研究者詳細 - 水口　徹

論文 - 水口　徹

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Hyperbaric oxygen stimulates cell proliferation and normalizes multidrug resistance protein-2 protein localization in primary rat hepatocytes

T Mizuguchi, H Oshima, H Imaizumi, H Kohara, M Kawamoto, T Nobuoka, H Kawasaki, K Harada, Y Masuda, Y Kikkawa, T Mitaka, Y Asai, K Hirata

WOUND REPAIR AND REGENERATION ( BLACKWELL PUBLISHING ) 13 ( 6 ) 551 - 557 2005年11月 [査読有り]

Hyperbaric oxygen therapy (HBO) has been used for many clinical treatments, including primary liver nonfunction. However, the cellular mechanism by which HBO treatment ameliorates liver function is not understood. Therefore, the purpose of this study was to elucidate this cellular mechanism using primary cultured rat hepatocytes in in vitro studies. Hepatocytes were treated with HBO at 1 day after plating, and the morphological and functional characteristics of bile canaliculi formed in cultured hepatocytes were observed by time-lapse microscopy. Multidrug resistance protein-2 localization was observed by confocal laser microscopy. In cultured hepatocytes, the labeling index in the HBO group at 2 days after treatment was significantly higher than that in the control group. In addition, the proliferating cellular nuclear antigen level in the HBO group was significantly higher than that in the control group. The contraction of the bile canaliculi in the HBO group was slower than in the control group and the dilatation of bile canaliculi in the HBO group was much larger than in the control group. Multidrug resistance protein-2 in the HBO group was localized at the apical membrane. These results show that HBO stimulates hepatocytes to proliferate and HBO normalizes Multidrug resistance protein-2 localization to the apical membrane, which could dilate bile canaliculi.

DOI PubMed

Hepatic organoid formation in collagen sponge of cells isolated from human liver tissues

S Sugimoto, K Harada, T Shiotani, S Ikeda, N Katsura, Ikai, I, T Mizuguchi, K Hirata, Y Yamaoka, T Mitaka

TISSUE ENGINEERING ( MARY ANN LIEBERT INC ) 11 ( 3-4 ) 626 - 633 2005年03月 [査読有り]

We examined whether small hepatocytes (SHs), which are hepatic progenitor cells, could be isolated from a normal human liver and whether human hepatic cells could form hepatic organoids in a collagen sponge. Normal liver tissues were obtained from resected specimens from nine patients who underwent hepatic resection. Isolated hepatic cells were plated on dishes and a collagen sponge. More than 1 month later, SH-like cells appeared and proliferated on the dishes, whereas cell aggregates were formed in the sponge and showed characteristic tissue architecture: columnar and/or cuboidal epithelial cells lined the surface of the sponge. Clusters of epithelial cells with a large cytoplasm and ductular structures were observed under the lining cells. The lining and ductular cells were positive for cytokeratins 7 and 19, which indicated they were biliary epithelial cells (BECs), and the epithelial cells forming clusters were positive for the anti-human hepatocyte antibody, identifying them as hepatocytes. Some lining cells were positive for both the hepatic marker and the BEC markers. The cells in the collagen sponge actively proliferated and the hepatocytes excreted albumin into the medium. Thus, hepatic organoids could be reconstructed in a collagen sponge by normal human liver cells.

DOI PubMed

Hepatocyte transplantation for total liver repopulation.

Mizuguchi T, Mitaka T, Katsuramaki T, Hirata K

Journal of hepato-biliary-pancreatic surgery ( 5 ) 12 378 - 385 2005年 [査読有り]

DOI PubMed

Biliary tract malignancy and chronic inflammation from the perspective of pancreaticobiliary maljunction

Y Kimura, N Nishikawa, K Okita, T Furuhata, T Mizuguchi, T Nobuoka, H Nishimori, H Zenbutsu, M Satoh, T Katsuramaki, K Hirata

ONCOLOGY ( KARGER ) 69 41 - 45 2005年 [査読有り]

This is a brief review of pancreaticobiliary maljunction. The basic treatment for this condition includes either cholecystectomy or extrahepatic bile duct resection. When the condition is accompanied by malignancies, a radical operation should be the first treatment option. Knowledge on molecular pathogenesis is gradually increasing. However, studies should be expanded to include larger patient cohorts, and other types of molecules should be carefully investigated and analyzed. Copyright (C) 2005 S. Karger AG, Basel.

