【作者】 袁颖；

【导师】 顾晓松；

【作者基本信息】 苏州大学 ， 人体解剖与组织胚胎学， 2009， 博士

【摘要】 目的研究牛膝多肽(Achyranthes bidentata Blum Polypeptides,ABPP)对体外培养的大鼠海马神经元突起生长的促进作用及其对GAP-43和NF-H基因表达的影响,以及与ERK1/2信号转导通路的关系。研究ABPP诱导PC12细胞神经元性分化的作用,初步探讨ABPP对PC12细胞的ERK1/2信号转导途径的影响。观察ABPP对小鼠坐骨神经夹伤后神经再生的影响。方法1.以体外原代培养的胎鼠海马神经元为研究模型,通过MTT检测ABPP对海马神经元细胞活性的影响,通过免疫荧光细胞化学法,采用Image-Pro Express软件测量不同浓度ABPP(0.1μg/ml、0.5μg/ml、1.0μg/ml)加药24 h,对海马神经元神经突起生长的影响;通过实时荧光定量PCR法定量分析不同浓度ABPP加药6 h,对海马神经元GAP-43和NF-H基因表达的影响。同时应用ERK特异性拮抗剂PD98059与ABPP共培养海马神经元分析ABPP对海马神经元的作用与ERK1/2信号转导通路的关系。2.以PC12细胞为研究模型,观察在低血清的情况下不同浓度ABPP(0.25μg/ml、0.5μg/ml、1.0μg/ml)诱导PC12细胞发生的形态学改变,第7 d和第14 d时分别观察细胞分化的形态学改变。通用免疫荧光细胞化学法,观察NF-H在ABPP诱导分化的PC12细胞中的表达,从而鉴定ABPP诱导的分化细胞;为了研究与ABPP促分化有关的信号转导通路,采用Western blotting法观察不同浓度ABPP加药不同作用时间后对低血清培养的PC12细胞ERK活性的影响,同时应用ERK特异性拮抗剂PD98059与ABPP共培养PC12细胞,分析ERK1/2信号转导通路在ABPP对PC12细胞影响中的作用。3.ICR小鼠50只,雌雄各半,行左侧坐骨神经夹伤术。术后随机分为5组:ABPP低、中、高剂量组(剂量分别为1,4,16 mg/kg),弥可保组(阳性对照,剂量为65μg/kg)及生理盐水组(空白对照,给予等体积生理盐水)。经尾静脉注射给药,每日一次,连续给药21 d。术后定期做足迹试验,损伤后3周记录复合肌动作电位,计算运动神经传导速度;取损伤远端5mm处坐骨神经,进行免疫组织化学染色及透射电镜观察,测定再生神经纤维密度、再生神经纤维直径和髓鞘厚度;取腓肠肌做石蜡切片,染色后测定肌纤维横截面积。结果1.MTT结果显示在0.01到10μg/ml范围内,ABPP对海马神经元没有明显毒性。不同浓度ABPP(0.1μg/ml、0.5μg/ml、1.0μg/ml)加药24 h,海马神经元神经突起长度明显增加,存在时间依赖性和剂量反应关系,加药24 h后作用最强,剂量为1μg/ml时作用最佳。ABPP(0.5μg/ml、1.0μg/ml)培养6 h后,海马神经元的GAP-43和NF-H的mRNA表达量与阴性对照组相比是明显升高的。PD98059抑制了ABPP(0.5μg/ml)对海马神经元突起的生长,同时降低了NF-H和GAP-43基因mRNA和蛋白质的表达。并且ABPP促神经生长的作用可能是通过ERK通路产生的。2.ABPP处理PC12细胞3 d后,部分PC12细胞开始出现神经元样的形态:细胞胞体的皱缩、平展以及可见有明显的类似于神经元突起的生长,细胞停止增殖。随着加药时间延长具有神经元样的细胞逐渐增多,到第7 d时,NGF和ABPP处理组PC12细胞的突起都显著增多,能形成网络;到第14 d时,这种现象愈发显著。加药后第7 d和第14 d,ABPP各浓度组的细胞分化率以及细胞突起长度均明显提高,且存在明显的剂量反应关系。加药第7 d和第14 d,ABPP高剂量组与NGF组的PC12细胞均出现NF-H标记阳性的分化细胞。ABPP在0.25μg/ml、0.5μg/ml、1.0μg/ml剂量范围内作用于低血清培养的PC12细胞2 d,对ERK1/2的激活作用存在明显的剂量反应关系,以1.0mg/ml者作用为最大。当用PD98059抑制MEK1/2的活化时,ABPP对ERK1/2的激活作用被部分阻断。3.ABPP对小鼠坐骨神经夹伤后的神经功能恢复有促进作用,对SFI的改善存在剂量反应关系。能提高损伤后神经近、远端复合肌动作电位幅度和运动神经动作电位,再生髓鞘的密度、厚度、轴突直径及腓肠肌肌纤维横截面积,均显著优于空白对照组,超微结构观察显示,ABPP组小鼠坐骨神经夹伤段有髓神经纤维的髓鞘形态、厚度及成熟度均优于对照组。结论1.ABPP能促进体外培养的海马神经元神经突起的生长,其作用与GAP-43和NF-H基因表达的升高相关,可能是通过ERK1/2信号转导途径实现的。2.ABPP具有诱导PC12细胞神经元性分化的作用,该作用可能是通过ERK1/2信号转导途径实现的。3.ABPP能促进小鼠坐骨神经夹伤后的神经再生和功能恢复。更多还原

