Kibira S, Dudek R, Narayan K S, Bing R J
Huntington Medical Research Institutes, Pasadena, CA 91105.
Mol Cell Biochem. 1991 Nov 13;108(1):75-84. doi: 10.1007/BF00239544.
Cultured endothelial cells have been used in the past as a source of endothelium-derived relaxing factor (EDRF) and of prostacyclin (PGI2). Although cell cultures are essential for observation of prolonged exposure to media or when there is delayed response, they are time consuming and sterile conditions are essential. In the present study, we report that endothelial cells, freshly harvested from bovine aortas, readily attached themselves to cytodex-3 microcarrier beads and released an endothelium-derived relaxing factor (EDRF), prostacyclin (PGI2) and increased the amount of cyclic GMP in vascular smooth muscle. Attachment to microcarrier beads was essential since it increased the surface area and the number of attached cells and permitted collection of cell free filtrates because of the formation of dense networks of cells and beads. As a result superfusion of cells and beads on the filter did not dislodge bound cells which remain on the filter. Conditioned filtrates from freshly harvested endothelial cells attached to microcarrier beads caused marked relaxation of endothelium-deprived bovine pulmonary artery strips. The degree of relaxation depended on the number of cells; maximal relaxation occurred with 50 million cells at ED50 of 14 million. High values of cyclic GMP were found in vascular smooth muscle exposed to conditioned filtrate. The calcium ionophore A23187 further increased the amount of cyclic GMP. Large amounts of PGI2 were released by freshly harvested endothelial cells particularly after stimulation with the calcium ionophore. In contrast, endothelin production by freshly harvested cells attached to microcarrier beads was barely detectable after 30 min incubation and was beyond the limit of detection by bioassay procedures. Freshly harvested endothelial cells attached to microcarrier beads appear to be a useful adjunct to tissue cultures under specific experimental conditions.
过去，培养的内皮细胞一直被用作内皮源性舒张因子（EDRF）和前列环素（PGI2）的来源。尽管细胞培养对于观察长时间暴露于培养基的情况或出现延迟反应时至关重要，但它耗时且无菌条件必不可少。在本研究中，我们报告从牛主动脉新鲜收获的内皮细胞很容易附着在Cytodex - 3微载体珠上，并释放内皮源性舒张因子（EDRF）、前列环素（PGI2），并增加血管平滑肌中环鸟苷酸（cGMP）的含量。附着在微载体珠上至关重要，因为它增加了表面积和附着细胞的数量，并且由于细胞和珠子形成密集网络而允许收集无细胞滤液。结果，在滤器上对细胞和珠子进行灌流不会使结合的细胞脱落，这些细胞仍留在滤器上。来自附着在微载体珠上的新鲜收获的内皮细胞的条件滤液可引起去内皮牛肺动脉条明显舒张。舒张程度取决于细胞数量；在5000万个细胞时出现最大舒张，半数有效量（ED50）为1400万个细胞。在暴露于条件滤液的血管平滑肌中发现了高值的环鸟苷酸（cGMP）。钙离子载体A23187进一步增加了环鸟苷酸（cGMP）的量。新鲜收获的内皮细胞大量释放前列环素（PGI2），特别是在用钙离子载体刺激后。相比之下，附着在微载体珠上的新鲜收获细胞在孵育30分钟后几乎检测不到内皮素的产生，并且超出了生物测定程序的检测限。在特定实验条件下，附着在微载体珠上的新鲜收获的内皮细胞似乎是组织培养的有用辅助手段。
