小白菊内酯抑制CD34^+CD38^-KG-1a白血病细胞的增殖并增强其被allo-NK细胞杀伤的敏感性

Parthenolide inhibits proliferation of CD34^+CD38^- KG-1a leukemia cells and enhances susceptibility of CD34^+CD38^- KG-1a leukemia cells to the cytotoxicity of allo-NK cells

目的:探讨小白菊内酯(parthenolide,PTL)对CD34+CD38-KG-1a白血病细胞的增殖、Bcl-2表达及其被同种异体NK(allo-NK)细胞杀伤敏感性的影响。方法:免疫磁珠法从KG-1a细胞中分离CD34+CD38-KG-1a细胞。XTT法检测PTL对CD34+CD38-白血病细胞增殖的影响,Real-time PCR和Western blotting分别检测PTL对CD34+CD38-KG-1a细胞Bcl-2mRNA和蛋白表达的影响,LDH释放法观察PTL对CD34+CD38-KG-1a细胞被allo-NK细胞杀伤敏感性的影响。结果:PTL对CD34+CD38-KG-1a细胞增殖的抑制呈剂量(2.5~80μmol/L)依赖性。PTL浓度为2.5μmol/L时,PTL组CD34+CD38-KG-1a细胞的增殖抑制率显著高于对照组[(4.89±1.07)%vs 0,P<0.01];PTL杀伤CD34+CD38-KG-1a细胞的IC50为20μmol/L;PTL组CD34+CD38-KG-1a细胞Bcl-2 mRNA[(0.105±0.007)vs(0.307±0.013),P<0.01]与蛋白表达均显著低于对照组。在效靶比为10∶1,20∶1和40∶1时,allo-NK细胞对PTL组CD34+CD38-KG-1a细胞杀伤率逐步升高,且均高于阴性(无PTL处理)对照组[(19.76±1.01)%,(30.14±0.96)%和(51.48±3.15)%vs(12.50±1.42)%,(16.90±0.93)%和(31.70±1.53)%(均P<0.01)];效靶比为40∶1时,allo-NK细胞对PTL组细胞的杀伤效率显著高于阳性(Bcl-2抑制剂ABT-737处理)对照组[(51.48±3.15)%vs(43.08±2.81)%,P<0.05]。结论:PTL能抑制CD34+CD38-KG-1a细胞的增殖并增强其被allo-NK细胞杀伤的敏感性,这可能与PTL下调Bcl-2的表达有关。

Objective：To investigate the effects of parthenolide （PTL） on the proliferation, bcl-2 expression and sus ceptibility of CD34 ~ CD38- KG-1 a leukemia cells to eytotoxicity of allogeneic natural killer cells （allo-NK cells）. Meth ods： CD34 ＋ CD38- KG-Ia cells were separated from KG-la cells by magnetic activated cell sorting （MACS）. The effect of trFL on the proliferation of CD34 ^＋ CD38 - KG-1 a cells was assessed by XTT assay. The expression of Bcl-2 mRNA and protein in CD34 ^＋ CD38- KG-1 a cells regulated by parthenolide were evaluated by real-time reverse transcriptase-polymer ase chain reaction analysis （RT-PCR） and Western blotting, respectively. LDH-releasing assay was performed to observe the effect of PTL on the cytotoxicity of allo-NK cells against CD34 ^＋ CD38 - KG-1 a ceils. Results： PTL inhibited the pro liferation of CD34＋ CD38- KG-la cells with a concentration-dependent manner. As the concentration of PTL was 2.5 umol/L, the cellular proliferation inhibition rate of CD34 ＋ CD38 - KG-1 a cells in PTL group was significantly higher than that of the control group （ [4.89 ± 1.07 ] % vs 0, P 〈 0.01 ）. The ICs0 of PTL to inhibit the proliferation of CD34 ±CD38 KG-la leukemia cells was 20 pumol/L. By treated with 20 Ixmol/L PTL, the expression of bcl-2 mRNA （ [0. 105 ＋ 0. 007 ] vs [ 0. 307 ＋ 0.013 ] P 〈 0.01 ） and protein in CD34 ＋ CD38- KG-1 a ceils were significantly lower than those of the control group. At effect-to-target ratio of 10： 1, 20：1 and 40： 1, the cytotoxicity rate of allo-NK cell against CD34~ CD38 - KG-1 a cells in the FrL group and in the positive control group was increasing and was higher than that of the nega- tive control group （[19.76＋1.011%, [30.14-＋0.961% and E51-48 ＋3.151% vs [12.50：tl.421%, [16.90＋ 0.93 ] % and [ 31.70 ＋ 1.53 ] % ; all P 〈 0.01 ）. In addition, at effect-to-target ratio of 40： I, the cytotoxicity rate of al- lo-NK cell against CD34 ＋CD38- KG-Ia cells in the PTL group was significantly higher than that of the positive control group （ [ 51.48 _＋ 3.15 ] % vs [ 43.08 _＋ 2.81 ] %, P 〈 0.05 ）. Conclusion ： FFL can suppress the proliferation of CD34 ~ CD38 - KG-1 a cells and enhance its susceptibility to the cytotoxicity of allo-NK cells, which may be related with the down- regulation of Bcl-2 by PTL.