通络救脑注射液对离体胚鼠皮层神经干细胞增殖、分化作用的实验研究

【作者】 张丽娟；

【导师】 司银楚；

【作者基本信息】 北京中医药大学 ， 中西医结合基础， 2006， 硕士

【摘要】 神经干细胞是具有潜在的分化能力的细胞,即能在一定的条件下能增殖并定向分化,当成体神经组织受损或神经退行性变后,通过定向诱导神经干细胞向相应的神经组织或细胞分化,进而替代损伤组织或细胞。本研究拟探讨通络救脑注射液对神经干细胞增殖、分化的影响,拓宽中医祛瘀生新法在脑病治疗中的现代医学理论基础,希望通过对其深入研究,为外源性神经干细胞的替代治疗提供证据。1胚鼠皮层神经干细胞的培养、鉴定及通络救脑注射液对神经干细胞增殖影响选取E17胎鼠皮层细胞,通过原代细胞培养、传代和免疫荧光方法及MTS/PMS法离体研究通络救脑注射液对胚鼠皮层神经干细胞增殖的影响。结果显示,细胞有Nestin表达,本研究中分离的细胞具备了神经干细胞的基本属性,属于中枢神经系统的干细胞。应用MTS/PMS法测定不同浓度、同一时间点通络救脑注射液对神经干细胞增殖的影响和不同时间点、同一浓度通络救脑注射液对神经干细胞增殖的影响。结果显示通络救脑注射液在0.77～123.2μg/ml剂量时,对神经干细胞的增殖有促进作用,在15.4μg/ml时的作用最强;当药物浓度达到492.8μg/ml时,开始对神经干细胞的增殖有抑制作用;当药物浓度在246.4μg/ml时,给药组和对照组没有显著差异,可能是因为药物对神经干细胞增殖的促进作用和抑制作用达到了一个平衡。在15.4μg/ml浓度时,给予通络救脑注射液6h后给药组与对照组OD值有明显差异,说明给予通络救脑注射液6h后才能起到促进神经干细胞增殖的作用。2通络救脑注射液对离体胚鼠皮层神经干细胞增殖、分化的影响本实验采用免疫荧光双标的方法和RT-PCR法研究通络救脑注射液对胚鼠皮层神经干细胞增殖、分化、自我更新的作用。结果显示,与对照组相比,通络救脑注射液组Nestin/Brdu、Nestin/Vimentin、Nestin/Tuj-1表达的阳性细胞面密度、光密度和细胞个数比对照组明显增多,统计具有显著性差异。Tuj-1、Vimentin、Nestin三种基因的mRNA条带均在200～300bp之间且接近300bp,给药组均比空白对照组条带较宽;且每个指标给药组和对照组组间的积分光密度值经统计学处理,具有显著性差异。提示通络救脑注射液能够促进离体胚鼠皮层神经干细胞的存活和增殖,并且能够促进离体胚鼠皮层神经干细胞分化为新的神经元和胶质细胞。综上所述,无血清培养方法可培养Nestin阳性的神经干细胞;通络救脑注射液在一定浓度范围内可以促进神经干细胞的增殖、自我更新,并可促进其分化为神经元和胶质细胞。可能是因为此药物有类生长因子的作用或者通过刺激细胞产生生长因子,由此促进神经干细胞的增殖和分化。更多还原

【Abstract】 Neural stem cells have the ability of potential differentiation, that is orientation differentiation under some conditions. When the adult nerve tissue is destroyed or degenerated, it is possible to replace the injured nerve tissue or cells with the homologous nerve tissue or cells produced through the orientation inducement of neural stem cells. In this study we try to investigate the influences of Tong-luo-jiu-nao Injection on neural stem cells, extend the basic theories and methods of modern medical science which promoting tissue regeneration after removing blood stasis in treatment of brain disease, and hoped that through the in-depth study of neural stem cells lay the groundwork for alternative treatment.1 Neural stem cell culture and the cell multiplication effect of Tong-luo-jiu-nao Injection on neural stem cells using MTS/PMS ColorimetryWe selected cortical cells of E17 embryonic rat, and investigated the effects of Tong-luo-jiu-nao Injection on the cell proliferation by primary cell culture, passage, immunofluorescence and MTS/PMS colorimetry. The results showed that cells have Nestin expression and the basic attributes (properties) of neural stem cells which belong to the stem cells in the central nervous system. We adopted MTS/PMS colorimetry to measure the cell proliferation effects of Tong-luo-jiu-nao Injection different in concentrations at the same time point and those in the same concentration at different time points. The results showed that Tong-luo-jiu-nao Injection promotes cell proliferateion at 0.77-123.2μg/ml, the most at 15.4μg/ml, and has inhibitory action on the proliferation when the concentration is more than 492.8μg/ml. When the concentration is 246.4μg/ml, there is no significance between the treatment and the control group which possibly caused by the reason that the promoting and prohibiting effects are on the balance. Until the Tong-luo-jiu-nao Injection administrated for 6h, can it promote the neural stem cells to proliferate.2. Effects of Tong-luo-jiu-nao Injection on the proliferation and differentiation of the neural stem cellsIn this study, we adopted the immunofluorescence double labeling and RT-PCR to investigate the effects of Tong-luo-jiu-nao Injection on the proliferation, differentiation and self-renew of the neural stem cells. The results shown that compared with the control group, the area density, optical density and numbers of the Nestin/Brdu、Nestin/Vimentin、Nestin/Tuj-1 positive cells in the Tong-luo-jiu-nao Injection group are much higher with significance. The mRNA bands of the three genes of Tuj-1、Vimentin、Nestin are in the range of 200-300bp, nearing 300bp. The bands in the treatment group are wider than those in the control group. The value of the integral optical density of these three更多还原