40000/微囊化细胞低温保存的研究进展

背景：微胶囊是一种有效的免疫隔离工具,低温冷冻方便了微囊的保存与运输,有利于微囊化技术在临床的推广应用。 目的：综述微囊低温保存技术中微囊低温保存特点、不同保存方法优缺点、研究方法及微囊低温保存实验现状,提出其中存在的问题,并对其发展进行展望。 方法：1980/2010 PubMed数据库（网址http://www.ncbi.nlm.nih.gov/PubMed）,1991/2010万方数据库（网址http://www.wanfangdata.com.cn）,维普数据库（网址http://www.vmis.net.cn/yixue/index.asp）,有关微囊低温保存研究,低温保存工艺理论及专用仪器,微囊低温保存技术医学应用的文献,英文关键词为“microencapsulation,microencapsulated cell,cryopreservation,transplantation”。中文关键词为“微囊化细胞,低温保存”。 结果与讨论：微囊化细胞在低温保存过程中的损伤特点与细胞、组织有很大不同。微囊相对较大的尺寸与复杂的囊壁半透膜结构,使得微囊结构与囊内细胞更容易受到溶质损伤与冰晶损伤,因此不能简单的套用单细胞悬液的低温保存方案。慢速冷却法与玻璃化法是两种常见的微囊化细胞低温保存方法,各有利弊。微囊化细胞低温保存技术尚未成熟,在开发新型微囊低温保存设备,优化设计微囊低温保存方案的同时,需进一步加强对微囊冻存机理的更深层次的探索。     

微囊化肾上腺细胞移植与生物可降解材料的临床应用

摘要：肾上腺细胞移植是治疗肾上腺皮质功能不全的一种新方法,但免疫排斥反应是限制细胞移植广泛应用的主要问题,微囊免疫隔离技术是克服免疫排斥反应的有效方法之一。微囊化细胞一方面有较好的免疫隔离作用,另一方面其半透膜表面可以容许小分子物质的通过,保证了囊内移植物的存活。海藻酸钙-聚赖氨酸-海藻酸钙是应用比较成熟的微囊材料,具有较好的生物相容性和机械稳定性,能提高细胞的生物活性和分泌功能。微囊的外径大小是影响其强度的主要因素,一般认为体积小且表面光滑的微囊比体积大的微囊强度高,不易破裂。但海藻酸钙-聚赖氨酸-海藻酸钙作为膜材料随着时间的延长,其囊本身有降解的可能,易引起囊周纤维化反应,选择合适的膜材料成为目前研究的焦点。随着组织工程技术的发展,以生物可降解材料聚β-羟基丁酸酯为载体的肾上腺细胞移植已经进入实验研究,研究发现该材是一种较好的细胞载体,可以促进移植细胞的生存、功能的恢复,有着广阔的应用前景。关键词：细胞移植;肾上腺细胞移植;微囊化技术;可生物降解材料     

微囊化技术在医学领域中的应用与进展

背景：微囊的免疫隔离保护和可控缓释的特性得到了医学领域的重视,并已广泛应用于糖尿病、帕金森症、肝衰竭、镇痛、肿瘤等疾病治疗过程中。 目的：综述微囊化技术的特性以及在医学研究领域中的应用。 方法：由第一作者检索1964/2009PubMed数据库(网址http://www.ncbi.nlm.nih.gov/PubMed),2000/2009万方数据库(网址http://www.wanfangdata.com.cn)有关微囊化移植,微囊化可控缓释,微囊化技术在医学应用方面的应用方面的文献,英文关键词为“microencapsulated,transplantation,controlled release”。中文关键词为“微囊化”。 结果与结论：微囊作为一种载体,为细胞提供了一个三维培养基质,改善细胞间的作用,提高细胞的功能;为异体细胞提供了免疫屏障,阻挡免疫细胞的识别,但又不妨碍营养物质、氧气及代谢产物运输;为机体提供可控的长期稳定缓释有效成分治疗疾病。微囊化技术在糖尿病方面研究比较多,在帕金森症、肝衰竭、脊髓损伤、甲状旁腺功能低下、骨科领域、肿瘤癌症等方面也有一定的探索,但微囊化技术从个别动物试验成功到真正临床广泛应用还有很漫长的历程,在拓宽微囊化技术在医学领域运用的同时,需进一步加强理想刚性结构、良好生物相容性和可控半透膜性的探索。 关键词：微囊化;生物人工肝;胰岛;免疫隔离;组织工程 doi:10.3969/j.issn.1673-8225.2010.29.041     

