【作者】 廖光军；

【导师】 陈安民；

【作者基本信息】 华中科技大学 ， 外科学， 2007， 博士

【摘要】 脊髓损伤(Spinal Cord Injury ,SCI)是一种比较常见的严重创伤,SCI可导致运动功能障碍,常遗留严重伤残。长期以来认为,成年哺乳动物中枢神经系统的神经元是不可能再生的,损伤后只能由胶质细胞代替。目前各种细胞移植和组织移植这完全改变了中枢神经系统损伤后无法再生修复的传统观念,使SCI治疗成为当今神经科学的一个热点。将各种干细胞、组织、神经营养因子移植到损伤的脊髓,能替代、补充因损伤而死亡的神经元细胞,刺激轴突再生,重建神经元回路,同时又能自主释放神经递质,分泌大量治疗性神经营养因子或神经保护因子,抑制神经变性、死亡,并促进神经再生,从而能改善脊髓运动功能障碍。成熟神经元再生能力低下,由于内环境的改变,脊髓继发性损伤,如神经元的变性、坏死,神经元兴奋性增加,神经递质的改变等使的轴突生长受抑。脊髓损伤后导致星形胶质细胞增生,反应性星形胶质细胞增生是神经组织损伤的修补愈合反应,最后成为胶质瘢痕。反应性星形胶质细胞同时又产生各种抑制轴突生长的因子。因而对脊髓损伤的研究,成为基础和临床医学工作者所面临的一大课题。早在1911年,Allen曾用重物坠击法(Weight-Dropping,WD)首次在实验动物身上复制SCI模型。这是实验性脊髓损伤研究的开始,此后,出现了各种各样的损伤模型,为SCI的研究提供了有利的保障。SCI的动物模型按其致伤时间分为急性和慢性损伤,按其病理进程分为不完全性损伤和完全性损伤模型。一般认为,自SCI后,脊髓各种组织细胞的功能状态常有个继发性,进行性的与病理变化相一致的变化过程。从单纯细胞损伤到不可逆性组织坏死以及胶质瘢痕形成之间有个时间过程。如能找出其与病理变化相对应的变化趋势,阻断这一继发性变化过程,则可能会得到较好的功能恢复。Propentofylline,化学名称是3-Methyl-1(5-oxohexyl)-7-propylxanthine, (PPF)是Hoechst公司开发的一种能抑制磷酸二酯酶和腺苷摄取的黄嘌呤的衍生物,分子式为C15H22N4O3,分子量306.36目前更多的实验已经证明PPF在治疗痴呆症中的作用是可靠和有效的。PPF是一种具有神经保护作用的胶质细胞的调制剂。本品Ⅲ期临床试验中显示了它具有确切的痴呆症状改善作用且有良好的安全性,且有长效神经保护作用。本研究旨在SCI早期利用药物PPF调整星形胶质细胞活性,改善内环境,减轻脊髓继发性损伤,延缓或避免最后成为胶质瘢痕,为各种细胞移植和组织移植后轴突生长提供一个时间窗。本试验共分三个部分:1.探讨PPF对大鼠SCI后损伤区脊髓组织一般病理变化、神经细胞凋亡表达的影响及神经功能恢复的影响。2.探讨PPF对大鼠SCI后损伤区脊髓组织中GFAP、VIM基因表达的影响。3.探讨PPF对体外模拟大鼠SCI后星形胶质细胞GFAP、VIM表达的影响。第一部分动物模型的建立及脊髓组织的观察目的:建立起急性SCI的动物模型,观察Propentofylline (PPF)干预对急性脊髓损伤后的神经功能评分、一般组织病理学变化及脊髓组织细胞的凋亡(Programmed cell death,PCD)影响。材料和方法:取SD大鼠66只随机分为3组:A组为药物干预组;B组为手术对照组;C组为正常对照组,用改良Allen法建立急性脊髓损伤动物模型,药物干预组SD大鼠腹腔注射PPF,手术对照组腹腔注射等体积生理盐水,损伤后各时间段,进行神经功能评分(BBB评分、Rivlin斜板功能试验),用HE、尼氏(Nissl)染色法观察脊髓的一般组织病理学变化,用原位末端标记法( Terminal deoxynucleotidy transferase mediadted deoxyuridine-triphosphate nick-end labeling-TUNEL)观察细胞凋亡情况,结果:1.大鼠SCI后,BBB评分和Rivlin斜板功能明显下降,而药物干预组使用PPF后3天内BBB评分和Rivlin斜板功能无明显上升,但在3天后有明显上升,即大鼠神经功能评分显著提高;2.大鼠SCI后,损伤脊髓有明显出血、水肿、变性、空泡等,而使用PPF可明显改善损伤脊髓的病理形态及超微结构;3.大鼠SCI后,神经细胞凋亡明显增加,而药物干预组使用PPF后可使神经细胞凋亡显著减少(P<0.01).结论:1.结合脊髓的一般组织病理学变化和神经胶质细胞凋亡的变化规律,急性脊髓损伤动物模型建立成功,可应用于急性脊髓损伤的实验研究。2.神经胶质细胞的凋亡可能是脊髓损伤继发病变的机制之一。3.PPF干预可减少急性脊髓损伤后大鼠的神经细胞凋亡,有效保护神经细胞。提高急性SCI大鼠的神经功能评分。总之,对大鼠急性脊髓损伤早期的脊髓具有保护作用。第二部分Propentofylline对大鼠脊髓损伤后GFAP、VIM基因表达的影响目的:探讨Propentofylline (PPF)对大鼠SCI后脊髓组织中胶质纤维酸性蛋白(GFAP)和波形蛋白(VIM)的表达的影响。材料与方法:采用逆转录-聚合酶链式反应(RT-PCR)法和Western印记分析(Western blot)法检测SCI后不同时间点损伤区脊髓组织中GFAP和VIM mRNA和蛋白含量的表达。免疫组化法检测SCI后不同时间点损伤区脊髓组织中GFAP和VIM阳性细胞数及蛋白表达情况。结果:1. GFAP mRNA在无损伤脊髓组织中稍有表达,GFAPmRNA脊髓损伤后6h后即有明显表达。VIM mRNASCI在无损伤脊髓组织中没有表达,脊髓损伤3d后VIMmRNA表达,5d后明显上调,PPF组与对照组比差异非常显著(P<0.01); 2. GFAP蛋白表达在无损伤脊髓组织中低,VIM蛋白表达在无损伤脊髓组织中没有表达。SCI后12h,GFAP蛋白表达明显上调,PPF组与对照组比差异显著(P<0.05);VIM蛋白表达脊髓损伤3d后有表达,5d后明显表达,与PPF组比差异显著( P<0.05),脊髓损伤后各时间点对照组GFAP表达阳性细胞明显多于PPF治疗组,与PPF组比差异显著(P<0.01);伤后3d PPF治疗组VIM阳性表达细胞与对照组比较差异显著(P<0.01)。结论:1.急性SCI3d后,损伤区脊髓组织中GFAP、VIMmRNA及蛋白水平明显升高,与细胞增殖有明显的相关性,提示GFAP、VIM在胶质瘢痕形成过程中起重要作用。2.PPF对SCI后的神经保护作用一个可能的机制是:通过抑制SCI后损伤区脊髓组织中GFAP、VIM的表达,减轻SCI后星形胶质细胞过度增殖,从而促进损伤脊髓功能的恢复。第三部分Propentofylline对体外培养的星形胶质细胞GFAP、VIM表达的影响目的:观察体外培养的星形胶质细胞损伤后,Propentofylline(PPF)对其胶质纤维酸性蛋白(GFAP)和波形蛋白(VIM)表达的改变,探讨PPF对损伤星形胶质细胞的影响。方法:建立体外培养大鼠纯化星形胶质细胞机械损伤模型,利用免疫细胞化学方法观察PPF干预后星形胶质细胞GFAP、VIM的变化。星形胶质细胞机械损伤后星形胶质细胞随机分为2组:药物干预组、对照组。药物干预组予PPF处理,用免疫细胞化学染色的方法和图像分析测定法观察星形胶质细胞GFAP、VIM蛋白表达的变化及星形胶质细胞的数量。结果:机械损伤后12h后损伤边缘区GFAP、VIM细胞数目增加、胞体肥大,GFAP、VIM表达增强,PPF干预后GFAP、VIM表达增强被抑制,胞体肥大程度减轻,GFAP、VIM阳性细胞数减少。结论:PPF对损伤后的体外培养的星形胶质细胞的活性具有明显的调节作用。更多还原

