フローサイトメトリー法を用いたEBER1mRNA発現の解析と臨床的意義の検討

取得学位：博士(医学), 学位授与番号：医博乙第1534号, 学位授与年月日：平成13年4月18日, 学位授与年：200

Knockdown of the bovine prion gene PRNP by RNA interference (RNAi) technology

<p>Abstract</p> <p>Background</p> <p>Since prion gene-knockout mice do not contract prion diseases and animals in which production of prion protein (PrP) is reduced by half are resistant to the disease, we hypothesized that bovine animals with reduced PrP would be tolerant to BSE. Hence, attempts were made to produce bovine <it>PRNP</it> (b<it>PRNP</it>) that could be knocked down by RNA interference (RNAi) technology. Before an in vivo study, optimal conditions for knocking down b<it>PRNP</it> were determined in cultured mammalian cell systems. Factors examined included siRNA (short interfering RNA) expression plasmid vectors, target sites of <it>PRNP</it>, and lengths of siRNAs.</p> <p>Results</p> <p>Four siRNA expression plasmid vectors were used: three harboring different cloning sites were driven by the human U6 promoter (hU6), and one by the human tRNA^Valpromoter. Six target sites of bovine <it>PRNP </it>were designed using an algorithm. From 1 (22 mer) to 9 (19, 20, 21, 22, 23, 24, 25, 27, and 29 mer) siRNA expression vectors were constructed for each target site. As targets of siRNA, the entire b<it>PRNP </it>coding sequence was connected to the reporter gene of the fluorescent EGFP, or of firefly luciferase or <it>Renilla </it>luciferase. Target plasmid DNA was co-transfected with siRNA expression vector DNA into HeLaS3 cells, and fluorescence or luminescence was measured. The activities of siRNAs varied widely depending on the target sites, length of the siRNAs, and vectors used. Longer siRNAs were less effective, and 19 mer or 21 mer was generally optimal. Although 21 mer GGGGAGAACTTCACCGAAACT expressed by a hU6-driven plasmid with a <it>Bsp </it>MI cloning site was best under the present experimental conditions, the corresponding tRNA promoter-driven plasmid was almost equally useful. The effectiveness of this siRNA was confirmed by immunostaining and Western blotting.</p> <p>Conclusion</p> <p>Four siRNA expression plasmid vectors, six target sites of b<it>PRNP</it>, and various lengths of siRNAs from 19 mer to 29 mer were examined to establish optimal conditions for knocking down of b<it>PRNP </it>in vitro. The most effective siRNA so far tested was 21 mer GGGGAGAACTTCACCGAAACT driven either by a hU6 or tRNA promoter, a finding that provides a basis for further studies in vivo.</p

Nutrient Condition in the Microenvironment Determines Essential Metabolisms of CD8(+) T Cells for Enhanced IFN gamma Production by Metformin

Metformin (Met), a first-line drug for type 2 diabetes, lowers blood glucose levels by suppressing gluconeogenesis in the liver, presumably through the liver kinase B1-dependent activation of AMP-activated protein kinase (AMPK) after inhibiting respiratory chain complex I. Met is also implicated as a drug to be repurposed for cancers; its mechanism is believed identical to that of gluconeogenesis inhibition. However, AMPK activation requires high Met concentrations at more than 1 mM, which are unachievable in vivo. The immune-mediated antitumor response might be the case in a low dose Met. Thus, we proposed activating or expanding tumor-infiltrating CD8(+) T cells (CD8TILs) in a mouse model by orally administering Met in free drinking water. Here we showed that Met, at around 10 mu M and a physiologically relevant concentration, enhanced production of IFN gamma,TNF alpha and expression of CD25 of CD8(+) T cells upon TCR stimulation. Under a glucose-rich condition, glycolysis was exclusively involved in enhancing IFN gamma production. Under a low-glucose condition, fatty acid oxidation or autophagy-dependent glutaminolysis, or both, was also involved. Moreover, phosphoenolpyruvate carboxykinase 1 (PCK1), converting oxaloacetate to phosphoenolpyruvate, became essential. Importantly, the enhanced IFN gamma production was blocked by a mitochondrial ROS scavenger and not by an inhibitor of AMPK. In addition, IFN gamma production by CD8TILs relied on pyruvate translocation to the mitochondria and PCK1. Our results revealed a direct effect of Met on IFN gamma production of CD8(+) T cells that was dependent on differential metabolic pathways and determined by nutrient conditions in the microenvironment

Frequent Loss of Genome Gap Region in 4p16.3 Subtelomere in Early-Onset Type 2 Diabetes Mellitus

A small portion of Type 2 diabetes mellitus (T2DM) is familial, but the majority occurs as sporadic disease. Although causative genes are found in some rare forms, the genetic basis for sporadic T2DM is largely unknown. We searched for a copy number abnormality in 100 early-onset Japanese T2DM patients (onset age <35 years) by whole-genome screening with a copy number variation BeadChip. Within the 1.3-Mb subtelomeric region on chromosome 4p16.3, we found copy number losses in early-onset T2DM (13 of 100 T2DM versus one of 100 controls). This region surrounds a genome gap, which is rich in multiple low copy repeats. Subsequent region-targeted high-density custom-made oligonucleotide microarray experiments verified the copy number losses and delineated structural changes in the 1.3-Mb region. The results suggested that copy number losses of the genes in the deleted region around the genome gap in 4p16.3 may play significant roles in the etiology of T2DM

