功能化自组装多肽水凝胶与嗅鞘细胞生物相容性研究

Olfactory ensheathing cell (OEC) transplantation is a promising or potential therapy for spinal cord injury (SCI). However, the effects of injecting OECs directly into SCI site have been limited and unsatisfied due to the complexity of SCI. To improve the outcome, proper biomaterials are thought to be helpful since these materials would allow the cells to grow three-dimensionally and guide cell migration. In this study, we made a new peptide hydrogel scaffold which named GRGDSPmx by mixing the pure RADA16 and designer-peptide RADA16-GRGDSP solution, and we examined the molecular integration of the mixed nanofiber scaffolds using Atomic force microscopy (AFM). In addition, we have studied the behavior of OECs in GRGDSPmx condition as well as on RADA16 scaffold by analyzing their phenotypes such as cell proliferation, apoptosis, survival and morphology. The experimental results showed that GRGDSPmx could be self-assembled to form a hydrogel. Inverted optical microscope and Scanning electron microscope (SEM) analysis showed that OECs are viable and they proliferate within the nanostructured environment of the scaffold. MTT assay demonstrated that OECs proliferation rate was increased on GRGDSPmx scaffold compared to the pure RADA16 scaffold. In addition, OECs on GRGDSPmx scaffolds also showed less apoptosis and maintained the original spindle-shape morphology. Calcein-AM/PI fluorescence staining revealed that OECs cultured on GRGDSPmx grew well and the viable cell count was 95%. These results suggested that this new hydrogel scaffold provided an ideal substrate for OEC 3D culture and suggested its further application for SCI repair.     

In vitro evaluation of the compatibility of a novel collagen-heparan sulfate biological scaffold with olfactory ensheathing cells

Background Stroke and traumatic injury to the nerve system may trigger axonal destruction and the formation of scar tissue, cystic cavitations and physical gaps. Olfactory ensheathing cells （OECs） can secrete neurotrophic factors to promote neurite growth and thus act as a prime candidate for autologous transplantation. Biological scaffolds can provide a robust delivery vehicle to injured nerve tissue for neural cell transplantation strategies, owing to the porous three-dimensional structures （3D）. So transplantation of the purposeful cells seeded scaffolds may be a promising method for nerve tissue repair. This study aimed to evaluate the compatibility of a novel collagen-heparan sulfate biological scaffold with olfactory ensheathing cells in vitro. Methods Collagen-heparan sulfate （CHS） biological scaffolds were made, and then the scaffolds and OECs were co-cultured in vitro. The viability of OECs was tested by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyl tetrazolium （MTT） assay at days 1, 3, 5 and 7. Statistical analysis was evaluated by student＇s ttest. Significance was accepted at P 〈0.05. OECs were labeled with carboxyfluorescein diacetate succinimidyl ester （CFSE）, and the CFSE-labeled OECs were seeded into CHS scaffolds. The attachment and growth of OECs in CHS scaffolds were observed and traced directly by fluorescent microscopy and environmental scanning electron microscope （ESEM）. Results CHS biological scaffolds had steady porous 3D structures and no cytotoxicity to OECs （F=-0.14, P=-0.9330）. CHS biological scaffolds were good bridging materials for OECs attachment and proliferation, and they promoted the axonal growth. Conclusion The compatibility of CHS biological scaffolds with OECs is pretty good and CHS biological scaffold is a promising cell carrier for the implantation of OECs in nerve tissue bioengineering.     