【Abstract】 Spinal cord injury (SCI) is one kind of serious trauma. SCI may cause the movement function barrier, often leaves behind the serious disability. Since long ago believed that, neuron of central nervous system in the grown-up mammal is not impossible to be regenerated after the damage only can be replaced by the spongiocyte. At present each kind of cell transplantation and histological transplantation change the traditional ideas of the central nervous system damage to be unable completely to regenerate and repair, which causes treatment of the spinal cord damage into a hot spot in neuroscience now. Each kind of stem cell、the organization、the nerve nutrition factor transplanted to the damage spinal cord, substitute and fill up defect because of the neuron cell which were damaged to die. To stimulate axon regeneration, reconstruct the neuron return route, simultaneously can release the nerve to hand over the nature independently, to secrete massive medical nerve nutrition factor or the nerve protection factor, to inhibit nerve denaturation、the death, and to promote the neuranagenesis, thus can improve the spinal cord movement function barrier. Reproductive property of mature neuron is low,and becauseof changing in inner environment, the spinal cord secondary damage, like the neuron denaturation、necrosis, the neuron excitability increasing,and the changing of neurotransmitter make axon growth damped. After SCI causes the astrocyte proliferation, the reactivity astrocyte (Ast) proliferation is the response of cicatrization for the nervous tissue damaged, finally becomes the sol scar. The reactivity Ast simultaneously has the factor which each kind of suppression axon grows. Thus the research on SCI, becomes a big topic which the foundational and the clinical medicine workers face. As early as in 1911, Allen once used the heavy item to fall (Weight-Dropping, WD) for the first time to duplicate SCI model on the experimental animal. This is the start on experimental SCI research.hereafter, appeared various damage model for the spinal cord to provide the advantageous safeguard for study. According to the time, the animal models of SCI were divided into acute and the chronic damage, and divided into the incomplete damage and the complete damage model according to its pathology advancement. Believed generally, after SCI, function condition of the spinal cord commonly carries on consistent pathological change process. From the pure cell damage to the irreversibility organization necrosis as well as the sol scar forms have the time process. If can discover its change tendency which corresponds with the pathological change, block this sequential change process, then possibly can obtain the good function restoration.Propentofylline(PPF),that chemistry name is 3-Methyl-1 (5-oxohexyl) -7- propyl- xanthine, is one kind medicine which Hoechst Corporation develops can suppress the phosphoric acid diesterase and the gland glucoside ingestion xanthine derivative.The molecular formula was C15H22N4O3, molecular weight 306.36 .At present more experiments have already proved that PPF in the treatment dementia sickness function was reliable and effective. The PPF is a kind of moderator of the nerve spongiocyte. ThisⅢclinical