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

取离体犬胸导管远端用聚乙烯导管插管，灌注含有250 U/ml胶原酶和0.5 mM EGTA的Rinaldi溶液。用该溶液孵育10分钟后，用含有20%胎牛血清的最低必要培养基冲洗胸导管，然后离心灌注，获得剥离的内皮细胞。将微球重悬于添加胎牛血清的MEM中，并置于包被I型胶原蛋白的组织培养皿中。淋巴内皮细胞生长时间约为58小时，呈倍增期。多角形细胞在汇合处呈典型的“鹅卵石”形态。大多数培养细胞表达因子viii相关抗原，并通过荧光显微镜显示通过“清道夫细胞途径”吸收乙酰化LDL。将培养的淋巴内皮细胞加载fura- 2am，测量Ca^<2+>-瞬态。10^<-5>M组胺可显著增加培养细胞内Ca^<2+>浓度。在给药1 min后，[Ca^<2+>]i增加了10倍。给药后[Ca^<2+>]i逐渐降低，4 ~ 5 min后维持在恒定水平。猴腘窝淋巴结中存在5- ht_1样受体。刺激这些受体通过激活细胞内的胞质鸟苷酸环化酶在淋巴结平滑肌中产生内源性一氧化氮依赖性松弛。未受刺激的大鼠腹腔巨噬细胞引起一氧化氮和血管扩张性前列腺素的共同释放，导致动脉平滑肌明显松弛

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