【作者】 陈巍；

【导师】 周国民；

【作者基本信息】 复旦大学 ， 医学信息学， 2009， 硕士

【摘要】 1.目的人工视觉是指由植入视觉假体产生电信号,刺激并激活视觉系统,从而使失明或濒临失明的患者重新获得部分有用视力。我国则于2005年批准了首个“973”项目,对人工视觉功能修复的基础理论与关键科学开展研究。先期,我们应用计算机技术,已初步成功重建了大鼠视神经内微血管的三维模型,确定了视觉假体电极接入视神经的最佳窗口。而生物相容性则是决定视觉假体材料是否能应用于临床的先决条件之一,本课题从神经胶质细胞的体外培养入手,参照有关标准采用了多种生物学试验方法,研究视神经胶质细胞与聚酰亚胺薄膜电极的黏附性及生物相容性、最佳体外电脉冲刺激参数,为人工视觉假体材料的植入及刺激参数的选择提供实验依据。2.方法本研究采用植块培养方法获得大鼠的视神经胶质细胞,用免疫荧光染色方法对细胞进行鉴定,选用传至3-5代的细胞用于生物相容性进行检测。通过制备聚酰亚胺薄膜电极浸提液,用CCK-8法检测其细胞毒性,计算细胞相对增殖率,并绘制细胞增值曲线。采用6级毒性分级法评价材料毒性,即0级为≥100%;1级为75%～99%;2级为50%～74%;3级30%～49%;4级为15%～29%;5级0～14%。将细胞接种在底部放置有聚酰亚胺薄膜电极材料的培养板,分别于第1,3,5,24小时观察细胞贴壁程度,检测其短期黏附性;将聚酰亚胺薄膜电极固定在培养板的底壁,接种剪碎的视神经组织块或经传代的视神经胶质细胞,倒置显微镜和扫描电镜下观察细胞在聚酰亚胺薄膜电极上的黏附及生长情况。建立电刺激神经胶质细胞体外模型,观察脉冲电流对神经胶质细胞生物学特性的影响。3.结果体外培养视神经组织块至第6-10天,可见细胞从组织块中爬出,多为长梭形,呈束状或条索状排列,核呈圆形或卵圆形。细胞培养9-14天后进行传代,经过3-5代扩增后,应用免疫荧光染色显示95%以上细胞为GFAP阳性。CCK-8法检测聚酰亚胺薄膜电极浸提液的细胞毒性,其OD值和细胞RGR与完全培养液比较差异不具有显著性,且表现出较高的细胞增殖率,浸提液组细胞毒性级为0-1级,即无细胞毒性作用,且随着培养时间的延长,细胞毒性趋于0级。短期黏附测试显示接种1、3小时薄膜电极组黏附性低于空白对照组,而接种5小时后,薄膜电极组与空白对照组比较差异无显著性意义,表明随时间的延长薄膜电极组的黏附性增加。聚酰亚胺薄膜电极表面的细胞黏附生长良好,该材料对体外培养的细胞形态无损害,对细胞的生长和增值均无明显抑制作用。扫描电镜下神经胶质细胞紧密贴附在材料表面,铺展良好并连接成片。将电脉冲刺激的电压与宽度分别固定为2.4V与5ms,给神经胶质细胞施以不同频率的刺激,发现当频率为10Hz时,脱落和死亡细胞较多,而当频率为1Hz则较少,两者之间的差异具有显著性(P<0.05),提示当刺激频率越低,对细胞的影响则越小。4.结论原代混合胶质细胞的培养纯化后,获得的星形胶质细胞纯度较高、活力较好,达到生物相容性测试要求。通过体外实验证实,聚酰亚胺薄膜电极与视神经胶质细胞有较好的黏附性和生物相容性,是一种优良的视神经植入材料。建立的体外电脉冲刺激模型能够使薄膜电极有效的对细胞产生刺激作用,并能使细胞在持续培养条件下受到薄膜电极电场的刺激,因而具有通用性。由于预设刺激方案是在体条件下响应的刺激参数,用此参数测试时发现细胞在各种频率下均有脱落和死亡,因此有必要对参数进行进一步的筛选。更多还原