琼脂糖微囊化猪胰岛异种移植治疗糖尿病的实验观察

背景：以往研究表明体外琼脂糖微囊化成猪胰岛具有一定的有效生物活性。但移植到动物体内后的生物活性还有待明确。 目的：观察实验性糖尿病大鼠腹腔内移植琼脂糖微囊化成猪胰岛治疗效果。 设计、时间及地点：对比观察实验,于2007-12/2008-12在郑州大学中心实验室完成。 材料：以琼脂糖作为制备微囊的材料,用相分离方法包被成猪胰岛。 方法：①将纯化后未微囊化胰岛及微囊化胰岛分置于RPMI1640 培养液培养, 隔日换液。收集培养第1天及第5天培养液上清,检测胰岛素含量。②解剖镜下挑选同一时间微囊的胰岛细胞,分别置于5.6 mmol/L 葡萄糖、16.6 mmol/L葡萄糖 和10.0 mmol/L茶碱溶液中,做静态孵育下的胰岛素刺激释放试验,放射免疫法测定胰岛素含量。③27只糖尿病大鼠随机分为3 组：对照组、未微囊化胰岛移植组、微囊化胰岛移植组,分别注入生理盐水、(0.9~1.6)×103个未微囊化成猪胰岛、(0.8~1.7)×103个微囊化成猪胰岛。 主要观察指标：微囊化胰岛培养液中基础胰岛素含量变化,微囊化胰岛对高糖与茶碱刺激的反应性,胰岛移植后糖尿病大鼠的生存期。 结果：微囊化胰岛在1个月的培养期间可持续分泌胰岛素。囊内细胞生长良好,微囊没有溶解和破碎。培养2 d的微囊化胰岛对高糖与茶碱刺激有明显反应,胰岛素释放量分别为低糖的1.96倍和2.58倍(P < 0.01)。移植后 7 d 时,3 组血糖分别为(21.61±1.21) mmol/L,(19.11±2.39) mmol/L 和(13.67±1.43) mmol/L,微囊化胰岛移植组与前2组相比差异均有显著性意义,且生存期明显长于前2组。 结论: 琼脂糖微囊化成猪胰岛可存活于糖尿病大鼠体内,并且具有有效生物活性。     

微囊胰岛移植的实验研究进展

微囊胰岛细胞移植后能改善糖尿病的糖代谢异常,并实现对血糖的持续监测和调节,且微囊的免疫隔离作用能消除或减轻受体对异体或异种免疫排斥反应,减少或消除对免疫抑制剂的依赖。合适的囊材、合理的工艺、先进的制囊设备和胰岛分离提纯技术、适宜的移植位置能增强微囊胰岛细胞的抗机械强度,提高生物相容性,减少囊周纤维化现象,改善人工胰岛细胞的功能,延长存活时间。资料综合：国内外研究者为了延长微囊化人工胰岛细胞功能起效和存活时间,使微囊化人工胰岛细胞早日应用于临床治疗糖尿病,从囊材、胰岛分离提纯和移植部位等各方面着手加以研究。结论：微囊胰岛细胞移植后能改善糖尿病的糖代谢异常,并实现对血糖的持续监测和调节,且微囊的免疫隔离作用能消除或减轻受体对异体或异种免疫排斥反应,减少或消除对免疫抑制剂的依赖。合适的囊材、合理的工艺、先进的制囊设备和胰岛分离提纯技术、适宜的移植位置能增强微囊胰岛细胞的抗机械强度,提高生物相容性,减少囊周纤维化现象,改善人工胰岛细胞的功能,延长存活时间。     

微囊化PC12细胞移植治疗帕金森病的实验研究

目的 研究治疗帕金森病的一种新方法。方法 采用免疫隔离技术—微囊技术将能分泌多巴胺的PC12细胞包裹后，移植到帕金森病大鼠脑内，观察阿朴吗啡诱发病鼠旋转行为变化，高效液相色谱电化学法测定移植区多巴胺含量，酪氨酸羟化酶(TH)抗体免疫组织化学染色观察移植区阳性细胞表达。结果 帕金森病大鼠阿朴吗啡诱发旋转行为明显改善，不同时相点的移植区多巴胺含量较对照组明显升高，而未行微囊化的PC12细胞移植则会有致死性肿瘤形成。移植后3月，大鼠脑内微囊完整，仍有TH阳性PCl2细胞存活。结论 微囊化PC12细胞移植，可避免药物治疗、主体定向毁损手术、胎脑移植等方法的诸多弊端，因此是一种新的有效的治疗帕金森病的方法。     