Methylxanthine sensitization of human colon cancer cells to 186Re-labeled monoclonal antibody

金沢大学大学院医学系研究科Tumor cells lacking the functional p53 suppressor gene may arrest at the G2 phase of the cell cycle after exposure to ionizing radiation, resulting in increased radioresistance. Methylxanthines (MTXs), such as pentoxifylline (PTX) or caffeine (CAF), can inhibit the G2-phase checkpoint arrest of damaged cells and thus radiosensitize them. However, the effect of MTX in cells irradiated with low-dose-rate β-emission is not well understood. Methods: A clonogenic assay was performed with LS180 human colon cancer cells lacking the functional p53 suppressor gene. Cells were irradiated with increasing concentrations of 186Re-mercaptoacetyltriglycine (186Re-MAG3)-labeled A7 monoclonal antibody against colorectal cancer (0-925 kBq/mL) at 37°C in 5% CO2 for 24 h in the presence or absence of PTX (0-2 mmol/L) or CAF (0-5 mmol/L). The enhancement ratio (ER) with MTX was calculated as a ratio of 50% cell-killing concentration of 186Re-MAG3-A7 in control cells to that in cells treated with PTX or CAF. The cell cycle distribution was analyzed with a flow cytometer. Results: The concentration of 50% cell kill was 474 kBq/mL 186Re-MAG3-A7. Both PTX and CAF dose dependently enhanced the cytotoxicity of 186Re-MAG3-A7: ERs of 0.5 mmol/L PTX, 2 mmol/L PTX, 1 mmol/L CAF, and 5 mmol/L CAF were 1.50, 2.18, 1.54, and 2.63, respectively. Flow cytometry showed that the percentage nonirradiated cells in the G2/M phase of the cell cycle was 11.3% ± 1.66%. On the other hand, cells exposed to 186Re-MAG3-A7 accumulated in the G2/M phase of the cell cycle (40.2% ± 1.46%), which was inhibited by the presence of 1 mmol/L PTX (19.8% ± 8.12%) or 2 mmol/L CAF (26.9% ± 6.21%). Conclusion: Cellular modulation of the cell cycle with PTX and CAF radiosensitized LS180 colon cancer cells exposed to 186Re radiation

トウニョウビョウ ケア ノ リスク マネージメント

The number of diabetics has been increasing in recent years. The diabetics are under varioustreatments, including the improvement of life habit and the medication for diabetes with insulin.Our hospital set a team of diabetic care, which is composed of a diabetic specialist, certified diabeteseducators（CDEs）, nurses, dietricians and pharmacists. This team takes great care of the diabetics.For medical safety measures, the department of risk management was organized in our hospital.The department investigated the cases of Hiyari-Hatto within 1 year and 3 months, from 2005to 2006, and found that 3% of them was the diabetic case, which was caused by the nurses exceptCDEs. Therefore the department made the manual of diabetic therapy in cooperation with theCDEs. All the staffs in our hospital were educated by the seminars according to the manual. Theknowledge about the diabetic therapy proved to be mostly accurate one year after the last seminar.For the improvement of medical safety, the department of risk management helps the CDEswith holding the educational seminars by giving the informations after analyzing the cases of Hiyari-Hatto and the questionnaires following the seminars

Increased Plasma mRNAs of Placenta-specific 1 (PLAC1) and Glial Cells-missing 1 (GCM1) in Mothers with Pre-eclampsia

In this study we have investigated whether quantitative analysis of placental mRNAs in maternal plasma provides a way to monitor placental status. We measured plasma concentrations of human chorionic gonadotropin /)-subunit (/3hCG) and human placental lactogen (hPL) mRNAs as previously reported mRNAs and pregnancy associated plasma protein A (PAPP-A), placenta-specific 1 (PLACl) and glial cells-missing 1 (GCMl) mRNAs, which have not been measured during the course of normal pregnancy. Firstly, peripheral blood was obtained at various times from healthy pregnant women to clarify the time course of placental mRNAs. Secondly, blood was obtained from women with pre-eclampsia and gestational age-matched controls to examine whether placental mRNAs change in pre-eclampsia. Plasma was separated from these samples for extraction of RNA, followed by reverse transcription polymerse chain reaction analysis. Median concentrations of PLACl and GCMl mRNA in plasma of pre-eclamptic subjects respectively were 1625 and 2141 copies/ml, significantly higher than 195 and 881 copies/ml, the values for controls (Mann-Whitney test, p<0.001). No significant difference was seen in hPL, f3hCG, or PAPP-A mRNA concentration between pre-eclamptic and control groups. Plasma PLACl and GCMl mRNAs appear promising as noninvasively measurable molecular markers for pre-eclampsia