抗TGF-β抗体对人脐血CD34+细胞体外扩增及黏附分子表达的影响

本研究的目的是证实抗TGF-β抗体在人脐血CD34 细胞体外扩增过程中可以增加CD34 细胞的扩增效率,并观察其对脐血CD34 细胞粘附分子表达的影响。从6份新鲜脐血标本中富集CD34 细胞,将其接种于含抗TGF-β抗体 SCF Flt-3L TPO IL-3因子组合的SFEM无血清培养体系中,培养6天后,细胞计数,并用FACS检测CD34、c-kit(CD117)、CD11a、CD49d、CD33表达情况,同时进行集落分析。结果表明:①实验组有核细胞数、CD34 细胞数、CD34 c-kit 细胞数及CFU-GEMM分别扩增了41.82±13.49,15.62±6.95,13.36±6.12,11.07±4.05倍,明显高于对照组(P分别=0.001,0.002,0.003,0.002),其中实验组中更为早期的CD34 c-kit-亚群的扩增倍数更多(69.10±41.06),多于有核细胞、CD34 细胞、CD34 c-kit 细胞的扩增倍数(P=0.024)。②TGF-β抗体对CD11a和CD49d的表达无明显影响(P值均大于0.05)。结论:抗TGF-β抗体可协同早期干细胞生长因子有效扩增脐血CD34 细胞,同时不降低CD34 细胞黏附分子的表达。

Effect of Anti-TGF-β Antibody on ex vivo Expansion and Expression of Adhesion Molecules of Human Cord Blood CD34 + Cells

TGF-beta, as an inhibitor of hemopoiesis, excreted by hematopoietic stem and progenitor cells, down-regulates the expression of cytokines such as Flt-3 ligand, SCF, IL-3 etc on the stem and progenitor cells. The effect of anti-TGF-beta antibody on ex vivo expansion and expression of adhesive molecules on cord blood CD34(+) cells was studied in this research. The CD34(+) cells from six units of fresh umbilical cord blood were enriched by density gradient sedimentation and purified by miniMACS cell isolation system, and plated them into the SFEM serum free culture system which containing SCF, Flt-3L, TPO and IL-3 in the condition of 37 degrees C, 5% CO2, and saturated moisture. There were three groups in this experiment: (1) blank group: same as the culture system described above; (2) control group: added with normal rabbit IgG into the mentioned culture system; (3) test group: the same culture system with anti-TGF-beta1 antibo-dy. Cultured for 6 days, the number of mononuclear cells (MNC) was counted, the expression of CD34 antigen, CD117 (c-kit) antigen, CD11a antigen, CD49d antigen and CD33 antigen was tested with FCM. Meanwhile, cells of the three groups were plated in the methylcellulose culture system for 14 days, the number of CFU-GEMM, BFU-E, CFU-GM was counted. The results indicated that the expansion multiples of MNC, CD34(+) cells, CD34(+)c-kit(+) cells, CFU-GEMM in the test group (41.82 +/- 13.49, 15.62 +/- 6.95, 13.36 +/- 6.12, 11.07 +/- 4.05) were significantly higher than in the control group (28.86 +/- 9.03, 10.40 +/- 4.98, 9.04 +/- 4.40, 6.36 +/- 2.37) (P = 0.001, 0.002, 0.003, 0.002) respectively. The expansion multiple of more primitive CD34(+)c-kit(-) subpopulation in the test group (69.10 +/- 41.06) was even higher than in the control group (27.29 +/- 10.40) (P = 0.024). Adhesion molecule expression on the CD34(+) cells after short-term expansion: the expression of CD11a on the CD34(+) cells of the original cord blood was (61.73 +/- 4.13)%, and CD49d was (55.12 +/- 5.22)%. After expansion in each group the expression of CD11a on the CD34(+) cells did not change with statistical significance (P > 0.05), the expression of CD49d increased (P < 0.05). Compared with blank group and control group, anti-TGF-beta antibody did not impact on the expression of CD11a and CD49d (P > 0.05). It is concluded that anti-TGF-beta antibody can synergize other cytokines to effectively enhance the proliferation of cord blood NC, CD34(+) cells, progenitor subpopulation of CD34(+)c-kit(-) cells, and increase the output of more primitive progenitor colony, CFU-GEMM and BFU-E. At the same time, anti-TGF-beta antibody did not depresss the expression of adhesion molecules on CD34(+) cells.