β淀粉样肽前体蛋白N端328-332(APP5肽)类似物神经营养作用的体外实验研究

The in vitro experimental research on neurotrophic effect of the analogs of APP 5-mer peptide

目的研究β淀粉样肽前体蛋白N端328-332(APP5肽)及其类似物对人神经母细胞瘤株SY5Y生长的影响,在体外寻找具有神经营养作用又能抵抗胃蛋白酶消化能力的5肽类似物。方法合成多个5肽类似物,以MTT代谢率进行筛选,选出最佳的5肽类似物,以人神经母细胞瘤株SY5Y作为体外实验模型,以MTT代谢率、细胞计数,LDH漏出率,Westernblotting检测作为观察指标,进一步证实5肽类似物的神经营养作用。应用胃蛋白酶对APP5肽和类似物165进行消化实验,观察两者的酶解率。结果(1)APP5肽及由此基础上改造的多种类似物均可使SY5Y细胞MTT代谢率增高,而类似物165的效果最好,40μmol/L为最佳有效浓度。(2)APP5肽及类似物165可使SY5Y细胞MTT代谢率增高,LDH漏出率减少,细胞数量增加,SY5Y细胞P-CREB、Bcl-2蛋白表达增加。(3)胃蛋白酶消化实验显示,胃蛋白酶浓度为40g/L时,APP5肽和类似物165的酶解率分别为96.4%和16.9%;胃蛋白酶量为100g/L时,APP5肽和类似物165的酶解率为99.1%和58.6%,两者差异非常显著。5肽类似物165的抗酶解能力明显大于APP5肽。结论APP5肽及类似物165对SY5Y细胞具有神经营养作用,而APP5肽类似物165具有更强的抗胃蛋白酶消化能力。

Objective To observe the effects of APP 5-mer peptide and its analogs on the growth of human neuroblastoma strain SY5Y, and to find the best analog which has not only neurotrophic effect but also the ability to resist pepsin. Methods Several analogs of APP 5-mer peptide were synthesized and the best one was picked out by MTT metabolic rate screening. The human neuroblastome strain SY5Y was used as the in vitro experimental model; and cell count, MTT metabolic rate, LDH leakage rate and Western blotting were used as indicators to confirm the neurotrophic action of this analog of APP 5-mer peptide. The digesting rates of the analogs of APP 5-mer peptide and the APP 5-mer peptide were observed by the digestion test of the pepsin. Results （1）APP 5-mer peptide and several analogs of APP 5-mer peptide could increase the MTT metabolic rate of SY5Y cells. The analog 165 was the best one among the analogs and 40μmol/L was the optimal concentration. （2）APP 5-mer peptide and the analog 165 enhanced MTT metabolic rate, the cell count, the expression of Bcl-2 and p-CREB of SY5Y ceils, and reduced the LDH leakage rate. （3）The result of the digestion test indicated that the rate of digestion of APP 5-mer peptide was 96.4 % and the rate of digestion of the analog 165 was 16.9 % when the concentration of pepsin was 40g/L. When the concentration of pepsin was 100g/L, the rate of digestion of APP 5-mer peptide was 99.1% and the rate of digestion of the analog 165 was 58.6 % , with statistical significance between them. The analog 165 of APP 5-mer peptide had higher ability to resist pepsin digestion than APP 5-mer peptide obviously. Conclusion APP 5-mer peptide and analog 165 had neurotrophic effect on SY5Y cells and could increase the livability of human neuroblastoma cells SY5Y. The ability of the ananlog 165 to resist pepsin digestion was better than that of APP 5-mer peptide.