MicroRNA-21促进大鼠雪旺细胞增殖的实验研究

目的:探讨神经损伤后microRNA-21(miR-21)促进雪旺细胞增殖的分子机制。方法:通过实时荧光定量PCR检测miR-21的表达。通过脂质体介导将人工合成的miR-21模拟物(mimic)及其阴性对照转染大鼠雪旺细胞,CCK-8法检测转染miR-21的雪旺细胞的增殖。流式细胞术分析miR-21对雪旺细胞细胞周期的影响。使用Western blotting实验分析转化生长因子β诱导蛋白(TGFBI)和cyclin D1的表达水平。结果:miR-21在损伤模型组中的表达分别为假手术组和正常神经组织的(7.87±0.75)和(7.75±0.80)倍(P0.01)。miR-21 mimic组miR-21的表达分别为对照组和空白组的(2.21±0.14)和(2.29±0.21)倍(P0.05)。转染48 h后miR-21 mimic组CCK-8实验的A450值较阴性对照组和空白对照组增高(P0.05)。实验组细胞增殖指数高于阴性对照组和空白对照组(P0.01)。同时与对照组相比,转染miR-21后48 h实验组细胞TGFBI明显降低,cyclin D1表达增多(P0.05)。结论:miR-21可促进雪旺细胞增殖,其机制可能与其下调TGFBI的表达有关。     

miR-21在周围神经损伤时调控雪旺细胞凋亡

目的:探讨周围神经损伤时,微小RNA-21(microRNA-21,miR-21)与雪旺细胞(SC)凋亡的关系及相关分子机制。方法:采用实时荧光定量PCR(real-time PCR)检测动物模型中miR-21以及人第10号染色体缺失的磷酸酶及张力蛋白同源基因(phosphatase and tensin homologue deleted on chromosome 10,PTEN)的表达情况;通过将miR-21类似物(miR-21-mimic)、miR-21抑制物(miR-21-inhibitor)和阴性对照miRNA(negative control miRNA,NC-miRNA)转染入RSC96细胞中,构建出过表达miR-21的雪旺细胞株(MI-SC)、抑制miR-21表达的雪旺细胞株(IN-SC)及表达对照miRNA的雪旺细胞株(NC-SC);采用流式细胞仪检测3组细胞的凋亡情况;real-time PCR检测3组细胞中miR-21以及PTEN的表达情况;Western blot检测3组细胞中PTEN及凋亡相关蛋白激活型caspase-3(cleaved caspase-3)的表达情况。结果:神经损伤组miR-21的表达量与对照组相比明显升高,神经损伤组PTEN mRNA的表达水平与对照组相比明显降低;与NC-SC相比,上调miR-21组细胞的凋亡比例减少,PTEN mRNA及蛋白的表达水平降低,cleaved caspase-3的表达水平降低,下调miR-21组细胞的凋亡比例增加,PTEN mRNA及蛋白的表达水平升高,cleaved caspase-3的表达水平升高(P0.05)。结论:miR-21可能通过下调PTEN的表达抑制雪旺细胞的凋亡,从而可能在周围神经的损伤修复中发挥作用。     

雪旺细胞促进周围神经再生机制的研究进展

雪旺细胞(SCs)具有强大的可塑性。周围神经损伤后,雪旺细胞去分化为修复表型,主导了修复过程。c-Jun氨基末端激酶、有丝分裂原激活的蛋白激酶(MAPK)等多种信号通路和转录调节因子参与调控雪旺细胞介导的修复程序。本综述讨论了雪旺细胞促进周围神经再生的主要信号通路与转录调节因子。     

NT-3基因修饰施万细胞促进神经干细胞体外分化为神经元样细胞

目的：观测神经营养素-3（NT-3）基因修饰施万细胞(SCs)对神经干细胞体外分化为神经元样细胞的影响。方法：神经干细胞(NSCs)分别与NT-3基因修饰SCs（NT-3-SCs）、LacZ基因基因修饰SCs（LacZ-SCs）和SCs在体外共培养，7d后用免疫组化方法观测NSCs的分化并计算其中神经元样细胞的分化率。结果：NSCs在体外可分化为神经元样细胞（NF阳性）和神经胶质样细胞（GFAP阳性），与未基因修饰的SCs相比，NT-3-SCs能更有效地提高神经元样细胞的分化率，而LacZ-SCs与SCs没有明显区别。结论：NT-3-SCs能促进NSCs向神经元样细胞分化。     

