成年大鼠雪旺细胞的快速扩增

采用接种雪旺细胞的可降解导管修复外周神经损伤是一种有望替代自体神经移植的方法。如何在短期内利用病人少量的神经碎片获得大量雪旺细胞是该方法用于临床的关键。以大鼠坐骨神经为模型,利用雪旺细胞增殖的内在机制,探索出一种快速增殖成年雪旺细胞的方法。采用预变性7d的坐骨神经,用酶消化分离出雪旺细胞,接种在层粘连蛋白包被的培养瓶中,经过7d的培养,获得纯度为96％、细胞密度为600个／mm^2的雪旺细胞,雪旺细胞的纯度和密度明显高于对照的新鲜神经。未使用霍乱毒素、毛喉素等促有丝分裂剂和抑制成纤维细胞的基因毒素,符合临床使用要求。结果表明,可以利用少量的损伤神经碎片在短期内获得大量可用于临床的雪旺细胞。     

成年猴雪旺细胞的在体增殖和体外迁移的研究

为了探讨成年猴雪旺氏细胞的在体增殖和体外迁移的能力,我们对用神经结扎术结扎的A组6只3-13岁雄性恒河猴的腓肠神经进行植块培养,部分细胞培养在聚酯纤维上,2-4周后作抗S-100抗体免疫组化染色和电镜观察;B组2只未做结扎的新生猴腓肠神经培养作为对照.结果显示A组雪旺氏细胞平均在培养的第5天从神经段中迁出,年幼者早于成年猴;细胞在纤维上以螺旋状向前迁移;雪旺氏细胞抗S-100蛋白抗体染色阳性;电镜显示,雪旺氏细胞包卷纤维,但是,未见髓鞘形成.B组神经段培养2周仍无雪旺氏细胞迁出.研究表明,结扎神经使其发生瓦勒氏变性,经植块培养、纯化,能够获得可用于移植的成年猴的雪旺氏细胞.     

新生SD大鼠皮质神经元的体外培养和鉴定

目的：建立高纯度的新生SD大鼠皮质神经元原代培养方法。方法：取24h内的新生SD大鼠皮质,用木瓜酶和DNaseⅠ共同消化,5％胎牛血清终止消化,吹打分离组织获得单细胞悬液,进行细胞计数,用无血清DMEM／F12种植培养,4h后换成用无血清Neurobasal配制的维持培养液继续培养,尼氏小体染色和免疫荧光法鉴定神经元的纯度。结果：培养第10d,神经元胞体饱满,结构清晰完整,光晕明显,折光性强,可见粗长的树突和轴突,相邻细胞形成紧密网状联系,神经元纯度达到96％以上。结论：经改良和优化,无须添加阿糖胞苷抑制胶质细胞的生长即能够获得生长状态良好、高纯度的神经元。     

雪旺氏细胞与同种异体骨支架的体外共培养研究

目的:探讨雪旺细胞(Schwann’s cells,SCs)在同种异体骨支架上的生物相容性,体外构建组织工程骨神经化模型。方法:利用新鲜人体骨骼制备同种异体骨支架材料,检测其物理性能;采用优化方法提取新生SD大鼠坐骨、臂丛神经培养SCs,实验分为三维培养实验组(SCs+同种异体骨)、二维培养对照组(SCs+胶原玻片),S-100抗体免疫荧光染色鉴定SCs纯度;细胞计数法检测两组细胞增殖特点;细胞接种后第3、7天取样,扫描电镜观察。结果:同种异体骨支架具有良好的三维孔隙结构,适宜细胞贴附生长;S-100免疫荧光染色证实SCs纯度95%;扫描电镜检测显示两组SCs均可正常粘附增殖,细胞间排布规律相似,培养早期实验组SCs胞体更加细长,伪足更加明显,随着培养时间的延长表现出较强的迁移能力;细胞增殖检测:两组SCs生长曲线特征基本一致,支架材料对SCs无毒性作用。结论:同种异体骨支架SCs具有良好的生物相容性,其三维立体多孔结构有利于SCs的粘附与迁移,初步构建了体外组织工程骨神经化模型。     

神经生长因子与冻干异体神经桥接大鼠神经缺损的研究

实验采用冻干处理的异体神经与外源性神经生长因子（ＮＧＦ）结合来桥接大鼠的坐骨神经１．０ｃｍ的缺损。用雄性Ｗｉｓｔａｒ大鼠进行的四组实验结果表明：冻干处理的异体神经可降低其抗原性,但处理后并不损害雪旺氏细胞（ＳＣ）基底膜的完整性,在移植后可能成为轴突再生的通道和支架;外源性ＮＧＦ与冻干神经结合形成的复合体,可为神经的再生提供一个较好的微环境,具有成为理想桥接材料的可能性     

