兔膀胱移行上皮细胞的体外培养

Culture of rabbit bladder transitional epithelial cells in vitro

目的:对兔膀胱移行上皮细胞进行原代及传代培养,探索用于泌尿系统组织工程的膀胱移行上皮种子细胞的最佳培养方法。方法:实验于2005-09/2006-01在吉林大学药学院生物工程实验室完成。①取1月龄新西兰大耳白兔,耳缘静脉行空气栓塞处死,超净台内无菌取膀胱。②采用中性蛋白酶分离膀胱各层组织。③胰蛋白酶消化获得膀胱移行上皮细胞。④将获取的膀胱移行上皮细胞分别接种到含体积分数为0.01,0.05,0.1血清的DMEM-F12培养液进行培养。④每日在倒置相差显微镜下观察细胞形态及生长状况;分别在第1,3,5,7,9天以四甲基偶氮唑盐法作细胞生长曲线。结果:①原代移行上皮细胞于接种后34～48h贴壁,培养六七天时可达80%融合,呈典型铺路石状。②传代后的细胞生长稳定快速,24～36h贴壁,五六天可达80%融合。③应用四甲基偶氮唑盐法检测不同体积分数血清对移行上皮细胞增殖的影响,显示膀胱移行上皮细胞在含有体积分数为0.05血清的DMEM-F12培养中生长状态最佳。结论:采用中性蛋白酶与胰蛋白酶联合消化法,对兔膀胱移行上皮细胞进行原代及传代培养,是一种稳定、快速的膀胱移行上皮细胞的培养方法。

AIM： The bladder transitional epithelial cells are subjected to primary culture, passage and expansion. This paper is aimed to investigate the best culture method of acquiring bladder transitional epithelial cells, which will be used for urinary system tissue engineering. METHODS： The experiment was completed at the Department of Bioengineering in Jilin University from September 2005 to January 2006. （1）New Zealand male rabbit of one-month old was sacrificed by aeroembolism in entotic vein and fook the bladder without germ under the super-clean bench. （2）Bladder mucosa was separated by dispase. （3）Bladder mucosa was digested into single bladder epithelial cells by trypsin. （4）The bladder transitional epithelial cells were primarily cultured in DMEM-F12 media which contained 0.01, 0.05, 0.1 serum respectively. RESULTS： （1）Primary bladder transitional epithelial cells adhered with culture board after 34-48 hours, monolayers attained confluence about 80% with a typical cobble like appearance after 6-7 days. （2）Subcultured cells adhered with culture board after 24-36 hours, achieved confluence about 80% within 5-6 days. （3）MTT method was adopted to examine the effect of serum on bladder transitional epithelial cells and the result showed that the optimized culture media was DMEM-F12 containing 0.05 serum. CONCLUSION： Bladder transitional epithelial cells can be acquired by primary culture and passage in rabbits that are digested with the combination of dispase and trypsin, and the effect is stable and rapid.