用于移植的嗅鞘细胞的纯化培养方法研究

目的 探讨移植用成年大鼠嗅球嗅鞘细胞(OECs)的培养和纯化方法.方法 取成年SD大鼠嗅球,将分离消化所得细胞悬液进行接种.然后分别于接种后2 h、6 h和18 h 3个时间点进行单步骤差速贴壁法去除成纤维细胞,纯化嗅鞘细胞,纯化培养约11 d时采用神经生长因子受体P75(NGFRP75)和碘化丙啶(PI)双染OECs并对其纯度及细胞数量进行免疫荧光鉴定分析.结果 纯化培养11 d后在共聚焦显微镜下呈双染阳性的细胞为OECs,数量级达106/mL,形态以梭形、双极或三极突起为主,亦有少量多极或单极细胞.2 h、6 h和18 h组嗅鞘细胞纯度分别为(53±5)%、(73±8)%和(69±13)%,组间差异有统计学意义(F=11.766,P<0.001). 结论分别经2、6、18 h单步骤差速贴壁法纯化的OECs在纯度和数量级上均可满足移植用细胞的要求,且方法简单、经济、快捷.     

以差速贴壁与化学药物并胰酶限时消化法纯化新生大鼠嗅鞘细胞：优于单一差速贴壁和化学药物法吗？★

背景：目前国内外对嗅鞘细胞培养条件的报道各不相同，个别方法重复性较差，不利于实际应用。 目的：观察差速贴壁+化学药物+胰酶限时消化法纯化大鼠嗅鞘细胞的效果，并与化学药物法、差速贴壁法培养结果进行比较。 设计、时间及地点：细胞学体外对照观察，于2007-06/2008-06在福建医科大学人体解剖与组织胚胎学系实验室完成。 材料：新生2 d的SD大鼠8只，由福建医科大学试验动物中心提供。 方法：无菌条件下完整取出大鼠双侧嗅球，剥除嗅球表面的软脑膜及毛细血管网，剪成0.5 mm3的小块进行胰蛋白酶消化，过筛后按4.0×108 L-1密度种植于包被多聚左旋赖氨酸的培养瓶中进行原代培养。设立4组：①未经纯化的常规对照组。②化学药物组加入5 μmol/L阿糖胞苷抑制成纤维细胞分裂。③差速贴壁组采用Nash差速贴壁法纯化嗅鞘细胞。④差速贴壁+化学药物+胰酶限时消化组首先采用Nash差速贴壁法去除大部分成纤维细胞，而后对于少量残留的成纤维细胞加入阿糖胞苷处理，培养6 d贴壁细胞大部分融合后，加入1.25 g/L胰蛋白酶消化1 min，显微镜下见突起回缩、细胞变圆、少量细胞浮起时终止消化。 主要观察指标：嗅鞘细胞的形态，NGFR p75免疫细胞荧光染色结果。 结果：体外培养的新生大鼠嗅球嗅鞘细胞主要为双极或三极细胞，其突起细长；未经纯化的常规对照组培养7 d时视野内成纤维细胞已达60%以上，至14 d成纤维细胞完全长满；经过纯化处理的另外3组始终以嗅鞘细胞居多，嗅鞘细胞的形态与原代基本相同。存活的双极和3极嗅鞘细胞呈NGFRp75阳性反应，但化学药物组、差速贴壁组嗅鞘细胞纯度偏低，仅为75%；差速贴壁+化学药物+胰酶限时消化组嗅鞘细胞纯化效率明显增高，达85%以上。 结论：实验结果证实了差速贴壁+化学药物＋胰酶限时消化法的三合一操作技术，是一种高效率的纯化培养嗅球嗅鞘细胞的方法。     