雪旺氏细胞与中枢神经再生

雪旺氏细胞是周围神经系统的胶质细胞,具有非常活跃的生理功能,它能表达数种神经营养因子,防止受损神经元胞体的死亡,为轴突再生提供先决条件;SC分泌细胞外基质成分和细胞粘着分子,为轴突提供良好的再生环境,支持轴突再生并引导再生的轴突重新支配靶组织。离体实验表明,SCs本身及其分泌的生物活性物质能支持中枢神经突起的生长,将SCs作为移植材料进行移植能促进视神经、脊髓、隔-海马通路等再生。由于雪旺氏细胞有可能从自体获得,将为中枢神经损伤的修复开辟一条新途径。     

骨髓间充质干细胞与施万细胞联合移植对大鼠周围神经 损伤端侧吻合的修复效果研究

目的:探究骨髓间充质干细胞(MSCs)与施万细胞(SCs)联合移植对大鼠周围神经损伤端侧吻合的修复效果。方法:选取SD雌性大鼠60只均制作成坐骨神经损伤端侧吻合模型,并将其随机分为联合移植组、MSCs组和SCs组,分别对吻合端进行骨髓间充质干细胞与SCs联合移植、MSCs移植、SCs移植。观察分析三组大鼠的神经电生理学指标和腓神经功能指数(PFI)和神经传导速度(NCV)。结果:三组大鼠的PFI和NCV均有所改善,且联合移植组的PFI和NCV均优于其他两组,并随着时间推移损伤坐骨神经功能恢复越来越好。结论:MSCs与SCs均具有促进大鼠周围神经身上修复的功能,且两种细胞联合移植效果更加明显。     

无血清原代培养大鼠海马神经元的形态与膜电位

分离新生Wistar鼠海马,采用添加B27的无血清培养液进行海马神经元原代培养,动态观察海马神经元形态学变化;通过免疫荧光细胞化学法检测神经纤丝(NF)的表达,进行神经元鉴定及纯度计算;采用电位敏感的荧光探针标记神经元,在激光扫描共聚焦显微镜上动态监测去极化剂KCl作用前后膜电位的变化,观察神经元电生理反应。结果表明:此方法培养的大鼠海马神经元可在体外存活20天以上,9～14天为发育最成熟阶段,培养7天神经元纯度达90%。KCl作用于细胞后胞内荧光强度增强,细胞迅速去极化。本培养方法在体外获得高纯度的海马神经元并延长体外存活时间,且显示出神经元的电生理反应特性。     

乳鼠外周神经雪旺氏细胞的体外培养和纯化的研究

本文取５～７天ＳＤ新生大鼠坐骨神经剪碎,用０．２５％胰蛋白酶及０．０３％胶原酶在３７℃消化４５分钟,吹打分散后接种于培养瓶。培养基为含１０％胎牛血清的Ｆ１０。接种后４８小时,加入阿糖胞苷（Ａｒａ－ｃ,Ｓｉｇｍａ）１０－５ｍｏｌ／Ｌ处理,４８小时后换每毫升含神经生长因子（ＮＧＦ）１００μｇ的培养液,刺激雪旺氏细胞（ＳＣ）生长５～７天。此时根据成纤维细胞去除的情况,可再次加入Ａｒａ－Ｃ１～２天或转为无血清培养基一周。这种抗有丝分裂法结合无血清培养法,既能有效去除成纤维细胞,又能减少对ＳＣ的损伤,获得的ＳＣ纯净度可达９８％。     

神经脊来源许旺细胞的体外培养研究

目的:探讨坐骨神经中神经脊来源的许旺细胞所占的比率。方法:将Wnt1-Cre+/-与Rosa-EGFP+/-小鼠杂交,获取Wnt1/EGFP小鼠,其所有起源于神经脊的细胞都有EGFP蛋白表达。取其坐骨神经,经过消化分离纯化,获得许旺细胞。进行抗GFP免疫荧光染色和流式分析的检测。结果:根据P1代许旺细胞的形态学观察,其纯度约为60%。抗GFP免疫荧光染色显示,并非所有的许旺细胞均呈阳性。P3代许旺细胞的纯度约为99%,流式细胞术分析显示约65%左右的GFP阳性率。结论:小鼠坐骨神经中的许旺细胞在体外培养提纯后,神经脊起源的许旺细胞占总许旺细胞的比例约为65%。     

