on the differentiation of human embryonic stem cell-derived NCSCs towards the Schwann cell lineage. Using electrospun fibers of different diameters and alignments we demonstrated that aligned fiber matrices effectively induced cell alignment, and that fiber matrices with average diameters of 600 nm and 1.6 μm most effectively promoted NCSC differentiation towards the Schwann cell lineage compared with random fibers and two-dimensional tissue culture plates. More importantly, human NCSCs that were predifferentiated in Schwann cell medium for 2 weeks exhibited higher sensitivity to the aligned fiber topography than undifferentiated NCSCs. This study provides an efficient protocol for Schwann cell derivation by combining an aligned nanofiber matrix and an optimized differentiation medium, and highlights the importance of matching extrinsic matrix signaling with cell intrinsic programming in a temporally specific manner.     

雪旺细胞增殖的研究进展

随着周围神经组织工程学的发展,雪旺细胞的研究越来越受到重视。在周围神经损伤后,雪旺细胞对周围神经再生过程中的形态和功能的修复起着不可替代的作用,因此雪旺细胞的增殖情况对于周围神经损伤后的修复就尤为重要。其增殖速度的提高可大大改善神经桥接体移植的存活率,为临床神经移植术的成功赢得宝贵的时间。本文从雪旺细胞的功能、增殖机制及其影响因素入手,为雪旺细胞的临床应用提供理论依据。     

外周血基质干细胞培养鉴定及其向施万细胞的诱导分化

目的研究体外分离培养大鼠外周血基质干细胞(PBMSCs)并诱导分化为施万细胞的潜能。方法从SD大鼠取血分离培养PBMSCs,采用流式细胞术和免疫细胞化学法对其细胞表面抗原进行检测与鉴定,并用免疫细胞化学法检测BrdU作用4h后BrdU的阳性率。PBMSCs经β-巯基乙醇、全反式维甲酸及复合条件培养基等三个步骤向施万细胞定向诱导后,用免疫细胞化学法检测S-100和P75的表达。结果流式细胞术显示培养获得的PBMSCs中CD11b、CD29、CD45、CD49d、CD90及CD106的阳性率分别为19.97%、99.96%、46.62%、5.46%、71.22%和10.76%。免疫细胞化学法显示PBMSCs呈CD34阴性,而BrdU阳性率为(34.1±4.3)%。PBMSCs经定向诱导后S-100和P75的阳性率分别是(75.2±4.1)%和(78.9±4.6)%。结论从外周血分离获得的干细胞符合基质干细胞的特性,这些细胞在特定的条件下可诱导分化成为施万细胞。     

周围神经组织工程研究进展

组织工程化的有生物活性的神经替代物称为“人工神经”,当前正被尝试用来替代来源有限的自体神经移植修复周围神经缺损。“人工神经”由支架材料和细胞外基质、种子细胞以及诱导和促进生长的因子等几部分组成。近年来人们使用各种材料,并以不同的模式构建人工神经支架。施万细胞仍是最常使用的种子细胞,但干细胞已开始被用作种子细胞。人们不但研究可促进神经再生的各种因子的作用,也研究了一些抑制因子对神经再生的影响。     

成年大鼠雪旺细胞增殖和纯化研究

目的：探索成年大鼠雪旺细胞的体外培养和纯化方法。方法：取成年大鼠背根神经节采用植块法培养，坐骨神经采用酶消化法分散培养。各分三组，第一组为阿糖胞苷处理组；第二组为免疫溶解处理后加垂体浸出液组；第三组为对照组。以活细胞计数和S-100标记相结合判断雪旺细胞增殖和纯化程度。结果：采用植块培养法至第2周末，雪旺细胞纯度在第一、二和三组分别为90％、97％和50％。分散培养至第18天，雪旺细胞纯度第一组94％，第二组大于97％，第三组80％。细胞数量第一组增加1．4倍，第二组增加5．1倍，第三组增加2．4倍。结论：采用免疫溶解处理后加垂体浸出液方法培养成年大鼠来源的雪旺细胞，可得到数量多纯度高的雪旺细胞。     