原位定型微囊化载体制剂在糖尿病大鼠皮肤溃疡中的作用

背景：原位定型微囊化载体制剂成分之一胰岛素可以促进溃疡愈合。 目的：观察原位定型微囊化载体制剂在糖尿病大鼠皮肤溃疡中的疗效。 方法：腹腔内注射链脲佐菌素建立糖尿病大鼠模型,应用外科方法建立全层皮肤缺损模型。根据皮肤溃疡处干预方式将实验动物分为4组。①空白对照组用生理盐水处理创面。②一般制剂组应用甲硝唑+山莨菪硷1+普通短效胰岛素处理创面。③单纯微囊组创面外敷不含有效药物成分的微囊化载体膜。④微囊化有效制剂组溃疡处外涂微囊化载体制剂膜,内含药物成分与一般制剂组相同。定时测量溃疡面积,记录溃疡愈合时间,取创面全层组织进行组织学观察,测定表皮生长因子受体、纤维连接蛋白阳性细胞数量。 结果与结论：微囊化有效制剂组大鼠溃疡愈合时间短于其他3组(P < 0.05或P < 0.01),微囊化有效制剂组表皮生长因子受体和纤维连接蛋白阳性细胞数目高于其他各组(P < 0.05或P < 0.01)。结果表明,原位定型微囊化载体制剂能够缩短愈合时间和促进糖尿病大鼠皮肤溃疡愈合。     

微囊化成年兔许旺细胞的体外培养*

背景：研究发现将体外培养扩增的大量许旺细胞种植到神经导管上来引导周围神经再生效果明显,而目前备受关注的微囊化免疫隔离技术,在克服免疫排斥问题上具有明显的优势与实用性。 目的：微囊包裹兔许旺细胞并进行体外培养,观察许旺细胞形态及增殖变化。 设计、时间及地点：体外细胞水平观察实验,于2005-09/2006-05在南昌大学第二附属医院分子实验室完成。 材料：健康成年家兔由南昌大学医学院动物中心提供,微囊发生器由南昌大学医学院神经生物学研究室研制。 方法：使双侧坐骨神经发生Wallerian变性,经改良后反复差速贴附法进行培养,快速分离纯化许旺细胞。海藻酸钠-氯化钡一步成囊法包裹生长良好的第3代许旺细胞。 主要观察指标：倒置显微镜下观察培养过程中微囊化许旺细胞形态变化,MTT检测不同时期微囊化许旺细胞及游离许旺细胞的增殖改变。 结果：在培养过程中可见微囊及其囊内许旺细胞形态均保持完整,微囊未见破损。微囊化后的许旺细胞总体呈现悬浮生长的趋势。培养0,1,3,5,7 d时 MTT检测微囊许旺细胞组与游离许旺细胞组吸光度值差异有显著性意义(P < 0.05)。 结论：微囊包裹的许旺细胞体外可存活较长时间,微囊化许旺细胞相比游离许旺细胞增殖速度有所减慢。     

微囊化PC12细胞的分泌特征及其致瘤性

背景：有报道微囊化细胞移植不仅能避免免疫排斥反应，而且肿瘤细胞微囊化后可以消除其致瘤性。 目的：观察大鼠肾上腺嗜铬细胞瘤细胞PC12 细胞微囊化前后的分泌功能变化，及微囊化PC12 细胞植入大鼠蛛网膜下腔后的存活情况和致瘤性。 设计、时间及地点：动物观察实验，于2007-07/12在中山大学实验动物中心完成。 材料：用微囊包裹PC12 细胞。 方法：将微囊化后第3~5天的PC12细胞植入12只雄性SD大鼠蛛网膜下腔。 主要观察指标：检测微囊化前后PC12 细胞去甲肾上腺素和甲硫氨酸脑啡肽的分泌情况；移植术后8周，回收移植的微囊化PC12细胞，检测存活情况和分泌功能；移植术后12 周，对大鼠腰段脊髓予病理切片检查，观察移植物对脊髓组织的影响。 结果：①PC12细胞经微囊包裹后，体外培养生长良好，除微囊化后第1天外，细胞微囊化前后去甲肾上腺素和甲硫氨酸脑啡肽分泌水平差异无显著性意义(P > 0.05)。②回收移植术后的微囊化PC12细胞，再培养，其去甲肾上腺素和甲硫氨酸脑啡肽的分泌水平与移植前比较差异无显著性意义(P > 0.05)。③移植术后12 周，大鼠脊髓大体观察均未见新生物形成, 病理切片显示神经细胞和髓鞘结构完整,少量淋巴细胞浸润，无纤维增生和坏死。 结论：PC12细胞微囊化后可保持其活性和分泌功能；同种移植于蛛网膜下腔后，仍保持活性和分泌功能，无致瘤性。     

