硼替佐米联合柔红霉素对成人急性白血病原代细胞生长、凋亡的影响

Effects of Bortezomib Combined with Daunorubicin on Proliferation and Apoptosis in Primary Adult Acute Leukemia Cells

目的探讨蛋白酶体抑制剂硼替佐米单药以及联合柔红霉素对成人急性白血病原代细胞生长、凋亡和Bcl-2 mRNA表达的影响。方法用Ficoll液分离各型成人急性白血病患者骨髓单个核细胞,分别加入不同浓度硼替佐米(5、10、20、50 nmol/L)、柔红霉素(50、100、200、500 nmol/L),以及低浓度硼替佐米(5、10 nmol/L)联合不同浓度柔红霉素(50、100、200、500 nmol/L)进行细胞培养;采用MTT方法检测培养48 h后细胞增殖活性,计算抑制率、半抑制浓度(IC50)和相互作用系数(CDI)。流式细胞术检测培养24 h后细胞凋亡率,RT-PCR检测培养48h后凋亡基因Bcl-2 mRNA表达。结果所测各型急性白血病细胞生长抑制率随硼替佐米或柔红霉素单药浓度增高而增高;柔红霉素联合小浓度硼替佐米(5、10 nmol/L)后,柔红霉素的IC50由(102±27)nmol/L分别减小为(73±26)、(55±22)nmol/L;柔红霉素200 nmol/L联合硼替佐米10 nmol/L时协同作用最为显著,CDI=0.17。柔红霉素100 nmol/L联合硼替佐米20 nmol/L组与未加药组或单药组比较细胞凋亡率增高、Bc-l2 mRNA表达下降(P<0.05)。结论硼替佐米联合柔红霉素对成人急性白血病原代细胞有协同促凋亡和抑制白血病细胞生长的作用,有一定的临床应用前景。

Objective To assess effects of proteasome inhibitor Bortezomib(Bor) in combination with Daunorubicin(DNR) on proliferation,apoptosis and the expression of Bcl-2 mRNA in primary leukemia cells in vitro.Methods Primary leukemia cells were isolated from bone marrow of adult acute leukemia patients using Ficoll liquid,then the primary leukemia cells were treated with different concentration of these two drugs(Bor 5,10,20,50 nmol/L,DNR 50,100,200,500 nmol/L,and Bor 5,10 nmol/L combined with DNR 50,100,200,500 nmol/L respectively).Cells proliferation,IC50 and CDI were analyzed by MTT assay,cellular apoptosis was observed by flow cytometry,Bcl-2 mRNA was analyzed by RT-PCR.Results Growth inhibition ratio of all the types of acute leukemia cells were increased with the treatment of DNR and Bor in dose-dependent manner.Combined with Bor(5,10 nmol/L),the IC50 of DNR decreased from(102±27) nmol/L to (73±26),(55±22) nmol/L respectively.DNR 200 nmol/L combined with Bor 10 nmol/L showed a better synergism(CDI=0.17).Compared with control group and single drug(DNR or Bor) group,there were obvious increase of apoptosis ratio and obvious decrease of Bcl-2 in the group of DNR 100 nmol/L combined with Bor 20 nmol/L after 24 h or 48 h cultivation(P<0.05).Conclusion Bor combined with DNR shows synergetic effect in promoting the apoptosis of adult acute leukemia primary cells as well as inhibitory effect on the proliferation of leukemia cells.