周围神经组织工程研究新进展

应用组织工程学原理和技术方法研制组织工程化人工神经是解决周围神经缺损创新的治疗方法。综述了周围神经组织工程种子细胞、支架材料及结构、人工神经4个方面的最新研究进展。     

周围神经组织工程研究新进展

应用组织工程学原理和技术方法研制组织工程化人工神经是解决周围神经缺损创新的治疗方法。综述了周围神经组织工程种子细胞、支架材料及结构、人工神经4个方面的最新研究进展。     

Mechanical enhancement and in vitro biocompatibility of nanofibrous collagen-chitosan scaffolds for tissue engineering

The collagen–chitosan complex with a three-dimensional nanofiber structure was fabricated to mimic native ECM for tissue repair and biomedical applications. Though the three-dimensional hierarchical fibrous structures of collagen–chitosan composites could provide more adequate stimulus to facilitate cell adhesion, migrate and proliferation, and thus have the potential as tissue engineering scaffolding, there are still limitations in their applications due to the insufficient mechanical properties of natural materials. Because poly (vinyl alcohol) (PVA) and thermoplastic polyurethane (TPU) as biocompatible synthetic polymers can offer excellent mechanical properties, they were introduced into the collagen–chitosan composites to fabricate the mixed collagen/chitosan/PVA fibers and a sandwich structure (collagen/chitosan-TPU-collagen/chitosan) of nanofiber in order to enhance the mechanical properties of the nanofibrous collagen–chitosan scaffold. The results showed that the tensile behavior of materials was enhanced to different degrees with the difference of collagen content in the fibers. Besides the Young’s modulus had no obvious changes, both the break strength and the break elongation of materials were heightened after reinforced by PVA. For the collagen–chitosan nanofiber reinforced by TPU, both the break strength and the Young’s modulus of materials were heightened in different degrees with the variety of collagen content in the fibers despite the decrease of the break elongation of materials to some extent. In vitro cell test demonstrated that the materials could provide adequate environment for cell adhesion and proliferation. All these indicated that the reinforced collagen–chitosan nanofiber could be as potential scaffold for tissue engineering according to the different mechanical requirements in clinic.     

Re-utilization of Schwann cells during ingrowth of ventral root afferents in perinatal kittens

Ventral roots in all mammalian species, including humans, contain significant numbers of unmyelinated axons, many of them afferents transmitting nociceptive signals from receptive fields in skin, viscera, muscles and joints. Observations in cats indicate that these afferents do not enter the spinal cord via the ventral root, but rather turn distally and enter the dorsal root. Some unmyelinated axons are postganglionic autonomic efferents that innervate blood vessels of the root and the pia mater. In the feline L7 segment, a substantial proportion of unmyelinated axons are not detectable until late in perinatal development. The mechanisms inducing this late ingrowth, and the recruitment of Schwann cells (indispensable, at this stage, for axonal survival and sustenance), are unknown. We have counted axons and Schwann cells in both ends of the L7 ventral root in young kittens and made the following observations. (1) The total number of axons detectable in the root increased throughout the range of investigated ages. (2) The number of myelinated axons was similar in the root's proximal and distal ends. The increased number of unmyelinated axons with age is thus due to increased numbers of small unmyelinated axons. (3) The number of separated large probably promyelin axons was about the same in the proximal and distal ends of the root. (4) Schwann cells appeared to undergo redistribution, from myelinated to unmyelinated axons. (5) During redistribution of Schwann cells they first appear as aberrant Schwann cells and then become endoneurial X-cells temporarily free of axonal contact. We hypothesize that unmyelinated axons invade the ventral root from its distal end, that this ingrowth is particularly intense during the first postnatal month and that disengaged Schwann cells, eliminated from myelinated motoneuron axons, provide the ingrowing axons with structural and trophic support.     