微囊胎垂体下丘脑黑质细胞移植研究

采用2.2％的海藻酸钠将胎垂体下丘脑黑质混合细胞进行微囊包膜后移植于去垂体兔皮下。在12周的观察期内,移植兔血清T_4水平与正常兔无明显差异,各靶器官无明显萎缩。结果提示:胎垂体下丘脑黑质细胞微囊具有免疫隔离功能,可在颅外移植而重建激素分泌功能12周以上。     

壳聚糖与大鼠嗅鞘细胞的生物相容性

背景：众多研究证实,嗅鞘细胞移植和生物导管在修复中枢神经系统损伤中发挥了显著的作用,课题组前期研究已证实了壳聚糖在大鼠体内神经系统具有良好的生物相容性。 目的：观察壳聚糖与大鼠嗅鞘细胞的生物相容性。 设计、时间及地点：体外细胞学对比观察,于2005-12/2006-05在武汉协和医院泌尿外科实验室完成。 材料：将壳聚糖粉溶于10 g/L的乙酸溶液中,配制成10 g/L的壳聚糖溶液;显微剥离成年大鼠嗅球表面层,按常规方法行原代细胞培养。 方法：实验分为3组,每组10孔。壳聚糖组将制备好的壳聚糖溶液0.1 mL加入到培养板中,50 ℃烘箱至成膜干燥,加入40 g/L氢氧化钠溶液中和乙酸,双蒸水漂洗,晾干即可。多聚赖氨酸组按常规方法赖氨酸包被培养板。未包被组不用任何材料包被。将原代培养的嗅鞘细胞分别接种至壳聚糖包被、多聚赖氨酸包被和未包被的培养板上进行培养。 主要观察指标：于培养第3,5天显微镜下观察细胞形态学变化,MTT比色实验检测细胞存活和增殖状况。 结果：嗅鞘细胞可在壳聚糖膜上贴壁生长,细胞形态及数量与未包被组相比差异无显著性意义,但与多聚赖氨酸组相比细胞数量偏低。 结论：嗅鞘细胞与壳聚糖有良好的生物相容性。     

Astrocytes, but not olfactory ensheathing cells or Schwann cells, promote myelination of CNS axons in vitro

We have examined the interaction between olfactory ensheathing cells (OECs), Schwann cells (SC), oligodendrocytes, and CNS axons using cultures generated from embryonic rat spinal cord. Oligodendrocyte process extension and myelination in these cultures was poor if the cells were plated on OECs or SCs. Myelin internodes and nodes of Ranvier formed frequently if these cultures were plated onto monolayers of neurosphere-derived astrocytes (NsAs). In the myelinated fibers generated on NsAs, Nav channels, caspr, and neurofascin molecules were correctly assembled at the nodes of Ranvier. The density of neurites, survival, and antigenic differentiation of oligodendrocytes was similar on OEC and NsAs monolayers. However, on OEC monolayers, despite a transient increase in the number of endogenous oligodendrocytes, there was a decrease in oligodendrocyte process extension and axonal ensheathment when compared with cultures plated on NsAs monolayers. To determine if these changes were due to axonal or glial factors, spinal cord oligodendrocytes were plated onto monolayers of OECs, NsAs, and poly-L-lysine in the absence of neurons. In these cultures, process extension and myelin-like membrane formation by oligodendrocytes was improved on monolayers of OEC. This suggests that inhibition of process extension is mediated via cross-talk between OECs and neurites. In cultures containing axons plated on OEC monolayers, oligodendrocyte process formation, axonal ensheathment, and myelination occurred albeit lower if the cultures were supplemented with NsAs conditioned medium. These data suggest OECs can permit neurite extension and oligodendrocyte proliferation, but lack secreted factor(s) and possible cell-cell contact that is necessary for oligodendrocyte process extension and myelination.     

