CAJ | 학술논문

目的了解应用全骨髓贴壁法分离的大鼠骨髓间充质干细胞(bone mesenchymal stem cells,BMSCs)的生长、增殖特点,以及接种密度、培养时间对细胞增殖的影响,为预防和治疗机体退行性疾病等提供多潜能的种子细胞.方法通过全骨髓贴壁法分离SD大鼠BMSCs,在体外条件下培养,应用倒置显微镜观察细胞形态学特征及流式细胞仪鉴定、检测大鼠BMSCs表面标志抗原,并通过分析对接种于96孔板不同接种密度(2×103、5×103、1×104个细胞/ml)连续培养10 d细胞生长曲线的特点,研究接种密度和培养时间对BMSCs生长、增殖的影响.结果流式细胞仪分析CD29细胞阳性率为97.68%(7607/7788),CD34、CD45细胞阳性率分别为7.93%(661/8340)、2.76%(215/7788),符合BMSCs表面标记物表达特征.通过换液及消化传代,3代以上的BMSCs较为均一纯化,呈典型的旋涡或放射状生长,约传代至7～10代时出现细胞衰老并衰老细胞逐渐增多;中等密度(5×103个细胞/ml)接种细胞的生长曲线呈较典型的S形曲线.第1、2天为潜伏期;第3～5天为对数生长期;第6天细胞数目进入平台期;第8～10天细胞数量稍有下降.结论全骨髓贴壁法是获得BMSCs较为简便有效的方法,且中等量接种密度更有利于细胞增殖,可获得更多的种子细胞.
목적 료해응용전골수첩벽법분리적대서골수간충질간세포(bone mesenchymal stem cells,BMSCs)적생장、증식특점,이급접충밀도、배양시간대세포증식적영향,위예방화치료궤체퇴행성질병등제공다잠능적충자세포.방법 통과전골수첩벽법분리SD대서BMSCs,재체외조건하배양,응용도치현미경관찰세포형태학특정급류식세포의감정、검측대서BMSCs표면표지항원,병통과분석대접충우96공판불동접충밀도(2×103、5×103、1×104개세포/ml)련속배양10 d세포생장곡선적특점,연구접충밀도화배양시간대BMSCs생장、증식적영향.결과 류식세포의분석CD29세포양성솔위97.68%(7607/7788),CD34、CD45세포양성솔분별위7.93%(661/8340)、2.76%(215/7788),부합BMSCs표면표기물표체특정.통과환액급소화전대,3대이상적BMSCs교위균일순화,정전형적선와혹방사상생장,약전대지7～10대시출현세포쇠로병쇠로세포축점증다;중등밀도(5×103개세포/ml)접충세포적생장곡선정교전형적S형곡선.제1、2천위잠복기;제3～5천위대수생장기;제6천세포수목진입평태기;제8～10천세포수량초유하강.결론 전골수첩벽법시획득BMSCs교위간편유효적방법,차중등량접충밀도경유리우세포증식,가획득경다적충자세포.
Objective This study was to investigate the growth and proliferation characteristics of rat bone mesenchymal stem cells (BMSCs) isolated by the method of whole bone marrow culture and to explore the effect of cell inoculation density and incubation period on cell proliferation,with an aim to provide multipotential seed cells for preventing from degenerative disease. Methods Bone mesenchymal stem cells were isolated by the method of whole bone marrow culture and then culturedin vitro. The cell morphologic features were observed by inverted microscope. The cell surface antigens were identified by flow cytometry. The effect of cell inoculation density and culture period on cell growth and proliferation was explored by analyzing the characteristics of a ten-day cell growth curve in 96-well plates. Results Flow cytometry results showed the detection rates for CD29, CD34 and CD45 were 97.68% (7607/7788) ,7.93%(661/8340) and 2. 76% (215/7788)respectively, which was consistent with the expression characteristics of BMSCs surface antigens. BMSCs became uniform after three cell passages, existing in a typical shape of whirlpool or radial colony. The senescent cells started to appear at 7th passage,and more senescent cells were found at 10th passage. The growth curve for moderate inoculation density was typically S-shaped. Lag phase was found during the first two days, and logarithm growth phase was in the following three days. Plateau phase started from the 6th day and cell numbers decreased slightly from the 8th day. Conclusion The whole bone marrow culture is an effective way to obtain BMSCs. A moderate inoculation density was more advantageous to cell proliferation, by which more seed cells could be obtained.