DOI

動脈合併切除再建, 肝外胆管切除再建にて切除しえた肝エキノコックス症の1例

信岡隆幸, 桂巻正, 水口徹, 島宏彰, 木村康利, 古畑智久, 向谷充宏, 平田公一

胆道 = Journal of Japan Biliary Association ( 日本胆道学会 ) 18 ( 1 ) 81 - 87 2004年03月

肝左葉に発生し,肝動脈・胆管浸潤を認めた肝エキノコックス症に対して,拡大肝左葉切除, 肝外胆管切除, 肝動脈合併切除・再建にて治癒切除できた症例を経験した.症例は58歳女性で,黄疸を主訴に近医受診し,腹部USおよび腹部CTで肝左葉全体に腫瘤を認め,血清学的検査にて肝エキノコックス症と診断された.胆管造影では腫瘤の浸潤により,左肝管閉塞,肝門部胆管狭窄を認め,減黄目的にERBDチューブが挿入された.減黄後,手術目的に当科紹介され拡大左葉切除を行なった.開腹時の腹腔内所見は,肝十二指腸間膜で右肝動脈は胆管に浸潤した腫瘤と一塊となって剥離不能であったため,右肝動脈の一部を合併切除し,右肝動脈と固有肝動脈を端々吻合して再建した.本症は発見時にすでに脈管・胆管浸潤を伴って広範囲に進展し,切除不能な場合もある.本症例は肝門部に高度に浸潤していたものの, 肝外胆管切除, 肝動脈再建にて切除できた, 稀な症例と思われ報告した.

DOI CiNii

A case of giant hepatic hemangioma with spontaneous intratumoral bleeding <Case Report>

Nobuoka Takayuki, Katsuramaki Tadashi, Mizuguchi Toru, Shima Hiroaki, Kimura Yasutoshi, Mukaiya Mitsuhiro, Hirata Koichi

Tumor research : experimental and clinical ( 札幌医科大学 ) 39 1 - 5 2004年

Cavernous hemangioma is the most common benign tumor of the liver, and most of them remain asymptomatic. However, a giant hemangioma may rupture spontaneously and present severe complications such as shock and anemia, and need emergency operation. We report a case that spontaneous intratumoral bleeding of a giant hemangiona of the liver that occurred in a 55-year-old woman. She presented with severe abdominal pain, anemia, and shock at 3 and a half years after a transcatheter arterial embolization (TAE) treatment. Although the bleeding was under control, a surgical procedure was considered to reduce the risk for rerupture. The general outcome of surgical treatment for giant hemangiomas is satisfactory. In contrast, especially in our case. TAE was unable to reduce the risk of rupture over the long term. Therefore, surgery for giant hemangioma of the liver should be considered as the first treatment to avoid the risk of rupture even in "healthy" case.

DOI CiNii

Effects of bone marrow stromal cells on the structural and functional polarity of primary rat hepatocytes

T Mizuguchi, K Palm, T Hui, T Aoki, Y Mochizuki, T Mitaka, AA Demetriou, J Rozga

IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY-ANIMAL ( SOC IN VITRO BIOLOGY ) 38 ( 2 ) 62 - 65 2002年02月 [査読有り]

