【作者】 何玉宝；

【导师】 杨有庚；

【作者基本信息】 吉林大学 ， 外科学， 2010， 博士

【摘要】 脊髓损伤(SCI)机制复杂,目前尚未完全明确,是困扰医学界的难题之一,也是导致SCI治疗效果欠佳的原因。本研究旨在探讨热休克蛋白47(HSP47)在大鼠脊髓中的表达情况,揭示其在大鼠SCI中的作用,完善SCI损伤机制,同时对中药单体成分苦参素发挥治疗作用的机制进行研究。首先建立大鼠钳夹型SCI模型,然后通过不同时间点(3天、1周、3周、5周和8周)观察Basso, Beatlie, Bresnahan (BBB)评分,应用苏木精-伊红(HE)染色、尼氏染色、Masson染色及免疫组织化学染色等形态学技术,半定量逆转录聚合酶链式反应(RT-PCR)的分子生物学技术及蛋白质印迹(WB)的蛋白定量技术,对目的蛋白HSP47的空间和时间表达进行定性和定量分析,确定HSP47存在于大鼠脊髓中,且SCI后表达增加。说明HSP47参与到SCI的病理改变过程中,其在SCI胶质瘢痕形成中具有重要的作用,为SCI修复提供新的治疗靶点,开辟治疗SCI的新领域。在第8周给予苦参素(Oxymatrine)治疗后,通过对上述指标的检测,提示HSP47及瘢痕表达明显下降,说明苦参素对SCI具有治疗作用,其发挥治疗作用的机制可能是通过干预HSP47 mRNA的病理表达,进一步降低胶原表达治疗SCI,为苦参素应用于SCI领域奠定了理论基础。通过SCI后HSP47表达分析,对SCI的机制作了重要的补充和完善,为从基因及分子水平开展针对HSP47的治疗提供了直接的理论依据,为SCI的治疗提出了一个新的课题。更多还原

