Parise E R, Summerfield J A, Hahn E, Wiedmann K H, Doenhoff M J
Trans R Soc Trop Med Hyg. 1985;79(5):663-70. doi: 10.1016/0035-9203(85)90186-5.
The livers of female CBA mice were examined 9 to 10 weeks after subcutaneous infection with Schistosoma mansoni. Cryostat liver sections and isolated liver cells were examined by indirect immunofluorescence using specific antibodies against basement membrane proteins (laminin, fibronectin and type IV collagen and type III collagen precursor. Liver cells were isolated by collagenase digestion, purification on Percoll density gradients and centrifugal elutriation to yield enriched fractions of hepatocytes, endothelial and Kupffer cells (Fractions I, II, III respectively). Infected animals yielded more than three times the control number of non-parenchymal cells; electron microscopy revealed that the increase in Fraction II was due mainly to eosinophilic leucocytes and in Fraction III due to Kupffer cells and macrophages from the schistosomal granulomata. Studies of cryostat liver sections showed that the schistosomal granulomata contained dense deposits of type III collagen precursor and fibronectin in the distribution of the reticulin fibres but laminin and type IV collagen were conspicuous only in new vessels in the periphery of the granuloma. Isolated liver cells showed fibronectin on their surface. Immunofluorescence studies could not be performed on Kupffer and endothelial cell fractions because of marked non-specific fluorescence. These experiments indicate that centrifugal elutriation is a useful method for isolating the constituent cells of murine schistosomal granulomata.
在雌性CBA小鼠皮下感染曼氏血吸虫9至10周后,对其肝脏进行检查。使用针对基底膜蛋白(层粘连蛋白、纤连蛋白、IV型胶原和III型胶原前体)的特异性抗体,通过间接免疫荧光法对低温恒温器肝脏切片和分离的肝细胞进行检查。通过胶原酶消化、在Percoll密度梯度上纯化以及离心淘析来分离肝细胞,以获得富含肝细胞、内皮细胞和库普弗细胞的组分(分别为组分I、II、III)。受感染动物产生的非实质细胞数量是对照动物的三倍多;电子显微镜显示,组分II的增加主要归因于嗜酸性白细胞,而组分III的增加归因于来自血吸虫性肉芽肿的库普弗细胞和巨噬细胞。对低温恒温器肝脏切片的研究表明,血吸虫性肉芽肿在网状纤维分布区域含有密集的III型胶原前体和纤连蛋白沉积物,但层粘连蛋白和IV型胶原仅在肉芽肿周边的新血管中明显可见。分离的肝细胞表面有纤连蛋白。由于明显的非特异性荧光,无法对库普弗细胞和内皮细胞组分进行免疫荧光研究。这些实验表明,离心淘析是分离小鼠血吸虫性肉芽肿组成细胞的一种有用方法。