DOI PubMed

Regulation of c-Met expression in rats with acute hepatic failure

T Mizuguchi, Y Kamohara, T Hui, T Neuman, T Mitaka, AA Demetriou, J Rozga

JOURNAL OF SURGICAL RESEARCH ( ACADEMIC PRESS INC ) 99 ( 2 ) 385 - 396 2001年08月

Background. Earlier we described a model of fulminant hepatic failure (FHF) in the rat where partial hepatectomy is combined with induction of right liver lobe necrosis. In FHF rats, lack of hepatocyte proliferation was associated with delayed expression of HGF and HGF receptor c-met. Since the c-met promoter region has Sp1 binding sites, we decided to examine whether in FHF rats down-regulation of c-met is associated with decreased Spl function and whether changes in blood HGF, IL-6, and TGF beta1 levels might be responsible for these effects. Materials and methods. Induction of FHF, partial (2/3) hepatectomy (PH), and sham hepatectomy (SH) was performed in adult Sprague-Dawley rats. The levels of c-met mRNA and Spl DNA binding activity were studied in rat liver remnants at different time points after surgery. Blood levels of HGF, IL-6, and TGF beta1 were also measured in these rats. Additionally, the effects of treatment with TGF-beta1, IL-6, or a combination of both on c-met expression and Sp1 DNA binding were studied in HGF-induced rat hepatocyte cultures. Results. Compared to SH rats, in PH rat livers c-met was up-regulated after 6 h and Sp1 DNA binding was at or only slightly lower than levels at all time points studied. In FHF rat livers, c-met expression was markedly reduced after 2 and 6 h, moderate after 12 h, and undetectable after 24 h. At the same time, Sp1 DNA binding was detected at 2 h postinduction only. In FHF rats, blood levels of all three cytokines showed early and sustained elevation. In vitro, IL-6 had no effect on c-met expression, whereas TGF beta1 upregulated c-met. When used alone, none of the cytokines affected Spl DNA binding activity. In contrast, a combination of IL-6 and TGF beta1 down-regulated c-met expression as well as Sp1 DNA binding activity. These effects were dependent on the IL-6 concentration used. This study suggests that following massive loss of hepatocyte mass in rats, early increase in blood IL-6 and TGF beta1 levels may weaken the expression of HGF receptor c-met in surviving hepatocytes through suppression of Spl DNA binding. (C) 2001 Academic Press.

DOI

Effects of nicotinamide-related agents on the growth of primary rat hepatocytes and formation of small hepatocyte colonies

F Sato, T Mitaka, T Mizuguchi, Y Mochizuki, K Hirata

LIVER ( MUNKSGAARD INT PUBL LTD ) 19 ( 6 ) 481 - 488 1999年12月

Aims/Background: We report in this study that, 10 mM nicotinamide can stimulate the proliferation of primary rat hepatocytes in serum-free Dulbecco's modified Eagle's medium supplemented with 10 ng/ml epidermal growth factor and that small hepatocyte colonies appear from 4 to 5 days after plating. We examined the effects of nicotinamide-related agents on the growth and differentiation of primary rat hepatocytes and on the appearance of small hepatocyte colonies. Methods: As nicotinamide is an aqueous vitamin named niacin and known to act as an inhibitor of poly (ADP-ribose) polymerase (PARP), we therefore chose to examine the effects on hepatocytes of three nicotinamide-related agents, nicotinic acid (NA) which is also a niacin, 3-aminobenzamide (3-AB) which is a strong inhibitor of PARP but is not a niacin, and 3-acetylpyridine (3-AP) which is a weak inhibitor of PARP and also not a niacin. To examine their effects on the growth of the cells and on the formation of the colony, immunocytochemistry for BrdU was carried out. Expression of albumin, tryptophan 2,3-dioxygenase (TO), and connexin 32 (Cx32) mRNAs were used as marks of hepatic differentiation. Intracellular NAD(+) content was also measured. Results. At concentration of 10 mM, NA could not enhance the proliferation of mature hepatocytes but induced the appearance of small hepatocyte colonies. At concentration of 5 mM, 3-AB enhanced the proliferation of the hepatocytes but did not induce small hepatocyte colonies. On the other hand, although 10 mM 3-AP remarkably inhibited the DNA synthesis of the cells, the expression not only bf albumin but also of TO and Cx32 mRNAs in the cells was well maintained for more than one week, The intracellular NAD(+) concentration was correlated with the proliferation of the hepatocytes. Conclusion: These results suggest that the intracellular NAD(+) content may be correlated with the proliferation of primary hepatocytes and that the supplementation of niacin in the medium may be important for the appearance of small hepatocyte colonies.