【Abstract】 The acute spinal cord injury (SCI) incidence and the rate of disability ascend followed by the development of vehicle and increase of highrise building year by year. The SCI therapy appears urgent and significant. Uncounted scholars have tried to conquer this tough task. However, SCI mechanism is complicated, the therapeutic results are unsatisfactory. SCI includes primary injury and secondary physiopathologic changes. Secondary injury is the main factor in SCI. The physic barrier formed by glial scar is an important environmental factor, while the pathological foundation is hyperplasia of cells which are mainly astrocyte and over deposition of extracellular matrix (ECM). The collagen is the most important ingredient of ECM.Heat shock protein (HSP) 47 is a collagen-specific chaperone. It has been shown to be present in the endoplasmic reticulum (ER) and numerous studies have shown its expression in collagen-producing cells and tissues. HSP47 plays a key role in the processing, secretion, folding and assemble of collagen. HSP47 has a single substrate protein, collagen. HSP47 binds specifically to various types of collagens including types I～V in vitro. The biochemical properties, intracellular localization and tissue distribution of HSP47, suggest its role in post-transcriptional regulation of procollagens. Recent studies have convincingly shown a pathogenic role of HSP47 in the development and progression of various human and experimental fibrotic diseases. Therefore, HSP47 provides a selective target to manipulate collagen production.The oxymatrine is an anti-fibrotic traditional Chinese drug. It has been applied widespread in scar treatment. The therapeutic action is obviously, while the side effect is minute. To promote the traditional medicine, the study about the therapeutic mechanism of active ingredient monomer has been a hot subject of research in domestic and abroad. We used a clip-compression injury modle of spinal cord in rats, evaluated Basso, Beatlie, Bresnahan (BBB) Locomotor Rating Scale and applied Hematoxylin-Eosin (HE), Nissl staining, Masson staining, immunohistochemistry, Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Western Blot techniques to analyze the space and time regularity of HSP47. At the same time, we observed the changes of these indexes after treatment with oxymatrine. We intend to perfect the SCI mechanism, explain the oxymatrine therapeutic mechanism, find a new SCI therapeutic target and open up a new SCI field.PartⅠ: Establishment of a clip-compression injury modle of spinal cord in ratsObjective: To establish a reliable, reduplicate and detectable modle of SCI in rats to observe the behavior and pathological changes, found a base for subsequent research.Method: A clip-compression injury modle of spinal cord in rats was used. 54 Wistar rats were divided to normal group, sham-operated group, SCI group randomly. The sham-operated group only received T10 decompression laminectomy;the SCI group received T10 spinal cord injury. The BBB Locomotor Rating Scale were assessed at 3 days and every week after SCI. They were sacrificed to study the morphous of spine and cells using HE staining, the changes of Nissl’s body using Nissl staining, GFAP and NF using immunohistochemistry technique at 1, 3, 5 and 8 weeks.Results: The Wistar rats BBB scores was 21 in normal and sham-operated group, while the BBB scores was (0.67±0.48) in SCI group at 3 days. The gray matter distribution in spinal cord was typical butterfly shape in normal and sham-operated group. The demarcation of white and gray matter was clear. The cellular morphous was normal and the Nissl’s body in cytoplasm was granular. While the normal spinal morphous disappeared, cavitation formed, the demarcation of white and gray matter vanished, the cellular body was smaller, the cytoplasm condensed, the Nissl’s body was ambiguity, reduced, sparse and even vanished in SCI group. The expression of GFAP in normal and sham-operated group was increased, while the expression of NF was decreased. But, the expression of GFAP after SCI was increased significantly, while the expression of NF was decreased. Compared with these at 1 and 3 weeks, the expression of GFAP was increased obviously, while the expression of NF was decreased at 5 weeks after injury. Conclusions: A clip-compression injury modle of spinal cord in rats is established successful.PartⅡ: HSP47 increased in rats with acute SCIObjective: To investigate the expression and tanscription of collagen and HSP47 in ratswith acute SCI. Method: Wistar rats modle of SCI established by clip-compression, setting normal rats and those receiving sham-operated as controls. 90 Wistar rats were divided to normal group, sham-operated group and SCI group randomly. BBB Locomotor Rating Scale were assessed at 3 days and every week after SCI. 9 animals each group were sacrificed to analyse the changes of collagen and HSP47 using Masson staining, immunohistochemistry, semi-quantitative RT-PCR and Western Blot technique at 1, 3 and 5 weeks. Type I collagen, typeⅣcollagen and HSP47 mRNA tanscription levels in 3 rats each group were determined by semi-quantitative RT-PCR 8 weeks after SCI respectively.Results: The Wistar rats BBB scores were 21 in normal and sham-operated group, while the BBB scores were (0.70±0.46) in SCI group at 3 days. The expression of collagen and HSP47 were increased significantly after lesion. Compared with these at 1 and 3 weeks, the expression of collagen and HSP47 were increased obviously at 5 weeks after injury. However, type I collagen, typeⅣcollagen and HSP47 mRNA tanscription levels increased significantly, especially typeⅣcollagen and HSP47 mRNA tanscription levels at the 8th week after SCI.Conclusions: The expressions of collagen and HSP47 increase after SCI, mainly distribute around indiscriminate hyperplastic blood vessel and spinal dura mater in rats. The tanscription levels of collagen and HSP47 mRNA of Wistar rats are up-regulated after SCI and last for 8 weeks. HSP47 increases after SCI, indicating that HSP47 might be involved in the pathogenic change of SCI.PartⅢ: The study about the therapeutic mechanism of oxymatrine in rats with acute SCIObjective: To discuss the therapeutic effect and mechanism of oxymatrine in rats with acute SCI.Method: A modle of clip-compression injury of spinal cord in rats was used. 45 Wistar rats were divided to normal group, sham-operated group, SCI group, control group and therapeutic group randomly. They received intraperitoneal injection with oxymatrine 150 mg/(kg·d) every day after SCI in therapeutic group, while those received intraperitoneal injection with sodium chloride in the equivalent dosage in control group. BBB Locomotor Rating Scale were assessed at 3 days and every week after SCI. They were sacrificed to study the morphous of spine and cells, the changes of Nissl’s body, collagen, GFAP, NF and HSP47 using HE, Nissl staining, Masson staining, immunohistochemistry, RT-PCR and Western Blot technique at 8 weeks.Results: Wistar rats BBB scores were 21 in normal and sham-operated group, while the BBB scores were (3.56±1.29) in SCI group, (3.22±1.52) in control group, (4.56±1.20) in therapeutic group at 1 week. The normal spinal morphous disappeared, cavitation formed, the demarcation of white and gray matter vanished, the cellular body was smaller, the cytoplasm condensed, the Nissl’s body was ambiguity, reduced, sparse and even vanished in control group. Compared with those in control group, there were not obvious in therapeutic group. The expressions of GFAP and HSP47 after SCI were increased significantly, while the expression of NF was decreased. The expressions of GFAP and HSP47 in therapeutic group were reduced obviously, while the expression of NF was raised.Conclusions: SCI in Wistar rats can be treated with oxymatrine. It probably suppresses the expression of HSP47, and reduces the formation of collagen.更多还原