改良差速贴壁+无血清条件培养液纯化培养大鼠嗅鞘细胞*

背景：细胞移植是脊髓损伤的一种有效治疗手段,嗅鞘细胞被认为是最适合的种子细胞之一,但目前对其培养纯化的方法尚无公认标准。 目的：运用改良差速贴壁法+含同源嗅鞘上清液的无血清条件培养液来纯化培养嗅鞘细胞,以期建立一种新颖、经济、简单、实用的纯化培养成年大鼠嗅鞘细胞的方法。 设计、时间和地点：观察性对照实验。实验于2008-02/06在苏州大学干细胞与组织工程实验室完成。 材料：健康成年SD大鼠6只,体质量150~180 ｇ。 方法：①无菌条件取大鼠嗅球组织,消化、离心去除上清液后,用含体积分数为0.2胎牛血清的DMEM/F-12培养基重悬,稀释的单细胞混悬液均分成3份,分别用于单纯改良差速贴壁、单纯无血清条件培养液纯化和改良差速贴壁+无血清条件培养液纯化的实验。②单纯改良差速贴壁：将单细胞混悬液以8 000个/cm²密度种植于无包被的25 cm²培养瓶中。经12 h+ 24 h两次差速培养后,吸出细胞上悬液,计数板计算浓度后,按1×109 Ｌ-1浓度重新种植于载有多聚赖氨酸包被盖玻片的35 mm培养皿中继续培养3周。③单纯无血清条件培养液纯化：将单细胞混悬液用预先收集并制备的含同源嗅鞘培养上清液的无血清条件培养液直接种植于载有多聚赖氨酸包被盖玻片的35 mm培养皿中,孵育两三天后再换成含体积分数为0.1胎牛血清DMEM/F12培养基继续培养3周。④改良差速贴壁+无血清条件培养液纯化：经12 h +24 h两次差速纯化,吸出上悬液接种于培养皿中继续培养2.0~3.0 d后,改换成无血清条件培养液,孵育36~48 h后再换成含体积分数为0.1胎牛血清DMEM/F-12培养基继续培养。以后视情况每3~5 d半量换液1次,培养3周。 主要观察指标：运用倒置显微镜观察各组不同时间的嗅鞘细胞生长状况和形态学特征。采用GFAP、NGFRp75和Hoechst33258等抗体,于分离培养14 d后进行细胞免疫荧光染色并检测嗅鞘细胞的纯度。 结果：①倒置显微镜观察：差速后3 d内有大量细胞贴壁生长,细胞形态较混杂,辨认嗅鞘细胞较困难。差速后再运用无血清条件培养液2 d后,可见纯度较高的典型嗅鞘细胞形态,以双极梭形和多极为主,细胞突起细长。伴少量扁平状“油煎蛋样”细胞。继续培养至14 d可见细胞立体感及折光性最佳,数量和纯度最高。培养3周后3组细胞开始老化,活力明显下降,成纤维等杂细胞开始挤占原嗅鞘细胞生长空间。②免疫荧光染色显示嗅鞘细胞特异性表达GFAP和NGFRp75,单纯差速后嗅鞘细胞纯度仅有72%~75%,单纯无血清条件培养液纯化后仅为48%~52%,改良差速贴壁+无血清条件培养液纯化组纯度可达88%~92%。 结论：实验建立了完善的成年大鼠嗅鞘细胞培养纯化新方法。     

