本文選題：雪旺細(xì)胞 + 神經(jīng)生長(zhǎng)因子��； 參考：《大連醫(yī)科大學(xué)》2008年碩士論文

【摘要】： 神經(jīng)再生室技術(shù)的出現(xiàn),解決了短距離神經(jīng)再生的問題。但是除自體神經(jīng)移植外,其他人工修復(fù)技術(shù)尚無法解決長(zhǎng)距離神經(jīng)缺損的問題。盡管組織工程技術(shù)在不斷發(fā)展,但體外構(gòu)建的“人工神經(jīng)”還沒有從本質(zhì)上超越再生室技術(shù)。其原因是組織工程中作為種子細(xì)胞的細(xì)胞老化、分泌能力下降等諸多問題。雪旺細(xì)胞是周圍神經(jīng)最主要的膠質(zhì)細(xì)胞,也是最重要的種子細(xì)胞。雪旺細(xì)胞分泌多種神經(jīng)營(yíng)養(yǎng)因子在神經(jīng)再生中發(fā)揮關(guān)鍵作用,但其分泌能力受諸多因素影響,甲基強(qiáng)地松龍(MP)被認(rèn)為是一種通過抑制脂質(zhì)過氧化而對(duì)神經(jīng)細(xì)胞及其突起發(fā)揮保護(hù)作用的藥物。 目的: 1.采用新生SD大鼠的雪旺細(xì)胞作為周圍神經(jīng)組織工程的種子細(xì)胞,在體外分離培養(yǎng),探索體外培養(yǎng)及增殖雪旺細(xì)胞的方法。 2.選取生長(zhǎng)狀況和純度較好的第二代細(xì)胞,將經(jīng)胰酶消化下來的細(xì)胞接種到六孔板中,以不同濃度的MP作用于雪旺細(xì)胞,以研究MP對(duì)雪旺細(xì)胞分泌功能影響。 方法: 1.取SD大鼠的新生鼠雙側(cè)的坐骨、臂叢神經(jīng),剪碎至1mm3大小的組織塊,雙酶消化法進(jìn)行分離培養(yǎng),阿糖胞苷抑制成纖維細(xì)胞生長(zhǎng),低濃度胰酶快速消化傳代進(jìn)一步純化雪旺細(xì)胞,用光鏡進(jìn)行形態(tài)學(xué)觀察、S-100蛋白進(jìn)行鑒定。 2.取生長(zhǎng)狀況較好的第二代細(xì)胞,將經(jīng)胰酶消化下來的細(xì)胞接種到六孔板中,以不同濃度的MP(10~(-3)、10~(-4)、10~(-6)、10~(-8) mol/L)作用于雪旺細(xì)胞,作用24、48和72h,設(shè)立不加藥物的空白對(duì)照組,并通過RT-PCR的方法半定量檢測(cè)其分泌的NGF,以研究MP對(duì)雪旺細(xì)胞分泌功能的影響。 結(jié)果: 1.通過上述方法分離培養(yǎng)獲得了經(jīng)S-100蛋白鑒定純度為88%的第二代雪旺細(xì)胞,細(xì)胞數(shù)量為3.128×107/ml,細(xì)胞形態(tài)多數(shù)為梭形,細(xì)胞排列成典型的漩渦狀或柵欄狀。 2. RT-PCR方法檢測(cè)后,經(jīng)Lab works 4.60軟件進(jìn)行分析處理,以內(nèi)參基因β-actin為基準(zhǔn)分析各條帶的相對(duì)密度值,應(yīng)用Spass 10.0軟件進(jìn)行統(tǒng)計(jì)分析得出結(jié)論:10-8 mol/L的MP作用72h分泌的NGF與空白對(duì)照組比較及與其它濃度、時(shí)間組比較表達(dá)增加(p0.05); 10-3 mol/L的MP在各時(shí)間段分泌的NGF與空白對(duì)照組及其它濃度、時(shí)間組比較表達(dá)減少(p0.05)。 結(jié)論: 1.新生SD大鼠坐骨、臂叢神經(jīng)中分離培養(yǎng)雪旺細(xì)胞來源確實(shí),取材方便,可以作為周圍神經(jīng)組織工程試驗(yàn)研究的種子細(xì)胞來源,采用雙酶消化法、阿糖胞苷抑制成纖維細(xì)胞生長(zhǎng),可以獲得較高純度的雪旺細(xì)胞。 2.不同濃度的MP對(duì)雪旺細(xì)胞的分泌能力有不同的影響:高濃度(10~(-3) mol/L)的MP抑制雪旺細(xì)胞分泌NGF;長(zhǎng)時(shí)間、低濃度(72 h,10~(-8) mol/L)的MP則促進(jìn)雪旺細(xì)胞分泌NGF,故低濃度的MP可以提高雪旺細(xì)胞分泌NGF的能力,更好發(fā)揮雪旺細(xì)胞作為種子細(xì)胞的能力。
[Abstract]:The technique of nerve regeneration chamber solves the problem of short-distance nerve regeneration. But with the exception of autologous nerve transplantation, other artificial repair techniques can not solve the problem of long-distance nerve defect. Despite the development of tissue engineering technology, the in vitro construction of artificial nerve has not surpassed the regenerative chamber technology in essence. The reasons are the aging of cells as seed cells and the decline of secretion ability in tissue engineering. Schwann cells are the most important glial cells and seed cells of peripheral nerve. Schwann cells secrete a variety of neurotrophic factors that play a key role in nerve regeneration, but their secretion capacity is affected by many factors. MPM is considered to be a protective drug against nerve cells and its processes by inhibiting lipid peroxidation. Objective: 1. Schwann cells from newborn SD rats were isolated and cultured in vitro as seed cells for peripheral nerve tissue engineering to explore the methods of culture and proliferation of Schwann cells in vitro. 