Adhesion and growth of human Schwann cells on trimethylene carbonate (co)polymers

Seeding of artificial nerve grafts with Schwann cells is a promising strategy for bridging large nerve defects. The aim of the present study was to evaluate the adhesion and growth of human Schwann cells (HSCs) on 1,3-trimethylene carbonate (TMC) and epsilon-caprolactone copolymers, with the final goal of using these materials in the development of an artificial nerve graft. The adhesion, proliferation, and morphology of HSCs on copolymers containing 10 and 82 mol % of TMC and on the parent homopolymers were investigated. HSCs adhered faster and in greater numbers on the copolymer with 82 mol % of TMC and on the TMC homopolymer compared with the other (co)polymers. On all polymer films, cell adhesion was lower than on gelatin (positive control). Despite differences in cell adhesion, cells displayed exponential growth on all tested surfaces, with similar growth rates. Cell numbers doubled approximately every 3 days on all substrates. When the polymer films were coated with fibronectin, no significant differences in cell adhesion and proliferation were observed between coated polymer surfaces and gelatin. The results indicate that all tested materials support the adhesion and proliferation of HSCs and can in principle be used for the preparation of flexible and slowly degrading nerve guides.     

Adhesion and proliferation of human endothelial cells on photochemically modified polytetrafluoroethylene

We studied the adhesion and proliferation of human endothelial cells on photochemically modified polytetrafluoroethylene samples. The polymer surfaces were modified by exposure to the ultraviolet light of a Xe₂^*-excimer lamp at a wavelength of 172 nm in an ammonia atmosphere. Treatment times were between 10 and 20 min. The endothelial cell density was determined 1, 3 and 8 days after seeding by image analysis. Surface modification of the samples resulted in a significant increase in the number of adhering cells and in the formation of a confluent cell layer after 3–8 days. The results were comparable than those obtained on polystyrene Petri dishes, which are used as standard substrates in cell cultivation. Thus modified PTFE appears to be a promising material for the fabrication of artificial vascular prostheses coated with endothelial cells.     

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。     

利用贴壁特点提取和纯化乳鼠雪旺细胞

目的主要利用乳鼠的雪旺细胞比成纤维细胞贴壁快的特点,介绍简单而快速提取和纯化乳鼠雪旺细胞的方法。方法取3天SD大鼠双侧坐骨神经,在解剖镜下剥离去除神经外膜,剪碎后用Ⅰ型胶原酶消化,差速法去除成纤维细胞,分小皿培养进一步纯化细胞;光镜下计算细胞纯度;S-100免疫荧光鉴定细胞性质。结果获取的雪旺细胞纯度大于96%;S-100免疫荧光鉴定细胞为阳性。结论本方法可以简单而快速地获得高纯度雪旺细胞。     

Adhesion and proliferation of human dermal fibroblasts on collagen matrix

The purpose of this study was to evaluate adhesion and growth of human dermal fibroblasts on a 0.150 mm-thick matrix of reconstituted collagen isolated from horse tendon. Collagen was extracted and polymerized according to the standard procedures (Opocrin, Corlo, Modena, Italy). By light microscopy, the bottom surface of the matrix appeared linear and compact, whereas the superficial one was indented and less homogeneous. By scanning electron microscopy, the collagen fibrils had different diameters and the great majority of them was oriented parallel to the surface of the gel. By transmission electron microscopy, collagen fibrils showed the typical banding. Human dermal fibroblasts were seeded on the collagen matrix, previously equilibrated in growth medium. Fibroblast proliferation stopped in the second week and was always significantly lower than that of the same cell strain seeded on plastic and cultured in parallel. By light microscopy, after six days culture, cells formed a confluent multilayer on the surface of the gel. By scanning and transmission electron microscopy, fibroblasts appeared flat and adherent to the matrix. Contacts of cells among themselves and with the collagen fibrils were observed. Fibroblasts never moved into the collagen gel. In conclusion, human dermal fibroblasts can be grown in a three-dimensional matrix made by horse tendon that, on the other hand, seems to condition their proliferation rate.     