改良Nash差速贴壁联合阿糖胞苷法体外分离培养大鼠嗅球及嗅黏膜源性嗅鞘细胞*☆

背景：目前常用的嗅鞘细胞培养方法有差速贴壁法、化学抑制法、抗体亲和吸附法、补体法等,各自均存在优缺点,单一使用某种方法时细胞纯化率较低。 目的：拟采用改良Nash差速贴壁+阿糖胞苷法体外分离纯化大鼠嗅球及嗅黏膜来源的嗅鞘细胞。 设计、时间及地点：细胞学体外对照观察,于2007-11/2008-05在武汉大学人民医院骨科实验室和消化内科实验室完成。 材料：10周龄Sprague-Dauley大鼠10只,由武汉大学人民医院实验动物中心提供,阿糖胞苷由武汉大学人民医院制备。 方法：完整取大鼠双侧嗅球及剪取鼻中隔后1/3嗅黏膜,剪碎后胰酶消化,制成单细胞悬液,按1×109 L-1密度接种于未包被的25 cm2玻璃培养瓶中,18~20 h后将细胞悬液转移至另一未包被的25 cm 2玻璃培养瓶中（第1次差速贴壁）,24 h后再将细胞悬液转移至经多聚右旋赖氨酸包被的25 cm2塑料培养瓶或经多聚右旋赖氨酸包被的6孔培养板中（第2次差速贴壁）,接种培养48 h后半量换液以去除杂细胞。在差速贴壁培养后二三天,加入3.0~5.0 pmol/L阿糖胞苷去除残余成纤维细胞。 主要观察指标：嗅鞘细胞的形态观察、分裂增殖情况、免疫荧光染色鉴定结果及纯度测定。 结果：体外培养24 h嗅球源性嗅鞘细胞即可贴壁,而嗅黏膜源性嗅鞘细胞多在培养四五天后贴壁,两种来源的嗅鞘细胞形态相似,以双极或梭形细胞为主,少量为3极及多突起形多级细胞,同时夹杂扁平、煎鸡蛋形细胞。纯化培养10 d的嗅鞘细胞,胶质纤维酸性蛋白、神经生长因子受体p75免疫荧光染色及神经生长因子受体p75+hoechs免疫荧光双染后大部分双极或多极细胞膜、胞体、突起呈阳性,细胞纯度可达90%以上。 结论：改良Nash差速贴壁+阿糖胞苷法可在体外成功分离培养出高纯度的嗅球及嗅黏膜源性嗅鞘细胞,嗅球源性嗅鞘细胞贴壁时间及分裂增殖程度优于嗅黏膜源性嗅鞘细胞。     

分离、培养及纯化嗅鞘细胞的简便方法

目的：建立一种免胰酶消化的剪切分散法提取嗅鞘细胞（olfactory ensheathing cells,OECs）并优化其纯化培养方法。方法：从成年SD大鼠解剖分离嗅球,显微镜下剥离软脑膜、剔除中央白质,将灰质成分置于含10%胎牛血清的DMEM/F12全培养基,眼科剪剪切至组织成糜状,吸管吹打15-20次, 混悬液免胰酶消化直接接种培养,每三天半量换液。原代培养至细胞大部分融合后,依次以低浓度胰酶（0.05%）有限消化法、30min短时差速贴壁法、抗有丝分裂法三步顺序纯化细胞,每步纯化结果作免疫荧光染色鉴定。结果: 原代培养中嗅鞘细胞快速贴壁成为优势细胞、生长旺盛,三步顺序纯化过程细胞纯度逐步提高,最终纯度可达95%以上并长时间维持。结论:剪切分散法提取OECs及其三步顺序法纯化培养经济实用、易重复,细胞培养周期短,能收获大量可控纯度的嗅鞘细胞供进一步移植实验及基础研究。     

改良成年大鼠嗅鞘细胞的原代培养

目的研究成年大鼠嗅鞘细胞(OECs)分离与培养的方法,为进一步进行基础研究以及临床应用提供高纯度的细胞.方法采用改良差速贴壁法从成年大鼠的嗅球培养出OECs,并用Forskolin和碱性成纤维细胞生长因子(bFGF)促增殖,最后进行OECs特异标记物NGFRp75、S-100的免疫细胞化学染色鉴定OECs.结果改良差速贴壁法成功培养出高纯度的OECs.结论此法简便、经济、纯化效率高,Forskolin和bFGF能够促进OECs的增殖.     

人嗅粘膜嗅鞘细胞的分离、培养及生物学特性

目的细胞移植是目前的研究热点,本文主要研究人嗅粘膜嗅鞘细胞(OM-OECS)的分离、培养、纯化、鉴定,来观察其生长情况,分析其生物学特性,为具有神经功能障碍的患者实行同种细胞移植提供一种可行的方法。方法选取意外交通事故或意外疾病、事故急性死亡3h内及经蝶窦手术的患者,征得家属同意,取鼻中隔后1/3鼻粘膜,免疫吸附法结合采用P75抗体包被的培养皿进行纯化人OM-OECs,免疫组化方法进行细胞的鉴定。结果 OM-OECs体外培养过程中,采用免疫吸附法使抗体与间充质来源的成纤维细胞进行特异结合从而将其去除,P75抗体包被的培养皿促进了OECs的贴壁使其与杂质细胞进一步分离,在体外培养10d后,细胞增殖最快,经P75、GFAP免疫组化双标染色显示呈双阳性,计数阳性细胞率达90%以上,说明从人嗅粘膜分离培养OECs具有较高的纯度。结论从人鼻粘膜取材,可以分离培养出OECs,体外生长增殖旺盛,并且经纯化后具有较高的纯度。     

