重组人可溶性TRAIL的制备及其联合硼替佐米诱导肿瘤细胞的凋亡作用

发布时间：2018-08-06 10:53

【摘要】：目的:构建重组人肿瘤坏死因子相关的凋亡诱导配体(tumor necrosis factor-related apoptosis-inducing ligand,TRAIL)原核表达质粒p ET-28a(+)-TRAIL114-281,优化蛋白表达和纯化条件,制备重组人可溶性TRAIL并鉴定其活性。方法:使用CCK-8初步验证TRAIL是否具有抑制肿瘤细胞生长的生物活性;将制备的TRAIL单独或联合50 nmol/L硼替佐米应用于H460细胞(对TRAIL敏感)和K562细胞(对TRAIL抵抗)24 h,流式细胞术检测细胞凋亡率,比色法检测caspase-8、-9、-3的活化程度,Western blot分析细胞中Bax、Bcl-2和c FLIP蛋白的表达。流式细胞术检测硼替佐米处理H460细胞和K562细胞24 h后DR4和DR5的表达量变化。结果:制备了具有生物学活性且性质稳定的重组人可溶性TRAIL,且成功诱导H460和K562细胞凋亡。不同浓度TRAIL处理H460细胞后其凋亡率随着TRAIL浓度升高而显著升高(P0.05),但K562细胞凋亡率并未随着TRAIL浓度明显升高。联合用药组的H460和K562细胞凋亡率均显著高于单独用药组(P0.05),凋亡过程中caspase-8、-9、-3均被活化,药物处理组的Bcl-2和c FLIP表达量均比对照组下降,尤其联合用药组表达量下降最为显著(P0.05),而Bax表达量无明显变化。硼替佐米处理H460和K562细胞后DR4和DR5表达量均上调(P0.05)。结论:硼替佐米能协同TRAIL启动内源性凋亡途径诱导H460和K562细胞凋亡,其可能机制是通过上调死亡受体DR4和DR5的表达量、下调抗凋亡蛋白Bcl-2和c FLIP的表达量来实现的。
[Abstract]:Aim: to construct the prokaryotic expression plasmid p ET-28a () -TRAIL114-281, a recombinant apoptosis-inducing ligand (tumor necrosis factor-related apoptosis-inducing ligand trail) associated with human tumor necrosis factor (TNF), to optimize the expression and purification conditions, to prepare recombinant human soluble TRAIL and to identify its activity. Methods: CCK-8 was used to verify whether TRAIL had the biological activity of inhibiting the growth of tumor cells. The prepared TRAIL was applied to H460 cells (sensitive to TRAIL) and K562 cells (resistant to TRAIL) for 24 h either alone or in combination with 50 nmol/L bortezomil. The apoptosis rate was detected by flow cytometry. The activation of caspase-8, caspase-9 and caspase-9 was detected by colorimetric method. The expression of BaxanBcl-2 and c FLIP protein was detected by Western blot. The expression of DR4 and DR5 in H460 cells and K562 cells were detected by flow cytometry. Results: Recombinant human soluble trail with biological activity and stable property was prepared and apoptosis of H460 and K562 cells was induced successfully. After H460 cells were treated with different concentrations of TRAIL, the apoptosis rate of H460 cells increased significantly with the increase of TRAIL concentration (P0.05), but the apoptosis rate of K562 cells did not increase with the concentration of TRAIL. The apoptotic rate of H460 and K562 cells in the combination group was significantly higher than that in the control group (P0.05). The expression of Bcl-2 and c FLIP in the drug treated group was lower than that in the control group. Especially, the expression of Bax decreased most significantly in the combination group (P0.05), but the expression of Bax did not change significantly. The expression of DR4 and DR5 in H460 and K562 cells were up-regulated by bortezomib (P0.05). Conclusion: bortezomib can induce apoptosis of H460 and K562 cells through endogenous apoptosis pathway induced by TRAIL, which may be achieved by up-regulating the expression of DR4 and DR5, and down-regulating the expression of Bcl-2 and c FLIP.
【作者单位】：广州医科大学金域检验学院;
【基金】：广州市属高校重点学科建设经费资助项目(穗教高教[2011]34号)
【分类号】：R96;R94

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