Adhesion and proliferation of corneal epithelial cells on self-assembled monolayers

The effect of surface chemistry on the proliferation and adhesion of SV-40 human corneal epithelial cells was investigated. The surface chemistry of substrates was controlled by the deposition of self-assembled monolayers (SAMs) terminated with the following functional groups: -CF3, -CH3, -CO(2)H, and -NH(2). SAMs of alkanethiols on gold and of alkylsiloxanes on SiOx were included in the study. Comparisons are made between different types and functionalities of SAMs and between SAM-covered substrates and tissue culture polystyrene. Adhesion assays were performed after incubation of the cells for 1 h in 10% fetal bovine serum and in serum-free conditions. The cellular response was found to be a function of surface chemistry and the presence of exogenous proteins. The number of cells that adhered to most of the SAMs in 10% serum and in serum-free conditions was not significantly different from the number of cells that adhered to TCPS. Proliferation assays were carried out in 10% serum and in 0.5% serum. Cell behavior was influenced by surface chemistry but did not deviate significantly from the behavior on TCPS for most of the SAMs. Serum level did not play a major role in cell proliferation. Our data establish the expected behaviors for a corneal epithelial cell line under defined conditions on specific surfaces.     

不同浓度人羊膜匀浆上清液培养大鼠许旺细胞的增殖

背景：许旺细胞是周围神经修复过程的重要细胞,而研究发现人羊膜细胞分泌的多种细胞因子能够促进许旺细胞增殖。 目的：观察不同浓度人羊膜匀浆上清液对鼠许旺细胞(RSC96)生长的影响。 方法：使用含体积分数20%胎牛血清的高糖DMEM培养基原代培养RSC96细胞株,传代至第2代用于实验研究。根据人羊膜匀浆上清液在培养基中的不同体积分数(0,10,15,20,25%)分组。 结果与结论：人羊膜匀浆上清液的总蛋白浓度为675 mg/L,表皮生长因子、碱性成纤维生长因子和血管内皮生长因子浓度分别为(470.625±2.546),(4.121±0.026)和(0.172±0.002) ng/L。在培养第1-7天,10%和15%人羊膜匀浆上清液组的增殖率大于20%和25%人羊膜匀浆上清液组(P < 0.05);10%、15%人羊膜匀浆上清液组显示出促进细胞增殖的作用,而20%、25%人羊膜匀浆上清液组显示出抑制细胞增殖的作用;各实验组的细胞活力与对照组接近(P > 0.05)。提示人羊膜匀浆上清液低浓度时(10%和15%)具有促进RSC96增殖作用,高浓度时(20%和25%)抑制RSC96增殖。 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：     

法舒地尔和RhoA沉默对许旺细胞增殖的影响

背景：许旺细胞在周围神经损伤和修复再生过程中发挥重要作用。 目的：观察Rho激酶抑制剂法舒地尔和RNAi介导的RhoA基因沉默对大鼠许旺细胞增殖的影响。 方法：体外培养Wistar大鼠许旺细胞,分6组干预：对照组,5,10,15,20 μmol/L 法苏地尔组,siRNA沉默RhoA基因组。处理后第3天,RT-PCR、Western blot检测各组许旺细胞RhoA基因和蛋白的表达。连续5 d用细胞计数法测定生长曲线。应用MTT比色法观察许旺细胞增殖情况;采用流式细胞术测定许旺细胞周期分布的变化。 结果与结论：法苏地尔浓度增加到20 μmol/L时对细胞的作用并非随浓度的增加而增强,与15 μmol/L组的差异无显著性意义(P > 0.05)。Rho激酶抑制剂法舒地尔和RNAi介导的RhoA基因沉默在体外实验均能促进许旺细胞,法舒地尔最佳作用浓度为15 μmol/L,沉默RhoA基因效果较好。     

