无血清培养脐带间充质干细胞的研究

本研究观察无血清培养液培养的人脐带间充质干细胞（UC-MSC）,并比较与含10％胎牛血清培养液培养的人脐带间充质干细胞的异同.采集正常足月剖宫产胎儿脐带,用MesenCult-XF无血清培养液或含10％胎牛血清培养液培养.观察不同培养液培养的间充质干细胞细胞的形态、免疫表型、细胞周期、增殖分化潜能及对混合淋巴细胞反应的抑制作用.结果表明,采用无血清MesenCult（R）-XF培养液培养的MSC平均传代倍增6.57±0.7倍,含血清培养液培养的MSC平均传代倍增4,59±0.45倍（P＜0.05）；两种培养液培养的MSC均表达CD44、CD90、CD73、CD105抗原,不表达CD31、CD45、HLA-DR及CD34等抗原,表达程度无明显统计学差异；无血清培养液培养的MSC（65±5.2）％均为G0/G1期细胞,含血清培养液培养的MSC（62±3.1）％为G0/G1期细胞（P＞0.05）；两种培养液培养的人脐带MSC均可向成脂细胞、成骨细胞分化；无血清培养液培养的脐带MSC按1 000、5 000、10 000、20 000个细胞/孔接种密度与反应细胞和刺激细胞共培养的每分钟闪烁值（CPM）分别为（6.43±0.47）×104、（4.30 ±0.38）×104、（1.97±0.13） ×104和（0.24±0.03）×104,含血清培养液培养的MSC与不同密度的混合淋巴细胞共培养的CPM值分别为（7.85 ±0.07） ×104、（5.64 ±0.12）×104、（3.09±0.18）×104和（1.73±0.05）×104.结论：无血清培养液培养的细胞均为MSC,有传代增殖潜能,传代可达临床治疗MSC细胞量,并可避免异种蛋白致敏.

Serum-free Culture of Umbilical Cord Mesenchymal Stem Cells

This study was purposed to observe the culture of umbilical cord mesenchymal stem cells （UC-MSC） with serum-free medium, and compared it with the medium containing 10% fetal bovine serum（FBS）. The normal umbilical cords were acquired during cesarean section, and then were cultured with MesenCult-XF serum-free medium or medium containing 10% fetal bovine serum （FBS）. The monphology, immunophenotype, cell cycle, proliferation and differentiation potential of mesenchymal stem cells and the inhibition of mixed lymphocyte reaction were observed through different medium culture method. The results showed that the MSC cultured with serum-free MesenCult-XF medium could transfer and multiply for average of 6.57 ± 0.7 times, and the serum medium-cultured MSC could transfer and multiply for average of 4.59 ± 0.45 times （ P 〈 0. 05 ）. Two kinds of medium cultured MSC all expressed CD44, CD90, CD73, CD105 antigen, but did not expressed CD31, CIM5, HLA-DR and CD34 antigen, and their expression levels were not significantly different. The serum-free medium-cultured MSC （65 ± 5.2% ） were all at G0/G1 phase, and the serum-contained medium-cultured MSC （62 ±3.1% ） were at G0/G1 phase（P 〉0. 05） ; the 2 kinds of media- cultured MSC all could differentiate into fat and ossification; when serum-free medium cultured umbilical cord MSC were innoculated at the the density of 103 , 5 × 103 , 104, and 2 × 104 cells/well, then co-cultured with the reactant and stimulating cells, the CPM were （6. 43 ± 0. 47 ） × 104, （4. 30 ± 0. 38 ） × 104, （ 1.97 ± 0. 13 ） × 104 and （ 0. 24 ± 0. 03 ） × 104, respectively, and the serum-containing medium-cultured MSC were incubated with different density of mixed lymphocyte, displaying CPM that were （7.85 ±0.07） × 104 , （5.64 ±0.12） × 104, （3.09 ±0.18） × 104 and （ 1.73 ± 0. 05）× 104. It is concluded that the serum-free medium has been confirmed to culture MSC, which have potential of transfer and differentiation with count for clinical application, and can avoid foreign protein sensitization.