摘要
目的进行大鼠肾小球内皮细胞体外培养和鉴定,并观察高糖对其分泌一氧化氮(nitricoxide,NO)的影响。方法利用不同目数的筛网从大鼠肾皮质分离肾小球,Ⅳ型胶原酶消化后用内皮细胞培养液培养。待细胞自肾小球内长出,以套环或挑克隆的方法纯化,在倒置显微镜下观察细胞形态,分别用Ⅷ因子相关抗原抗体和CD31荧光抗体对纯化后的细胞进行鉴定。不同浓度的高糖作用于培养的肾小球内皮细胞,Griess法检测各时相点培养液中NO的含量。结果倒置显微镜下观察纯化后的细胞为单层排列,似鹅卵石状,Ⅷ因子相关抗原抗体免疫组化染色和CD31荧光抗体染色阳性。高糖可以引起肾小球内皮细胞NO分泌减少,并呈浓度和时间依赖性。结论利用过筛法分离肾小球并在特定条件下进行培养、纯化,成功培养大鼠肾小球内皮细胞。高糖可以导致肾小球内皮细胞NO释放减少,这可能与糖尿病肾病的发生有关。
Objective To culture and identify endothelial cells of rat glomeruli in vitro and investigate the effects of high glucose concentrafion on its nitric oxide secretion. Methods Through a series of sieving, the rat glomeruli were isolated from cortex, treated with collagenase Ⅳ, and cultured in endothelial cell medium. Outgrowths from individual glomeruli were purified by ring cloning or clone picking. Then, the purified cells were observed by inverted microscopy and identified by using anti-factor Ⅷ-related antigen antibody and CD31 fluorescent antibody. The cultured glomerular endothelial cells were incubated with different concentrations of high glucose. Whereafter, the content of nitric oxide was detected by Griess method. Results The purified cells were arranged in monolayers, like cobble-stone, and positively stained with anti-factor Ⅷ-related antigen antibody and CD31 fluorescent antibody. After being incubated with high concentration of glucose, the release of nitric oxide from glomerular endothelial cells decreased in concentration- and time-dependent manner. Conclusion Rat glomerular endothelial cells can be obtained by graded sieving, conditional culturing, and purification; high concentration of glucose can reduce the secretion of nitric oxide from glomemlar endothelial cells, which may be related to the pathogenesis of diabetic nephropathy.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2007年第1期13-15,共3页
Immunological Journal
关键词
肾小球内皮细胞
高糖
一氧化氮
Glomerular endothelial cells
High glucose concentration
Nitric oxide
