THE EXPERIMENTAL STUDY OF FUNCTION FOR SEPTAL CELL IN LUNG

肺间隔细胞功能的实验研究

• 批准号: 08670002
• 负责人: AOKI Takeo
• 金额: $ 1.22万
• 依托单位: GUNMA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1996
• 资助国家: 日本
• 起止时间: 1996 至 1997
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08670002/
• 关键词: septal cell; caveolin; inositol 1,4,5-trisphosphate receptor; Ca pump; endothelin receptor; endothelin-1; tunicamycin; vimentin-related antrigen; カベオラ; エンドセリン; イノシトール3燐酸レセプター; CaATPase; α-平滑筋型アクチン

Establishment of separation and culture method for septal cell in rat lungIt was known that, during the process of rat postnatal development, the number of septal cell increase to 5-7 day after birth and decrease after the these days. We digested to disperse these cell with collagenase and attempted to two separate method ; Centrifugal cell fractionation to cell suspension with Percoll density solution and 7% Nycodenz solution. We were able to gain septal cell rich fraction with both two method. Cell was cultured with 1O% fetal calf serum containing medium F-10 or F12.The distributional change and relationship between caveola component, receptor to biologically active substance and culture time1)The expression of alpha-smooth muscle actin was weak on day l and positive cekll was 20%, but, on day 3, became strong and positive cell arrive to 75% with immunofluorescence study. This tendency was also confirmed on immunoblotting. 2)Caveolin (cavela specific protein), IP3R (inositoll, 4,5-tr … More isphosphate receptor) and CaATPase (Ca pump) was similar distributed in cultured day 1 septal cell. But the distribution of caveolin became be different localization of IP3R and CaATPase in day 3 septal cell. 3)Using with antibodies to a peptide corresponding to the carboxyl terminus of human ETA and ETB (gifted by Dr.Takimoto, Ciba-Geigy Japan), immunofluorescent study showed the existence of ETA and ETB in septal cell.4) ET-1-Cy3 induced the construction of the cell and the internalized ET-1-Cy3 localized with dot-like structure in the cell. 5)These antibodies recognized many bands on Western blot, but in tunicamycin treated cell, antibodies recognized the correct bands for ETA and ETB.The pseudoband was concluded the results of glycosylation to ETR.In ultrathin cryosection and immunoelectron microscopy, ETA partly localized caveola in septal cells and ETB did caveola in endothelial cellsVimentin-related antien and fodrin around lipid drolets in septal cells1) Antibody to the antigen around lipid droplets (gifted by Dr.SM-Wang, Taiwan Natl.Univ.) was rim pattern around lipid droplets and filamentous structure in septal cells. 2) On immunoblot, this antibody recognized 190,160 and 57 kDa band. 3) This antibody recognized 57 kDa band as anti-vimentin antibody in the homogenate of rat eye ball lens. 4) Colcemid and Cytochalasin Binduced the disappearance of the rim Pattern. This was similar to that of fodrin. 5) These results showed that this antigen associate with actin, vimentin and fodrin localized around lipid droplets in septal cells. Less

大鼠肺间隔细胞分离培养方法的建立可知，在大鼠出生后发育过程中，出生后5 ~ 7天间隔细胞数量增加，出生后5 ~ 7天间隔细胞数量减少。用胶原酶消化分散这些细胞，并尝试两种分离方法；用Percoll密度溶液和7% Nycodenz溶液对细胞悬浮液进行离心分离。两种方法均能获得中隔细胞丰富的部分。细胞用含培养基F-10或F12的10%胎牛血清培养。小窝成分、生物活性物质受体与培养时间的分布变化及关系1)免疫荧光研究显示，α -平滑肌肌动蛋白在第1天表达较弱，阳性细胞数为20%，而在第3天表达较强，阳性细胞数达到75%。免疫印迹也证实了这一趋势。2) CaATPase （Ca泵）、Caveolin （cavela特异性蛋白）、IP3R（肌醇、4,5-tr…More isphosphate受体）和CaATPase （Ca泵）在培养第1天的隔膜细胞中分布相似。但在第3天的间隔细胞中，小窝蛋白的分布与IP3R和CaATPase的定位不同。3)利用人ETA和ETB羧基端对应肽段的抗体（由Dr.Takimoto， Ciba-Geigy Japan提供），免疫荧光研究表明ETA和ETB在间隔细胞中存在。4) ET-1-Cy3诱导细胞的构建，内化的ET-1-Cy3在细胞内以点状结构定位。5)这些抗体在Western blot上识别了许多条带，但在tunicamycin处理的细胞中，抗体识别了ETA和ETB的正确条带。伪带是糖基化到ETR的结果。超薄切片和免疫电镜观察发现，ETA部分定位于间隔细胞内的小窝，ETB在内皮细胞内也有小窝。在间隔细胞内脂滴周围可见维门蛋白相关抗原和fodrin。1)脂滴周围抗原的抗体（dr . m . wang, natl . university）在间隔细胞内脂滴周围呈边缘状，呈丝状结构。2)在免疫印迹上，该抗体识别190,160和57kda条带。3)该抗体识别大鼠眼球晶状体匀浆中57 kDa条带为抗vimentin抗体。4)秋碱和细胞松弛素诱导边缘图案的消失。这与fodrin相似。5)结果表明，该抗原可与位于间隔细胞脂滴周围的肌动蛋白、波形蛋白和蛋白结合。少

项目成果

Hagiwara, H. ほか: "Development of Striated RootLets.during cilrogenesis in the Human oviduct eplthelium" Cell 8 Tissue Res.290. 39-42 (1997)

Hagiwara, H. 等人：“人输卵管上皮纤维形成过程中条纹根的发育”Cell 8 Tissue Res.290 (1997)。

Hagiwara, H., Aoki, T., Ohwada, N.and Fujimoto, T.: "Development of striated rootlets during ciliogenesis in the human oviduct epithelium" Cell Tissue Res. 290. 39-42 (1997)

Hagiwara, H.、Aoki, T.、Ohwada, N. 和 Fujimoto, T.：“人类输卵管上皮纤毛发生过程中横纹细根的发育”细胞组织研究。

Hagiwara., H., Aoki, T., and Fujimoto, T.: "Ultrastructural observation on 'transitional tubules'in human oviductal ciliogenic cells" J.Anat. 191. 285-90 (1997)

Hagiwara., H.、Aoki, T. 和 Fujimoto, T.：“人输卵管纤毛细胞‘移行小管’的超微结构观察”J.Anat。

Aoki, T., Hagiwara, H.and Fujimoto, T.: "Peculiar distribution of fodrin in fat-storing cells" Exp.Cell Res. 234. 313-20 (1997)

Aoki, T.、Hagiwara, H. 和 Fujimoto, T.：“脂肪储存细胞中胞质蛋白的特殊分布”Exp.Cell Res。