摘要
目的探讨X连锁凋亡抑制蛋白(XIAP)在纤维连接蛋白(Fn)黏附介导的HL-60细胞耐药中的作用。方法用Fn包被培养板,以牛血清白蛋白(BSA)包被培养板作为对照。通过CCK-8 实验检测Fn黏附对柔红霉素(DNR)敏感性的影响,流式细胞仪检测HL-60细胞内DNR浓度的变化, RT-PCR、Western blot检测XIAP、bcl-2、MRP和mdr1基因mRNA和蛋白表达变化。将XIAP反义寡核苷酸通过脂质体法转染Fn黏附培养的HL-60细胞,计算DNR对HL-60细胞的IC50值。结果 HL-60 细胞与Fn黏附后,对DNR的敏感性下降,Fn组IC50值为0．526μmol/L,明显高于BSA组(0．132 μmol/L)和悬浮培养组(0．123μmol/L)(P<0．05);Fn组XIAP mRNA和XIAP蛋白表达水平均较BSA 组和悬浮培养组明显上调(P<0．05);Fn组细胞内的DNR浓度与BSA组和悬浮培养组比较差异无统计学意义(P>0．05)。XIAP反义寡核苷酸可使Fn黏附介导的耐药HL-60细胞XIAP表达明显下调, 对DNR的敏感性增加。结论 XIAP可能参与了Fn黏附介导的HL-60细胞耐药,应用XIAP反义寡核苷酸能够逆转Fn黏附介导的HL-60细胞耐药。
Objective To explore the role of X-linked inhibitor of apoptosis protein（XIAP） in the fi- bronectin （Fn）-adhesion mediated drug resistance of HL-60 cells. Methods Immunosorp plates were coated with Fn and bovine serum albumin （BSA） （as control）, respectively. Colorimetrie CCK-8 assay was used to determine the effects of Fn on the cytotoxicity of DNR to HL-60 ceils. Intracellular DNR accumulation was assayed with flow cytometry. Reverse transcription-PCR and Western blot were used to examine the mRNA expression and XIAP, bcl-2, MRP and mdrl proteins, respectively. HL-60 cells were added to Fn coated immunosorp plates. The fully phosphorothioated antisense oligonucleotide （AS-ODNs） and the control ODNs of XIAP were delivered into HL-60 cells in the form of liposome-ODN complexes. IC50 was calculated by linear regression of survival percent versus drug concentration. Results HL-60 cells adhered to Fn-coated plates had a significant survival advantage over those grown on BSA coated plates and in suspension when exposed to DNR, the IC50 of Fn group being significantly higher than that of BSA group and suspension group （0. 526 μmol/L vs 0. 132μmol/L, 0. 123μmol/L, respectively, P 〈 0.05）. XIAP was up-regulated significantly in Fn group compared with BSA group and suspension group （ P 〈 0.05 ） , whereas there was no difference in the expressions of bcl-2, MRP and mdrl among the three groups （P 〉 0.05 ）. The intracellular concentration of DNR in Fn-adhered HL-60 cells was similar to that in BSA group and suspension group （ P 〈 0.05 ）. AS-ODNs of XIAP down-regulated the XIAP expression in Fn-adhered HL-60 cells. In addition, AS-ODNs sensitized HL-60 ceils to the cytotoxic effects of DNR. Conclusion The increased XIAP protein level contributes to the drug resistance induced by adhesion to Fn. AS-ODNs of XIAP might reverse the drug resistance.
出处
《中华血液学杂志》
CAS
CSCD
北大核心
2006年第1期1-5,共5页
Chinese Journal of Hematology
