CAJ | 학술논문

背景：骨髓间充质干细胞被认为是构建组织工程骨修复骨与软骨缺损中较为常用的种子细胞，在其基本操作过程中注意常见问题并及时避免，对后期细胞学及组织工程学实验很有意义。目的：通过作者实验操作过程中所遇问题的总结和分析，为初学者和科研人员提供可靠的骨髓间充质干细胞分离培养鉴定方法，减少操作过程中的人为失误和易犯问题。方法：取16只幼年新西兰大白兔作为实验对象，进行髂骨穿刺抽取骨髓液。采用密度梯度离心法联合贴壁培养法体外筛选纯化细胞，并且通过倒置相差显微镜观察其形态学特点、生长曲线和流式细胞术鉴定骨髓间充质干细胞表型。结果与结论：实验过程中前5只兔骨髓抽吸、骨髓间充质干细胞分离过程中遇到不同的问题和困难，经认真总结和分析，后11只兔骨髓抽吸、骨髓间充质干细胞分离均获成功，在细胞培养过程中未发现细菌污染和细胞老化，第3代骨髓间充质干细胞高表达CD29、CD44抗原，而CD14、CD34抗原低表达，MTT测细胞生长曲线显示P3和P5增殖活性较高。尽管骨髓间充质干细胞分离培养鉴定技术已较为成熟，但是如果操作过程中不注意细节问题，也将会导致实验困难重重或失败。严格执行常规操作步骤可以得到纯度较高的骨髓间充质干细胞，提高成功率，为后续相关细胞实验和动物实验做好准备。
배경：골수간충질간세포피인위시구건조직공정골수복골여연골결손중교위상용적충자세포，재기기본조작과정중주의상견문제병급시피면，대후기세포학급조직공정학실험흔유의의。목적：통과작자실험조작과정중소우문제적총결화분석，위초학자화과연인원제공가고적골수간충질간세포분리배양감정방법，감소조작과정중적인위실오화역범문제。방법：취16지유년신서란대백토작위실험대상，진행가골천자추취골수액。채용밀도제도리심법연합첩벽배양법체외사선순화세포，병차통과도치상차현미경관찰기형태학특점、생장곡선화류식세포술감정골수간충질간세포표형。결과여결론：실험과정중전5지토골수추흡、골수간충질간세포분리과정중우도불동적문제화곤난，경인진총결화분석，후11지토골수추흡、골수간충질간세포분리균획성공，재세포배양과정중미발현세균오염화세포노화，제3대골수간충질간세포고표체CD29、CD44항원，이CD14、CD34항원저표체，MTT측세포생장곡선현시P3화P5증식활성교고。진관골수간충질간세포분리배양감정기술이교위성숙，단시여과조작과정중불주의세절문제，야장회도치실험곤난중중혹실패。엄격집행상규조작보취가이득도순도교고적골수간충질간세포，제고성공솔，위후속상관세포실험화동물실험주호준비。
BACKGROUND:Bone marrow mesenchymal stem cells are considered as commonly used seed cells to construct tissue-engineered for repair of bone and cartilage defects. It is of great significance for cytology and tissue engineering experiments to study the common problems existing in the basic operation and how to avoid these problems in a timely manner. OBJECTIVE:To summarize the common problems existing in the process of operation in order to provide reliable methods about separation, culture and identification of bone marrow mesenchymal stem cells for beginners and researchers. These can reduce or avoid some errors and problems during operation. METHODS:Sixteen New Zealand white rabbits were selected as experiment objects, and bone marrow mesenchymal stem cells were separated from rabbits by iliac puncture, purified and augmented by using density gradient centrifugation combined with adherent culture method. Then cellmorphology was observed by inverted phase contrast microscope, growth curve detected by MTT method and cellphenotype identified by flow cytometry. RESULTS AND CONCLUSION:We encountered some problems in the process of separation and culture, when we operated the first five rabbits. After careful y summarizing and analysis of the reasons, the operation was successful y completed on the rest 11 rabbits. Bacteria pol ution and cellaging were not found in the process of cellculture. What is more, the cells at passage 3 appeared with high-expression of CD29, and CD44, but low expression of CD14 and CD34. The cellgrowth curve showed that the proliferation activity of cells at passages 3 and 5 was higher than that at passage 10. Although the technology of separation, culture and identification of bone marrow mesenchymal stem cells is mature, the failure wil be happen if we do not pay attention to the details of operation. By strictly carrying out normal operations, we can get high purity of bone marrow mesenchymal stem cells, which lays a good foundation for celland animal experiments in the future.