Identification of specific surface proteins in small hepatocytes and reconstruction of hepatic organoids ex vivo.

小肝细胞中特定表面蛋白的鉴定和离体肝类器官的重建。

基本信息

批准号：
14370393
负责人：
MITAKA Toshihiro
金额：
$ 8.9万
依托单位：
Sapporo Medical University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

项目摘要

We found three genes, CD44, BRI3, and D6.1A, with much higher expression in small hepatocytes (SHs)than in mature hepatocytes (MHs)and possessing transmembrane domain. Using specific antibody to CD44, BRI3, and D6.1A, we examined the localization of SHs in a rat liver. Although none of expressions of the proteins were observed in hepatic lobules of a normal liver, the hepatocytes reacting with the antibodies existed in the periportal lesion of severely injured liver treated with galactosamine. Using the antibody with magnetic beads (MACS), CD44+ cells were sorted from the galactosamine-treated livers and then cultured. The CD44+ cells could proliferate and form colonies, which are morphologically similar to SHs. Although hepatic marker genes were expressed in both sorted and cultured CD44+ cells, neither bile duct cell nor oval cell marker genes were detected by PCR. When cultured SHs were transplanted into congenic rat livers in combination with radiation and partial portal ligation, SHs were inserted into hepatic trabecules and proliferated.To reconstruct hepatic organoids, we tried three different methods. (1)When SHs and NPCs were plated on collagen sponge, SHs could proliferate and expand to form a hepatic organoid in the sponge. The structure includes MHs with bile canaliculi, bile ducts, and capillary-like structures. Furthermore, we also observed that isolated human hepatic cells could form hepatic organoid in the sponge. (2)After SHs were cultured on two multiporous membranes until they formed large colonies, one membrane were stacked on the other membrane as cells faced each other. The stacked cells could differentiate into MHs with forming bile canalicular networks. (3)When both SH colonies and the connective tissues of the remnant after the hepatocyte isolation were cultured in the microgravity rotating culture system, a relatively large size of hepatic organoids was formed with a few weeks.

我们发现了三个基因CD44，BRI3和D6.1a，在小肝细胞（SHS）中的表达高于成熟的肝细胞（MHS），并且具有跨膜结构域。使用对CD44，BRI3和D6.1A的特异性抗体，我们检查了SHS在大鼠肝脏中的定位。尽管在正常肝脏的肝小叶中没有观察到蛋白质的任何表达，但在用半乳糖胺处理的严重损伤肝脏的外周病变中，肝细胞与抗体反应。使用带有磁珠（MAC）的抗体，将CD44+细胞从半乳糖胺处理的肝脏中分类，然后培养。 CD44+细胞可以增殖并形成菌落，在形态上与SHS相似。尽管在分类和培养的CD44+细胞中表达肝标记基因，但通过PCR检测到胆管细胞和椭圆形细胞标记基因。将培养的SHS与辐射和部分门户连接结合使用到先天大鼠肝脏中时，将SH插入肝脏小梁中并增殖。为了重建肝癌，我们尝试了三种不同的方法。（1）当将SHS和NPC铺在胶原蛋白海绵上时，SHS可以扩散并膨胀以在海绵中形成肝脏类动物。该结构包括带有胆管的MHS，胆管和毛细管样结构。此外，我们还观察到分离的人肝细胞可以在海绵中形成肝癌。（2）将SHS培养在两个多膜膜上之前，直到它们形成大菌落，当细胞彼此面对时，将一个膜堆叠在另一个膜上。堆叠的细胞可以通过形成胆管网络的MHS分化为MHS。（3）当在微重力旋转培养系统中培养肝细胞分离后的SH菌落和残留物的结缔组织时，几周就形成了相对较大的肝癌。

项目成果

期刊论文数量（231）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Mesangial cells organize the glomerular capillaries by adhering to the G domain of laminin alpha5 in the glomerular basement membrane

系膜细胞通过粘附在肾小球基底膜中层粘连蛋白α5的G结构域来组织肾小球毛细血管

DOI：
发表时间：
2003
期刊：
Journal of Cell Biology 161(1)
影响因子：
0
作者：
Kikkawa Y;et al.
通讯作者：
et al.

Meguro M, 他8名: "A novel inhibitor of inducible nitric oxid synthase (ONO-1714) prevents critical warm ischemia-reperfusion injury in the pig"Transpiantation. 73(9). 1439-1446 (2002)

Meguro M 和其他 8 人：“一种新型诱导型一氧化氮合酶抑制剂 (ONO-1714) 可预防猪的严重热缺血再灌注损伤”73(9) 1439-1446 (2002)。

DOI：
发表时间：
期刊：
影响因子：
0
作者：
通讯作者：

Keisuke Harada, et al.: "Rapid formation of hepatic organoid in collagen sponge by rat small hepatocytes and hepatic nonparenchymal cells"Journal of Hepatology. 39(11). 716-723 (2003)

Keisuke Harada 等人：“大鼠小肝细胞和肝非实质细胞在胶原海绵中快速形成肝类器官”肝脏病学杂志。

DOI：
发表时间：
期刊：
影响因子：
0
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通讯作者：

Hitoshi Kimura, et al.: "Role of inducible nitric oxide synthase in pig liver transplantation"Journal of Surgical Research. 111(1). 28-37 (2003)

Hitoshi Kimura 等人：“诱导型一氧化氮合酶在猪肝移植中的作用”外科研究杂志。

DOI：
发表时间：
期刊：
影响因子：
0
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Opposing roles of integrin alpha6Abetal and dystroglycan in laminin-mediated extracellular signal-regulated kinase activation

整合素 α6Abeta 和肌营养不良聚糖在层粘连蛋白介导的细胞外信号调节激酶激活中的相反作用

DOI：
发表时间：
2003
期刊：
Mol Biol Cell 14(5)
影响因子：
0
作者：
Ferletta M;Kikkawa Y;Yu H;Talts JF;Durbeej M;Sonnenberg A;Timpl R;Campbell KP;Ekblom P;Genersch E.
通讯作者：
Genersch E.