MicroRNA和雪旺细胞自噬与周围神经再生修复的研究进展

正周围神经损伤会使神经支配区域的运动、感觉机能下降和缺失,严重影响生产和生活质量。现代显微外科技术的应用已大大提高了修复效果,但由于周围神经的特殊结构和功能,目前的修复技术对于神经功能的恢复效果仍然有限,这引起了研究者们对于周围神经损伤后再生修复机制的探索。周围神经损伤后,损伤处残存雪旺细胞(Schwann cells)的数量和增殖分化能力是影响神经再生修复的关键因素[1]。在周围神经损伤后的雪旺细胞内发现大量溶     

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。     

过表达NT-3SCs和过表达TrkC NSCs移植减少脊髓损伤处瘢痕形成和促进神经再生

目的 探讨神经营养素-3(NT-3)基因修饰雪旺细胞(SCs)和NT-3受体(TrkC)基因修饰神经干细胞(NSCs)联合移植能否减少大鼠脊髓全横断损伤处的瘢痕组织,并促进下行神经纤维再生.方法 用NT-3基因的重组腺病毒(Ad-NT-3)感染培养的SCs(NT-3-SCs),同时用含TrkC基因的重组腺病毒(Ad -...     

Fas,FasL, and cleaved caspases 8 and 3 in glioblastomas: A tissue microarray-based study

This investigation analyzed the immunoexpression of FasL, Fas, cleaved caspase-8, and cleaved caspase-3 in glioblastomas. Formalin-fixed and paraffin-embedded glioblastoma tissues and control brain tissues from 97 patients were analyzed by tissue microarrays and immunohistochemistry. Patients with glioblastomas that were negative or weakly stained (<50% of cells positive) for cleaved caspase-8 had worse cancer-specific overall survival (median = 8.5 months) than did patients with tumors that highly expressed cleaved caspase-8 (median = 11.7 months; P = 0.0325), independent of clinical variables. There was no association of other markers with survival, treatment, sex, age, tumor size, and primary site. Among the tumors, there were reasonable to good positive correlations between the expression of FasL and Fas (r = 0.47) and between Fas and cleaved caspase-8 (r = 0.41), and there were poor positive correlations between Fas and cleaved caspase-3 (r = 0.26), FasL and cleaved caspase-8 (r = 0.22), and cleaved caspase-8 and -3 (r = 0.31). Our results suggest that Fas-Fas-ligand signal transduction could be inhibited, especially at the stage of caspase-8 activation, thereby establishing a major mechanism for evasion of apoptosis by these tumors. The absence or low expression of cleaved caspase-8 in the tumors was a negative prognostic indicator for patient survival.     

ABCA2蛋白在大鼠面神经组织内雪旺细胞成熟过程中的表达

目的本研究通过检测面神经发育过程中ATP结合盒式转运蛋A2(ABCA2)的表达方式,探讨ABCA2蛋白在周围神经系统髓鞘形成过程中的作用。方法应用多重免疫荧光方法检测发育过程中面神经组织内ABCA2和特异雪旺细胞标志抗体的表达。结果 ABCA2蛋白由出生后第8d在成髓鞘雪旺细胞特异抗体-锌指端转录因子Krox20和S100阳性的细胞中开始少量表达;在出生后第14d,近50%髓鞘化的该类细胞中可检测到ABCA2蛋白;在成熟期,所有该类细胞中都可检测到ABCA2蛋白。结论 ABCA2在生后雪旺细胞的表达随髓鞘化的发育成熟而增加。     

Re-activation of atrophic motor Schwann cells after hypoglossal-facial nerve anastomosis