test has demonstrated it has the accurate function also has the good security.This study in SCI ,early time want to use medicine PPF to adjust Ast activeness, improve the inner environment, reduce the spinal cord secondary damage, delay or avoid finally becoming the sol scar,which provides a time window for the axon growth after each kind of cell transplantation and the histological transplantation. This experiment altogether is divided three parts: 1. To produce an animal model of acute SCI using the modified Allen’s method and to observe the effect of PPF on the BBB’s motor scales, Rivlin’s inclined plane scales, programmed cell death and the histopathological changes after acute SCI in rats. 2. To investigate the effect of PPF on GFAP、VIM in the rat spinal cord after SCI. 3. To investigate the effect of PPF on GFAP、VIM in vitro cultured Ast of mechanical injury.PartⅠThe Animal Model Establishment and the Spinal Cord Organize ObservationAbstract: Objective To produce an animal model of acute spinal cord injury (SCI) using the modified Allen’s method and to observe the effect of propentofylline (PPF) on the BBB’s motor scales, Rivlin’s inclined plane scales, programmed cell death and the histopathological changes after acute SCI. Material and methods 66 healthy rats were randomly divided into 3 groups. Group A: PPF treatment group; Group B: operation group; Group C: normal group. An acute SCI model of rats was made by the modified Allen’s weight dropping method. The rats in group A (test group n=30) were treated with PPF after SCI. The second group (the control group n=30) were treated with 0.9%NS after SCI. The third group (n=6) were normal group. To study BBB’s scales, Rivlin’s inclined plane scales,the spinal cord blocks were cut in cross section 10μm thick and stained with HE、Nissl stain,TUNEL technique was used to mark the apoptosis cells. The histopathological changes and the apoptosis cells were observed with Light microscope. Results 1 .There was significantly haemorrhage, edema, degeneration, vacuole and so on in spinal cord after SCI in rats. But the pathological and ultrastlvctural changes of spinal cord were significantly improved in PPFgroups. 2. The BBB’s scales and Rivlin’s inclined plane scales decreased significantly after SCI. The neurological function of rats in PPFgroups was significantly improved at 72-hour after SCI. 3. The number of apoptosis in spinal cord significantly increased after SCI but significantly decreased in PPF groups (P<0.01).Conclusion 1. According to change rule of the common organization pathology change and the neuroglia cell apoptosis in the spinal cord, the acute spinal cord damage animal model established successfully, may be applied to the experimental study of the acute spinal cord injury. 2. The neuroglia cell apoptosis possibly is one of mechanisms of the secondary spinal cord injury. 