【Abstract】 ObjectiveArtificial vision applies the implanted visual prosthesis to produce the electrical signals which stimulate and activate the visual system,so that the blind or the brink of blindness in some patients can restore partial useful vision.China approved the research in 2005 through the first "973" project for visual function repair’s basic theory and the key scientific questions.Earlier,we have acquired the 3-D reconstruction of microvasculature of optic nerve successfully by computer technology which determined the best window for optic nerve implantation. Biocompatibility is one of the preconditions which the visual prosthesis material can be applied in clinical.The research began with the culturing glial cells in vitro, performed manifold biological tests to evaluate the adhesion,biocompatibility between glial cell and polyimide(PI) film electrode and the best parameters of electrical pulse in vitro,providing scientific basis for the implant and the choice of stimulation parameters of artificial vision substrate material.MethodsThe research used the explant culture method to obtain the optic nerve glial cells of rat.We identified the cells by the immunofluorescent staining and selected 3-5 generation’s cells to carry on the examination for biocompatibility test.Through the preparation of the polyimide film electrode extraction,we examined the cytotoxicity with the CCK-8 assay,calculated the relative cell proliferation rate and draw value-added curve.Using 6 levels of toxic staging to evaluate material toxicity, that is 0-level for＞= 100%;1 level for 75%-99%;2 level for 50%-74%;3 level for 30%-49%;4 level for 15%-29%;5 level for 0-14%.The cells were inoculated on the polyimide film electrode which was placed in the bottom of culture plate and the short-term adhesion had been observed separately in the 1st、3rd、5th、24th hour.We fixed the polyimide film electrode to the bottom of culture plate,inoculated the optic nerve tissue or the generated optic glial cells on it,observed the adhesion and growth of glial cells on the polyimide film electrode by inverted microscope and scanning electron microscope.The electrical stimulation model of glial cells of rat in vitro would be established and the biology characteristic’s effect of pulse current to the glial cells would be observed.ResultsAfter 6-10 day’s explant culture,cells moved out of the optic nerve tissue.Most of them were long-spindle,cord-like or fascicular arrangement.Nucleuses were round or oval.The cells cultured for 9-14 days to generate,after passing through 3-5 generations,the immunity fluorescence dyeing demonstrated that more than 95%of the cells were GFAP positive.CCK-8 assay was used to evaluate the cytotoxicity of the polyimide film electrode extraction.The OD value and cellular RGR did not have significant difference compared with the complete culture medium and displayed high Cell proliferation rate.The cytotoxicity level of extraction group was 0-1 level,namely non-cytotoxicity function,and along with the culture time’s extension,the cytotoxicity tend to 0 level.The short-term adhesive test demonstrated that vaccinated at 1st＞3rd hour the adhesion of film electrode group was lower than the control group. Vaccinated for 5 hours later,there were no significant difference,indicating the adhesion of film electrode group to be increased along with the time’s extension.The adhesion was fine and no obvious changes in cultured cells attached to the surface of film electrode.The growth and proliferation of the cultured cells were not significantly inhibited with the substrate materials.Under the scanning electron microscope,the glial cells were observed closely attached to the surface of film electrode,the good spreading and the connecting into a film.A regular voltage and pulse width was fixed at 2.4V,5ms respectively as well as the stimulation time.The glial cells were subjected to different frequencies of stimulation and found that the 10Hz group cells showed significant detachment and necrosis while the 1Hz group cells showed lower.The difference was significant compared to normal control group(P＜0.05).The results suggested that the frequency was lower, the impact on cell was smaller.ConclusionThe glial cells were isolated and purified after primary glial culture.It become possible to obtain higher purity and active astrocytes which meet the biocompatibility testing requirements.In-vitro experiments confirmed that the polyimide film electrode was well adhesion and biocompatibility with the glial cell of rat,which was appropriate for optic nerve implant.The established models of in-vitro pulse current stimulation could cause the film electrodes to stimulate the cell effectively and caused the cell receive continual electrical pulse stimulation under the culture conditions.As the pre-installation stimulation plan was based on the body’s stimulation parameters.With these parameters,we discovered that the cells showed significant detachment and necrosis under variety of frequency,therefore it was necessary to carry on further screening under these parameters.更多还原