Preparation and characterization of gelatin/hyaluronic acid cryogels for adipose tissue engineering: In vitro and in vivo studies

Macroporous elastic scaffolds containing gelatin (4% or 10%) and 0.25% hyaluronic acid (HA) were fabricated by cryogelation for application in adipose tissue engineering. These cryogels have interconnected pores (～200 μm), high porosity (>90%) and a high degree of cross-linking (>99%). The higher gelatin concentration reduced the pore size, porosity and swelling ratio of the cryogel but improved its swelling kinetics. Compressive mechanical testing of cryogel samples demonstrated non-linear stress–strain behavior and hysteresis loops during loading–unloading cycles, but total recovery from large strains. The presence of more gelatin increased the elastic modulus, toughness and storage modulus and yielded a cryogel that was highly elastic, with a loss tangent equal to 0.03. Porcine adipose-derived stem cells (ADSCs) were seeded in the cryogel scaffolds to assess their proliferation and differentiation. In vitro studies demonstrated a good proliferation rate and the adipogenic differentiation of the ADSCs in the cryogel scaffolds, as shown by their morphological change from a fibroblast-like shape to a spherical shape, decreased actin cytoskeleton content, growth arrest, secretion of the adipogenesis marker protein leptin, Oil Red O staining for triglycerides and expression of early (LPL and PPARγ) and late (aP2 and leptin) adipogenic marker genes. In vivo studies of ADSCs/cryogel constructs implanted in nude mice and pigs demonstrated adipose tissue and new capillary formation, the expression of PPARγ, leptin and CD31 in immunostained explants, and the continued expression of adipocyte-specific genes. Both the in vitro and in vivo studies indicated that the gelatin/HA cryogel provided a structural and chemical environment that enabled cell attachment and proliferation and supported the biological functions and adipogenesis of the ADSCs.     

施万细胞的层黏连蛋白表达变化及两者关系的初步研究

目的:观察层黏连蛋白(laminin,LN)在坐骨神经发育过程中的表达情况,以及LN和施万细胞(Schwann cells,SCs)的相互关系。方法:取E14、E17、P1和成年SD大鼠坐骨神经,免疫荧光组织化学染色检测LN表达情况;体外培养大鼠来源的SCs,经过外源性LN处理后,免疫荧光细胞化学染色检测LN、nidogen、type IVcollagen等细胞外基质成分的表达,酸性磷酸酶法检测SCs的黏附能力。结果:E17大鼠坐骨神经SCs有LN阳性免疫反应;经过外源性LN处理后,SCs有LN、nidogen、type IV collagen阳性免疫反应,且黏附能力增加。结论:在E17大鼠中,SCs开始分泌LN;LN具有促进SCs合成细胞外基质成分,并在其黏附过程中发挥作用。     

PCL基电纺纤维膜的抑菌、相容及降解性能研究﹡

目的探讨不同材料组成对PCL基电纺纤维膜的表面形貌、亲水性能、抑菌性能、生物相容性、屏蔽和降解性能的影响。方法电纺丝法制备了PCL、PCL/甲硝唑、PCL/明胶/甲硝唑以及PCL/明胶/甲硝唑/醋酸纳米纤维膜,对应P0,P30,PG30及PGH30。扫描电镜(SEM)观察不同膜的表面结构。通过测量载药膜周围抑菌圈的直径来表征膜的抗菌性能。四唑盐比色法(MTT)测试测试细胞毒性。通过兔皮下埋植,伊红苏木素(H&E)染色切片法观察不同膜的组织相容性,降解性能及细胞屏蔽性能。结果甲硝唑的引入赋予膜良好的抑菌性能。明胶引入显著提高了膜的组织相容性及降解速率。电纺液中微量醋酸(0.1%v/v聚合物溶液)能够有效提高电纺液的均一性,从而得到结构及性能稳定的纤维膜。高药物含量及微量醋酸的加入对于膜的细胞及组织相容性均无明显副作用。P0及P30在24周内均能够维持对成纤维细胞的屏蔽作用,PGH30能够维持8周,而PG30的细胞屏蔽期小于8周。结论不同组分对纳米纤维膜的结构和性能具有不同影响。本研究将为设计广泛应用于骨科疾病治疗的膜材料奠定基础。