本文選題：單唾液酸神經(jīng)節(jié)苷脂　切入點：提取　出處：《西北大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：單唾液酸神經(jīng)節(jié)苷脂(Monosialoganglioside)位于生物體的細(xì)胞膜上,主要分布于哺乳動物的中樞神經(jīng)系統(tǒng)。神經(jīng)節(jié)苷脂作為一種特異性受體,與一些活性物質(zhì)相互作用,可以促進大腦發(fā)育,調(diào)節(jié)神經(jīng)系統(tǒng)功能,并且有再生神經(jīng)組織的作用。臨床研究表明,單唾液酸四己糖神經(jīng)節(jié)苷酯(Monosialotetrahexosylganglioside, GM1)對神經(jīng)細(xì)胞有定的保護修復(fù)作用,可以用于癱瘓、癡呆、生活能力喪失、老年癡呆、心腦血管意外創(chuàng)傷和帕金森等神經(jīng)性疾病的治療。目前的GM1提取分離及純化工藝復(fù)雜,所得產(chǎn)品中也通常殘留一些高分子物質(zhì)雜質(zhì)及其他神經(jīng)節(jié)苷脂雜質(zhì)。因此,建立一個方便、快捷的提取方法,獲得高純度的GM1,具有重要的社會意義和經(jīng)濟價值。本課題以鮮豬腦為原材料分離純化得到GM1,提取分離過程包括丙酮粉的制備、神經(jīng)節(jié)苷脂的萃取、弱陰離子交換層析及凝膠排阻層析,并用氨基鍵合柱和二醇基柱兩種高效液相色譜法和染色法對得到的樣品與Sigma公司生產(chǎn)的GM1標(biāo)準(zhǔn)品進行了對照。結(jié)果顯示,本方法得到的樣品與標(biāo)準(zhǔn)品的物理化學(xué)性質(zhì)一致,樣品純度高且不含毒害物質(zhì)。本論文的主要研究內(nèi)容如下：①提取分離。將鮮豬腦在冷丙酮中勻漿處理,去除組織中包含的水分和極性水溶性小分子物質(zhì),得到丙酮不溶物。丙酮不溶物經(jīng)一定比例的氯仿-甲醇-水抽提除去中性脂類,上層甲醇-水溶液經(jīng)旋轉(zhuǎn)蒸發(fā)得到粗提取的樣品水溶液,粗提取樣品產(chǎn)率約為3.36%±0.64%。將所得的樣品水溶液脫鹽后,進行DEAE Sepharose Fast Flow弱陰離子交換層析。平衡系統(tǒng)為pH7.420mmol/L Tris-HCl緩沖液,洗脫系統(tǒng)為0-70%1.0mol/L NaCl+pH7.420mmol/L Tris-HCl, GM1提取率約為(4.59±0.49)×10-2%。所得GM1樣品進一步進行Sephacryl S-100HR凝膠排阻層析分離,洗脫系統(tǒng)為pH7.420.0mmol/L PBS緩沖液,得到純品GM1,總提取率約為5.02mg/500g濕組織。②樣品檢測。將得到的GM1樣品分別在氨基鍵合高效液相上進行純度分析,并附以特征顯色反應(yīng)和二醇基柱高效液相檢測,以Sigma公司GM1標(biāo)準(zhǔn)品為對照。結(jié)果表明,所得的GM1樣品與標(biāo)準(zhǔn)品的物理化學(xué)性質(zhì)一致,并具有較高的純度。
[Abstract]:Monosialoganglioside (Monosialoganglioside) is located on the cell membrane of organisms and is mainly distributed in the central nervous system of mammals. Ganglioside, as a specific receptor, interacts with some active substances to promote brain development. Clinical studies have shown that Monosialotetrahexosylganglioside (GM1) has a definite protective and repair effect on nerve cells, which can be used for paralysis, dementia, and loss of life ability. Treatment of neuropathies such as dementia, cardio-cerebrovascular injury and Parkinson's disease. The current extraction, isolation and purification of GM1 are complicated, and some macromolecule impurities and other ganglioside impurities are usually retained in the obtained products. It is of great social significance and economic value to establish a convenient and fast extraction method to obtain high purity GM1.In this paper, GM1 was isolated from fresh pig brain, and the extraction and separation process included the preparation of acetone powder. Extraction of ganglioside, weak anion exchange chromatography and gel exclusion chromatography, Amino-bonded column and diol column were used to compare the obtained samples with the GM1 standard produced by Sigma. The results showed that the physical and chemical properties of the samples obtained by this method were consistent with those of the standard. The main contents of this thesis are as follows: 1. The fresh pig brain was homogenized in cold acetone to remove the water and polar water-soluble small molecule in the tissue. Acetone insoluble substance was obtained. Acetone insoluble substance was extracted by chloroform-methanol-water extraction to remove neutral lipids, and the upper methanol-water solution was rotated to obtain crude sample aqueous solution. The yield of crude extracted samples was about 3.36% 鹵0.64. DEAE Sepharose Fast Flow weak anion exchange chromatography was performed after desalination of the sample solution. The equilibrium system was pH7.420mmol/L Tris-HCl buffer. The elution system was 0-70mol / L NaCl pH7.420mmol/L Tris-HCl, the extraction rate of GM1 was about 4.59 鹵0.49 脳 10-2.The GM1 sample was further separated by Sephacryl S-100HR gel exclusion chromatography, and the elution system was pH7.420.0mmol/L PBS buffer. The purified GM1 was obtained and the total extraction rate was about 5.02 mg / 500g. The purity of the obtained GM1 samples was analyzed in amino bonded high performance liquid phase, followed by characteristic color reaction and high performance liquid phase detection on diol column. The results showed that the physical and chemical properties of the GM1 samples were consistent with those of the standard samples, and the purity of the GM1 samples was higher than that of the standard samples.
【學(xué)位授予單位】：西北大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R96

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