Olfactory ensheathing cells promote migration of Schwann cells by secreted nerve growth factor

Transplantation of Schwann cells (SCs) and olfactory ensheathing cells (OECs) have emerged as very promising therapies for spinal cord repair. The important features of interaction between SCs and OECs are beginning to be appreciated, although the underlying mechanism remains unclear. In the present study, we tested the effects of OECs on SCs migration using a range of in vitro migration assays. We found that SCs migrated abundantly upon OECs monolayer, and the migration-promoting effects were identified to be due to the secreted diffusible factors in OEC-derived conditioned medium (OEC-CM). Furthermore, neutralizing nerve growth factor (NGF) in OEC-CM with NGF antibody could block this effect. Moreover, we found that NGF promotes SCs migration even on astrocyte monolayer. Taken together, these findings provide the first evidence that OECs can promote SCs migration in astrocytic environment by secreted NGF.     

Acute transplantation of olfactory ensheathing cells or Schwann cells promotes recovery after spinal cord injury in the rat

We compared the neurological and electrophysiological outcome, glial reactivity, and spared spinal cord connectivity promoted by acute transplantation of olfactory ensheathing cells (group OEC) or Schwann cells (group SC) after a mild injury to the rat spinal cord. Animals were subjected to a photochemical injury of 2.5 min irradiation at the T8 spinal cord segment. After lesion, a suspension containing 180,000 OECs or SCs was injected. A control group (group DM) received the vehicle alone. During 3 months postsurgery, behavioral skills were assessed with open field-BBB scale, inclined plane, and thermal algesimetry tests. Motor (MEPs) and somatosensory evoked potentials (SSEPs) were performed to evaluate the integrity of spinal cord pathways, whereas lumbar spinal reflexes were evaluated by the H reflex responses. Glial fibrillary acidic protein and proteoglycan expressions were quantified immunohistochemically at the injured spinal segments, and the preservation of corticospinal and raphespinal tracts caudal to the lesion was evaluated. Both OEC- and SC-transplanted groups showed significantly better results in all the behavioral tests than the DM group. Furthermore, the OEC group had higher MEP amplitudes and lower H responses than the other two groups. At the injury site, the area of spared parenchyma was greater in transplanted than in control injured rats. OEC-transplanted animals had reduced astrocytic reactivity and proteoglycan expression in comparison with SC-transplanted and DM rats. Taken together, these results indicate that transplantation of both OEC and SC has potential for restoration of injured spinal cords. OEC grafts showed superior ability to reduce glial reactivity and to improve functional recovery.     

Isolation, culture, and purification of olfactory mucosa-derived olfactory ensheathing cells using modified differential attachment with low concentration serum

BACKGROUND:Studies have demonstrated that olfactory mucosa can promote the regeneration and formation of axonal medullary sheath of injured neurons. To date, olfactory ensheathing cells (OECs) utilized in basic and clinical research arise primarily from the olfactory bulb mucosa. However, little is known regarding culture, purification, and biological properties of OECs . OBJECTIVE: To isolate and culture OECs utilized modified, differential attachment in combination with neurotrophic factor 3 (NT3) and low...     

Isolation, culture, and purification of olfactory mucosa-derived olfactory ensheathing cells using modified differential attachment with low concentration serum*☆

BACKGROUND：Studies have demonstrated that olfactory mucosa can promote the regeneration and formation of axonal medullary sheath of injured neurons. To date, olfactory ensheathing cells （OECs） utilized in basic and clinical research arise primarily from the olfactory bulb mucosa. However, little is known regarding culture, purification, and biological properties of OECs .
OBJECTIVE： To isolate and culture OECs utilized modified, differential attachment in combination with neurotrophic factor 3 （NT3） and low concentration serum to explore an optimal in vitro culture method for OECs.DESIGN, TIME AND SETTING： Single-sample observation was performed at the Medical Experimental Center of Stomatology College, Xi＇an Jiaotong University between March 2006 and December 2007. MATERIALS： Twelve samples from aborted embryos, 4-6 months, were used to isolate OECs; rabbit-anti-human p75NTR and glial fibrillary acidic protein （GFAP） antibody were provided by Sigma, USA. METHODS： The differential time was six hours. This was repeated twice, based on Nash＇s differential attachment. Attached OECs were cultured in DMEM-F12 culture medium containing 10% fetal bovine serum （FBS） or 2.5% FBS and NT3. MAIN OUTCOME MEASURES： OEC morphology was observed, and p75NTR and GFAP immunocyto-chemistry was used for identification and purity detection. RESULTS： Some cells attached after three days in culture. Several cells possessed short neurites with good refractivity. Some shuttle-shaped fibroblasts could be seen. On day six, more cells attached, exhibiting a three-dimensional appearance. Many cells appeared dipolar or tripolar, with slender neurites, and fibroblasts were clustered. On day nine, the number of dipolar or tripolar cell bodies with slender neurites was increased, and fibroblasts were clustered. On day 15, fibroblasts occupied the majority of the bottom of the culture bottle, with several OECs surviving at the upper layer. OECs were positive for P75NTR and GFAP expression, as identified by an im     