2. In order to study the effect of MP on the secretory function of Schwann cells, the second generation cells with good growth status and purity were inoculated into the Six-well plate with different concentrations of MP to study the effect of MP on the secretion of Schwann cells. Methods: 1. The sciatic bones and brachial plexus nerves of newborn SD rats were isolated and cultured by double enzyme digestion method, which were cut to the size of 1mm3. Cytarabine inhibited the growth of fibroblasts, and Schwann cells were further purified by rapid digestion and subculture of low concentration trypsin. The S-100 protein was identified by light microscope. 2. The cells digested by trypsin were inoculated into the six-well plate and treated with different concentrations of MPO10OOOON-3OOOTRONIDAE (10OOOTHYL) and Schewan cells (2448 and 72 h, respectively), and the control group was set up as a blank control group, and the control group was treated with different concentrations of MPO10OOOOOTHYL, and the control group did not add drugs to the cells for 2448 and 72 hours, and the cells were treated with different concentrations. The effect of MP on secretory function of Schwann cells was studied by semi-quantitative detection of NGF secreted by RT-PCR. Results: 1. The second generation of Schwann cells with the purity of 88% identified by S-100 protein was obtained by the method mentioned above. The number of cells was 3.128 脳 107 / ml, and most of the cells were fusiform, and the cells were arranged in a typical whirlpool or palisade shape. 2. After detection by RT-PCR method, the relative density of each band was analyzed by Lab works 4.60 software, using 尾 -actin as reference. Using Spass 10.0 software, it was concluded that the NGF secreted by 10 ~ (-8) mol/L at 72 h was higher than that in the control group and compared with other concentrations, and the expression of NGF in the time group was higher than that in the control group, and the NGF secreted by 10 ~ (-3) mol/L was higher than that in the blank control group and other concentrations. In the time group, the expression of P0. 05 was decreased. Conclusion: 1. Schwann cells isolated and cultured from the sciatic and brachial plexus of newborn SD rats can be used as seed cells for peripheral nerve tissue engineering research. Cytarabine can inhibit the growth of fibroblasts by double enzyme digestion. High purity Schwann cells can be obtained. 2. Different concentrations of MP have different effects on the secretory ability of Schwann cells: high concentration of MP can inhibit the secretion of NGF in Schwann cells for a long time. The low concentration of MP increased the secretion of NGF by Schwann cells, and the ability of Schwann cells as seed cells was improved by low concentration of MP.
【學(xué)位授予單位】：大連醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R329.2

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