Extraepithelial enterochromaffin cells and Schwann cells in the human appendix

To test earlier theories on the subject, extraepithelial enterochromaffin (EEEC) cells were sought in the mucosa of 500 appendixes and in the solid axial cores of 283 others whose lumina were obliterated over part or all of their lengths. The EEEC cells were lying free in the lamina propria of 53% of the specimens with intact lumina and in the axial cores of 49% of the obliterated specimens. In both locations they were invariably accompanied by Schwann cells and neurites, which were often present in markedly increased numbers. Therefore, because they occur so frequently in nonepithelial locations, enterochromaffin cells can no longer be considered as exclusively epithelial. Their close association with Schwann cells and neurites suggests that they may have some sort of neural function.     

The serum of dysautonomia patients enhances proliferation and signaling in Schwann cells

Disorders of the autonomic nervous system, or dysautonomias, affect a large segment of the population, especially women, and represent a diagnostic challenge. Identification of biomarkers for autonomic disorders, and the subsequent development of screening methods, would benefit diagnosis and symptom management. We studied the effect of sera from fifteen well-characterized dysautonomia patients (mean age 49 ± 16 years, 10 females, 5 males) and ten control subjects (mean age 31 ± 14 years, 5 females, 5 males) on the proliferation of cultured Schwann cells and activity of mitogen-activated protein kinases (MAPKs) in these cells. We correlated characteristics of patients with the effects on cell proliferation and signaling. Overall, we observed a significant increase in proliferation when Schwann cells were incubated with sera from female dysautonomia patients when compared to control subjects and male patients. Interestingly, removal of IgGs significantly reduced the proliferative effect of patient sera. We also observed significant activation of p38 MAPK following incubation with both male and female patient sera. These results suggest that patient sera contain factors that contribute to aberrant Schwann cell proliferation and signaling and may ultimately lead to autonomic nerve dysfunction. Our observations represent a promising first step in the identification of dysautonomia biomarkers.     

HMGB1-induced autophagy in Schwann cells promotes neuroblastoma proliferation

Neuroblastoma inflicts mostly on children, and the pathogenesis remains elusive. Clinical diagnosis and therapeutic approaches are still on the incipient stage, so further understanding of the molecular and cellular mechanisms of the disease is necessary. Inflammation has been commonly regarded as a hallmark in tumorigenesis and development, and we identified a new inflammatory factor, HMGB1, is considerably increased in neuroblastoma. Our study shows that HMGB1 induces autophagy in Schwann cells through activation of TLR4, and knockdown of TLR4 obviates the HMGB1-induced autophagy. The HMGB1-induced autophagy is through classical pathway, as deficiency of Beclin 1 deprived autophagy in Schwann cells. Coculture of neuroblastoma with Schwann cells pretreated with HMGB1 promoted the proliferation of neuroblastoma cells, and if Beclin 1 is knocked down in Schwann cells, no promotion effects is observed. Taken together, our study demonstrates that HMGB1-induced autophagy in Schwann cells contributes to neuroblastoma cell proliferation, thus providing a potential therapeutic approach on neuroblastoma development.     

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。     

Cisplatin-induced apoptosis in cultures of human Schwann cells

To investigate the sensitivity of human Schwann cells to cisplatin (cis-DDP), different approaches to estimate DNA damage were used: the comet assay, morphological evaluation of the granular condensation of nuclear chromatin and the terminal transferase-mediated dUTP nick-end-labelling (TUNEL) method. The number of micronuclei (MNi), as a sign of cisplatin-induced genotoxicity, was counted. DNA damage assessed by the comet assay was already evident after 1.5 microM cisplatin treatment at all exposure times (24, 48, and 72 h). Initial morphological changes characterised by the granular condensation of nuclear chromatin were detectable after 24 h exposure to 25 microM cis-DDP, while an increased number of apoptotic cells, determined by the TUNEL method, was noted after 48 h exposure to the same concentration. The first significant increase in the number of MNi was observed in cells treated with 75 microM cis-DDP for 24 h. We demonstrate that the comet assay is a highly sensitive method for measuring cisplatin induced DNA damage. Morphological observation revealed advanced as well as less prominent alterations in the nuclear chromatin. In contrast, the TUNEL method detected only those cells with advanced DNA fragmentation.     