【Abstract】 ObjectiveTo determine the effects of ABPP(Achyranthes bidentata Blum Polypeptides) on neurite growth in cultured hippocampal neurons of rats.To study the potential ability of ABPP to induce the neuronal differentiation of PC12 cells and to preliminary investigate the activation of ERK1/2 cascade induced by ABPP.To study the repair effects of ABPP on the crushed sciatic rat nerve in mice.Methods1.The cytotoxicity of ABPP was tested with MTT.After 24 h of incubation by different concentrations(0.1μg/ml,0.5μg/ml,1.0μg/ml) of ABPP,the hippocampal neurons were photographed by TCS SP2 confocal microscope with fluorescent immunocytochemistry,and the neurite length was analyzed using the Image-Pro Express software.Real-time quantitative RT-PCR was performed to examine the mRNA levels of GAP-43 and NF-H after 6 h with different concentrations of ABPP.Western blotting were performed to further examine the protein levels of GAP-43 and NF-H in cultured hippocampal neurons after 24 h incubation with different concentrations of ABPP,and detected the activities of ERK1/2 in hippocampal neurons co-culture with ABPP and the MEK1/2 inhibitor,PD98059.2.By cell culture in the low-concentration of serum,the morphological changes of PC12 cells treated with of ABPP at different concentration(0.25μg/ml、0.5μg/ml、1.0μg/ml) was detected.Fluorescent immunocytochemistry was performed to examine the expression of NF-H,a well-known neuronal marker,in differentiated PC12 cells induced by 1.0μg/ml ABPP.In order to study the signaling pathways involved in the differentiation induced by ABPP,Western blotting was performed to detect the acitvities of ERK1/2 in PC12 cells activated by 1.0μg/ml ABPP after different periods(0h、6h、12h、1d、2d、3d and 7d) using the activated(Diphosphorylated ERK1&2) antibody and mitogen activated protein kinase(MAP Kinase,MAPK,ERK-1 & ERK-2) antibody when PC12 cells were cultured in low-concentration of serum medium.3.Fifty mice were performed sciatic nerve crush injury operation.After that the animals were randomly divided into 5 groups of 10 animals each and treated tail vein administration as follows:ABPP groups(low,middle and high dose,1,4,16 mg/kg, respectively),Methycobal group(served as positive control) at 65μg/kg and saline group (served as negative control).The treatment lasted for 21d.At different time after nerve crush,a combination of experiments,including walking track analysis, electrophysiological assessments,immunohistochemistry and electron microscopy to the regenerated sciatic nerve,masson trichome staining to the gastrocnemius muscle,as well as morphometric analyses,was carried out to evaluate the regenerative outcomes of ABPP administration.Results1.ABPP promoted the neurite growth of cultured hippocampal neurons.Real-time quantitative RT-PCR showed that the mRNA level of GAP-43 and NF-H in cultured hippocampal neurons was up-regulated by ABPP in a dose-dependent manner.Meanwhile, fluorescent immunocytochemistry and Western blotting showed that the protein level of GAP-43 and NF-H was up-regulated by ABPP.ABPP-induced phosphrylation of ERK1/2 was blocked by PD98059.2.After 3 days treated with ABPP(0.25μg/ml、0.5μg/ml、1.0μg/ml),neuron morphology was observed in PC12 cells:compaction of cell bodies,the outgrowth of neurites,unproliferation.As the time went on,the number of the differentiated PC12 cells increased.At day 7 the differentiated PC12 cells could produced the neurite network and at day 14 the phenomena became mo(?)e obvious.It has been shown that ABPP could promote the differentiation of PC12 by dose-effect relation.ABPP activated ERK1/2 in PC12 cells by dose-effect relation and time-dependent character,with the best concentration of 1.0μg/ml after being co-cultured with PC12 cells for 2 days.And PD98059 significantly inhibited ABPP-induced phosphorylation of ERK1/2.3.The results indicated that treatment with ABPP at a dose range(1-16 mg/kg) promoted histological regeneration and functional recovery of the injured sciatic nerve and its target muscle,yielding a desired efficacy greater than that by vehicle treatment and close to or even greater at some parameters than that by methylcobalamin,a positive control. Conclusion1.ABPP could promote the neurite growth and modulate the expression of GAP-43 and NF-H in cultured hippocampal neurons,suggesting neurotrophic effects of ABPP.2.ABPP could induce the neuronal differentiation of PC12 cells.The differentiation of PC12 cells induced by ABPP may be mediated through ERK1/2 phosphorylation cascade potentially.3.Plant polypeptides,ABPP,may be a potential agent in ameliorating the effects of neuropathy caused by sciatic nerve crash.更多还原