嗅鞘细胞诱导神经干细胞分化后神经元电生理特性的研究

目的探索大鼠嗅鞘细胞对神经干细胞(NSC)分化的影响,以及分化后神经元电生理特性。方法取新生鼠大脑皮质,原代培养大鼠NSC。NSC分为实验组和对照组,实验组将无血清培养的NSC中加入嗅鞘细胞条件培养液,对照组单纯无血清培养NSC。光镜下观察细胞分化情况,免疫组化法分别检测巢蛋白(nestin)、神经生长因子受体(NGFRp75)、神经丝蛋白(NF200)和胶质纤维酸性蛋白(GFAP)的表达,膜片钳检测神经元电生理特性。结果实验组嗅鞘细胞主要诱导NSC分化为神经元,少量分化为胶质细胞。对照组NSC逐渐萎缩,最终死亡。分化后的神经元记录到快速激活、快速失活能被河豚毒素特异阻断的钠电流,以及慢激活、慢失活能被四乙铵特异阻断的延迟整流性钾电流。结论嗅鞘细胞能诱导NSC分化成神经元,分化后的神经元具有活跃的电生理特性。     

Comparative gene expression profiling of olfactory ensheathing cells from olfactory bulb and olfactory mucosa

Olfactory ensheathing cells (OEC) have the ability to promote regeneration in the nervous system. Hence, they hold promise for cell therapy. Most of the experimental studies have investigated the role of OECs taken from olfactory bulb (OB). However, for a clinical human application, olfactory mucosa (OM) seems to be the only acceptable source for OECs. Many studies have compared the distinct ability of OECs from OB and OM to improve functional nerve regeneration after lesion of the nervous system. Nevertheless, the two populations of OECs may differ in several points, which might affect all fate after transplantation in vivo. We report here the first study which compares gene expression profiling between these two populations of OECs. It appears that OB‐OECs and OM‐OECs display distinct gene expression pattern, which suggest that they may be implicated in different physiological processes. Notably, OM‐OECs overexpress genes characteristic of wound healing and regulation of extra cellular matrix. In contrast, OB‐OECs gene profile suggests a prominent role in nervous system development. Hence, OB‐OECs and OM‐OECs fundamentally differ in their gene expression pattern, which may represent a crucial point for future clinical application. © 2010 Wiley‐Liss, Inc.     

Effects of olfactory ensheathing cells on the proliferation and differentiation of neural stem cells

BACKGROUND:Olfactory ensheathing cells can promote oriented differentiation and proliferation of neural stem cells by cell-secreted neural factors. OBJECTIVE:To observe the effect of olfactory ensheathing cells on the differentiation and proliferation of neural stem cells. DESIGN,TIME AND SETTING:Cytology was performed at the Department of Neurology,Tongji Medical College,Huazhong University of Science and Technology,China,from September 2007 to October 2008. MATERIALS:Mouse anti-nestin polyclonal antibo...     

The glycoprotein fibulin-3 regulates morphology and motility of olfactory ensheathing cells in vitro

The primary olfactory pathway in adult mammals has retained a remarkable potential for self-repair. A specialized glial cell within the olfactory nerve, called olfactory ensheathing cell (OEC), and their associated extracellular matrix are thought to play an important role during regenerative events in this system. To gain insight into novel molecules that could mediate the OEC-supported growth of axons within the olfactory nerve, gene expression profiling experiments were conducted which revealed high expression of the glycoprotein fibulin-3 in OECs. This observation was confirmed with quantitative PCR. In vivo, the distribution of all members of the fibulin family, fibulin-3 included, was localized to the lamina propria underneath the olfactory epithelium, in close association within olfactory nerve bundles. To manipulate fibulin-3 gene expression in cultured OECs, lentiviral vector constructs were designed to either transgenically express or knock-down fibulin-3. Experimental data showed that increased levels of fibulin-3 induced profound morphological changes in cultured OECs, impeded with their migratory abilities and also suppressed OEC-mediated neurite outgrowth. Knock-down of fibulin-3 levels resulted in reduced OEC proliferation. In conclusion, the data provide novel insights into a putative role for fibulin-3 in the regulation of cell migration and neurite outgrowth within the primary olfactory pathway.