体外评估硫酸肝素-胶原蛋白生物支架与嗅鞘细胞生物相容性的研究

背景：中风和神经系统的外伤和卒中可导致神经功能缺损,同时伴随轴突受损,形成胶质瘢痕、空腔和裂隙。嗅鞘细胞作为自体移植最佳的候选细胞,可以分泌神经营养因子促进轴突的生长。并且生物支架具有多孔的三维立体结构,能够运载细胞填充和桥连这些空隙。因此移植支架联合种子细胞是一种修复神经组织重要的策略。 方法：硫酸肝素-胶原蛋白生物支架经过一系列的程序而制作成功。将硫酸肝素-胶原蛋白生物支架与嗅鞘细胞在体外共培养,在第1、3、5和7天时用MTT法检测细胞活性。数据分析采用重复测量设计的方差分析,当P < 0.05时被认为具有显著性差异。之后将嗅鞘细胞用CFSE标记,通过流式细胞仪检测标记率为99.8%。用CFSE标记后的嗅鞘细胞以适宜的浓度种植于支架上。细胞在支架上的粘附和生长可以通过荧光显微镜和扫描电镜直接追踪和观察。 结果：硫酸肝素-胶原蛋白生物支架具有稳定的三维立体结构,对于嗅鞘细胞无毒性（F= 0.14,P= 0.9330）。除此之外,细胞及其轴突也能够在该支架中很好的黏附和生长。 结论：本实验表明硫酸肝素-胶原蛋白生物支架与嗅鞘细胞在体外具有很好的生物相容性。因此,硫酸肝素-胶原蛋白生物支架可能可以作为一种载体,种植嗅鞘细胞后应用于神经组织工程。     

功能化自组装多肽水凝胶支架促进小鼠骨髓间充质干细胞的粘附、增殖及成骨

目的 探讨功能化自组装纳米多肽DGEAmx水凝胶对小鼠骨髓间充质干细胞(mesenchymal stem cells,MSCs)的粘附、增殖和成骨分化的影响.方法 原子力显微镜观察功能化自组装多肽形成的纳米级结构特征;用倒置显微镜对贴壁细胞拍照计数,对比细胞粘附情况;用CCK-8法检测细胞增殖;激光共聚焦显微镜观察细胞在材料表面形貌;检测碱性磷酸酶(alkaline phosphatase,ALP)活性、成骨分化特异性基因Ⅰ型胶原(ICAM-1)和骨钙蛋白(osteocalcin,OCN)的表达来评价成骨效果.结果 原子力显微镜观察功能化自组装多肽DGEAmx可以形成纳米纤维网状结构;倒置显微镜对贴壁细胞拍照计数结果显示在30、60、90 min时DGEAmx组对比RADA16-Ⅰ组粘附细胞数差异有统计学意义(P＜0.05);CCK-8法检测结果显示在培养第3、5、7天,DGEAmx组MSCs增殖较RADA16-Ⅰ组明显增高(P＜0.05);DGEAmx组成骨诱导3、7、14 d后,ALP活性高于RADA16-Ⅰ组(P＜0.05);Real-time PCR结果显示成骨诱导3、7、14 d后DGEAmx组较RADA16-Ⅰ组有更高的ICAM-1和OCN的表达(P＜0.05).结论 功能化自组装多肽DGEAmx纳米纤维水凝胶支架能促进MSCs粘附、增殖和成骨分化,作为骨组织工程支架材料有良好的应用潜力.     

A novel biosynthetic hybrid scaffold seeded with olfactory ensheathing cells for treatment of spinal cord injuries

Background Implantation of tissue-engineered scaffolds is one of the most promising therapeutic strategies for inducing nerve regenerations following spinal cord injuries. In this paper, we report a novel bioengineered hybrid scaffold comprised of three major extracellular matrix （ECM） proteins. Methods ECM-scaffolds （ECM-S） were prepared by gelling fibrinogen, fibronectin and laminin using fresh rat plasma. Olfactory ensheathing cells （OECs） were isolated from fresh rat olfactory mucosa, purified under differential adhesion, and assessed by immunofluorescent staining. OECs were seeded onto ECM-S and cultured. The effects of the scaffolds on the seeded cells were detected using the immunofluorescent staining, Western blotting, scanning electron microscopy and transmission electron microscopy. Results Tissue-engineered ECM-S could be easily molded into mat-like or cylindrical shapes and gelled by addition of fresh plasma. Observations by electron microscopy show that the ECM-S forms a stable three-dimensional porous network. Studies on the effects of the ECM-S on the biological behaviors of OECs in vitro indicate that the scaffold can promote OEC adhesion, proliferation and process extensions. Additionally, OECs seeded on the scaffold maintained the expression of nerve growth factor, matrix metalloproteinase-3 and matrix metalloproteinase-9. Conclusion We developed a biosynthetic hybrid gel which could be used as a scaffold for OEC transplantation; this gel can promote nerve regeneration following spinal cord injuries. Chin Med J 2009; 122（17）：2032-2040     

