国产β-淀粉样蛋白聚合寡聚体的方法及效果初探

Preliminary study on the methods and effects of aggregating of domestic amyloid-beta peptide into amyloid-beta oligomer

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 methods of domestic synthetic amyloid-beta peptide （human sequence ） oligomerization and to preliminarily verify the effect of its aggre- gation. Methods Human synthetic Aβ40 and Aβ42 were suspen-ded in hexafluoroisopropanol （HFIP）, whose purity was ≥99. 0% to 1 mmol/L for 20 rain at room temperature, respectively. HFIP was evaporated with the stream of nitrogen. Then Aβ4o and Aβ42 were resuspended at a concentration of 6 mmol/L in dimethylsulfoxide, whose purity was ≥99. 5% and sonieated for 10 mln at 30℃. The pretreated Aβ40 and Aβ42 was dilu- ted in 0. 02 mol/L phosphate-buffered saline （pHT. 4） containing 0. 05% sodium dodecyl sulfate （w/v） to 120 pxnol/L. 120 μmol/L Aβ solution was diluted to 40 p.mol/L and 80 μmol/L by PBS described above to produce a gradient solution with three concentrations. The aggregation was performed in a 37℃ shaker at 100 rpm for 2 h, 12 h, 23, h, 48 h, 72 h, 96 h and 120 h. The aggregation effects on Aβ40, Aβ42 under the above conditions were surveyed by gradient gel electrophoresis with silver staining. Results The aggregation effect of AI340 at lower concentration （40 μmol/L and 80 μmol/L） was greatly different from that at 120 μ mol/L. Twenty-two-mers were generated when 40 μmol/L Aβ40 was aggregated for 48 h and 120 h, and 80 panol/L Aβ40 for 12 h. When the polymers from tetramers to decamers were produced when 120 μmol/L Aβ4o was aggregated for 12 h and more time. Therefore, for Aβ40 there were certain similar characters under three concentration condi- tions, namely, numerous monomers were retained and lots of dimers and trimers were generated. The oligomers and high-or- der oligomers had no significant increase with the increase of aggregation time. Regarding Aβ42, the effects of aggregation significantly differed in three concentration conditions. The amount of dodeeamers and twenty-one-mers of Aβ42 gradually de- creased with the increase of aggregation concentration. The amount of Aβ42 polymers from tetramers to decamers rose with the increase of the initial concentration within the same aggregation time. There were same characters under three Aβ42 content conditions. Plenty of monomers were maintained and many dimers and trimers were produced, and the amount of Aβ42 poly- mers from tetramer to decamers gradually increased with the increase of processing time, which were tremendously different from the character of Aβ40. Conclusion The oligomers could be generated with domestic amyloid-beta peptide and the methods adopted in this study. Generally, the initial aggregation concentration of Aβ has greater impact on the kinds of larger molecular weight oligomers compared with the aggregation time.