原代肾上皮细胞改良培养方法  

An improved method for primary culture of renal epithelial cells

作　　者：周峰[1] 杜贤进[1] 董长垣[2] 张杰[1,2] 

机构地区：[1]武汉大学人民医院泌尿外科,湖北省武汉市430060 [2]武汉大学病毒学国家重点实验室,湖北省武汉市430071

出　　处：《中国组织工程研究与临床康复》2009年第11期2101-2104,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

摘　　要：背景：目前国内对上皮细胞的研究多采用细胞株的方式,但其离体时间长,细胞易产生变异,而原代培养则与体内状态更相近,希望能为组织工程肾脏相关研究提供肾上皮细胞来源。目的：探索原代肾上皮细胞培养的条件和方法。建立一套高效快捷的原代肾上皮细胞培养方案。设计、时间及地点：单一样本观察,开放性实验,于2007-12/2008-03在武汉大学病毒学国家重点实验室分子病毒与癌研究室完成。材料：新生2~4dSPF级雄性SD大鼠鼠婴12只。方法：取SD大鼠,切取双侧肾脏组织,将其反复剪成约1mm×1mm×1mm块,加入2.5g/L的胰酶和0.4g/L乙二胺四乙酸,放入37℃恒温水浴箱消化,30min后移至200目钢网过滤,去除未消化组织块和细胞团,收集网下悬液1000r/min低温离心5min,用DMEM（pH7.2）培养基制成细胞悬液,以4×108L-1密度接种于培养瓶中,置CO2培养箱中培养。再将细胞悬液接种到一个培养瓶内,静止培养30min,镜下观察细胞部分贴壁,将培养液连同尚未贴壁细胞一起吸到另一瓶内,继续培养,重复上述操作一次,纯化肾上皮细胞。主要观察指标：以4×108L-1密度接种于培养瓶中培养。用4g/L锥虫蓝染色检测肾上皮细胞的存活率,不着色者为存活细胞。并观察肾上皮细胞的形态学变化、贴壁率。结果：肾上皮细胞存活率为95.20%,4h约60%的细胞贴壁,12h85%以上的细胞已经贴壁。24h后肾上皮细胞伸出伪足呈梭形、多角形,具有胞核一两个,细胞大小均匀,生长迅速,三四天后形成细胞簇,最后肾上皮细胞连接成片生长。结论：该肾上皮细胞培养方法存活率、贴壁率高,是一种可靠的肾上皮细胞培养方法。BACKGROUND： Cell strain of epithelial cells is focus of domestic studies. However, long period of ex vivo may contribute to cell variation. Primary culture is similar to in vivo status, and is hopeful to provide sources for renal epithelial cell in renal tissue engineering. OBJECTIVE： To explore the condition and approach for the culture of primary renal epithelial cell, so as to establish an effective method for renal epithelial cell culture. DESIGN, TIME AND SETTING： Single sample observation was performed at Laboratory of Molecular Virology and Cancer, State Key Laboratory of Virology, Wuhan University between December 2007 and March 2008. MATERIALS： Twelve neonatal SPF male SD rats at 2 4 days old were used. METHODS： Bilateral kidney tissues were harvested from SD rats, cut into pieces of 1 minx1 minx1 ram, digested with 2.5 g/L pancreatic enzyme and 0.4 g/L EDTA at 37 ℃ for 30 minutes. The cells were filtered by 200-mesh steel net to remove non-digested tissues and cell groups. The cell suspension was centrifuged at 1 000 r/rain for 5 minutes, and collected cells were cultured in DMEM （pH：7.2） to prepare cell suspension, seeded in culture flask at the density of 4 × 10^8/L, and incubated in CO2 at 37 %. In addition, cell suspension was seeded in a culture flask and cultured for 30 minutes. Cell attachment was observed. Cell culture solution and unattached cells were placed to another flask for continuous culture. This process was repeated to purify renal epithelial cells. MAIN OUTCOME MEASURES： The survival rate of renal epithelial cell was detected by 4 g/L trypan-blue staining. Those without staining were survival cells. The survival rate, morphological changes of renal epithelial cell and the rate of monolayer adherence were observed. RESULTS： The survival rate of the renal epithelial cell was 95.20%, and the adherence rate was about 60% after 4 hours and 85% alter 12 hours. After 24 hours, cells had pseudopodia, and exhibited fusiform or polygon-shaped with one or two nucle

关 键 词：肾上皮细胞 原代培养 组织构建 

分 类 号：R329.33[医药卫生—人体解剖和组织胚胎学]