Facial nerve lesions are common in humans and often require surgical intervention. If repair is delayed, reinnervation can be facilitated by transposing the freshly cut hypoglossal nerve end-to-end directly to the distal facial nerve, allowing for uncompromised hypoglossal axons to reinnervate the denervated facial musculature (hypoglossal-facial anastomosis, HFA). Schwann cells (SCs) in the distal nerve stump have an important function in promoting axonal regeneration by expressing multiple regeneration-associated proteins. Chronically denervated SCs cease to express those factors, but it is unknown whether they can be reactivated by fresh axonal sprouts and regain part of their function. We evaluated SC function and viability in distal facial nerve stump of rats at various time points after chronic denervation as well as following immediate or delayed HFA by assessing their expression of growth-associated protein 43 kDa (GAP-43) and the neuregulin receptors erbB2 and erbB4. Our results show that maximal upregulation of those factors in denervated SCs occurred a few weeks after nerve transection, indicating that a short period of denervation might even be beneficial before nerve repair. Motor SCs denervated for 32 weeks had downregulated their activity and ceased to express the regeneration-associated factors. SCs immediately re-expressed GAP-43, erbB2, and erbB4 following contact with fresh hypoglossal motor axons, demonstrating they are competent to promote regeneration even after long-term denervation.     

胃肠道间质瘤中LMTK3、caspase 3及caspase 7的表达意义

目的:探讨胃肠道间质瘤(GIST)中狐猴酪氨酸激酶3(LMTK3)、半胱氨酸天冬氨酸蛋白酶3(caspase 3)及半胱氨酸天冬氨酸蛋白酶7(caspase 7)蛋白的表达与该肿瘤临床病理特征的关系。方法:运用免疫组化EnVision二步法检测102例GIST患者肿瘤组织和相应正常组织中LMTK3、caspase 3及caspase 7蛋白的表达情况,分析LMTK3与caspase 3、caspase 7蛋白表达之间的关系,从而探讨3种蛋白表达与GIST临床病理特征之间的关系。结果:GIST肿瘤组织中LMTK3蛋白表达高于相应正常组织,差异具有统计学意义(P<0.05);LMTK3蛋白表达在不同肿瘤大小、核分裂计数及危险度分组之间比较差异有统计学意义(P<0.05),在不同患者性别、年龄及肿瘤发生部位分组之间差异无统计学意义(P>0.05);肿瘤组织中caspase 3和caspase 7蛋白表达均低于相应正常组织,差异具有统计学意义(P<0.05);caspase 3与caspase 7蛋白表达在患者不同性别、年龄、肿瘤发生部位、肿瘤大小、核分裂像计数及危险度...     

体外培养的大鼠Schwann细胞神经生长因子和脑源性神经营养因子的表达

目的：研究体外培养的Schwann细胞神经生长因子(NGF)和脑源性神经营养因子(BDNF)的表达。方法：用细胞培养技术，培养新生SD大鼠Schwann细胞，传代后用ABC免疫细胞化学法检测Schwann细胞的NGF、BDNF反应。结果：体外培养的Schwann细胞立体感强、折光性好，细胞为梭形、椭圆形或三角形，NGF、BDNF免疫反应为阳性。结论：体外培养的Schwann细胞能表达NGF、BDNF。     

组织工程化神经修复周围神经创伤的应用

背景：近年来,随着生物工程技术以及组织工程化神经的发展给周围神经缺损的治疗带来了新的希望,已逐渐成为研究的焦点。 目的：从种子细胞、生物材料以及构建周围神经组织技术3个方面综述组织工程方法修复周围神经损伤的新进展。 方法：由第一作者在2013年7月应用计算机检索PubMed 数据库及CNKI 数据库,英文关键词为“tissue engineering,peripheral nerves,nerve injuries,stem cells,schwann cells,scaffold,growth factor”,中文关键词为“组织工程,周围神经,神经损伤,干细胞,许旺细胞,支架,生长因子”。选择内容与神经组织工程、周围神经损伤修复相关的文章,同一领域文献则选择近期发表或发表在权威杂志文章,共纳入63篇文献。 结果与结论：现阶段组织工程方法修复周围神经损伤的研究虽已取得很大进展,但大多停留于实验探索阶段。将组织工程神经应用于临床尚存下列问题亟待解决：①种子细胞来源及伦理。②细胞扩增后移植的免疫排斥。③移植细胞稳定性问题及致瘤性。④神经支架材料的降解速度、最佳孔隙率、导管厚度、形状等。⑤体外神经构建后移植修复时机。⑥各种神经生物因子的局部释放与调控等等。随着科技的发展,期待上述问题的解决,从而使得众多临床神经损伤患者受益。     

