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Decreased Transketolase and Dysfunction of Pentose Phosphate Cycle are Involved in Pathogenesis of Brain Regionally Selective Damage Induced by Thiamine Deficiency

Author: ZhaoYanLing
Tutor: ZhongChunJiu；MaoYueShi；WangYang
School: Fudan University
Course: Neurology
Keywords: Pyruvate dehydrogenase α - ketoglutarate dehydrogenase Transketolase 6 - phosphate dehydrogenase Nicotinamide adenine dinucleotide phosphate Thiamine deficiency Lentivirus virus RNA interference Neural stem cells TK RNAi Hydroxy thiamine Neural Stem Cells
CLC: R747.9
Type: Master's thesis
Year: 2008
Downloads: 81
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Abstract

Wernicke encephalopathy due to thiamine (vitamin B1) deficiency caused by neurodegenerative diseases, typical clinical manifestations ophthalmoplegia, ataxia and brain function abnormalities. The lesion showed specific brain regions selective brain regions are mainly located in the midline structures. Wernicke's encephalopathy brain regions selectively damage the mechanism is still not clear. The current study suggests that thiamine deficiency lead to the thiamine dependent PDH and KGDH of decreased activity, causing energy metabolism and oxidative stress caused by damage to the brain tissue, but this does not explain why the energy metabolism more active cortex does not appear to damage. Thiamine-dependent enzyme - TK thiamine deficiency also significantly lowered. TK is a key enzyme involved in RNA and to reducing equivalents such as NAPDH the synthesis of non-oxidative pentose phosphate pathway pathway. TK activity will affect the synthesis of RNA and reducing equivalents, which damage the cells with the ability to add value, and this has been confirmed in the study of tumor cells. Midline on both sides of the structure is the more active parts of the cell proliferation, our group in the early study has found that mice thiamine deficiency early hippocampal dentate gyrus neurogenesis significantly down, whether caused by thiamine deficiency TK activity decreased due to the pentose phosphate pathway dysfunction involved in the mechanism of of this specificity selective damage? Thus, we prepared a mouse model of thiamine deficiency, research TD dependence of mouse brain tissue thiamine changes in enzyme activity and thiamine deficiency on the function of the pentose phosphate pathway; the preparation TKRNA interfere with Lentivirus viral vectors infected neural stem cells, neural stem cells observed the TK RNA interference, and hydroxyl thiamine. Part I: thiamine deficiency thiamine as a coenzyme activity of the pentose phosphate pathway function Objective: To research TD brain thiamine as a coenzyme PDH KGDH TK activity and pentose phosphate the the sugar pathway critical enzyme - G6PD and products NADPH / NADP to changes in investigate the specific brain regions TD lead damage the relationship between the occurrence mechanism. MATERIALS AND METHODS: According to the aforementioned methods, diet deprived of thiamine preparation TD mouse model, were divided TD9, TD14, TD21 and normal control group (n = 6) mice. Chloral hydrate anesthetized mice were decapitated and the brain rapidly removed. MRNA expression in situ hybridization studies in different brain regions TK. In accordance with the the Stephan methods to extract mitochondrial BCA assay mitochondrial protein concentration. KGDH activity was measured according to the method of Humphries et al, PDH activity was measured according to the method of Hinman et al, according to the literature it has been reported a method brain tissue TK and G6PD activity was measured and the content of NADP, NADPH, and calculate the NADP / NADPH ratio. Activity was detected in each repeated three times, all values ??are expressed as mean ± SEM, t-test statistical control group and TD9, TD14, TD21 group with or without statistical difference. Results: TD9 and TD14 brain tissue, the activity PDH and KGDH, no decreased the TD14 brain tissue TK activity was significantly decreased NADPH content in TD9 and TD14 were significantly decreased. Hippocampus TK mRNA expression was significantly higher than the cortex. TD9 and TD14 the hippocampus TK activity was significantly decreased, and no significant changes in the hippocampus and cortex of G6PD and cortical TK activity. Conclusion: the academics generally accepted PDH and KGDH of decreased activity related mechanism is difficult to fully explain the mechanism of TD lead on both sides of the midline tissue-specific damage and cognitive dysfunction; of TK lead to decreased activity of the pentose phosphate metabolic pathway function The obstacles may be involved in both sides of the specific brain area damage mechanisms of thiamine midline. Part II: the Transketolase RNA interference Lentivirus viral vector preparation and screening purposes: Preparation of the TK RNA interference the Lentivirus virus carrier, infected neural stem cells, screening the best interference target sequence. Materials and Methods: For the TK gene sequence, the use of public website designed in accordance with the design principles of the sequence of RNA interference, a plurality of RNA interference target sequence the interference sequence-containing double-stranded DNA oligonucleotide, the ends containing the sticky ends of the restriction sites , to connect it directly into the digested Lentivirus viral vector. The purpose of the product was ligated into the prepared bacteria competent cells, first identified by PCR on clones grow during sequencing ratio, identification of positive clones is successfully constructed of RNA interference Lentivirus viral vector. Cultured target cells grew well (ie rat neural stem cells), the purpose of cells divided into 12-well culture plate culture, RNAi Lentivirus virus particles were added to different MOI infection by experimental design group for the purpose of cell infection experiments. 3 days after infection GFP expression observed by fluorescence microscopy, cells were collected, using RT-PCR method to detect the target gene mRNA expression in 5 days after infection, and then judge the interference effect of different target sequences. Results: successfully constructed the TK RNA interference Lentivirus viral vectors; different siRNA-containing virus were infected neural stem cells, GFP expression was observed, the infection efficiency by up to 50%, was detected by RT-PCR the best TK interference effect of 65%. Conclusion: We successfully prepared the the TK RNA interference Lentivirus viral vectors, the expression of TK purpose cells better interference effect. Part III: transketolase alcohol the purpose of of the enzyme RNA interference and hydroxyl thiamine hippocampal neural stem cells, the observed inhibition of the TK the expression and / or activity of neural stem cells: two methods using RNA interference and TK inhibitors hydroxy thiamine, growth. Materials and Methods: Take the E15 rat fetal rat hippocampus, the cell suspension with neural stem cell culture medium. Nestin identification of neural stem cells. The cell culture medium with different concentrations of TK inhibitors hydroxy thiamine, and cultured for 1-7 days to observe the growth of neural stem cell spheres and CCK-8 and BrdU method to detect neural stem cell proliferation. Cell suspension was added the right amount of the Lentivirus virus, is divided into two groups, one to carry the TK RNAi the Lentivirus virus as a processing group, another group to carry negative RNAi sequence Lentivirus virus as the control group, after 3 days of culture of GFP expression determines transfection efficiency, transfection rate of more than 50%, then the next detection. Neural stem cells cultured for 5 days virus TK gene downregulation effect of neural stem cells was evaluated by RT-PCR. Size, number, and migration levels observed neurospheres formed after infection with CCK-8 evaluation of cell growth. Results: TK inhibitor hydroxy thiamine inhibit the ball the growth of neural stem cells, reduce the speed of neural stem cell proliferation, and the concentration of the inhibitor. The TK RNA interference Lentivirus infection neural stem cells as compared with the control group, the average neurospheres number, size, morphology and migration of water affected; CCK-8, the results also show that RNA interference inhibits proliferation of neural stem cells. Conclusion: TK RNA interference and inhibitors impair the proliferation and growth of neural stem cells in the hippocampus.

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