【作者】 李鹏；

【导师】 吕德成；

【作者基本信息】 大连医科大学 ， 中西医结合临床， 2008， 硕士

【摘要】 背景:脊髓损伤是骨科的常见多发伤,后果严重,预后差,临床治疗效果尚不满意。近年来人们逐渐认识到脊髓损伤后的永久性神经损害是原发损伤引起的一系列继发反应所致。通过大量研究,有学者认为可以通过神经干细胞移植治疗脊髓损伤,而谱系限制性神经前体细胞(NRPS细胞)是目前较好的选择。然而在不断改进移植方式的同时,人们发现损伤脊髓的局部微环境对移植的成败也起到了至关重要的作用。我校长期对“滋补脾阴方药”进行研究,发现其有保护脊髓继发损伤,提高脑神经线粒体膜抗氧化的能力。本药是否可以克服损伤脊髓微环境,提高移植细胞存活率是一崭新课题,有待于进一步研究。目的:研究滋补脾阴方药对过氧化氢(H2O2)诱导限制性神经元前体细胞(NRPS细胞)脂质过氧化损伤的保护作用,为进一步体内移植实验治疗脊髓损伤奠定基础。方法:取SD大鼠胚胎脊髓,行原位免疫荧光鉴定确认为NRPS细胞。将脊髓消化吹打后贴壁培养3-5天,利用流式细胞仪行抗Nestin、抗PSA-NCAM、抗A2B5免疫荧光鉴定,确认培养的细胞为NRPS细胞。利用红外光谱对含药血清和空白血清成分进行测定,确认含药血清和空白血清成分有所不同,滋补脾阴方药确实进入到血清中。建立实验模型,将细胞分为正常组、损伤组、空白血清干预组、中药血清干预组、空白血清干预后损伤组、中药血清干预后损伤组。其中血清浓度分别为0.25%、0.5%、1%、2%、4%,预保护时间为8小时;H2O2浓度分别为0.05、0.1、0.2mmol/L,损伤时间为4小时。将各组于倒置相差显微镜下对细胞进行一般形态学观察和分组比较;利用MTT法计算各组细胞存活率;并对各组进行Hoechst染色,于倒置荧光显微镜下观察细胞凋亡情况;检测细胞内SOD、MDA含量,得知细胞状态和受损程度,确定滋脾阴方药血清保护作用及最佳保护浓度。结果:H2O2可造成细胞MDA增高,SOD下降,符合脂质过氧化特征,并使细胞存活率降低,加快细胞凋亡,与H2O2浓度呈正性关系。滋补脾阴方药在0.25-2%浓度下对H2O2诱导的过氧化损伤细胞有保护作用,可以提高细胞存活率,预防NRPS细胞在脂质过氧化损伤下SOD的减少,并抑制MDA的生成,且在1%浓度时最保护能力最强。但含药血清大于4%浓度后便呈现出较明显的药理毒性作用,可以加快细胞的凋亡。结论:滋补脾阴方药血清在一定浓度下有抗脂质过氧化损伤作用,可以预防细胞SOD下降,减少MDA生成,提高了NRPS细胞在脂质过氧化条件下的存活率和存活状态,且在1%浓度时能发挥最佳保护作用。而空白血清对NRPS细胞无明显预保护及抗过氧化损伤作用,间接证明了滋补脾阴方药具有抗脂质过氧化作用。更多还原

【Abstract】 Background: Spinal cord injury is a common multiple injury in department of orthopaedics,of which the clinical efficacy is not perfect. For the past few years,people realized that the permanent nerve injury of spinal cord is caused by some secondary responses after the primary injury of the cord. Some scholars presumed that spinal cord injury could be cured by cellular transplant after much research,and the NRPS cell was a good choise so far.But when people improved the method of cellular transplant,they found the part microenvironment was also a key factor for the transplant.Nourishng Piyin Remedy was invested for a long time in our college,we found it could protect the injured spinal cord of the rabbit and improve the ability of mitochondria membrane of cerebral nerves against the lipid peroxidation.It is a new topic that if Nourishng Piyin Remedy can elevat the survival of the transplanted cells,which is needed to be invested.Objective: To invest the protection of Lineage-Restricted Neural Precursors peroxidized by hydrogen dioxide(H2O2) by Nourishing Piyin Remedy, and to establish the foundation of the remedy to the spinal injury by explantation in vivo.Methods: To confirm the NRPS cells after the spinal cord of the embryo of SD rats by immunofluorescence identification in situ.Then,made the cells of spinal cord for adherent culture for 3-5days.To establish them of the NRPS cells by immunofluorescence identified by Nestin、PSA-NCAM、A2B5 antibodies . To make sure the component of blank-blood serum was different from the remedy-blood serum by infrared spectra identification.So we knew that the Nourishing Piyin remedy was in the blood serum.Establish the lipid peroxidation analogs for six groups:normal group、pure damage group、blank-blood serum protected group、remedy-blood serum protected group、damage after blank-blood serum protected group、damage after remedy-blood serum protected group.The concentrations of the blood serum were 0.25%、0.5%、1%、2%、4%,and the protection time was 8h;the concentrations of H2O2 were 0.05、0.1、0.2mmol/L,and the damage time was 4h.Observed the cells under the inverted phase contrast microscope to compare the cell morphology change; calculated out the cell survival rates by the MTT method; fluorescein stained the cells with hoechst33258, and then observed the fluor and the apoptotic body under the inverted fluorescence microscope;detected the content of MDA and the archaeus of SOD in cells to get the proper protecting concentration of the remedy-blood serum.Results: H2O2 cuted down the cell survival rate and speeds up the apoptosis noteblely. It enhanced the content of MDA and decreased the archaeus of SOD in cells. After the interfering of Nourishing Piyin Remedy at a certain concentration, the cell survival rates were raised at the same damage condition. But, the noxious property of Nourishing Piyin Remedy appeared when the concentration above 4%.Conclusion: Nourishing Piyin Remedy could protect the Lineage-Restricted Neural Precursors against lipid peroxidation at a certain concentration,can and the best concentration is 1%.It could prevent the decrease of SOD,and dimish the MOD of cells.更多还原