大鼠背根神经节细胞的纯化培养

目的:在NB1培养基中建立体外胚胎大鼠背根神经节(DRG)细胞的纯化培养体系。方法:取胎鼠的背根神经节,用胰蛋白酶消化法分离成单细胞,通过差速贴壁法进行背根神经节神经元的分离纯化,在NB1中培养,用活细胞计数和神经元特异性的烯醇化酶(NSE)免疫组织化学染色相结合判定培养神经元的纯度。结果:纯化培养神经元生长状态良好,培养4天时神经元纯度为91％左右。结论:采用差速贴壁法可获得高纯度的神经元,可作为神经科学研究的重要工具。     

大鼠脊髓少突胶质前体细胞的培养与鉴定方法

目的 探讨大鼠脊髓少突胶质前体细胞（OPCs）的分离纯化及诱导分化方法.方法 取出生后3d内SD大鼠脊髓,采用0.125％胰蛋白酶消化获取原代混合胶质细胞,培养10 d左右在37℃恒温摇床采取180 r/min摇速振摇并差速贴壁40 min获得纯化的OPCs;取纯化后培养3d的OPCs免疫荧光鉴定细胞纯度或诱导分化.结果 采用胰蛋白酶消化法获取原代混合胶质细胞、振摇并差速贴壁法分离纯化能够得到纯度较高的OPCs,折光性强,呈双极或三极形态,免疫荧光染色显示95％细胞表达A2B5和NG2（OPCs标志物）,经诱导分化后表达O4及MBP（少突胶质细胞标志物）.结论 采用振摇差速贴壁法可从大鼠脊髓中分离纯化获得高纯度的OPCs,获得的OPCs体外生长稳定,经诱导可分化为成熟的少突胶质细胞.     

大鼠乳鼠和成鼠雪旺细胞提取纯化的对照研究*

背景：许旺细胞是神经创伤修复的种子细胞,获取大量高纯度、高活性许旺细胞是研究的关键。 目的：比较乳鼠和成年鼠许旺细胞的体外培养、纯化和形态学的差别,探讨简单可行的、可以获得高纯度许旺细胞的培养方法。 方法：出生1~3 d SD大鼠20只和成年大鼠10只(体质量150~200 g),实验按细胞的来源分为新生组和成年组。双酶分步消化,二次接种差速分离纯化细胞;倒置显微镜观察细胞形态、贴壁速度;细胞计数,纯度计算;MTT法检测细胞增殖能力,绘制两组许旺细胞的增殖曲线,判定增殖速度;S-100免疫化学法鉴定细胞。 结果与结论：乳鼠的许旺细胞贴壁速度快于成纤维细胞,成鼠的许旺细胞贴壁速度慢于成纤维细胞,两组许旺细胞纯度均达96%以上;MTT法检测两组许旺细胞增殖均活跃,由增殖曲线显示乳鼠许旺细胞增殖更快(P < 0.05);S-100免疫化学反应均呈阳性。提示双酶分步消化,二次接种差速分离纯化细胞,可以获取纯度高、活性良好的许旺细胞;乳鼠许旺细胞的增殖、贴壁能力更强。     

