视网膜色素上皮细胞脱离体外培养条件后细胞保存液的研究  

STUDY ON THE PROTECTIVE SOLUTIONS OF hRPE CELLS AFTER BREAKING AWAY CULTURE CONDITION IN VITRO

作　　者：孟书聪[1] 张君[1] 张小燕[1] 陆爱丽[1] 

机构地区：[1]北京大学医学部基础医学院细胞生物学系,干细胞研究中心,北京100083

出　　处：《解剖学报》2007年第6期768-770,共3页Acta Anatomica Sinica

基　　金：国家高技术研究发展计划重大专项基金资助项目(2003AA205007);教育部教育振兴行动计划专项基金资助"985工程"

摘　　要：目的研究人视网膜色素上皮细胞(hRPE细胞)离体或脱离体外培养条件后,维持细胞活性、且延长细胞存活时间最适宜的液体。方法将5代以上hRPE细胞(本实验室保存)按常规冷冻、复苏培养于DMEM/F12培养基添加10%胎牛血清,EGF20μg/L和bFGF20μg/L条件下,至80%以上汇合,经胰蛋白酶消化,收集细胞、计数,置于6种不同保存液中(平衡盐液、Quinn’sadvantage medium、生理盐水、18氨基酸液、5%葡萄糖液和人工脑脊液),用Annexin V/FITC Kit染色,利用流式细胞术(FACS)检测细胞死亡或凋亡。采用2h至8h之间3个时间点,检测hRPE细胞在6种不同细胞保存液中的细胞状态。结果室温状态下,hRPE细胞30min内均发生坏死。4℃状态下,2h平衡盐液组凋亡率最低(0.9%);各组分别与平衡盐液组相比,显示人工脑脊液组与平衡盐液组之间存在显著差异;4h至8h,hRPE细胞凋亡率从26.74%上升至41.36%。各组总死亡率与凋亡率基本一致。结论4℃状态下,6种溶液中,平衡盐液是维持hRPE细胞脱离培养条件后保持细胞活性,且延长细胞存活时间最适宜的溶液。Objective To investigate the best protective solution that could maintain the cell activity and prolong the survival time of the human retinal pigment epithelium （hRPE） cells after break away in vitro culture case. Methods hRPE cells （stored in our laboratory） culture in DMEM/F12, added 10% FBS, EGF 20μg/L and bFGF 20μg/L, which had been cultured for more than five generations. When these culture hRPE cells had been confluenced more than 80%, the hRPE cells were harvested and placed into six kinds of protective solutions （balanced salt solution, Quinn＇s advantage medium, physiological salt solution, 18-amino acid solution, 5% glucose solution, artificial cerebrospinal fluid）, then the cells were stained with the Annexin V / FITC, and the numbers of cell death and cell apoptosis were detected with the fluorescence activated cell sorter （FACS）. The hRPE cells were in six kinds of protective solutions and were investigated at three different times from 2 hours to 8 hours. Results The result presented as follows： At the room temperature, the hRPE cells in all six kinds of protective solutions occurred necrosis within 30 minutes. To investigate the state at 4℃, in a duration of 2 hours, the apoptosis rate in the group of balanced sahsolution was lowest （0.9%）; As compared with the group of balanced salt solution, the apoptosis rate of artificial cerebrospinal presented a great difference （P 〈 0.05）. The apoptosis rate in the group of artificial cerebrospinal fluid increased from 26.74% to 41.36% in a duration of 4 to 8 hours. The death rates in all six solutions were approximately the same with the apoptosis rate. Conclusion The preservative liquid of balanced salt solution at 4 ℃, as compared with the other 5 kinds of protective solutions, was the most suitable condition for the hRPE cells to maintain the cell activity and prolong the survival time after break away from culture case.

关 键 词：视网膜色素上皮细胞 细胞凋亡 细胞保护液 流式细胞术 

分 类 号：Q2-33[生物学—细胞生物学]