摘要
目的:探讨猪正常尿路上皮细胞分离、培养、扩增技术;观察利用纤维蛋白胶作为组织工程支架的可行性。方法:实验于2005-04/07在广东省人民医院医学研究中心完成。①采用酶消化法及无血清培养系统体外原代培养膀胱移行上皮细胞。②将体外扩增的膀胱上皮细胞以相同密度分别接种于普通培养板(对照组)和纤维蛋白凝胶包被的培养板(实验组),于接种后4d比较两组细胞克隆形成情况。③再将膀胱移行上皮细胞接种于纤维蛋白凝胶包被的培养板,在不同融合度(70%,100%)时将细胞消化传代,以相同的密度再次接种于纤维蛋白包被的培养板,比较生长于纤维蛋白凝胶表面的、不同融合度的细胞传代后的克隆形成情况。④取对数生长期膀胱移行上皮细胞,以相同密度接种于96孔板,以含不同浓度抑肽酶(0,37.5,75,150,300kIU/mL)的keratinocyte-SFM培养,4d后以四甲基偶氮唑盐法观察比较移行上皮细胞生长情况(不含抑肽酶者为对照组)。结果:①移行上皮细胞生长良好,多次传代后仍扩增迅速。②膀胱移行上皮细胞在培养板和纤维蛋白凝胶表面均生长良好,4d后细胞克隆形成率在纤维蛋白凝胶表面和培普通培养板无明显差异[(8.67±1.45)%,(9.33±1.76)%,P>0.05]。③生长于纤维蛋白凝胶表面的细胞传代后仍有较强的增殖能力,且70%融合组传代细胞克隆形成率显著高于100%组[(18.2±1.08)%,(12.8±1.35)%,P<0.05]。④抑肽酶在300kIU/mL时对细胞生长有显著抑制作用;而在其他浓度时则不明显。结论:①该法培养猪膀胱上皮细胞迅速可靠,多次传代后仍有较强的增殖能力。②细胞在纤维蛋白凝胶表面生长良好,传代后仍然有较强的增殖能力。③纤维蛋白凝胶具有作为泌尿系腔道组织工程支架的潜在运用价值。
AIM： To investigate the protocol for isolating, culturing, and proliferating bladder epithelial cells in vitro; then study the feasibility of fibrin glue for the scaffold of tissue engineering. METHODS： The experiment was conducted at the Medical Research Center of Guangdong Provincial People＇s Hospital from April 2005 to July 2005. ①ln vitro primary culture of bladder transitional epithelial cells with enzyme digestion method and serum-free culture system. ②Bladder epithelial cells proliferated in vitro were inoculated to normal culture plate （Control group） and fibrin glue-coated culture plate （Experimental group） at the same density. Cellular clone was compared between control group and experimental group 4 days after inoculation. ③Bladder transitional epithelial cells were inoculated to fibrin glue-coated culture plate . Cells were digested and cultured at different fusion degrees （70%, 100%）, then were re-inoculated to fibrin glue-coated culture plate at the same density . Clony-fortning efficiency （CFE） of the cells was compared between 70% and 100% fusion degrees after passage. ④Bladder transitional epithelial cells at logarithmic phase were chosen and inoculated to 96-well plate at the same density, then cultured with keratinocyte-SFM containing 0,37.5, 75,150,300 klU/mL aprotinin. Growth of transitional epithelial cells was observed with methylthiazolyl tetrazolium assay 4 days later （Control group without aprotinin）.
ESULTS：①Bladder transitional epithelial cells grew well in vitro, cells could be passaged 9 times without a noticeable decrease in rate of c.ell proliferation. ②Four days after the cells were inoculated on the fibrin glue coated and normal culture plates, CFE was （8.67±1.45）% and （9.33±1.76）% in the experimental group and control group , respectively, without significant difference （P 〉 0.05）. ③Cells released from fibrin glue-coated culture plate retained the power of proliferation, and the cells cultured on fibrin glue from subconfluent culture had a higher CFE than those from confluent ones, and CFE was（ 18.2±1.08）% and（ 12.8±1.35 ）% respectively （P 〈 0.05）. ④Aprotinin could notably restrain the proliferation of bladder epithelial cells at the concentration of 300 klU/mL, while it was not notable at other concentrations. CONCLUSION： It is reliable to culture and expand bladder epithelial cells with this protocol; the cells retain the power of proliferation even after several passages. Cells proliferate quickly on both normal and fibrin glue- coated culture plates; Cells released from the fibrin glue-coated plates also retain the power of proliferation. Therefore, the fibrin glue has the potential of being used as the scaffold of urology tissue engineering.
出处
《中国临床康复》
CSCD
北大核心
2006年第17期61-64,i0004,共5页
Chinese Journal of Clinical Rehabilitation
基金
广东省科技计划项目(2003C30301)~~