不同来源嗅鞘细胞修复脊髓损伤的效果比较

背景：研究表明嗅鞘细胞所分泌的细胞黏附分子和神经营养因子具有保护脊髓神经元和促进脊髓轴突再生的效应。 目的：比较嗅球及嗅黏膜固有层来源的嗅鞘细胞异体移植修复脊髓损伤的能力。 设计、时间及地点：随机对照动物实验,于2007-06/2008-06在西电集团医院中心实验室完成。 材料：随机选取雄性3月龄及23月龄SD大鼠各6只,分为实验组(23月龄)和对照组(3月龄),用于嗅鞘细胞的体外培养和纯化;SD大鼠30只随机分为乳鼠嗅球嗅鞘细胞移植组、正常嗅黏膜嗅鞘细胞移植组、对照组,每组10只。 方法：30只SD大鼠制造脊髓损伤模型,分别将体外培养的乳鼠和SD大鼠嗅鞘细胞进行脊髓损伤模型的异体移植,对照组不做移植。 主要观察指标：术后4,8周,进行BBB神经功能评分,诱发电位,组织病理学观察。 结果：实验过程中大鼠死亡7只,各组死亡率大致相同。移植后第4,8周时,乳鼠嗅鞘细胞移植组、正常嗅黏膜嗅鞘细胞组评分差异无显著性意义(P > 0.05),均显著高于空白对照组(P < 0.001);嗅鞘细胞移植2组评分8周高于4周(P < 0.01)。术后4周,各组动物均未引出运动诱发电位,移植后8周时,2组嗅鞘细胞移植组动物均可引出运动诱发电位,2组差异无显著性意义(P > 0.05),空白对照组动物仍未引出运动诱发电位(P < 0.001)。移植后8周,2组嗅鞘细胞移植组脊髓损伤区有较多细胞浸润,对照组细胞数目较少。 结论：来源于嗅球与嗅黏膜的嗅鞘细胞对脊髓损伤修复均有促进作用,且两者作用无明显差异。     

Olfactory ensheathing cells from the nasal mucosa and olfactory bulb have distinct membrane properties

Transplantation of olfactory ensheathing cells (OECs) is a potential therapy for the regeneration of damaged neurons. While they maintain tissue homeostasis in the olfactory mucosa (OM) and olfactory bulb (OB), their regenerative properties also support the normal sense of smell by enabling continual turnover and axonal regrowth of olfactory sensory neurons (OSNs). However, the molecular physiology of OECs is not fully understood, especially that of OECs from the mucosa. Here, we carried out whole-cell patch-clamp recordings from individual OECs cultured from the OM and OB of the adult rat, and from the human OM. A subset of OECs from the rat OM cultured 1–3 days in vitro had large weakly rectifying K⁺ currents, which were sensitive to Ba²⁺ and desipramine, blockers of Kir4-family channels. Kir4.1 immunofluorescence was detectable in cultured OM cells colabeled for the OEC marker S100, and in S100-labeled cells found adjacent to OSN axons in mucosal sections. OECs cultured from rat OB had distinct properties though, displaying strongly rectifying inward currents at hyperpolarized membrane potentials and strongly rectifying outward currents at depolarized potentials. Kir4.1 immunofluorescence was not evident in OECs adjacent to axons of OSNs in the OB. A subset of human OECs cultured from the OM of adults had membrane properties comparable to those of the rat OM that is dominated by Ba²⁺-sensitive weak inwardly rectifying currents. The membrane properties of peripheral OECs are different to those of central OECs, suggesting they may play distinct roles during olfaction.     

Olfactory ensheathing cells: Nitric oxide production and innate immunity

Olfactory nerves extend from the nasal cavity to the central nervous system and provide therefore, a direct route for pathogenic infection of the brain. Since actual infection by this route remains relatively uncommon, powerful endogenous mechanisms for preventing microbial infection must exist, but these remain poorly understood. Our previous studies unexpectedly revealed that the unique glial cells that ensheath olfactory nerves, olfactory ensheathing cells (OECs), expressed components of the innate immune response. In this study, we show that OECs are able to detect and respond to bacterial challenge via the synthesis of nitric oxide. In vitro studies revealed that inducible nitric oxide synthase (iNOS) mRNA and protein were present in Escherichia coli‐ and Staphylococcus aureus‐incubated OECs, but were barely detectable in untreated OECs. Neuronal NOS and endothelial NOS were not expressed by OECs pre‐ and post‐bacterial incubation. Nuclear translocation of nuclear factor kappa B (NFκB), detectable in the majority of OECs 1 h following bacterial incubation, preceded iNOS induction which resulted in the production of nitric oxide. N^G‐methyl‐L ‐arginine significantly attenuated nitric oxide (P < 0.001) and nitrite production (P < 0.001) by OECs. In rat olfactory mucosa which was compromised by irrigation with 0.17M zinc sulfate or 0.7% Triton X‐100 to facilitate bacterial infiltration, OECs contributed to a robust synthesis of iNOS. These data strongly support the hypothesis that OECs are an essential component of the innate immune response against bacterial invasion of the central nervous system via olfactory nerves. © 2009 Wiley‐Liss, Inc.     