3. PPF intervention may reduce nerve cell apoptosis of the acute spinal cord injury, protect the nerve cell effectively and enhance big mouse’s nerve function grading after acute SCI. In brief, PPF has the protective function to early time spinal cord of the rats after acute SCI.PartⅡEffect of Propentofylline on GFAP、VIM Gene Expression in the Rat Spinal Cord after SCIObjective To investigate the effect of propentofylline (PPF) on GFAP、VIM in the rat spinal cord after SCI Methods In the study, the mRNA expression of GFAP、VIM were measured using reverse transcription polymerase chain reaction(RT-PCR) and protein expression of GFAP、VIM were measured by western blot method and immunohistochemistry in the rat spinal cord after SCI,healthy rats were randomly divided into 3 groups. Group A: PPFtreatment group; Group B: operation group; Group C: normal group. An acute SCI model of rats was made by the modified Allen’s weight dropping method. The rats in group A were treated with PPF after SCI. The second group (the control group) were treated with 0.9%NS after SCI. The third group is normal group. The expressions of GFAP、VIMmRNA were measured by RT-PCR, as well as the protein expression of GFAP、VIM quantitated using western blot and immunohistochemistry (S-P) in the rat spinal cord after SCI. Results1.The mRNA expression of GFAP was detected in non-injured spinal cord. The expression of GFAP mRNA up-regulated at 6h postinjury (P<0.01) and peaked at 5d postinjury(P<0.01,vs. the control group), and dropped after 7 d; VIM mRNA expression up-regulated at 3d after SCI(P<0.01), and remained high level until 7d postinjury(P<0.01,vs. the control group); 2. The protein expression of GFAP was detected in non-injured spinal cord. The expression of GFAP protein up-regulated at 12h postinjury (P<0.05,vs. the control group), VIM protein expression was detected at 3 d, remained high level until 7d postinjury(P<0.05,vs. the control group). Conclusions 1. The level of GFAP、VIM mRNA and protein increased significantly at the lesion area after SCI, and correlated with cell proliferation in the lesion area, which indicates that GFAP、VIM play an important role in cicatrization after SCI. 2. Nerve protective function possibility mechanism on PPF after SCI is: to suppress GFAP、VIM expression of the damage area spinal cord organization after SCI and multiply excessively of Ast, thus to promote function restoration of damage spinal cord.PartⅢEffect of Propentofylline on GFAP、VIM Expression in Astrocytes Cultured in VitroObjective: To investigate the effect of propentofylline(PPF) on GFAP、VIM expression in vitro cultured astrocytes(AS) of mechanical injury. Methods: The model of mechanical injury on in vitro cultured and purified AS was made. AS were randomly divided into three groups: PPFgroup(group A)、group B.Group A were treated with propentofylline after injury. Immunohistochemical technique and imagine analysis technique were used to detect the expression of GFAP、VIM and the number of GFAP、VIM positive cell. Results: The expression of GFAP、VIM and the number of GFAP、VIM positive cell showed increased tendency after injury. AS injured had hypertrophy, proliferation, and the expression of GFAP、VIM and the number of GFAP、VIM -positive cells were increased. After treated with propentofylline, the hypertrophy of AS was attenuated and the expression of GFAP、VIM and the number of the GFAP、VIM -positive cells were decreased. Conclusion: Mechanical injury can promote responsive proliferation. PPF could modulate the hypertrophy and proliferation of AS effectively after mechanical injury.更多还原