Experimental necrosis and arrest of proliferation of Schwann cells by cytosine arabinoside.

In developing rat cervical sympathetic trunks, Schwann cells proliferate intensely during the first week after birth but axonal populations do not increase. Thus, experimental inhibition of DNA synthesis should affect Schwann cells and spare axons whose cell bodies are not dividing. The present investigation was aimed at determining the effects of an inhibitor of DNA synthesis-cytosine arabinoside (ara-C)--on axons and Schwann cells in developing nerves. Ara-C (60 mg kg(-1) body weight) was injected subcutaneously to newborn rats every six hours for 36 hours. At intervals from 2--4 days of age, animals were given tritiated thymidine (4 muCi per gram body weight) to label Schwann cells in synthesis phase. One hour later rats were killed by systemic perfusion of phosphate buffered glutaraldehyde. Cervical sympathetic trunks (CST's) and sciatic nerves were removed and processed for radioautography and electron microscopy (EM). Labelled and unlabelled Schwann cell nuclei were counted to determine the labelling index (LI%) for each nerve. By the end of ara-C treatment there was almost complete absence of labelling but LI's rose sharply 24 hours after discontinuing ara-C. By EM axons appeared normal; Schwann cells, however, showed prominent nuclear and cytoplasmic changes consisting of nuclear degeneration, dilatation of rough endoplasmic reticulum and increased cytoplasmic density. Ara-C, by suppressing proliferation and causing necrosis of Schwann cells but sparing axons, results in a developmental alteration of axon-Schwann cell relationships. It is suggested that the pathogenetic mechanism involves an inbalance in DNA/RNA synthesis metabolism.     

The mood stabilizer valproic acid induces proliferation and myelination of rat Schwann cells

Schwann cells (SCs) within peripheral nerve respond robustly after exposure to neurotrophic factors. Recent results have revealed that valproic acid (VPA), at a clinically relevant therapeutic concentration, produces effects similar to neurotrophic factors, and promotes neurite growth and cell survival. We hypothesized that VPA could also induce Schwann cell response. In this study, we sought to determine how pure Schwann cells responded to VPA by evaluating for proliferation, expression of S-100, growth cone-associated protein 43 (GAP-43), myelin-associated glycoprotein (MAG), and myelin basic protein (MBP). Immunohistochemistry demonstrated that the Schwann cells were positive for S-100, GAP-43, MAG, and MBP greater than 99% of the experimental cells. The rate of proliferation was increased in experimental cells from MTT assay and Bromodeoxyuridine/DAPI double staining. Furthermore, Western blot showed an up-regulation in GAP-43, MAG and MBP protein expression in experimental cells, respectively. We also found that mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) 1/2 pathway was involved in the enhanced cell proliferation of Schwann cells evoked by VPA. This study provides novel information regarding Schwann cell response to VPA, which might help the understanding of VPA-based treatment for peripheral nerve injury.     

Adhesion and proliferation of bovine aortic endothelial cells on monoamine- and diamine-containing polystyrene derivatives.

Adhesion and proliferation of bovine aortic endothelial cells on polystyrene derivatives having monoamine or diamine side chain was investigated focusing on the chemical structure of amino groups. Copolymers, SE8.5, is composed of polystyrene with 8.5 mol% of monoamine side chains, and SED8, which is with 8 mol% of diamine side chains, were estimated to contain almost the same amount of protonated amino groups in bulk composition at physiological pH (pH 7.4). There observed significant difference in cellular spreading of attached endothelial cells between these two types of copolymer surfaces. Spreading-% of attached cells on SED8 surfaces was approximately 1.6 times greater than that on SE8.5 6 h after seeding. This difference in cellular spreading influenced to subsequent cell growth. Cellular growth on each polymer surface was featured by parameter k, which corresponds to the 'rate constant' of cellular proliferation. While the k-value for SE8.5 decreased with decreasing seeding density as well as the case for polystyrene, SED8 maintained a high k-value even at low seeding density as 2 x 10(3) cells/cm2. These results suggest that cells may recognize the difference in the chemical structure of amine side chains of SE and SED copolymers.     

