抗-CD36单克隆抗体对人CD34^+造血干(祖)细胞增殖和分化的影响

The effect of anti-CD36 monoclonal antibody on proliferation and differentiation of human CD34^+ hematopoietic stem (progenitor) cells

目的探讨抗-CD36单克隆抗体对人CD34^+造血干(祖)细胞体外增殖和分化的影响。方法选择无各种产科并发症的健康足月产妇3名,取脐带血20 mL/(人)份,以Ficoll细胞分离液(1.077 g/mL)密度梯度800 g离心30 min后,流式细胞仪分选CD34^+造血干(祖)细胞,培养2—3代,四唑盐(MTT)比色法检测抗-CD36单克隆抗体对CD34^+细胞生长的影响;流式细胞术检测细胞凋亡,Annexin V/PI双染法和碘化丙啶(PI)单染法检测细胞周期。观察抗-CD36单克隆抗体对CD34^+造血干(祖)细胞凋亡和细胞周期的影响、对造血干(祖)细胞红系分化能力以及对红系集落形成单位(CFU-E)和红系爆式集落形成单位(BFU-E)生成的影响。结果流式细胞仪分选出经Ficoll分离脐带血获得的单个核细胞中约0.44%CD34^+造血干(祖)细胞。2、8、32、64、128μg/mL抗-CD36单克隆抗体分别与CD34^+造血干(祖)细胞体外共培养:单纯CD34^+造血干(祖)细胞培养(正常)组、抗-CD36单克隆抗体2及32μg/mL组,IgG(对照)组的OD值分别为1.05±0.12 vs 0.9±0.15 vs 0.81±0.11 vs 1.06±0.18(P<0.01)。Annexin V流式检测凋亡率(%):正常组、对照组与2μg/mL抗-CD36单克隆抗体组分别为10.13SymbolqB@1.42 vs 10.51SymbolqB@1.75 vs 24.57SymbolqB@2.75(P<0.05)。G_1/S值:正常组、对照组与2μL/mL抗-CD36单克隆抗体组分别为3.95±0.25 vs 4.36±0.55 vs 7.35±0.65(P<0.05)。CD34^+造血干(祖)细胞定向分化红系(CFU-E/BFU-E克隆形成数):正常组、对照组与抗-CD36单克隆抗体组分别为169±12、172±12和85±6(P<0.05)。结论抗-CD36单克隆抗体诱导人CD34^+造血干(祖)细胞凋亡,细胞增殖减少和红系CFU-E/BFU-E克隆能力降低。

Objective To investigate the effect of anti-CD36 monoclonal antibody on proliferation and differentiation of human CD34^+ hematopoietic stem ( progenitor) cells in vitro. Methods Three healthy full-term maternal women without various obstetric complications were chosen,and cord blood 20 mL of each was taken. After density gradient centrifugation ( 800 g,30 mins) of Ficoll cell separation solution ( 1. 077 g /mL),CD34^+ hematopoietic stem ( progenitor) cells were sorted by flow cytometry and cultured for 2-3 generations. MTT was used to examine the effect of anti-CD36 monoclonal antibody on the growth of hematopoietic stem ( progenitor) cells. Flow cytometry analysis was used to detect the apoptosis and cell cycle of CD34^+ hematopoietic stem ( progenitor) cells. The effect of anti-CD36 monoclonal antibody on the formation of CFU-E/ BFU-E in hematopoietic stem ( progenitor) cells was analyzed by CFU-E/BFU-E account after 10-14 days culture.Results After umbilical cord blood was isolated by Ficoll to obtain mononuclear cells,the hematopoietic stem ( progenitor) cells of CD34^+ were sorted by flow cytometry,and about 0. 44% of CD34^+ hematopoietic stem ( progenitor) cells were isolated. Different concentrations of anti-CD36 monoclonal antibody( 2,8,32,64 and 128 μg /mL,respectively) and hematopoietic stem ( progenitor) cells were cultured in vitro. The OD value of CD36 monoclonal antibody group ( 2 μg /mL) was ( 0. 9±0. 15),lower than normal group ( 1. 05±0. 12)( P<0. 05);while the OD value ( 0. 81±0. 11) decreased significantly in the CD36 monoclonal group ( 32 μg /mL)( P<0. 01). There was no significant difference between the hematopoietic stem ( progenitor) cells culture group and the IgG control group ( P>0. 05). In the Annexin V flow detection,the apoptotic rate of anti-CD36 monoclonal antibody group ( 2 μg /mL) was 24. 57± 2. 75, which was statistically higher than that of the normal group ( 10. 13±1. 42)( P<0. 05). Anti-CD36 monoclonal antibody significantly induced hematopoietic stem ( progenitor) cells to undergo S phase cell reduction,G1 phase cells increased,and G1 /S phase cell arrest occurred. The G1 /S value of the normal group,control group and the anti-CD36 monoclonal antibody culture group ( 2 μg /mL) was 3. 95±0. 25 , 4. 36±0. 55 and 7. 35±0. 65,respectively. The number of CFU-E/BFU-E clones in the normal group,IgG control group and the anti-CD36 monoclonal antibody culture group ( 2 μg /mL) was 169±12,172 ±12,and 85±6,respectively;the number of colonies formed in the last group was significantly lower than the other two groups ( P<0. 05).Conclusion Anti-CD36 monoclonal antibody can reduce the proliferation of human CD34^+ hematopoietic stem ( progenitor) cells and reduce the ability of erythroid differentiation.