Cell physiological approach for evaulating effects of lymphatic endothelial cells on lymph formation and transport

分离的犬胸导管远端用聚乙烯导管插管，然后用含胶原酶250U/ml和0.5 mM EGTA的Rinaldi液灌流，孵育10min后，用含20%胎牛血清的最低基本培养液冲洗胸导管，然后用灌流液离心，获得剥离的内皮细胞。将颗粒重新悬浮在添加胎牛血清的MEM中，放置在涂有I型胶原的组织培养皿中。犬淋巴管内皮细胞的生长倍增时间约为58小时。多角形细胞汇合处呈典型的鹅卵石状形态。大多数培养的细胞表达因子VIII相关抗原，并在荧光显微镜下显示通过“清道夫细胞途径”摄取乙酰化的低密度脂蛋白。用Fura-2 AM负载培养的淋巴管内皮细胞，测定细胞内钙瞬变。给予10^&lt；-5&gt；M组胺可使培养细胞内钙离子浓度显著升高。给药1分钟后，细胞内[Ca~(2+)]i升高10倍。给药后4~5min，[Ca~(2+)]i逐渐降低，并维持在恒定水平。5-HT1受体存在于猴窝淋巴结内。这些受体的刺激通过激活细胞内的鸟苷环化酶，在淋巴结平滑肌上产生内源性的一氧化氮依赖的松弛。未受刺激的大鼠腹膜巨噬细胞引起一氧化氮和扩张血管的前列腺素的共同释放，导致动脉平滑肌显着松弛

The distal end of isolated dog thoracic duct was cannulated by a polyethylene catheter and then perfused with Rinaldi solution containing 250 U/ml collagenase and 0.5 mM EGTA.After a 10-min incubation with the solution, the thoracic duct was flushed with minimum essential medium containing 20% fetal bovine serum and then perfusate was centrifuged to obtain the denuded endothelial cells. The pellets were resuspended in MEM supplemented with fetal bovine serum and placed onto tissue culture dishes coated with type I collagen. The dog lymphatic endothelial cells grew with doubling time of about 58 hrs. The ploygonal cells had a classic "cobblestone " morphology at confluence. Most culyured cells expressed Factor VIII-related antigen and were shown to take up acetylated LDL via the "scavenger cell pathway " by use of fluorescence microscopy. The cultured lymphatic endothelial cells were loaded with fura-2 AM to measure Ca^<2+>-transient. An administration of 10^<-5>M histamine caused a significant increase of intracellular concentrations of Ca^<2+> in the cultured cells. A tenfold increase of [Ca^<2+>]i was observed 1 min after the administration. The [Ca^<2+>]i was gradually decreased and then kept at constant level by 4-5 min after the administration.5-HT_1-like receptors exist in the monkey popliteal lymph nodes. Stimulation of these receptors produces an endogenous nitric oxide-dependent relaxation in lymph node smooth muscle through an activation of cytosolic guanylate cyclase in the cells. Unstimulated rat peritoneal macrophages cause a corelease of nitric oxide and vasodilative prostaglandins resulting in a marked relaxation of arterial smooth muscles