静电纺丝聚乳酸-乙醇酸聚合物膜与大鼠嗅鞘细胞的生物相容性研究

目的 探索静电纺丝技术制作的聚乳酸-乙醇酸聚合物（PLGA）膜与大鼠嗅鞘细胞（olfactory ensheathing cells,OECs）的生物相容性。方法 体外培养纯化成年大鼠OECs,将细胞接种于静电纺丝PLGA膜上,使用相差显微镜观察静电纺丝PLGA膜上OECs的细胞形态与分布;用CFDA SE荧光染色检测接种后1～5 d内静电纺丝PLGA膜上OECs的增殖情况;以多聚赖氨酸（PLL）包被细胞做对照。将静电纺丝PLGA膜植入大鼠体内观察其与大鼠的组织相容性。结果 体外培养纯化所得的OECs的细胞纯度大于90%。相差显微镜下示OECs在静电纺丝PLGA膜孔隙中的纳米纤维上黏附良好,细胞状态佳,且特异性地沿纳米纤维方向生长。接种后1～5 d内OECs在静电纺丝PLGA膜上均正常增殖,细胞数目与对照组相比差异无统计学意义。静电纺丝PLGA膜局部移植后大鼠无死亡,PLGA膜与周围组织融合并部分降解。结论 静电纺丝PLGA膜具有良好的生物相容性,OECs在其膜上的黏附和增殖状况良好,是有潜力的神经修复组织工程化神经移植物。     

嗅球成鞘细胞的培养纯化及与功能相关的形态学研究

目的 培养纯化嗅球成鞘细胞(Olfactory ensheathing cells，OECs)并对其进行免疫细胞化学研究，探讨其相关功能。方法 分离嗅球嗅神经纤维层获得嗅球成鞘细胞，采用免疫溶解法对培养的嗅球成鞘细胞进行纯化，并利用免疫细胞化学方法和电镜技术进行形态学观察。结果 通过纯化可以使培养的OECs纯度达95％以上，免疫细胞化学显示OECs表达P75NGFr、GAP43、N—CAM和GFAP；电镜结果显示OECs呈分叶状核，胞浆电子致密，胞膜有伪足样皱褶。结论 免疫溶解法可以较好的纯化OECs并保持其良好的生长状态，培养的OECs表达P75NGFr、GAP43、N-CAM可能与其促进轴突再生的功能相关。     

嗅鞘细胞与神经修复支架材料的相互作用

目的：探讨一种提取高纯度和一定数量的嗅鞘细胞(OECs)的新方法,并研究其与神经支架修复材料PLGL的生物相容性。方法：从新生的 Wister 大鼠身上分离制备嗅鞘细胞并与PLGL共同培养。测量其接触角、吸附率、存活率等。结果：PDLLA的接触角（84.5°±1.5°）明显大于PLGL的接触角（52.6°±0.8°）。PLGL的吸附率（80%）明显高于PDLLA（57%）。PLGL的细胞存活率（88%）明显高于PDLLA（76%）。结论：PLGL比PDLLA具有更好的亲水性、存活率以及良好的细胞生长状况。     

大鼠嗅黏膜嗅鞘细胞的分裂增殖和免疫细胞化学特性

目的:体外培养大鼠嗅黏膜嗅鞘细胞(olfactoryensheathingcells,OECs),观察其形态、分裂增殖和免疫细胞化学特性,探索自体嗅黏膜嗅鞘细胞作为OECs来源的可能性。方法:采用差时贴壁的方法分离培养成年大鼠嗅黏膜嗅鞘细胞,相差显微镜下观察细胞形态和分裂增殖;用免疫组化方法观察GFAP、NGFRp75、S鄄100、NGF、BDNF、NT鄄3的表达。结果:培养的细胞根据形态来分,可分为双极、多极和偏平状;细胞分裂时先回缩成球形,分裂成两个子球,再伸出突起,变成梭形或多极形。GFAP、NGFRp75、S鄄100、BDNF和NT鄄3表达阳性。结论:在含血清培养基上,大鼠嗅黏膜嗅鞘细胞能正常分裂增殖和分泌神经营养因子。     

