【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。更多还原

【Abstract】 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. ①In vitro primary culture of bladder transitional epithelial cells with enzyme digestion method and serum-free culture system.② Bladder 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. ③ Bladder 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-forming efficiency (CFE) of the cells was compared between 70% and 100% fusion degrees after passage. ④ Bladder 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 kIU/mL aprotinin. Growth of transitional epithelial cells was observed with methylthiazolyl tetrazolium assay 4 days later (Control group without aprotinin). RESULTS: ①Bladder transitional epithelial cells grew well in vitro, cells could be passaged 9 times without a noticeable decrease in rate of cell 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 kIU/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.更多还原