不同浓度的胎牛血清对骨髓间充质干细胞纯度及周期的影响  

Effect of different concentrations of fetal bovine serum on bone marrow mesenchymal stem cell purity and cycle

作　　者：陈小丹[1] 何家才[1] 

机构地区：[1]安徽医科大学口腔医学院、安徽医科大学附属口腔医院、安徽省口腔疾病研究中心实验室,合肥 230032

出　　处：《安徽医科大学学报》2014年第6期742-747,共6页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:81371114);安徽省自然科学基金(编号:11040606M173);安徽医科大学博士科研资助基金(编号:XJ201034);安徽高校省级自然科学研究重点项目(编号:KJ2013A154)

摘　　要：目的比较含有体积分数为0.10、0.15、0.20的胎牛血清(FBS)的完全培养基对体外培养的大鼠骨髓间充质干细胞(BMSCs)纯度及细胞周期的影响,寻求适宜干细胞培养的FBS浓度。方法用颈椎脱臼法处死SD大鼠,采用全骨髓贴壁法分离培养大鼠BMSCs。实验分为A、B、C 3组,分别为FBS体积分数是0.10、0.15、0.20的完全培养基培养的BMSCs,比较各组细胞在第2、3、4、5代(P2,P3,P4,P5)的CD45、CD29、CD90、CD44的表达情况;消化A组P3细胞,用3种不同浓度的完全培养基连续培养4 d,用流式细胞仪测其在培养后的24、48、72、96 h的细胞周期;收集P3 BMSCs制备细胞悬液接种到6块96孔板,每块板接种的BMSCs分别用FBS体积分数是0.10、0.15、0.20的完全培养基进行培养,每天取1块培养板用CCK-8检测其吸光度值(OD)。结果 CD45阴性表达,CD29、CD90、CD44阳性表达,3种完全培养基培养的BMSCs的表面标志物在前2代时差别较大,但在P3,P4时差别已较少,P4时均可以获得较纯的BMSCs;细胞周期结果显示,在同一时间点,G0/G1期:A、B、C 3组随着FBS浓度的增加,G0/G1期降低且差异无统计学意义;G2/M期:在培养后的24 h,A、B、C 3组差异有统计学意义(P<0.05,F=12.412),但随着时间的延长,差异消失;S期,3组均无差别;S+G2/M期随FBS浓度的增加而增高。细胞活力结果显示,在24 h时A、B、C 3组的OD值依次增大,差异有统计学意义(P<0.05,F=5.002),随时间的延长3组之间无明显差异。结论这3种培养基在P4能获得较纯的BMSCs;细胞周期及活力结果显示,3种完全培养基均能促进BMSCs的增长,3组之间不存在差异。体积分数为0.10的FBS已满足BMSCs的分离和扩增,要想在短期内获得较纯的BMSCs,原代培养使用体积分数为0.15,传代用0.10 FBS的完全培养基。Objective To compare the influence of three kinds of complete media with 0. 10 ,0. 15 ,0. 20 fetal bo-vine serum（ FBS） on purity and cycle of rat bone marrow mesenchymal stem cells （ BMSCs） cultured in vitro and to seek suitable FBS concentration for the cultivation of the stem cells. Methods SD rats were executed by cervical dislocation method and used whole bone marrow adherence to isolate rat BMSCs. Experiment was divided into A, B,C,3 groups. Compared the expression of CD45, CD29, CD90, CD44 in the three groups in 2,3,4,5 passages （ P2 , P3 , P4 , P5 ）;the cells of P3 in group A were digested and cultured in three different concentration of com-plete culture media for four days, measuring cell cycle in 24,48,72,96 h by flow cytometry instrument. BMSCs of P3 were collected and inoculated to 6 pieces of 96-well plates, then vaccinated with complete media with 0. 10, 0. 15,0. 20 FBS in every plate, one culture plate was taken out for optical density（OD） meaturement every day with CCK-8. Results CD45 was negative, CD29, CD90, CD44 were positive. The difference of BMSCs surface markers cultured in the three kinds of complete media was bigger in the first two passages, but the difference was less in P3 , P4 , all could obtain pure BMSCs in P4 relatively;according to the results of the cell cycle at the same time, G0/G1 phase：with the increase of concentration of fetal bovine, G0/G1 phase reduced and had no diference in A, B, C groups;G2/M phase：there was difference between them after 24 h（P〈0. 05,F=12. 412）, but with the extension of time, the differences disappeared;S phase：there was no difference in the three groups;S＋G2/M phase increased with the concentration of FBS. According to the result of cell vitality, the OD of ABC three groups increased in turn in 24 h, and there was difference（P〈0. 05,F=5. 002）, but with the extension of time, there was no obvious difference between them. Conclusion The three kinds of culture media in P4 can obtain pure BM-SCs, cell cycle and vitalit

关 键 词：骨髓间充质干细胞 胎牛血清 细胞周期 表面标志 

分 类 号：R33[医药卫生—人体生理学]