Role of Semaphorin 3E in liver fibrosis and regeneration

学位の種別: 課程博士審査委員会委員 : (主査)東京大学教授 深田 吉孝, 東京大学教授 宮島 篤, 東京大学教授 伊庭 英夫, 東京大学特任教授 渡邉 すみ子, 東京医科歯科大学教授 仁科 博史University of Tokyo(東京大学

Dielectrophoretic Assembly of Gold Nanoparticle Arrays Evaluated in Terms of Room-Temperature Resistance

Gold nanoparticles (GNPs) are often used as island electrodes of single-electron (SE) devices. One of technical challenges in fabrication of SE devices with GNPs is the placement of GNPs in a nanogap between two lead electrodes. Utilization of dielectrophoresis (DEP) phenomena is one of possible solutions for this challenge, whereas the fabrication process with DEP includes stochastic aspects. In this brief paper, we present our experimental results on electric resistance of GNP arrays assembled by DEP. More than 300 pairs of electrodes were investigated under various DEP conditions by trial and error approach. We evaluated the relationship between the DEP conditions and the electric resistance of assembled GNP arrays, which would indicate possible DEP conditions for fabrication of SE devices

Evaluation of the inter-particle distance of gold nanoparticles dispersed on silane-treated substrates to fabricate dithiol-connected arrays

Small tunnel junctions using gold nanoparticles (GNPs) as electrodes have been studied to fabricate single-electron devices. GNPs connected via dithiol molecules have been used as small tunnel junctions, and a two-stage dispersion method was used to fabricate dithiol-connected GNP arrays. In this process, the GNPs were fixed on silane-treated substrates by immersing the substrate in a colloidal gold solution. For fabricating dithiol-connected arrays, the inter-particle distance of the dispersed GNPs must be smaller than the GNP diameter. Consequently, the inter-particle distance controlled by the immersion time (T IM1) was evaluated. For T IM1 values exceeding 8 h, the inter-particle distance was less than the GNP diameter. A second dispersion of GNPs after treating samples with dithiol realized particle connections. For the GNP arrays produced with T IM1 values greater than 8 h, the I–V characteristics were measured at 77 K, and the yield of devices exhibiting nonlinear I–V curves was 23%

Expression and localization of sterile alpha motif domain containing 5 is associated with cell type and malignancy of biliary tree

<div><p>Cholangiocarcinoma (CC) is a type of relatively rare neoplasm in adenocarcinoma. The characteristics of CCs as well as biliary epithelial cells are heterogeneous at the different portion of the biliary tree. There are two candidate stem/progenitor cells of the biliary tree, i.e., biliary tree stem/progenitor cell (BTSC) at the peribiliary gland (PBG) of large bile ducts and liver stem/progenitor cell (LPC) at the canals of Hering of peripheral small bile duct. Although previous reports suggest that intrahepatic CC (ICC) can arise from such stem/progenitor cells, the characteristic difference between BTSC and LPC in pathological process needs further investigation, and the etiology of CC remains poorly understood. Here we show that Sterile alpha motif domain containing 5 (SAMD5) is exclusively expressed in PBGs of large bile ducts in normal mice. Using a mouse model of cholestatic liver disease, we demonstrated that SAMD5 expression was upregulated in the large bile duct at the hepatic hilum, the extrahepatic bile duct and PBGs, but not in proliferating intrahepatic ductules, suggesting that SAMD5 is expressed in BTSC but not LPC. Intriguingly, human ICCs and extrahepatic CCs exhibited striking nuclear localization of SAMD5 while the normal hilar large bile duct displayed slight-to-moderate expression in cytoplasm. <i>In vitro</i> experiments using siRNA for <i>SAMD5</i> revealed that SAMD5 expression was associated with the cell cycle regulation of CC cell lines. <i>Conclusion</i>: SAMD5 is a novel marker for PBG but not LPC in mice. In humans, the expression and location of SAMD5 could become a promising diagnostic marker for the cell type as well as malignancy of bile ducts and CCs.</p></div

Expression profiles of SAMD5 in human HCC and CC cell lines.

<p>(A) the relative expression of <i>SAMD5</i> gene in four CC cell lines (HuH28, TFK1, RBE and TKKK) and one HCC cell line (HuH7) to normal biliary epithelial cell (BEC) by quantitative RT-PCR. <i>SAMD5</i> mRNA was increased in all CC cell lines, but not expressed in HuH7. (n = 3; *<i>P</i> <0.05, compared to BEC) Data are mean ± standard error. n.d.: not detected (B). Immunocytochemical images of SAMD5 for CC cell lines. SAMD5 is visualized and localized at the nuclei of TKKK and RBE. Bars = 50 μm. (C and D) Images of exogenously introduced FLAG-tagged SAMD5 in HuH7 (C) and HuH28 (D) by Immunocytochemical staining. Overexpressed SAMD5 translocated to the nuclei of each cell. Bars = 50 μm.</p

Immunostaining of human liver tissue sections with anti-SAMD5 antibody.

<p>SAMD5 was stained for the paraffin-embedded sections of normal large bile duct at the hepatic hilum (A), intrahepatic CC (B), hilar CC (C and D), and extrahepatic CC (E). (A) Low or medial cytoplasmic staining of SAMD5 is observed in normal hilar large bile duct (asterisk). (Original magnification X200) (B) While the intrahepatic cholangiocytes show the cytoplasic staining of SAMD5 (arrow), the poorly-differentiated ICC exhibits striking nuclear staining of SAMD5. (Original magnification X100) (C) SAMD5 is stained in the nuclear of both poorly-differentiated ICC (p-ICC) and well-differentiated ICC (w-ICC) at the hepatic hilum. (Original magnification X200) (D) The cancerous cells invading hilar large bile duct show nuclear staining of SAMD5 (arrowhead). (Original magnification X200) (E) The papillary and moderately-differentiated tubular adenocarcinomas in the common bile duct exhibit nuclear staining of SAMD5. (Original magnification X200).</p

Primary antibodies.

<p>Primary antibodies.</p

Relationship between SAMD5 expression and cell cycle in CC cell line.

<p>(A) Real-time RT-PCR analysis of <i>SAMD5</i> mRNA in RBE cell line after 96 hours of knockdown using siRNA. n = 4 per each group. (B) Examination of RBE cell proliferation by WST-1 assay. n = 8 per each group. (C) Cell cycle analysis of RBE cell line by FACS. The knockdown of <i>SAMD5</i> in RBE cell line resulted in significant increase of cell population at S and M/G2 phase compared to the control. n = 3 per each group. (D) Real-time RT-PCR analysis of <i>SAMD5</i> mRNA in HuH28 cell line after 96 hours of overexpression. (E) Examination of HuH28 cell proliferation by WST-1 assay. n = 8 per each group. Data are mean ± standard error. *<i>P</i> <0.05; **<i>P</i> <0.01; ***<i>P</i> <0.001.</p

Result of cDNA microarray analysis of EpCAM⁺ cells between normal and DDC-fed mouse livers.

<p>Result of cDNA microarray analysis of EpCAM⁺ cells between normal and DDC-fed mouse livers.</p