人胚嗅粘膜嗅鞘细胞的分离、培养和纯化

目的 采用经我们改良的差速贴壁法结合神经营养因子3(NT3)和低浓度血清的培养方法对人胚嗅粘膜嗅鞘细胞进行分离和原代纯化培养,探讨建立嗅粘膜嗅鞘细胞体外培养的方法.方法 对差速贴壁后的人胚嗅粘膜嗅鞘细胞交替应用含10%胎牛血清和含2.5%胎牛血清、NT3的DMEM-F12培养基进行原代培养.观察嗅粘膜嗅鞘细胞的形态学变化.采用p75NTR和GFAP免疫细胞化学染色进行鉴定和纯度检测.结果 人胚嗅粘膜嗅鞘细胞形态多呈双极、三极,伴有细长的突起.p75NTR和GFAP染色均呈阳性反应,体外培养9 d时纯度可达83%.结论 差速贴壁法结合低浓度血清和NT3应用可以分离培养出人胚嗅粘膜嗅鞘细胞.     

嗅鞘细胞对活化的星形胶质细胞的作用

目的:研究体外培养的大鼠嗅鞘细胞(olfactory ensheathing cell,OEC)对活化的星形胶质细胞(astrocytes,AST)的作用.方法:体外培养嗅鞘细胞和星形胶质细胞.星形胶质细胞经纯化传代,细胞融合后利用划伤法得到活化的星形胶质细胞,将嗅鞘细胞与活化的星形胶质细胞共培养24 h后,观察星形胶质细胞增殖能力的改变,以及其表达胶质纤维酸性蛋白(GFAP)和硫酸软骨素蛋白多糖(CSPG)的变化.结果:活化的星形胶质细胞与嗅鞘细胞共培养后,MTT实验表明其增殖能力增强;免疫荧光染色、RT-PCR和蛋白质印迹法测定结果表明共培养后活化的星形胶质细胞表达的GFAP和CSPG降低.结论:嗅鞘细胞作用于活化后的星形胶质细胞以后,能够促进星形胶质细胞的分裂增殖,同时能够减少星形胶质细胞胶质化,降低抑制性细胞因子的表达.     

细胞外基质支架对大鼠嗅鞘细胞神经营养素表达的影响

目的:构建一种复合型细胞外基质支架,并探讨其是否影响大鼠嗅鞘细胞(olfactory ensheathing cells,OEC)合成神经营养素。方法:将纤维蛋白原、层黏连蛋白和纤黏连蛋白按一定比例混合,在新鲜大鼠血浆促凝作用下,构建细胞外基质支架,在支架表面种植大鼠OEC,观察其生长增殖情况和对神经营养因子表达能力的影响。结果:OEC在复合支架上贴壁良好,增殖旺盛,伸出较长的突起。生长于复合支架上的OEC较玻璃片上的细胞合成更多的神经营养素。结论:细胞外基质支架能促进OEC增殖,同时促进OEC神经营养素分泌。     

Culture and purification of human fetal olfactory bulb ensheathing cells

Objective：To obtain high purity of human fetal olfactory bulb ensheathing cells （OB-hOECs） in vitro and to develop a simple and effective method for primary culture of OB-hOECs. Methods： OB-hOECs were cultured based on the differential rates of attachment of the various harvested cell types. Then the method was combined with arabinoside cytosine （Ara-C）inhibition, serum-free starvation or intermittent neurotrophin 3 （NT3） nutrition method to observe cell states in different cultural environments. The purity of OB-hOECs was assessed with immunocytochemical analysis. Results： OB-hOECs appeared bipolar and tripolar shape, with slender processes forming network. The purity of OECs reached 88% with the selective attachment method on day 6, and then fibroblast proliferated quickly and reduced the purity. When combined with the starvation method, the purity of OECs was 91% on day 6 and 86% on day 9, however, OECs were in a poor state. While combined with the NT3 method, the purity reached 95% on day 9 and 83% on day 12, respectively. The cells still remained in a good state. Conclusion： A combination of selective attachment and intermittent NT3 nutrition is an effective method to obtain OECs with higher purity and quality.     