年轻成年GFP大鼠嗅鞘细胞的分离培养与形态学特征

目的 建立年轻成年GFP大鼠嗅鞘细胞(OECs)的分离培养方法 ,为OECs移植治疗脊髓损伤奠定基础. 方法 解剖显微镜下、无菌环境取2.5月龄的GFP大鼠嗅球最外层,经酶消化法分散后将细胞接种于含20%胎牛血清的DMEM/F12培养基中.定期进行光电镜观察并摄片.在培养第10天进行S-100和NGFRp75的免疫细胞化学染色鉴定,并计算OECs的纯度. 结果 培养的GFP-OECs在荧光显微镜下呈绿色强荧光,形态以双极、多突起形为主,呈S-100、NGFR p75阳性,纯度在95%以上.电镜下形态与光镜下一致,但细胞结构更为精细. 结论 本文所用方法 简便易行,可分离出高纯度的GFP-OECs,其形态学特征与生物学活性无变异.源自GFP转基因大鼠的OECs可以作为一种有效的工具细胞,广泛应用于OEC在脊髓损伤修复中的研究.     

Galanin inhibits the proliferation of glial olfactory ensheathing cells

The effect of galanin (GAL) on neural proliferation was studied in this article using olfactory ensheathing cells (OECs). OECs were isolated from newborn rat olfactory bulb and cultured in vitro. RT-PCR was used to determine the expression of GAL and its receptors in these cells. MTT analysis and LDH assay were used to detect the effects of GAL and the agonist, antagonist of GAL receptors on the proliferation of OECs. Results show that OECs express mRNAs for GAL and GAL receptor2 (GalR2) but not for the two other GAL receptors, GalR1 and GalR3. In addition, GAL and two receptor agonists, GAL1-11 and GAL2-11, can inhibit the proliferation of OECs significantly, but cause no cytotoxicity in the OECs population. Moreover, the influence can be blocked by M35, a nonspecific antagonist of GAL receptors. It is suggested that GAL is an inhibitory factor in regulating OECs proliferation.     

嗅鞘细胞移植损伤脊髓组织的超微结构变化

背景：脊髓损伤后神经功能难以自行恢复,嗅鞘细胞具有外周性和中枢性两种胶质细胞的成鞘功能,是修复受损神经最有前途的种子细胞。嗅鞘细胞移植到受损脊髓后的组织学和超微结构的变化可能帮助解释嗅鞘细胞发挥修复作用的机制。 目的: 验证嗅球源性嗅鞘细胞移植对脊髓损伤功能恢复的促进作用,并观察移植的嗅鞘细胞对神经元和轴突组织和超微结构的影响。 方法：将已制备脊髓模型的Wistar大鼠随机分为3组,对照组不做任何注射操作,DMEM/F12组注射DMEM/F12培养基,嗅鞘细胞组注射嗅鞘细胞悬液。每周进行肢体活动BBB评分,8周后取脊髓标本进行组织学和免疫组织化学观察,评价脊髓损伤的修复情况,并观察嗅鞘细胞移植对脊髓组织和超微结构的影响。 结果与结论：3组动物均出现后肢运动功能的恢复,嗅鞘细胞组优于对照组和DMEM/F12组,在4周后更为明显。组织学观察可见,在嗅鞘细胞组可见有神经纤维通过损伤处。损伤处附近,嗅鞘细胞组脊髓腹侧的神经纤维和神经元形态较好,损伤较轻。而对照组和DMEM/F12组神经纤维和神经元损害严重。嗅鞘细胞组的caspsase-3阳性细胞数少于对照组和DMEM/F12组。超微结构观察可见,嗅鞘细胞组的神经纤维和细胞形态均优于对照组和DMEM/F12组。结果表明嗅鞘细胞移植对大鼠脊髓损伤修复有明显的促进作用,并可恢复损伤神经的部分功能,对受损神经纤维和神经元有保护作用。     