自体神经桥接陈旧性周围神经缺损后雪旺细胞的组织学观察

目的:探索长时间失神经损伤后的雪旺细胞的促神经再生功能。方法:切除成年雌性SD大鼠左侧坐骨神经4 mm,分别饲养3、6个月后,修剪近、远侧神经断端制成不同持续时间的大鼠坐骨神经10 mm陈旧性缺损模型。实验组切取对侧正常坐骨神经桥接缺损,对照组缺损模型制备完成后不予任何修复。各组动物术后再饲养3个月取材,标本进行神经三色染色、免疫荧光染色,电镜等组织学方法观察。结果:各组损伤近端神经结构无明显差异,实验组桥接物段可观察到粗细不等的有髓神经纤维。电镜下,实验组远侧断端皆可观察到髓鞘形成良好的有髓神经纤维和存活的雪旺细胞。结论:长时间失神经损伤的雪旺细胞仍能在一定时间内存活并维持其促神经再生功能。     

神经营养因子3纳米微球载体基因工程转染许旺细胞

背景：纳米微球基因转染技术携带或增强种植细胞可持续稳定延长其释放,保持其活性和作用时间。 目的：观察纳米微球载体转染神经营养因子3基因修饰的许旺细胞对坐骨神经缺损的促进和修复作用。 方法：采用纳米微球载体将神经营养因子3基因转染入体外培养的新生Wistar大鼠许旺细胞。取Wistar成年大鼠80只制作坐骨神经缺损模型,将4种不同的移植物注入细胞外基质凝胶-聚乳酸聚羟基乙酸共聚物管桥接管内：空白对照组未注入其他任何物质,细胞组注入许旺细胞,基因组注入神经营养因子3基因,实验组注入神经营养因子3基因修饰的许旺细胞。 结果与结论：术后坐骨神经及肌纤维横截面积染色比较,实验组优于细胞组、基因组(P < 0.01),细胞组、基因组均优于空白对照组(P < 0.01),细胞组、基因组组间比较差异无显著性意义(P > 0.05)。表明神经营养因子3基因转染许旺细胞移植,可相互促进,再造神经再生的微环境,促进并修复缺损坐骨神经缺损。     

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。     

A complementary density gradient of zwitterionic polymer brushes and NCAM peptides for selectively controlling directional migration of Schwann cells

Selective enhancement of directional migration of Schwann cells (SCs) over fibroblasts (FIBs) plays a significant role in peripheral nerve regeneration, because this behavior facilitates neuron repair and avoids fibrosis. Herein a complementary density gradient of poly(3-dimethyl-methacryloyloxyethyl ammonium propane sulfonate) (PDMAPS, a zwitterionic polymer with antifouling property) and KHIFSDDSSE peptide (KHI, derived from neural cell adhesion molecule NCAM which mediates cell–cell adhesion) was fabricated. The gradient was visualized by fluorescent labeling, and further characterized by X-ray photoelectron spectrometry (XPS) and quartz crystal microbalance with dissipation (QCM-d). The SCs exhibited preferential orientation and enhanced directional migration on the gradient surface toward the region of lower PDMAPS density and higher KHI peptide density, while FIBs showed random migration. Moreover, the migration rate of the SCs was significantly enhanced to 2 folds, whereas that of the FIBs was reduced to 60% compared to their natural state on glass, leading to a faster migration rate of SCs than FIBs. The success of the complementary gradient relies on the appropriate interplay between the PDMAPS brushes and the cell-specific ligands, enabling the selective guidance of SCs migration.     