DOI PubMed

Reconstruction of hepatic organoid by rat small hepatocytes and hepatic nonparenchymal cells

T Mitaka, F Sato, T Mizuguchi, T Yokono, Y Mochizuki

HEPATOLOGY ( W B SAUNDERS CO ) 29 ( 1 ) 111 - 125 1999年01月

Hepatic cells isolated from an adult rat Liver, consisting of small hepatocytes (SHs), mature hepatocytes (MHs), liver epithelial cells (LECs), Kupffer cells, sinusoidal endothelial cells, and stellate cells, were cultured in a medium supplemented with 10% fetal bovine serum, 10 mmol/L nicotinamide, 1 mmol/L ascorbic acid 2-phosphate, 10 ng/mL epidermal growth factor, and 1% dimethyl sulfoxide. The SHs rapidly proliferated and formed a colony. About 10% of cytokeratin 8 (CK8)-positive cells formed SH colonies. All SHs at day 10 immunocytochemically showed positivity for albumin, transferrin, CK8, and CK18, which are markers for hepatocytes. In contrast, alpha-fetoprotein (AFP)-, CK14-, OC2-, and glutathione S-transferase placental type (GST-P)positive cells, which are thought to be markers for hepatic immature cells, were rarefy observed. At day 20 some cells in the colonies were positive for AFP, CK7, CK19, and GST-P LECs and stellate cells proliferated and surrounded the colonies. About 2 weeks after plating, piled up cells were often observed on the SH colonies. In those colonies LECs and stellate cells invaded under the colonies, The invasion of the cells and gradual deposits of extracellular matrix (ECM) such as type I collagen, type TV collagen, and laminin induced alteration of the shape of the SHs from relatively flat to cuboidal or rectangular. With the cellular structural changes, the expression of albumin, connexin 32 (Cx32), and tryptophan 2,3-dioxygenase (TO) messenger RNAs increased. In addition, overlapping nonparenchymal cells (NPCs) on the piled up cells induced the formation of duct- or cyst-like structures consisting of MHs. In the present experiment we showed that SHs could differentiate to MI-Is by interacting with NPCs and ECM. Thus, SHs may be "committed progenitor cells" that can further differentiate into MHs.

DOI PubMed CiNii

Growth and maturation of small hepatocytes

T Mitaka, T Mizuguchi, F Sato, C Mochizuki, Y Mochizuki

JOURNAL OF GASTROENTEROLOGY AND HEPATOLOGY ( BLACKWELL SCIENCE ) 13 S70 - S77 1998年09月

Proliferation of adult rat hepatocytes is observed in serum-free Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 mmol/L nicotinamide and 10 ng/mL epidermal growth factor (EGF). The proliferating cells are mainly mononucleate and form small cell colonies surrounded by mature hepatocytes. Although these cells in focal colonies have a less-differentiated appearance, immunocytochemically and ultrastructurally they possess hepatic characteristics. The size of small hepatocytes is one-third to half that of mature hepatocytes. Therefore, we call the cells forming a colony, small hepatocytes. The small hepatocytes can be subcultured for several passages. Furthermore, the cells are rich in the supernatant following 50 g centrifugation for 1 min after collagenase liver perfusion. When the cells are cultured in DMEM supplemented with 10% foetal bovine serum, 10 mmol/L nicotinamide, 1 mmol/L ascorbic acid 2-phosphate, 10 ng/mL EGF and 1% dimethyl sulphoxide, each small hepatocyte can clonally proliferate for more than 3 months. A small hepatocyte divides to form a colony and the number of cells reaches more than 100 within 20 days. With time in culture, cells with a large cytoplasm appear within a colony. They have many mitochondria and large peroxisomes with crystalline nucleoids and are typical, mature hepatocytes. Immunoreactivity to connexin 32 and well-developed bile canaliculus structures are often observed in the cell-cell borders. Thus, we suggest that small hepatocytes may be considered to be 'committed progenitor cells' that can further differentiate into mature hepatocytes.

