Tombaccini D, Mocali A, Paoletti F
Istituto di Patologia Generale, Università di Firenze, Italy.
Exp Mol Pathol. 1994 Apr;60(2):140-6. doi: 10.1006/exmp.1994.1013.
Cultured Alzheimer fibroblasts were found to present peculiar alterations of transketolase (TK) ascribed to enhanced proteolytic activities in these cells and tentatively proposed as a marker of the disease. TK abnormalities, consisting of enzyme forms (alkaline bands) with unusually high alkaline pI, were investigated with respect to the mechanism of their generation and modulation by culture conditions. Alzheimer fibroblasts propagated at different pH, within a range of 7.3-7.8, exhibited TK abnormalities whose expression correlated directly with increases in medium pH values. Alterations were mostly evident in cells grown at 5% CO2 saturation in the atmosphere and with 3.7 g/liter NaHCO3 in the medium to yield an initial pH of about 7.75. Alkaline bands were not detected in either Alzheimer fibroblasts incubated at 10% CO2 or in control cells under any of the other conditions tested. Changes in initial medium pH also affected the morphology of fibroblasts, which shifted from a relatively large to an elongated shape as the medium pH decreased. The formation of alkaline bands was abolished by the addition of E-64, a known cysteine protease inhibitor, to cells just prior to extraction. On the contrary, Alzheimer fibroblasts cultured for 2 days in the presence of the inhibitor maintained the typically altered TK pattern. The establishment of conditions suitable for the expression of TK alterations in Alzheimer fibroblasts might be of help for diagnostic purposes and provide information on still elusive pathogenetic mechanisms of the disease.
研究发现，培养的阿尔茨海默病成纤维细胞中转酮醇酶（TK）呈现出特殊变化，这归因于这些细胞中蛋白水解活性增强，并初步提出将其作为该疾病的一个标志物。针对TK异常（由具有异常高碱性pI的酶形式（碱性条带）组成）产生和受培养条件调节的机制进行了研究。在7.3 - 7.8范围内不同pH值下培养的阿尔茨海默病成纤维细胞表现出TK异常，其表达与培养基pH值的升高直接相关。这种变化在大气中5%二氧化碳饱和度且培养基中含有3.7 g/升碳酸氢钠以产生约7.75初始pH值的条件下生长的细胞中最为明显。在10%二氧化碳条件下培养的阿尔茨海默病成纤维细胞或在任何其他测试条件下的对照细胞中均未检测到碱性条带。初始培养基pH值的变化也影响成纤维细胞的形态，随着培养基pH值降低，细胞从相对较大的形态转变为细长形态。在提取前向细胞中添加已知的半胱氨酸蛋白酶抑制剂E - 64可消除碱性条带的形成。相反，在抑制剂存在下培养2天的阿尔茨海默病成纤维细胞维持了典型的TK改变模式。建立适合阿尔茨海默病成纤维细胞中TK改变表达的条件可能有助于诊断，并为该疾病仍然难以捉摸的发病机制提供信息。