坐骨神经瓦勒变性大鼠许旺细胞生物学特性及分泌功能变化

背景：研究表明外周神经损伤后,许旺细胞在基底膜管内形成Bunger带,引导再生轴突延伸,但具体作用机制目前尚不清楚。 目的：观察大鼠坐骨神经损伤后瓦勒变性对许旺细胞生物学特性及分泌功能的影响。 方法：建立大鼠坐骨神经横切模型,分为坐骨神经瓦勒变性组(坐骨神经横断组)和手术对照组。采用神经段单酶消化法分离培养许旺细胞,光镜下观察细胞形态变化,S-100免疫荧光鉴定。取第1代许旺细胞,利用计数法绘制14 d内许旺细胞的生长曲线,MTT法检测14 d内许旺细胞增殖活性,酸性磷酸酶法检测许旺细胞黏附能力,ELISA法检测神经生长因子浓度。 结果与结论：坐骨神经段培养第14天,坐骨神经横断组神经段边缘可见大量许旺细胞,呈线形排列;手术对照组许旺细胞数量少,呈散在分布,两组许旺细胞S-100均呈阳性表达。许旺细胞传代培养第3天,两组许旺细胞均进入对数增长期,随时间延长,细胞数及细胞增殖吸光度值均呈上升趋势,坐骨神经横断组细胞数及增殖吸光度值明显高于手术对照组(P < 0.05);坐骨神经横断组许旺细胞黏附能力明显高于手术对照组(P < 0.05);ELISA法检测示,坐骨神经横断组神经生长因子浓度在培养第4,6,8,10,12,14天时均高于手术对照组(P < 0.05)。结果表明大鼠坐骨神经损伤后两三周,瓦勒变性对许旺细胞生物学功能具有显著影响,可诱导许旺细胞幼稚化,促使许旺细胞在短期内迅速分裂增殖,并分泌大量神经营养因子及细胞外黏附成分,为再生轴突的延伸提供适宜的神经微环境;并增加细胞黏附能力,为外周神经损伤修复提供适宜的神经微环境。 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程全文链接：     

Adipose-derived stem cells promote peripheral nerve repair

Introduction

Recent evidence suggests that the implantation of bone marrow-derived mesenchymal stem cells improves peripheral nerve regeneration. In this study we aimed to investigate whether adipose-derived stem cells (ADSCs) can be used for peripheral nerve repair.

Material and methods

In a rat model, nerve regeneration was evaluated across a 15 mm lesion in the sciatic nerve by using an acellular nerve injected with allogenic ADSCs. The walking behaviour of rats was measured by footprint analysis, and electrophysiological analysis and histological examination were performed to evaluate the efficacy of nerve regeneration.

Results

Cultured ADSCs became morphologically homogeneous with a bipolar, spindle-like shape after ex vivo expansion. Implantation of ADSCs into the rat models led to (i) improved walking behaviour as measured by footprint analysis, (ii) increased conservation of muscle-mass ratio of gastrocnemius and soleus muscles, (iii) increased nerve conduction velocity, and (iv) increased number of myelinated fibres within the graft.

Conclusions

Adipose-derived stem cells could promote peripheral nerve repair in a rat model. Although the detailed mechanism by which ADSCs promote peripheral nerve regeneration is being investigated in our lab, our results suggest that ADSCs transplantation represents a powerful therapeutic approach for peripheral nerve injury.     

GFP共表达检测重组型Caspase-3对HeLa细胞凋亡的诱导作用

目的探讨由野生型人caspase-3大小亚基颠倒构建的重组型caspase-3的促细胞凋亡活性。方法用RT-PCR法获得人caspase-3基因。通过重组PCR进行改造,构建小亚基位于大亚基之前的两种重组型caspase-3基因,它们所对应的蛋白质的N端,分别带有或没有其自身识别的四肽序列。将上述基因克隆入绿色荧光蛋白(GFP)表达载体pIRES2-EGFP中,转染人HeLa细胞,在荧光显微镜和电镜下观察转染细胞的形态和结构。结果成功地获得了野生型caspase-3基因及两种重组型caspase-3基因。构建了重组型caspase-3基因的表达载体。转染HeLa细胞后,重组型caspase-3基因在细胞中得到表达,随后引起细胞荧光强度下降,生长状况变差甚至死亡。电镜观察显示,许多细胞呈现凋亡的典型特征。结论重组型caspase-3可促进HeLa细胞的凋亡。