人脐带干细胞来源的外泌体促进体外培养雪旺细胞迁移的研究

目的:外泌体是活细胞分泌的来源于多囊泡体的膜性囊泡,其主要作用包括携带与运输。雪旺细胞是周围神经再生中非常优秀的种子细胞,但其迁移能力较差,影响修复效果。本文旨在探讨外泌体和雪旺细胞共培养是否可以促进雪旺细胞迁移。方法:本实验通过分离纯化人脐带干细胞外泌体和大鼠坐骨神经雪旺细胞并鉴定,随后将其共培养于Transwell小室观察雪旺细胞迁移率。结果:通过人脐带干细胞超高速离心法得到的外泌体高表达干细胞标志物CD44(92.2±3.6%)、CD73(99.1±0.6%),并且低表达单核细胞表面抗原CD14(0.5±0.06%)以及造血干细胞表面抗原CD34(0.4±0.07%),外泌体鉴定高表达CD81和CD9;雪旺细胞培养鉴定纯度达(92.3±2.7)%;均符合实验要求。通过Transwell小室实验发现外泌体可以明显促进雪旺细胞的迁移,并且具有一定剂量关系。结论:外泌体可以提高雪旺细胞的迁移能力,从而使雪旺细胞在组织工程领域中的应用产生巨大突破。     

A method for obtaining Schwann cell cultures from adult rabbit nerve based on "in vitro" pre-degeneration and neuregulin treatment

Schwann cells (SCs) are basic elements for cell therapy and tissue engineering in the central and peripheral nervous system. Therefore, the development of a reliable method to obtain SC cultures is required. For possible therapeutic applications the cultures need to produce a sufficiently large number of SCs with a high level of purity in a relatively short period of time. To increase SC yield and purity we pre-degenerated pieces of 1-2 mm of adult rabbit sciatic nerves by incubating them for seven days in Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum, penicillin/streptomycin and NRG1-β1. Following pre-degeneration the nerve pieces were dissociated and then cultured for 6 or 15 days in the same culture medium. After 6 days of culture we obtained around 9.5x103 cells/mg with approximately 94% SCs (S-100 positive) purity. After 15 days of culture the yield was about 80x103 cells/mg and the purity was approximately 75%. Pre-degeneration and subsequent culture of small pieces of adult nerve with NRG1-β1 supplemented medium increased the number of SCs and restricted the overgrowth of fibroblast-like cells.     

低温保存许旺细胞对周围神经再生的作用

目的：比较原代培养许旺细胞（Schwann cells,SCs)和冷冻保存的SCs移植对损伤后坐骨神经再生的作用。方法：原代培养和液氮保存的SCs分别移植到桥接缺损坐骨神经的硅胶管内。在移植后不同时间（第6和8周末）,硅胶管远端神经干内注射HRP,逆行追踪背根神经节和脊髓前角的标记神经元数量;测量再生神经纤维的复合动作电位传导速度;电镜观察再生神经纤维的髓鞘形成。结果：原代培养和冷冻保存SCs在移植后不同时间其背根神经节和脊髓前角神经元HRP标记细胞数量、再生神经纤维的复合动作电位传导速度基本一致,再生神经纤维髓鞘的形成未见明显差别。结论：冷冻保存的SCs仍具有促进损伤后周围神经再生的能力。     

Differentiated human adipose-derived stromal cells exhibit the phenotypic and functional characteristics of mature Schwann cells through a modified approach

Background aimsTissue engineering technology is a promising therapeutic strategy in peripheral nerve injury. Schwann cells (SCs) are deemed to be a vital component of cell-based nerve regeneration therapies. Many methods for producing SC-like cells derived from adipose-derived stromal cells (ADSCs) have been explored, but their phenotypic and functional characteristics remain unsatisfactory.MethodsWe investigated whether human ADSCs can be induced to differentiate into mature and stable SC-like cells with the addition of insulin, progestero``ne and glucocorticoids. The phenotypic and functional characteristics of new differentiated ADSCs (modified SC-like cells) were evaluated by real-time quantitative polymerase chain reaction, enzyme-linked immunosorbent assay and immunocytochemistry in vitro. Cells loaded into collagen sponge biomaterials were implanted around transected sciatic nerves with a 10-mm gap in vivo. The axon regrowth and functional recovery of the regenerated nerves were assessed by immunohistochemistry and Walking footprint analysis.ResultsAfter differentiation induction, the modified SC-like cells showed significantly up-regulated levels of S100B and P0 and enhanced proliferative and migratory capacities. In addition, the modified SC-like cells showed increased secretion of neurotrophic factors, and their functional characteristics were maintained for more than 3 weeks after removing the induction reagents. The modified SC-like cells exhibited significantly enhanced axon regrowth, myelination and functional recovery after sciatic nerve injury.ConclusionsOverall, the results suggest that this modified induction method can induce human ADSCs to differentiate into cells with the molecular and functional properties of mature SCs and increase the promotion of peripheral nerve regeneration.     

