【摘要】：目的:構建重組人腫瘤壞死因子相關的凋亡誘導配體(tumor necrosis factor-related apoptosis-inducing ligand,TRAIL)原核表達質粒p ET-28a(+)-TRAIL114-281,優(yōu)化蛋白表達和純化條件,制備重組人可溶性TRAIL并鑒定其活性。方法:使用CCK-8初步驗證TRAIL是否具有抑制腫瘤細胞生長的生物活性;將制備的TRAIL單獨或聯合50 nmol/L硼替佐米應用于H460細胞(對TRAIL敏感)和K562細胞(對TRAIL抵抗)24 h,流式細胞術檢測細胞凋亡率,比色法檢測caspase-8、-9、-3的活化程度,Western blot分析細胞中Bax、Bcl-2和c FLIP蛋白的表達。流式細胞術檢測硼替佐米處理H460細胞和K562細胞24 h后DR4和DR5的表達量變化。結果:制備了具有生物學活性且性質穩(wěn)定的重組人可溶性TRAIL,且成功誘導H460和K562細胞凋亡。不同濃度TRAIL處理H460細胞后其凋亡率隨著TRAIL濃度升高而顯著升高(P0.05),但K562細胞凋亡率并未隨著TRAIL濃度明顯升高。聯合用藥組的H460和K562細胞凋亡率均顯著高于單獨用藥組(P0.05),凋亡過程中caspase-8、-9、-3均被活化,藥物處理組的Bcl-2和c FLIP表達量均比對照組下降,尤其聯合用藥組表達量下降最為顯著(P0.05),而Bax表達量無明顯變化。硼替佐米處理H460和K562細胞后DR4和DR5表達量均上調(P0.05)。結論:硼替佐米能協(xié)同TRAIL啟動內源性凋亡途徑誘導H460和K562細胞凋亡,其可能機制是通過上調死亡受體DR4和DR5的表達量、下調抗凋亡蛋白Bcl-2和c FLIP的表達量來實現的。
[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.
【作者單位】： 廣州醫(yī)科大學金域檢驗學院;
【基金】：廣州市屬高校重點學科建設經費資助項目(穗教高教[2011]34號)
【分類號】：R96;R94

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