【摘要】 通过检测转化生长因子 β1 (TGF β1 )对脐血单个核细胞增殖周期及凋亡的作用 ,探讨其对造血进行负调控的机制。采用 5′ 溴脱氧尿嘧啶核苷 (BrdU)掺入法检测TGF β1 对细胞DNA合成的影响 ;应用Westernblot检测TGF β1 对G1 期细胞周期蛋白cyclinA ,cyclinD1,CDK2和CDK4表达的影响 ;用姬姆萨染色和流式细胞分析法检测TGF β1 对细胞凋亡的作用。结果表明 :①培养细胞加入 10 %胎牛血清 (FBS)和 1,2及 5ng ml的TGF β1 12小时后 ,BrdU的OD值与对照 (10 %FBS)组相比有显著性差异 (P <0 .0 1) ;继续培养 2 4小时后 ,1ng mlTGF β1组的BrdU检测OD值与 10 %FBS组相比无显著性差异 (P >0 .0 5 ) ,而 2ng ml和 5ng mlTGF β1 组的OD值与10 %FBS组相比差异有显著性 (P <0 .0 5 )。②与对照组相比 ,2ng mlTGF β1 还可明显抑制cyclinA ,cyclinD1,CDK2和CDK4的表达。③与 2ng mlTGF β1 共培养 12和 2 4小时后 ,细胞出现凋亡特征性改变 ,凋亡率分别为14 .4 2 %和 31.98% ;而对照组未见明显形态变化 ,凋亡率分别为 4 .71%和 5 .76 %。结论 :TGF β1 可通过抑制造血细胞DNA的合成和G1 期细胞周期蛋白的表达 ,从而阻止细胞进入S期 ,抑制细胞增殖。TGF β1 还可促进造血细胞凋亡 ,因而是一个重要的造血负性调控因子。更多还原

【Abstract】 To explore the mechanism of transforming growth factor-β 1 (TGF-β 1)effect on umbilical cord blood mononuclear cells proliferation and a poptosis, 5-bromo-2′-deoxyurine (BrdU) incorporation assay was adopted to d etect effect of TGF-β 1 on synthesis of DNA in cells. Westem blot method was used to examine effect of TGF-β 1 on expression of cyclin A, Cyclin D1, CDK2 and CDK4 in G 1 phase of cell cycle. Giemsa staining and flow cytometry (FCM) w ere performed to detected effect of TGF-β 1 on cell apoptosis. The results showed that (1) after culture of cells with IMDM containi n g 10% FBS, 10% FBS+1 ng/ml TGF-β 1, 10% FBS+2 ng/ml TGF-β 1 or 10% FBS+5 n g/ml TGF-β 1 for 12 hours the OD values of TGF-β 1 group were significantly lower than control group(P<0.01); after culture for 24 hour s the OD values of 1 ng/ml TGF-β 1 group had no significant difference compa red wi th control group (P>0.05), but the OD values of 2 ng/ml and 5 ng/ml TGF-β 1 groups we re significantly lower than control group(P<0.05). (2) 2 ng/ml TGF -β 1 could significantly inhibite the production of cyclin A, cyclin D1, C DK2 and CDK4, the protein levels were significantly lower than control group. (3) when the cells were co-cultured with 2 ng/ml TGF-β 1 for 12 and 24 hours, Giemsa staining and FCM detection could display typical apoptosis, the apoptosis rates were 14.42% and 31.98%, while apoptosis rate in control were 4.71% and 5.76%. It is concluded that TGF-β 1 can inhibit production of G 1 cyclins and CDKs of umbilical cord blood mononuclear cells, arrest cells in the G 1 phase of cell cycle and induce cell apoptosis. Thus, TGF-β 1 may be an important ne gative modulator in hematopoiesis.更多还原