Gruber-Bzura Beata M, Krzysztoń-Russjan Jolanta, Bubko Irena, Syska Jarosław, Jaworska Małgorzata, Zmysłowski Adam, Rosłon Magdalena, Drozd Janina, Drozd Ewa, Majorczyk Edyta, Anuszewska Elżbieta L
Department of Biochemistry and Biopharmaceuticals, National Medicines Institute, Warszawa, Poland.
Adv Clin Exp Med. 2017 Aug;26(5):751-760. doi: 10.17219/acem/63091.
Oxidative stress accompanies neurodegeneration and also causes abnormalities in thiaminedependent processes. These processes have been reported to be diminished in the brains of patients with several neurodegenerative diseases.
The aim of this work was to conduct a comparative analysis of the impact of supplemented thiamine on the viability of human B lymphocytes with CAG abnormal expanded huntingtin gene (mHTT) (GM13509) and control, B lymphocytes without mHTT (GM14467) through the following studies: determination of the supplemented thiamine concentrations, which are effective for cell growth stimulation after incubation in thiamine deficit conditions; determination of cell capability to intake the exogenous thiamine; evaluation of exogenous thiamine influence on the profile of the genes related to thiamine and energy metabolism; determination of ATP synthesis and activities of thiamine-dependent enzymes, KGDHC and BCKDHC in the intact cells and upon the exogenous thiamine.
The following methods were used: EZ4U test for cell growth analysis; HPLC for determination of thiamine intake and ATP synthesis, qRT-PCR for evaluation of the gene profiles and spectrophotometric method for KGDHC and BCKDHC activities determination.
Maximal cell growth stimulation was observed at 2.5 mM in GM14467 up to 135% of the control culture and at 5.0 mM in GM13509 cells up to 165% of the control culture. Native levels of total ATP and KGDHC and BCKDHC activities in both cell types were comparable and did not changed upon thiamine deficit or supplementation. GM13509 cells showed more of an increase in growth stimulation upon thiamine supplementation than GM14467 cells and this effect was reflected in the increase of intracellular thiamine concentration.
The above results and reported changes in expression of GAPDH, IDH1 and SLC19A3 genes observed upon thiamine deficit conditions suggest that intracellular thiamine status and energy metabolism can have a role in HD pathogenesis.
氧化应激伴随着神经退行性变，并且还会导致硫胺素依赖性过程出现异常。据报道，在几种神经退行性疾病患者的大脑中，这些过程有所减弱。
本研究旨在通过以下研究对补充硫胺素对携带CAG异常扩增亨廷顿基因（mHTT）的人B淋巴细胞（GM13509）和对照、无mHTT的B淋巴细胞（GM14467）活力的影响进行比较分析：确定在硫胺素缺乏条件下孵育后对细胞生长刺激有效的补充硫胺素浓度；确定细胞摄取外源性硫胺素的能力；评估外源性硫胺素对与硫胺素和能量代谢相关基因谱的影响；测定完整细胞及外源性硫胺素作用下的ATP合成以及硫胺素依赖性酶、α-酮戊二酸脱氢酶复合体（KGDHC）和支链α-酮酸脱氢酶复合体（BCKDHC）的活性。
采用以下方法：EZ4U试验用于细胞生长分析；高效液相色谱法用于测定硫胺素摄取和ATP合成；定量逆转录聚合酶链反应用于评估基因谱；分光光度法用于测定KGDHC和BCKDHC的活性。
在GM14467细胞中，2.5 mM时观察到最大细胞生长刺激，可达对照培养物的135%；在GM13509细胞中，5.0 mM时可达对照培养物的165%。两种细胞类型中总ATP、KGDHC和BCKDHC活性的天然水平相当，在硫胺素缺乏或补充时均未改变。与GM14467细胞相比，GM13509细胞在补充硫胺素后生长刺激增加更多，且这种效应反映在细胞内硫胺素浓度的增加上。
上述结果以及在硫胺素缺乏条件下观察到的甘油醛-3-磷酸脱氢酶（GAPDH）、异柠檬酸脱氢酶-1（IDH1）和溶质载体家族19成员3（SLC19A3）基因表达的变化表明，细胞内硫胺素状态和能量代谢可能在亨廷顿舞蹈病发病机制中起作用。