Mechanism of metastasis and tumor invasion in gastric carcinoma

Ryuichi Denno, Koichi Hirata, Takahiro Yasoshima, Takayuki Shishido, Hideki Ura, Koji Yamaguchi, Toru Mizuguchi, Fumihiko Sato

Japanese Journal of Gastroenterological Surgery ( Japanese Society of Gastroenterological Surgery ) 31 ( 4 ) 974 - 978 1998年

Surgery should be a curative and minimally invasive treatment for early gastric cancer. Therefore we need to know the biology of malignancy of gastric carcinoma. We established a new cell line, AZ-H5c, which has a high potential of metastasis to the nude mice liver, from the human gastric carcinoma line AZ521. Experimental studies revealed that, compared with AZ521 cells. AZ-H5c cells moved more actively, had stronger enzymatic activity of MMP-9, and expressed higher levels of integrin alpha 1, alpha 2, alpha 3, slpha 4, alpha 5, integrin beta 1 and CD44v3. Based on the above information, we investigated the clinico-pathologic characteristics of patients with carcinoma of the stomach. There were significant correlations of integrin alpha 2 and lymph node metastasis and integrin alpha 3 and liver metastasis. There was no correlation of clinico-pathologic characteristics and the expression of MMP-9, nor was the MMP-9 staining pattern related to survival. To foster the development of more effective, minimally invasive surgery, we should continue to accumulate data on gastric carcinma so as to improve diagnostic accuracy regarding lymph node metastasis, the pattern of recurrence and other factors.

DOI

Subculture of proliferating adult rat hepatocytes in medium supplemented with nicotinamide and EGF

T Mitaka, T Kojima, T Mizuguchi, Y Mochizuki

IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY-ANIMAL ( SPRINGER ) 32 ( 8 ) 469 - 477 1996年09月 [査読有り]

To establish parenchymal hepatocyte cell lines, we tried to subculture the primary hepatocytes isolated from adult rats. The hepatocytes were cultured in serum-free modified Dulbecco's modified Eagle's medium supplemented with 10 mM nicotinamide and 10 ng/ml epidermal growth factor. When 6 x 10(5) cells were plated on 35-mm dishes coated with rat tail collagen, the cells proliferated and reached confluence at Day 6 to Day 8. The first subculture was carried out at Day 8 using 0.005% collagenase and gentle pipettings. Most cells were recovered and plated on the new dishes coated with the collagen (first passage). The attached cells could proliferate and reached near confluence when the cells occupied more than two-thirds of the dish surface. About a week after the first subculture, the second one was conducted. Although the number of the recovered cells was smaller than at the first passage, the cells could attach and proliferate to a certain extent. Thereafter, they were maintained for more than 2 mo. but they never overgrew. Albumin secretion into the culture medium was confirmed in the subcultured cells. Ultrastructurally, these subcultured cells possessed hepatic characteristics such as peroxisomes with a crystalline nucleiod and bile-canaliculus structures. When 10% fetal bovine serum and ascorbic acid 2-phosphate were added to the cells of the second passage, they began to proliferate very slowly. These proliferating cells were mainly mononucleate and had a small cytoplasm. In addition, some of them could differentiate into typical mature hepatocytes by forming a three-dimensional structure interacting with nonparenchymal cells. In this experiment, we showed the successful subculturing of parenchymal hepatocytes isolated from adult rats and provided evidence that the subcultured cells still have the potential to proliferate and to differentiate.

Changes in cellular distribution of connexins 32 and 26 during formation of gap junctions in primary cultures of rat hepatocytes

T Kojima, M Yamamoto, H Tobioka, T Mizuguchi, T Mitaka, Y Mochizuki

EXPERIMENTAL CELL RESEARCH ( ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS ) 223 ( 2 ) 314 - 326 1996年03月 [査読有り]

