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

用聚乙烯导管将犬离体胸导管远端插管，用含250 U/ml胶原酶和0.5 mM EGTA的Rinaldi液灌注，孵育10 min后，用含20%胎牛血清的最低必需培养基冲洗胸导管，离心，获得裸内皮细胞。将沉淀重悬于补充有胎牛血清的MEM中，并置于涂有I型胶原的组织培养皿上。犬淋巴管内皮细胞生长倍增时间约为58小时。融合时，多聚体细胞具有典型的“鹅卵石“形态。大多数培养的细胞表达因子VIII相关抗原，并通过使用荧光显微镜显示通过“清道夫细胞途径“摄取乙酰化LDL。用Fura-2 AM负载培养的淋巴管内皮细胞以测量Ca^<2+>-瞬时。给予10 μ <-5>M组胺可导致培养细胞内Ca^2+浓度显著增加。给药后1分钟观察到[Ca^&lt;2+&gt;]i增加10倍。[Ca^&lt;2+&gt;]i在给药后4-5 min逐渐下降并维持在一恒定水平。猴腘淋巴结内存在5-HT 1样受体。这些受体的刺激通过细胞内胞质鸟苷酸环化酶的激活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胰高血糖素的共同释放，导致动脉平滑肌显著松弛

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