A new protocol for cultivation of predegenerated adult rat Schwann cells

The purpose of this study was to optimize the methodology of cultivation of predegenerated Schwann cells (SCs). SCs were isolated from 7-day-predegenerated sciatic nerves of adult rats. We applied commercially available culture medium for cultivation of endothelial cells endothelial cell culture medium (EBM-2) instead of Dulbecco’s Modified Eagle’s Medium commonly used to culture adult Schwann cells. Additionally, cell culture medium was supplemented with factors specifically supporting SCs growth as: bovine pituitary extract (5 μg/ml), heregulin (40 ng/ml) and insulin (2.5 ng/ml). Similarly to the reports of others authors, we did not observe any beneficial effects of Forskolin application, so we didn’t supplement our medium with it. Cell culture purity was determined by counting the ratio of GFAP, N-Cadherin and NGFR p75-positive cells to total number of cells. About 94–97 % of cells were confirmed as Schwann cells. As a result, we obtained sufficient number and purity of Schwann cells to be applied in different experimental models in rats. EBM-2 medium coated with fibronectin was the best for cultivation of adult rat Schwann cells.     

Deletion of p75NTR impairs regeneration of peripheral nerves in mice

AimsAfter peripheral nerve injury, p75NTR was upregulated in Schwann cells of the Wallerian degenerative nerves and in motor neurons but down-regulated in the injured sensory neurons. As p75NTR in neurons mediates signals of both neurotrophins and inhibitory factors, it is regarded as a therapeutic target for the treatment of neurodegeneration. However, its physiological function in the nerve regeneration is not fully understood. In the present study, we aimed to examine the role of p75NTR in the regeneration of peripheral nerves.Main methodsIn p75NTR knockout mice (exon III deletion), the sciatic nerves and facial nerves on one side were crushed and regenerating neurons in the facial nuclei and in the dorsal root ganglia were labelled by Fast Blue. The regenerating fibres in the sciatic nerve were also labelled by an anterograde tracer and by immunohistochemistry.Key findingsThe results showed that the axonal growth of injured axons in the sciatic nerve of p75NTR mutant mice was significantly retarded. The number of regenerated neurons in the dorsal root ganglia and in the facial nuclei in p75NTR mutant mice was significantly reduced. Immunohistochemical staining of regenerating axons also showed the reduction in nerve regeneration in p75NTR mutant mice.SignificanceOur data suggest that p75NTR plays an important role in the regeneration of injured peripheral nerves.     

小鼠胃 Cajal 间质细胞分离与培养实验方法探讨

目的:尝试优化体外培养Balb/c小鼠胃Cajal间质细胞(interstitial cells of Cajal,ICC)的实验方法,为深入探索该细胞的生理病理作用机制提供基础。方法:无菌条件下取出小鼠胃组织,使用酶解法消化分离细胞,将细胞悬液接种于含有SCF(干细胞因子)的M199培养基中培养,并进行传代。倒置显微镜下观察不同时间段细胞生长状态,采用ICC特异性标志物c-Kit(酪氨酸激酶受体)进行免疫荧光鉴定。结果:细胞培养24 h后基本已贴壁,呈梭形或三角形,有短突起;72 h后细胞胞体变大,突起伸长;5 d后,细胞之间通过突起彼此相互连接,开始形成网状结构;传代后细胞依然保持其固有特征。免疫荧光鉴定可见细胞c-Kit抗体荧光染色阳性。结论:使用酶解法成功分离细胞,细胞数量较多但不增殖,传代后可见细胞纯度较好,稳定培养3周以上后细胞形态逐渐发生变化并开始凋亡。     

周围神经损伤后神经组织中Par-3蛋白表达和分布的变化

目的观察极性蛋白Par-3在损伤后神经组织中的表达和分布,探讨Par-3蛋白在周围神经损伤后髓鞘再生中的作用。方法 32只Sprague Dawley大鼠随机分为正常对照组、损伤组(坐骨神经损伤后第1、2、4、8周)。制备坐骨神经挤压伤模型,分别于损伤后各时间点,采用免疫组织化学法检测坐骨神经损伤远端Par-3蛋白的表达和分布。结果正常大鼠坐骨神经组织中即存在Par-3蛋白,但表达量少,且仅分布于Schwann细胞核内。坐骨神经损伤后,Par-3蛋白的表达和分布发生变化。损伤后1周,Par-3蛋白表达开始升高,Par-3散在分布于Schwann细胞核和细胞浆内。损伤后2周,神经组织中的Par-3蛋白达峰值,在Schwann细胞浆内呈不对称性分布似包绕轴突,呈新月形或C形。损伤后4周和8周,Par-3蛋白表达显著降低,神经组织中Par-3蛋白主要分布于Schwann细胞核内,胞浆内很少。结果 极性蛋白Par-3可能参与周围神经损伤后Schwann细胞的髓鞘再生。