CAJ | 학술논문

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。
양,매천취1괴배양판용CCK-8검측기흡광도치( OD)。결과 CD45음성표체,CD29、CD90、CD44양성표체,3충완전배양기배양적BMSCs적표면표지물재전2대시차별교대,단재P3,P4시차별이교소,P4시균가이획득교순적BMSCs；세포주기결과현시,재동일시간점,G0/G1기：A、B、C 3조수착FBS농도적증가,G0/G1기강저차차이무통계학의의；G2/M기：재배양후적24 h,A、B、C 3조차이유통계학의의( P<0．05,F=12．412),단수착시간적연장,차이소실；S기,3조균무차별；S+G2/M기수FBS농도적증가이증고。세포활력결과현시,재24 h시A、B、C 3조적OD치의차증대,차이유통계학의의(P<0．05,F=5．002),수시간적연장3조지간무명현차이。결론저3충배양기재 P4능획득교순적BMSCs；세포주기급활력결과현시,3충완전배양기균능촉진BMSCs적증장,3조지간불존재차이。체적분수위0．10적FBS이만족BMSCs적분리화확증,요상재단기내획득교순적BMSCs,원대배양사용체적분수위0．15,전대용0．10 FBS적완전배양기。
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 vitality show three complete media can promote the growth of BMSCs. There is no difference between them. Culture media with 0. 10 FBS can satisfy the isolation and amplification of BMSCs, in order to ob-tain pure BMSCs in the short term, using culture media with 0. 15 and 0. 10 FBS in the primary culture and subcul-ture respectively.