In the adult rat hepatocyte, gap junction proteins consist of connexin 32 (Cx32) and connexin 26 (Cx26), Previously, we reported that both Cx32 and Cx26 were markedly induced and maintained in primary cultures of adult rat hepatocytes, The reappearing gap junctions were accompanied by increases in both the proteins and the mRNAs, and they were well maintained together with extensive gap junctional intercellular communication (GJIC) for more than 4 weeks. In the present study, we examined the cellular location of the gap junction proteins and the structures in the hepatocytes cultured in our system, using confocal laser microscopy and immunoelectron microscopy of cells processed for Cx32 and Cx26 immunocytochemistry and freeze-fracture analysis, In immunoelectron microscopy, the size of Cx32-immunoreactive gap junction structures on the plasma membrane increased with time of culture, and some of them were larger than those in liver sections in vivo. Freeze-fracture analysis also showed that the size of gap junction plaques increased and that the larger gap junction plaques were composed of densely packed particles. These results suggest that in this culture system, not only the synthesis of Ox proteins but also the size of the gap junction plaques was increased markedly. In the adluminal lateral membrane of the cells, Cx32-immunoreactive lines were observed and many small gap junction plaques were closely associated with a more developed tight junction network. In the basal region of the cells, small Cx32- and Cx26-immunoreactive dots were observed in the cytoplasm and several annular structures labeled with the antibody to Cx32 were observed in the cytoplasm, These results indicated the formation and degradation of gap junctions in the cultured hepatocytes. (C) 1996 Academic Press, Inc.

Effects of oxygen radical scavengers on connexins 32 and 26 expression in primary cultures of adult rat hepatocytes

T Kojima, T Mitaka, T Mizuguchi, Y Mochizuki

CARCINOGENESIS ( OXFORD UNIV PRESS UNITED KINGDOM ) 17 ( 3 ) 537 - 544 1996年03月 [査読有り]

Although we recently reported our success in inducing and maintaining the gap junction proteins connexin 26 (Cx26) and connexin 32 (Cx32) in adult rat hepatocytes cultured in serum-free L-15 medium supplemented with epidermal growth factor, dimethylsulfoxide (DMSO) and glucagon, the mechanisms by which DMSO induces gap junctions are still not clear, It is known that DMSO is not only a differentiation reagent for various cells but also a powerful scavenger of oxygen radicals, In the present study, by using this culture system and the measurement of oxidative stress by the nitro blue tetrazolium (NBT) formazan assay, we have examined the effect of oxygen radical scavengers such as DMSO, dimethylthiourea (DMTU) and alpha-tocopherol on the expression of both Cxs and on gap junctional intercellular communication (GJIC), as compared to another differentiation reagent, hexamethylene-bis-acetamide (HMBA), DMSO and DMTU clearly inhibited the oxidative stress of the cultured hepatocytes, while alpha-tocopherol and HMBA did not, The expression of Cx26 and Cx32 in the cultured hepatocytes was markedly induced by DMSO and DMTU, Furthermore, extensive GJIC was also observed with DMSO and DMTU, These results suggest that the expression of gap junctions in the hepatocytes may be closely related to oxidative stress and that oxygen radical scavengers may be important substances in inducing this expression.

GROWTH AND MATURATION OF SMALL HEPATOCYTES ISOLATED FROM ADULT-RAT LIVER

T MITAKA, T KOJIMA, T MIZUGUCHI, Y MOCHIZUKI

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ( ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS ) 214 ( 2 ) 310 - 317 1995年09月 [査読有り]

Small hepatocytes existed in the supernatant following low-speed centrifugation of the cell suspension after collagenase liver perfusion. The cells proliferated for more than 2 months and formed colonies in the Dulbecco's modified Eagle's medium supplemented with 10 mM nicotinamide, 10% fetal bovine serum, 1 mM ascorbic 2-phosphate, and 10 ng/ml epidermal growth factor. One small cell finally proliferated to several hundred cells. In addition, some cells in the colonies were shown to differentiate into mature hepatocytes that had a large cytoplasm and sometimes two nuclei. The secretion of albumin in the medium by the hepatocytes increased with time in culture, and the cells possessed connexin 32 in their cell membrane and many peroxisomes. Thus, the small hepatocytes may be ''committed progenitor cells'' which can further differentiate into mature hepatocytes. (C) 1995 Academic Press. Inc.

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