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目的探讨国产人源性β-淀粉样蛋白(Aβ)聚合寡聚体的方法及其聚合效果。方法 Aβ40、Aβ42室温条件分别溶解于纯度≥99.0%的六氟异丙醇中,使Aβ浓度至1 mmol/L、20 min;高纯氮处理使六氟异丙醇完全挥发后,添加纯度≥99.5%的二甲基亚砜,使Aβ浓度至6 mmol/L,30℃水浴超声10 min;添加含0.05%十二烷基硫酸钠的磷酸盐缓冲液(0.02 mol/L,p H7.4),Aβ浓度至120μmol/L;再用上述缓冲液分别稀释Aβ至40、80μmol/L,形成3种浓度梯度。37℃恒温摇床100 r/pmin行聚合反应2、12、24、48、72、96和120 h;SDS-PAGE,银染处理,分别考察上述条件的聚合效果。结果 3种浓度的Aβ40寡聚体聚合效果有很大差异:40μmol/L聚合48、120 h,80μmol/L聚合12 h有较明显的二十二聚体出现,120μmol/L聚合≥12 h,有四聚体至十聚体形成。但3种浓度Aβ40形成寡聚体又有一定相似性:均有大量的单体、二聚体、三聚体存在,随着聚合时间延长,寡聚体总量及高分子量寡聚体形成量并未明显增加。3种浓度的Aβ42聚合效果有明显区别:随着聚合浓度升高,十二聚体、二十一聚体的形成量逐渐下降,相同聚合时间内,随着浓度的升高,四聚体至十聚体形成量逐渐增加;但3种浓度的Aβ42聚合也有相似处:均存在大量的单体、二聚体、三聚体,随着聚合时间的延长,四聚体至十聚体形成量有增加趋势,此与Aβ40聚合效果有明显差异。结论采用常用的方法均能使国产Aβ40、Aβ42形成寡聚体;总体来讲,相对于聚合时间,聚合浓度对形成高分子量的Aβ寡聚体种类影响较大。
Objective To investigate the method and the polymerization of domestic β-amyloid (Aβ) oligomers. Methods Aβ40 and Aβ42 were dissolved in hexafluoroisopropanol with the purity of ≥99.0% at room temperature, the concentration of Aβ was 1 mmol / L and 20 min respectively. When the purity of hexafluoroisopropanol was completely volatilized by high purity nitrogen, the purity of ≥ 99.5% dimethylsulfoxide, the concentration of Aβ to 6 mmol / L, 30 ℃ water bath ultrasonic 10 min; 0.05% sodium dodecyl sulfate in phosphate buffer (0.02 mol / L, p H7.4 ), Aβ concentration to 120μmol / L; and then the above buffer were diluted Aβ to 40,80μmol / L, the formation of three kinds of concentration gradient. The polymerization was carried out for 2, 12, 24, 48, 72, 96 and 120 h at 100 r / min with 37 ℃ constant temperature shaker. SDS-PAGE and silver staining were used to investigate the polymerization results. Results The aggregation effects of Aβ40 oligomer at three concentrations were quite different: 40μmol / L for 48,120 h, 80μmol / L for 12 h showed more obvious dodecamers, 120μmol / L for more than 12 h, Tetramer to decamer formed. However, the three concentrations of Aβ40 formed oligomers have a certain similarity: a large number of monomers, dimers, trimers exist, with the polymerization time, the total amount of oligomers and high molecular weight oligomer formation Not significantly increased. The aggregation effect of Aβ42 at three concentrations is obviously different: with the increase of the polymerization concentration, the formation of dodecamers and the nonadecamers decreased gradually. With the increase of the concentration, But the aggregation of Aβ42 at three concentrations is also similar: there are a large number of monomers, dimers and trimers. With the extension of polymerization time, the amount of tetramers to decamers There is an increasing trend, which has a significant difference with the Aβ40 polymerization effect. CONCLUSION: Domestic Aβ40 and Aβ42 can be oligomers formed by the commonly used methods. In general, the polymerization concentration has a great influence on the formation of high molecular weight Aβ oligomers relative to the polymerization time.