诱导人眼轮匝肌来源肌源干细胞向许旺细胞样细胞的分化

背景：肌源干细胞的优越性引导学者们尝试从人眼轮匝肌中分离该细胞,同时进行许旺细胞方向的诱导分化,为周围神经的修复提供新的种子细胞来源。 目的：诱导人肌源干细胞向具有许旺细胞特性的细胞分化。 方法：①收集重睑成形术中切除的上睑眼轮匝肌,经酶消化和细胞筛过滤,贴壁培养分离人肌源干细胞,予细胞特异标记物以免疫组织化学染色。②取大鼠坐骨神经分离培养许旺细胞,收集许旺细胞条件培养液。③将人肌源干细胞与许旺细胞条件培养液共培养,观察转化细胞的形态和免疫组织化学染色的变化。 结果与结论：①原代培养4周时可见人肌源干细胞,与传代培养之人肌源干细胞同样呈现明显的小圆形形态,折光性强,少量细胞呈现短梭形。所有人肌源干细胞表现为Desmin阳性,Sca-1染色阳性。②分离培养大鼠坐骨神经来源许旺细胞,S100染色阳性率为(97.4±0.7)%。③经与许旺细胞条件培养液共培养,人肌源干细胞分化后细胞表达许旺细胞特异标记物S100,GFAP和p75。结果提示分离获得人肌源干细胞与许旺细胞条件培养液共培养,可以诱导人肌源干细胞表达许旺细胞特异标记物,初步证实了人肌源干细胞可分化为许旺细胞样细胞。     

He-Ne laser irradiation affects proliferation of cultured rat Schwann cells in a dose-dependent manner

Summary Schwann cell proliferation is considered an essential part of Wallerian degeneration after nerve damage. Laminin, an important component of the extracellular matrix and produced by Schwann cells, provides a preferred substrate for outgrowing axons. To study whether low energy (He-Ne) laser irradiation may exert a positive effect on nerve regeneration through an effect on Schwann cells, its effect was evaluatedin vitro. Schwann cells were isolated from sciatic nerves of 4–5-day-old Wistar rates and cultured on 96-multiwell plates. The cells were irradiated by a He-Ne laser beam (632.8 nm, 5.98 mW) that was optically expanded to a beam width of 4 mm. During irradiation the plate was kept in an air-tight box equilibrated with humidified air containing 5% CO₂ and kept at 37 ° C. At three consecutive days, starting either at day 5 or day 8, cells were irradiated each day for 0.5, 1, 2, 5 or 10 min. Both cell number and laminin production were determined for each irradiation condition (n=5) within one experiment. Schwann cells that were irradiated from day 8 on were hardly affected by laser irradiation. However, the proliferation of cells that were irradiated starting on day 5 was significantly increased after 1, 2, and 5 min of daily irradiation, compared to non-irradiated control cultures. The laminin production per cell of these Schwann cells was not significantly altered. From these results we conclude that He-Ne laser irradiation can modulate proliferation of rat Schwann cellsin vitro in a dose-dependent manner.     

Embryonic Schwann cell development: the biology of Schwann cell precursors and early Schwann cells

The cellular events leading to the generation of Schwann cells from the neural crest have recently been clarified and it is now possible to outline a relatively simple model of the Schwann cell lineage in the rat and mouse. Neural crest cells have to undergo 3 main developmental transitions to become mature Schwann cells. These are the formation of Schwann cell precursors from crest cells, the formation of immature Schwann cells from precursors and, lastly, the postnatal and reversible generation of non-myelin- and myelin-forming Schwann cells. Axonal signals involving neuregulins are important regulators of these events, in particular of the survival, proliferation and differentiation of Schwann cell precursors.