THE EXPERIMENTAL STUDY OF FUNCTION FOR SEPTAL CELL IN LUNG

大鼠肺间隔细胞分离培养方法的建立在大鼠出生后发育过程中，间隔细胞的数量在出生后5-7天逐渐增加，此后逐渐减少。我们用胶原酶消化分散这些细胞，并尝试了两种分离方法：用Percoll密度液和7%Nycodenz液将细胞离心分离成细胞悬液。两种方法均能获得富含隔细胞的组分。用含10%胎牛血清的F-10或F-12培养液培养细胞，观察小窝成分、生物活性物质受体与培养时间的分布变化及其与培养时间的关系。1)免疫荧光检测发现，L天时α-平滑肌肌动蛋白表达较弱，阳性细胞数为20%，3天时表达增强，阳性细胞达75%。这一趋势在免疫印迹中也得到了证实。2)Caveolin(洞穴特异蛋白)，IP3R(肌醇，4，5-tR…在培养第1天的隔细胞中，CaATPase(钙泵)和异磷酸受体的分布相似。但小窝蛋白在第3天隔细胞中的分布与IP3R和CaATPase的定位不同。3)免疫荧光研究表明，ETA和ETB在间隔细胞中存在。4)ET-1-Cy3诱导细胞构建，内化的ET-1-Cy3定位于细胞内，呈点状结构。5)这些抗体在Western印迹上可识别许多条带，但在衣霉素处理的细胞中，抗体可识别正确的条带。假条带是ETR糖基化的结果。在超薄切片和免疫电子显微镜下，ETA部分定位于隔细胞中的凹陷，而ETB则定位于内皮细胞中的凹陷1)针对脂滴周围抗原的抗体(由台湾国立国立大学SM-Wang博士提供)隔细胞内脂滴周围呈环状排列，细胞内有丝状结构。2)在免疫印迹上，该抗体识别190、160和57 kDa条带。3)该抗体在大鼠眼球晶状体匀浆中识别57 kDa条带为抗波形蛋白抗体。4)Colcemid和Cellalasin B诱导边缘图案消失。这与胡芦巴碱相似。5)这些结果表明，该抗原与肌动蛋白、波形蛋白和Fodrin结合，定位于隔细胞的脂滴周围。较少

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