摘要
目的 建立纯度高、操作简便的大鼠近端肾小管上皮细胞分离纯化方法.方法 Wistar大鼠无菌取肾前先心脏抽血替代原位肾脏灌注,分离肾皮质,经Ⅰ型胶原酶消化后用45%Percoll分离液制备细胞悬液直接连续密度梯度离心.所得细胞用含10%胎牛血清的DMEM-F12培养基原代培养并传代,根据细胞形态、细胞角蛋白18（CK18）和水通道蛋白1（AQP1）免疫细胞化学染色及碱性磷酸酶化学染色进行鉴定.结果 此简化方法获得的肾小管细胞中肾小球掺杂明显减少,培养4～5 d后细胞融合长满,呈典型上皮样细胞的鹅卵石状,其CK18、AQP1免疫细胞化学染色及碱性磷酸酶化学染色几乎均呈阳性,证实所培养细胞为近端肾小管上皮细胞.结论 改良后的分离纯化方法可获得大量高纯度近端肾小管上皮细胞,操作简便.
Objective To establish the renal proximal tubular cells （PTCs） a pure and convenient method for the enrichment of Methods The both kidneys of Wistar rats were underlying hemospasia via cardiac apex substituted in situ kidney perfusion before taken out. The renal cortex was separated and sliced into small pieces which were digested in type Ⅰ collogen and resuspended in 45% Percoll gradient, then centrifuged by continuous density gradient. Cells were cultured and passaged in DMEM-F12 supplemented with 10% fetal bovine serum. Bred cells were identified by morphology, immunocytochemistry staining of cytokeratin-18 （CK18） and aquaporin 1 （AQP1）, and alkaline phosphatase staining. Results The tubular fragments obtained from the modified method had very little miscellany of renal glomerulus. After 4-5 days, the cultured cells reached confluence, displaying the typical cobblestone appearance. All the bred cells almost were positive expression in immunocytochemistry staining of CK18 and AQP1, and alkaline phosphatase staining. So they were validated as rat PTCs. Conclusion This modified method can yield large quantity of pure PTCs simply.
出处
《中华肾脏病杂志》
CAS
CSCD
北大核心
2010年第4期300-303,共4页
Chinese Journal of Nephrology
基金
教育部高校优秀青年教师奖励计划（201182）
吉林省卫生厅重点实验室基金
