MUSCLE RELAXANT DERIVED FROM HUMAN GINGIVAL MICROVASCULAR ENDOTHELIAL CELLS

本研究的目的是检测人牙龈微血管内皮细胞（HGECs）释放的平滑肌松弛剂（一氧化氮，NO）。机械刺激对HGECs释放NO的影响也与人脐静脉和动脉内皮细胞进行了比较。胶原酶处理后，压缩人牙龈样品以释放内皮细胞。将接种的细胞重悬于生长培养基中。将细胞悬浮液引入96孔板的每个孔中。收获具有鹅卵石外观的形态学均质的细胞。分离的细胞通过存在von Willebrand因子、摄取乙酰化LDL以及在Matrigel上形成毛细血管样结构来鉴定为HGECs。用荧光法测定NO，样品与DAN试剂反应生成1-（H）-萘并噻唑。在20 ℃下孵育10分钟后，用NaOH终止反应。使用荧光板读数器在360 nm激发和460 nm发射读数下测量终产物2，3-二氨基-萘并三唑。在基线时，没有测试的细胞释放NO。在Flexercell的机械刺激3天和5天后，在脐静脉和动脉内皮细胞培养液中检测到NO。然而，HGECs在整个实验期间不释放NO。可培养的HGECs为第4代，目前认为其不足以检测NO，进一步的研究将集中在更有效的HGECs培养方法上。

The purpose of this investigation was to detect the smooth muscle relaxant (Nitric oxide, No) released from human gingival microvascular endothelial cells (HGECs). The effect of mechanical stimulation on NO-release from HGECs was also compared with that from human umbilical vein and artery endothelial cells. Afer collagenase treatment, human gingival samples were compressed to release endothelial cells. Centrifuged cells were resuspended in the growth medium. The cells suspension was introduced into every well of a 96-well plate. Morphologically homogeneous cells with a cobblestone appearance were harvested. The isolated cells were identified as HGECs by the presence of von Willebrand factor, the uptake of acetylated LDL,and the formation of capillary-like structure on Matrigel. Fluorometric assay was used to detect the NO.The sample was reacted with DAN reagent to form 1- (H) -naphthotiazol. After 10-min incubation at 20 ﾟC,the reaction was terminated with NaOH.The end product, 2,3-diamino-naphthotriazol, was measured using a fluorescent plate reader with excitation at 360 nm and emission read at 460nm. At baseline, none of the cells tested released the NO.The NO was detected in the culture medium of the umbilical vein and artery endothelial cells after 3-day-and 5-day-mechanical stimulation by Flexercell. However, the HGECs did not release the NO during the whole experimental period. The cultivable HGECs were passage 4, which were thought not to be enough for detecting the NO.The further study will focus on more efficient method for cultivating the HGECs.