免疫吸附法纯化人胚嗅鞘细胞的实验研究

目的:探索分离培养人胚嗅鞘细胞以及纯化的方法。方法:分离人胚嗅球,采用胰酶消化获取单细胞悬液,应用含Forskolin、BPE的DMEM/F12培养基,接种于p75包被的培养皿,培养2周后细胞行免疫组化染色鉴定。结果:培养细胞形态多为2￣4极细胞,成纤维细胞少,嗅鞘细胞纯度非常高,嗅鞘细胞标志物p75阳性。结论:嗅鞘细胞体外分离培养条件要求极高,应用p75免疫吸附纯化培养的嗅鞘细胞效果好。     

人血清及神经生长因子对成人鼻黏膜嗅鞘细胞体外增殖的影响

目的:观察人血清及神经生长因子对成人鼻黏膜嗅鞘细胞体外增殖的影响.方法:取成人(自愿者)鼻腔嗅黏膜,制成细胞悬液,分别用DF12培养基(对照组)、10 ng/ml NGF DF12培养基、10%胎牛血清的DF12培养基、10 ng/ml NGF 10%胎牛血清的DF12培养基、10%人血清的DF12培养基和10 ng/mlNGF 10%人血清的DF12培养基培养细胞,并用差时贴壁法纯化嗅鞘细胞.采用MTT法观察上述各种培养液对嗅鞘细胞增殖的影响.结果:加入与不加入NGF培养液组之间的光密度无统计学意义(P＞0.05);胎牛血清组、人血清组与对照组之间的光密度有显著统计学意义(P＜0.01);胎牛血清组和人血清组之间的光密度无统计学意义(P＞0.05).结论:人嗅鞘细胞的体外培养对血清具有依赖性,人自体血清培养可替代胎牛血清用于嗅鞘细胞的体外培养、扩增.本研究结果为人嗅鞘细胞的自体移植治疗脊髓损伤提供了可行性基础.     

新型自组装多肽水凝胶对成骨细胞前体细胞作用的体外研究

目的 观察新型自组装肽TRSAWmx水凝胶对在体外小鼠成骨细胞前体细胞MC3T3-E1细胞粘附效果、增殖能力及成骨分化能力的影响.方法 通过原子力显微镜观察新型水凝胶的自组装性能;以倒置显微镜对贴壁细胞计数,比较不同材料细胞的粘附;以CCK-8检测和评价不同材料上细胞的增殖;通过激光共聚焦显微镜观察细胞在材料表面的生长;茜素红-S染色观察钙结节形成;检测材料上培养的细胞碱性磷酸酶(alkaline phosphatase,ALP)活性以及细胞ALP、骨钙蛋白(osteocalcin,OCN)、血管内皮生长因子(vascular endothelial growth factor,VEGF)等基因的表达.结果 TRSAWmx能自组装成纳米纤维结构;在接种30、60、90 min时,TRSAWmx组细胞粘附数明显高于空白组(P＜0.05);3、5、7d时TRSAWmx组细胞有着更佳的增殖活力(P＜0.05);在成骨诱导培养3、7、14 d时,TRSAWmx组ALP活性明显高于RADA16-Ⅰ组(P＜0.05);培养14 d后,TRSAWmx组茜素红-S染色可见钙结节形成多,成骨相关基因ALP、OCN、VEGF表达水平更高(P＜0.05).结论 新型自组装多肽纳米水凝胶TRSAWmx在体外能有效提高MC3T3-E1细胞粘附、增殖和成骨分化能力.     

