本文選題：基因工程 + 組織工程�。� 參考：《第三軍醫(yī)大學(xué)》2005年博士論文

【摘要】：神經(jīng)損傷的再生修復(fù)一直是神經(jīng)科學(xué)研究的難點(diǎn)和焦點(diǎn)。近年來,隨著交通事故和創(chuàng)傷的逐年上升,由此引起的神經(jīng)損傷,尤其是外周運(yùn)動(dòng)神經(jīng)損傷日益增多,其特點(diǎn)為致殘率高,危害性大。由于缺乏治療神經(jīng)損傷的理想藥物,臨床上僅能將斷裂的神經(jīng)吻合,其結(jié)構(gòu)及功能的恢復(fù)很不理想。如何有效的治療外周神經(jīng)損傷,特別是較長距離的神經(jīng)損傷,使之有效的實(shí)現(xiàn)功能恢復(fù),這也是當(dāng)前神經(jīng)科學(xué)研究中亟待解決的問題之一。 目前的研究表明,周圍神經(jīng)損傷后雖有一定的自我再生能力,但良好的再生修復(fù)更需要神經(jīng)營養(yǎng)因子(neurotrophic factors,NTFs)、基質(zhì)和神經(jīng)細(xì)胞這三方面因素的協(xié)同作用來實(shí)現(xiàn)。 膠質(zhì)細(xì)胞源性神經(jīng)營養(yǎng)因子(glial cell line-derived neurotrophic factor,GDNF)對(duì)多巴胺神經(jīng)元、運(yùn)動(dòng)神經(jīng)元等多種神經(jīng)元均具有促進(jìn)存活和損傷保護(hù)的作用,極有希望用來治療神經(jīng)損傷和神經(jīng)系統(tǒng)退行性病變。實(shí)施基因治療將GDNF植入損傷局部,使之持續(xù)表達(dá)分泌GDNF,就解決了外源性給藥作用時(shí)間短等的不足�；蛑委熞x擇適宜的載體細(xì)胞。神經(jīng)干細(xì)胞(neural stem cells,NSCs)能在體外大量增殖,并可誘導(dǎo)分化為神經(jīng)系統(tǒng)的主要細(xì)胞——神經(jīng)元和膠質(zhì)細(xì)胞。因此,NSCs成為基因治療理想的載體細(xì)胞。同時(shí)組織工程材料殼聚糖與組織有較好的相容性,能被組織吸收,更能抑制成纖維細(xì)胞的生長,防止瘢痕形成,因此它也是應(yīng)用于神經(jīng)損傷治療中的理想材料。 本實(shí)驗(yàn)運(yùn)用細(xì)胞培養(yǎng)技術(shù)、基因轉(zhuǎn)染技術(shù)、流式細(xì)胞計(jì)數(shù)、免疫組織(細(xì)胞)化學(xué)技術(shù)、HRP逆行示蹤、坐骨神經(jīng)功能指數(shù),膜片鉗電生理技術(shù)等方法,分離、培養(yǎng)大鼠大腦皮層NSCs,觀察NSCs與殼聚糖、雪旺細(xì)胞(Schwann's cells,SCs)共培養(yǎng)條件下生長、分化的情況。并將GDNF基因在體外轉(zhuǎn)染到NSCs中,篩選、擴(kuò)增后移植到經(jīng)過殼聚糖管套接的大鼠坐骨神經(jīng)離斷模型中,以滿足神經(jīng)再生修復(fù)的三大因素,觀察評(píng)價(jià)治療效果,為損傷神經(jīng)的治療探索新的可行性。主要結(jié)果如下: 1.本實(shí)驗(yàn)分離、培養(yǎng)胚胎大鼠大腦皮層NSCs。細(xì)胞呈球狀懸浮生長,細(xì)胞克隆
[Abstract]:The regeneration and repair of nerve injury has always been the difficulty and focus of neuroscience research. In recent years, with the increasing of traffic accidents and injuries, the nerve injury, especially the peripheral motor nerve injury, is increasing day by day, which is characterized by high disability rate and great harm. Due to the lack of ideal drugs for the treatment of nerve injury, the broken nerve can only be anastomosed clinically, and the recovery of its structure and function is not satisfactory. How to effectively treat peripheral nerve injury, especially the long distance nerve injury, so that it can effectively achieve functional recovery, which is one of the problems to be solved in the current neuroscience research. Current studies have shown that although the peripheral nerve injury has a certain ability of self-regeneration, good regeneration and repair need the cooperation of neurotrophic factor neurotrophic factors NTFsI, matrix and nerve cells. Glial cell line-derived neurotrophic factor (GDF) can promote the survival and protection of many kinds of neurons, such as dopamine neurons, motor neurons and so on. When gene therapy was carried out, GDNF was implanted into the injured area, and the expression and secretion of GDNFwere sustained, which solved the shortage of the short time of exogenous drug administration. Gene therapy should select suitable vector cells. Neural stem cells can proliferate in vitro and induce neural stem cells to differentiate into neuronal and glial cells. Therefore, NSCs have become the ideal vector cells for gene therapy. At the same time, chitosan has good compatibility with tissue, can be absorbed by tissue, can inhibit the growth of fibroblasts and prevent scar formation, so it is also an ideal material for the treatment of nerve injury. In this study, cell culture, gene transfection, flow cytometry, immunohistochemistry, HRP retrograde tracing, sciatic nerve function index, patch clamp electrophysiology, etc. The growth and differentiation of NSCs, chitosan and Schwann cells were observed. The GDNF gene was transfected into NSCs in vitro, screened, amplified and transplanted into the model of sciatic nerve transection by chitosan tube in order to satisfy the three factors of nerve regeneration and repair, and to observe and evaluate the therapeutic effect. To explore the new feasibility for the treatment of nerve injury. The main results are as follows: 1. In this experiment, NSCs were isolated and cultured in embryonic rat cerebral cortex. Cell suspension growth, cell cloning
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2005
【分類號(hào)】：R329;R318.08

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