嗅鞘细胞脑出血大鼠脑内移植的运动神经功能改变和形态学研究

目的 探索嗅鞘细胞脑出血大鼠脑内移植后运动神经功能及形态学改变.方法 差速贴壁法培养出较高纯度的嗅鞘细胞,用PI和P75荧光双染鉴定细胞,制作30只脑出血大鼠模型,分A、B、C 3组.A组10只为单纯脑出血组;B组10只为培养液移植组;C组10只为嗅鞘细胞移植组.3组大鼠分别在术前1h,术后7d、14d、21d、28d进行运动功能学评分,并于细胞移植后第4周、第8周取脑组织做冰冻切片,通过免疫荧光检测移植细胞在大鼠脑内形态学改变.结果 试验中分离、培养的嗅鞘细胞经标记移植入大鼠脑内后,经免疫荧光检测证实能在血肿腔边缘分化为成熟神经元细胞和星形胶质细胞.对3组大鼠术后进行评分,细胞移植组从第21天起其功能恢复明显优于其它两组(P<0.001).结论 嗅鞘细胞在脑出血大鼠脑内能存活并分化为成熟神经元及星形胶质细胞,能促进运动神经功能恢复.

Abstract：

Objective The olfactory ensheathing cells ( OECs) transplanted into cerebral hemorrhage mode to alter motor nerve function and the study of morphology. Methods Get high purification of olfactory ensheathing cells by using the method of the differing rates of attachment of the various cells type, use the technology of fluoresceinstain identificated the OECs by both PI and P75 ,make 30 rats of cerebral hemorrhage mode,and separate 3 groups. Ten rats in the A group are the blank group;Ten rats in the B group are the DPBS transplanting group;Ten rats in the C group are the OECs transplanting group. The three groups are all scored in 1 hour before operation and 7 days, 14 days,21 days and 28 days after operation by Rosenberg GA method. Evaluate the contribution of OECs transplanting curing rat cerebral hemorrhage by statistics method. And get brain of rat to make frozen section in 4weeks,8weeks after operation. Detection the morphology of transplanting cells by immunofluorescence technique. Results We get in experiment transplantd in cerebral hemorrhage rats brain. We found these OECs differentiating to riped neuron cell and horizontal cell unround cavity of blood tumor by immunofluorescence way. After scoring the three groups by Rosenberg GA method and statisticing them. We found that olfactory ensheathing cells injected significantly reduced motor deficits compared with control groups which cerebral hemorrhage after 21 days (P <0.001). Conclusions The OECs survival in the brain of rat cerebral hemorrhage, then differentiating to ripe neuron cell and horizontal cell. The treatment of OECs transplanting for rat cerebral hemorrhage could promote recovery of motor nerve function.     

Hepatocyte growth factor is a mitogen for olfactory ensheathing cells.

Hepatocyte growth factor (HGF) is a potent mitogen for mature hepatocytes, and it has multi-functional effects in a variety of cells in various organs. HGF stimulates DNA synthesis and promotes cell migration and morphogenesis in several cell types including the olfactory system. To characterize the potential mitogenic activity of HGF that might contribute to olfactory ensheathing cell (OEC) proliferation, we tested the ability of HGF to stimulate OEC division in vitro. OECs were obtained from adult rat olfactory bulbs and cultured in serum-free medium, and were identified by double immunostaining for p75 and S-100 antibodies. DNA synthesis assayed by pulsing BrdU for 24 hr showed that HGF at the concentration of 5-100 ng/ml elicited a 5-10-fold increase of OEC proliferation. By immunocytochemical analysis, we demonstrated that c-Met-immunoreactivity was present in cultured OECs, and c-Met anti-serum significantly sequestered the activity of HGF on OECs proliferation, suggesting that HGF-induced proliferation of OECs is mediated by the c-Met receptor. The mitogenic activity of HGF was potentiated by addition of heregulin (HRG), but inhibited by addition of forskolin. These results demonstrate that HGF is a novel mitogen for rat OECs in vitro, and HGF/c-Met system is involved in regulating OECs growth and development.