2014 Fiscal Year Research-status Report
Molecular Basis of Familial Glucocorticoid Deficiency
| Project/Area Number |
26461542
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| Research Institution | Hamamatsu University School of Medicine |
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Principal Investigator |
山口 理恵 浜松医科大学, 医学部, 特任研究員 (50420360)
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| Project Period (FY) |
2014-04-01 – 2017-03-31
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| Keywords | NNT / ROS / cortisol / redox status / HAC15 cells |
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| Outline of Annual Research Achievements |
Investigation of the effects of NNT knockdown on cellular redox status in HAC15 cells(an ACTH-responsive human adrenocortical cell line)
1.NNT knockdown was performed using two types of NNT-targeted siRNAs and confirmed with qPCR and western blot. 2. Both basal and ACTH-stimulated cortisol levels in the culture media of siNNT transfected cells were reduced. 3. H2O2(ROS) release with different glucose loadings was increased from NNT knockdown cells 4. Intracellular GSH/GSSG ratio, a biomarker of cellular redox status, was reduced in the siNNT cells.
The above data suggest that siRNA silencing of NNT in HAC15 cells results in an oxidized cellular redox status.
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| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
Investigation of the effects of NNT knockdownon on mitochondrial bioenergetics in HAC15 cells has not been performed as planned.
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| Strategy for Future Research Activity |
1. Investigation of the effects of NNT knockdown on mitochondrial bioenergetics
Assessment of mitochondrial oxygen consumption and intracellular ATP contents
2. Investigation of mitochondrial function and bioenergetics using the patient’s lymphocytes
(1)Assessment of NNT activity, NNT mRNA and protein expression in the patient’s lymphocytes.(2)Assessment of mitochondrial complex activities of the electron transport chain (reflecting mitochondrial function). (3)Measurement of lymphocytic mitochondrial ROS production rate. (4) Measurement of mitochondrial-dependent oxygen consumption (reflecting activity ofmitochondrial bioenergetics)
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| Causes of Carryover |
当初、研究計画していたもののうち、進行が遅れてしまったため、使用額に差が生じてしまった。
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| Expenditure Plan for Carryover Budget |
細胞導入実験にかかわるトランスフェクション試薬やsiRNAの購入を計画している。また、ミトコンドリア活性測定キット、ATP assay kit、ROS assay kit、シークエンス試薬なども購入予定である。
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