低氧诱导的大鼠嗅黏膜来源嗅鞘细胞自噬及其对细胞增殖能力的影响

目的: 探讨低氧诱导SD新生鼠嗅黏膜来源嗅鞘细胞(OECs)自噬的发生,分析自噬对细胞增殖能力的影响。方法: 采用酶消化法和改良的Nash差速贴壁法分离提纯培养SD新生鼠嗅黏膜来源OECs,利用免疫荧光法鉴定细胞属性和纯度。以低氧条件(氧浓度为2％)培养的SD新生鼠嗅黏膜来源OECs作为低氧处理组,以正常条件下培养的OECs作为对照组,采用自噬抑制剂3-MA处理后的OECs继续在低氧条件下培养作为3-MA低氧处理组。相差显微镜下观察各组细胞形态;Western blotting法检测对照组、4h低氧处理组和8h低氧处理组OECs中低氧诱导因子1α(HIF-1α)、自噬标志蛋白LC3Ⅰ/Ⅱ和自噬标志基因Beclin-1的蛋白表达水平;采用CCK-8法检测对照组、3-MA低氧处理组、低氧处理组和3-MA低氧处理组OECs的增殖能力。结果: 在对照组、低氧处理组和3-MA低氧处理组经分离提纯的OECs均呈梭形,表现为典型的双极、三极细胞形态,3组间无明显差异。OECs呈NGFRp75和GFAP阳性,纯度为87%;与对照组比较,低氧处理组OECs中HIF-1α表达水平升高(P<0.05),LC3Ⅰ/Ⅱ和Beclin-1表达水平升高(P<0.05);与低氧处理组比较,3-MA低氧处理组OECs增殖能力降低。结论: 低氧诱导的自噬可降低SD新生鼠嗅黏膜来源OECs的增殖能力。     

嗅鞘细胞上清液对抗过氧化氢诱导的星形胶质细胞损伤的线粒体机制

目的： 研究嗅鞘细胞上清液（olfactory ensheathing cell conditioned medium, OECCM）对星形胶质细胞的保护作用及其线粒体机制。方法： 先用500 μmol/L H₂O₂ 损伤星形胶质细胞20 min;再以50%的OECCM继续培养细胞24 h。MTT法检测星形胶质细胞的活性,Hoechst 33342 染色法和蛋白质印迹法检测细胞凋亡及凋亡相关蛋白caspase-3的表达;用JC-1染色法检测线粒体的膜电位。结果： OECCM能减少H₂O₂诱导的细胞凋亡,减少caspase-3的表达,并且使线粒体膜电位增高。结论： OECCM可以对抗500 μmol/L H₂O₂诱导的星形胶质细胞凋亡,其机制可能与稳定线粒体膜电位有关。     

成年大鼠嗅黏膜嗅鞘细胞的分离、培养、鉴定与纯化

目的:探讨嗅黏膜中嗅鞘细胞的培养方法.方法:取成年大鼠嗅黏膜,采用差速贴壁法获得较高纯度的嗅鞘细胞,并采用免疫荧光法进行鉴定与形态学观察.结果:通过差速贴壁法纯化,可获得纯度约为80%的嗅鞘细胞,并通过GFAP免疫荧光及P75和hoechst双重免疫荧光鉴定;相差显微镜下细胞形态以梭形、双极及三极细胞为主,有少量的多极细胞.结论:从成年大鼠嗅黏膜中成功分离培养出嗅鞘细胞,为从人鼻腔嗅黏膜中培养嗅鞘细胞提供重要的依据.     

Notch-1对大鼠嗅球成鞘细胞神经生长因子表达的影响

目的 观察Notch-1对嗅球成鞘细胞(olfactory ensheathing cells,OECs)神经生长因子(nerve growth factor,NGF)表达的影响.方法 原代培养大鼠OECs,用有活性的Notch质粒(NotchICV),无活性的Notch质粒(NotchVK)以及空质粒分别转染OECs,将实验对象分成NotchICV转染组,NotchVK转染组和空白质粒组,应用免疫荧光双标、Western blot及RT-PCR技术,检测各组OECs中NGF及其mRNA的表达变化.结果 转染Notch-1后,OECs中NGF的表达明显增高.免疫荧光双标结果显示,NotchICV转染组OECs 中NGF的表达较NotchVK转染组和空白质粒组均明显增高(P<0.05);RT-PCR结果显示,NotchICV转染组OECs中NGF mRNA的表达(0.496 1±0.026 9)均高于NotchVK转染组(0.244 5±0.013 4)及空白质粒组(0.245 9±0.016 0)(P<0.01);Western blot结果显示,NotchICV转染组OECs中NGF的表达(0.887 5±0.016 6)均高于NotchVK转染组(0.686 2±0.059 2)和空白质粒组(0.682 1±0.067 0)(P<0.01).结论 Notch-1转染OECs后,其NGF的表达上调,提示Notch-1能增强OECs中NGF的合成.