摘要
目的:讨两种浓度胎牛血清培养的不同传代次数的骨髓间充质干细胞探在生长特性及表面标志方面的差异性。方法:实验于2004年在中山大学附属第二医院林百欣医学研究中心进行。取健康Wistar大鼠5只,采用密度梯度离心法分离大鼠骨髓间充质干细胞,然后分别以体积分数为0.1和0.15的胎牛血清的L-DMEM培养基进行贴壁培养,观察原代和传代细胞的形态特征、生长曲线及表面标志CD45,CD11b,CD29和CD44的表达(骨髓间充质干细胞表达CD29和CD44,造血前体细胞表达CD45和CD11b)。结果:①两种浓度胎牛血清培养细胞均能获得贴壁梭形骨髓间充质干细胞,但体积分数为0.15的胎牛血清原代培养的细胞分裂增殖较快,集落融合较早,传代时间较短(平均10d)。②体积分数为0.1的胎牛血清传代培养第1,2代细胞均质性相对较差,CD45阳性率分别为23.4%,15.4%,CD11b阳性率分别为16.6%,10.3%,高于体积分数为0.15的胎牛血清培养组(P<0.05),至第3代后两组已无差异,CD45和CD11b阳性率均小于5%,而CD29和CD44阳性率均大于95%。③两组的骨髓间充质干细胞生长曲线相似,但体积分数为0.15的胎牛血清培养的骨髓间充质干细胞对数生长期峰值出现较早,为第6天,而体积分数为0.1的胎牛血清培养组为第7天。结论:体积分数为0.1和0.15的胎牛血清培养的骨髓间充质干细胞在生长特性和表面标志方面存在差异。使用体积分数为0.1的胎牛血清已可满足骨髓间充质干细胞的分离纯化和培养扩增,但要在短期内获得较纯的骨髓间充质干细胞,使用体积分数为0.15胎牛血清优于体积分数为0.1胎牛血清。
AIM： To explore the difference on growth property and surface markers in rat bone marrow mesenchymal stem cells （MSCs） cultured with two kinds of concentration of fetal bovine serum （FBS） with different passage time. METHODS： The experiment was conducted at the Linbaixin Medical Research Center of Second Affiliated Hospital of Sun Yat-sen University in 2004. Five healthy Wistar rats were gained. MSCs were isolated from the bone marrow of Wistar rats by density gradient centrifugation and respectively cultured by adhering in L-DMEM containing 10% and 15% FBS. The morphologic features, the growth curve and the surface markers CD45, CD11b,CD29 and CD44 （MSCs showed CD29 and CD44; Precursor of building blood expressed CD45 and CD11b） of MSCs from primary culture and subculture were observed. RESULTS： ①Adhering spindle-shaped MSCs could be obtained by using the two kinds of concentration of FBS. MSCs of primary culture in 15% FBS had more rapid proliferation, earlier colony confluence and shorter time for passage （about 10 days） than in 10% FBS. ②In passage 1 to 2, MSCs in 10% FBS had worse homogeneous relatively. The positive rate of CD45 was 23.4% and 15.4%, respectively. The positive rate of CDI lb was 16.6% and 10.3%, respectively, which was higher than that in the 15% FBS （P 〈 0.05）. After passage 3, there was no difference between the two groups. The positive rates of CD45 and CDI lb were both less than 5%, while the positive rates of CD29 and CD44 were more than 95%. ③The growth curves of MSCs showed a similar total cell number between the two groups, but the peak of cell duplication was more advanced in 15% FBS, which was at the 6m day, while it was the 7m day in the 10% FBS. CONCLUSION： The MSCs cultured in 10% FBS medium are partly different from those cultured in 15% FBS medium on growth property and surface markers. MSCs can be isolated and pored of MSCs by using 10% FBS. However, the 15% FBS can provide more pure MSCs in shorter period as compared with the 10% FBS.
出处
《中国临床康复》
CSCD
北大核心
2006年第1期28-30,i0001,共4页
Chinese Journal of Clinical Rehabilitation
基金
广东省自然科学基金(5001683)
广东省卫生厅医学科研基金(A2004200)~~
