CAJ | 학술논문

背景:骨髓中的间充质干细胞含量不高,且随着年龄增加或体质衰弱,骨髓间充质干细胞的数量会逐渐减少.目的:验证贴壁法分离培养大鼠骨髓间充质干细胞的效果.方法:大鼠麻醉后取双侧股骨和胫骨,剪去骨骺端,暴露骨髓腔,用含小牛血清的DMEM培养基冲洗骨髓腔,收集骨髓细胞,反复吹打制成单细胞悬液,接种后置于37 ℃、体积分数为5%的CO_2培养箱内孵育,24 h后全量换液,以后每周全量换液1次,筛选易贴壁但贴壁不牢的细胞进行传代培养.观察细胞形态,绘制细胞生长曲线,流式细胞仪及免疫细胞化学染色鉴定骨髓间充质干细胞表面标志的表达.结果与结论:培养24 h后细胞能够贴壁生长,呈梭形或三角形;第二三天贴壁细胞迅速增殖;培养15 d左右出现致密的贴壁细胞层,呈漩涡状生长或成簇生长.细胞在接种后2 d进入对数生长期,12 d左右进入平台期,约15 d细胞可铺满瓶底.分离培养的大鼠骨髓间充质干细胞CD90和CD54均呈阳性表达.结果验证了采用贴壁法可在体外成功分离培养大鼠骨髓间充质干细胞,操作简单,造成污染的环节和机会较少,不需离心,可以更好的保持细胞活性.
배경:골수중적간충질간세포함량불고,차수착년령증가혹체질쇠약,골수간충질간세포적수량회축점감소.목적:험증첩벽법분리배양대서골수간충질간세포적효과.방법:대서마취후취쌍측고골화경골,전거골후단,폭로골수강,용함소우혈청적DMEM배양기충세골수강,수집골수세포,반복취타제성단세포현액,접충후치우37 ℃、체적분수위5%적CO_2배양상내부육,24 h후전량환액,이후매주전량환액1차,사선역첩벽단첩벽불뢰적세포진행전대배양.관찰세포형태,회제세포생장곡선,류식세포의급면역세포화학염색감정골수간충질간세포표면표지적표체.결과여결론:배양24 h후세포능구첩벽생장,정사형혹삼각형;제이삼천첩벽세포신속증식;배양15 d좌우출현치밀적첩벽세포층,정선와상생장혹성족생장.세포재접충후2 d진입대수생장기,12 d좌우진입평태기,약15 d세포가포만병저.분리배양적대서골수간충질간세포CD90화CD54균정양성표체.결과험증료채용첩벽법가재체외성공분리배양대서골수간충질간세포,조작간단,조성오염적배절화궤회교소,불수리심,가이경호적보지세포활성.
BACKGROUND: A small number of mesenchymal stem cells (MSCs) present in bone marrow, which would gradually drop with age. OBJECTIVE: To verify the effect of adherent method on culture of bone marrow-derived MSCs. METHODS: Under anaesthesia, bone marrow cells were obtained from femur and tibia of rats, cultured by DMEM containing calf serum, placed in an incubator containing 5% CO_2 at 37 ℃. The culture medium was renewed after 24 hours, and remained periodical medium change with once per week. The weakly adherent cells were passaged. The cell morphology, growth curve, and the expression of cell-surface markers were identified by flow cytometry and immunocytochemical staining. RESULTS AND CONCLUSION: After 24 hours of culture, the cells could adhere to the walls with fusiform or triangle shapes, proliferated faster after 2-3 days, and presented whirlpool-like or clustering. The cells reached a logarithmic growth phase after 2 days, and into the late stationary phase after 12 days, which covered the bottle after 15 days. The cultured cells were positive to CD90 and CD54. The results verified that bone marrow-derived MSCs can be isolated by adherent method. This method is easy operation, and can maintain cell activity preferably.