【摘要】：目的:構(gòu)建重組人腫瘤壞死因子相關(guān)的凋亡誘導(dǎo)配體(tumor necrosis factor-related apoptosis-inducing ligand,TRAIL)原核表達(dá)質(zhì)粒p ET-28a(+)-TRAIL114-281,優(yōu)化蛋白表達(dá)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(yàn)證TRAIL是否具有抑制腫瘤細(xì)胞生長(zhǎng)的生物活性;將制備的TRAIL單獨(dú)或聯(lián)合50 nmol/L硼替佐米應(yīng)用于H460細(xì)胞(對(duì)TRAIL敏感)和K562細(xì)胞(對(duì)TRAIL抵抗)24 h,流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡率,比色法檢測(cè)caspase-8、-9、-3的活化程度,Western blot分析細(xì)胞中Bax、Bcl-2和c FLIP蛋白的表達(dá)。流式細(xì)胞術(shù)檢測(cè)硼替佐米處理H460細(xì)胞和K562細(xì)胞24 h后DR4和DR5的表達(dá)量變化。結(jié)果:制備了具有生物學(xué)活性且性質(zhì)穩(wěn)定的重組人可溶性TRAIL,且成功誘導(dǎo)H460和K562細(xì)胞凋亡。不同濃度TRAIL處理H460細(xì)胞后其凋亡率隨著TRAIL濃度升高而顯著升高(P0.05),但K562細(xì)胞凋亡率并未隨著TRAIL濃度明顯升高。聯(lián)合用藥組的H460和K562細(xì)胞凋亡率均顯著高于單獨(dú)用藥組(P0.05),凋亡過(guò)程中caspase-8、-9、-3均被活化,藥物處理組的Bcl-2和c FLIP表達(dá)量均比對(duì)照組下降,尤其聯(lián)合用藥組表達(dá)量下降最為顯著(P0.05),而B(niǎo)ax表達(dá)量無(wú)明顯變化。硼替佐米處理H460和K562細(xì)胞后DR4和DR5表達(dá)量均上調(diào)(P0.05)。結(jié)論:硼替佐米能協(xié)同TRAIL啟動(dòng)內(nèi)源性凋亡途徑誘導(dǎo)H460和K562細(xì)胞凋亡,其可能機(jī)制是通過(guò)上調(diào)死亡受體DR4和DR5的表達(dá)量、下調(diào)抗凋亡蛋白Bcl-2和c FLIP的表達(dá)量來(lái)實(shí)現(xiàn)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(xué)金域檢驗(yàn)學(xué)院;
【基金】：廣州市屬高校重點(diǎn)學(xué)科建設(shè)經(jīng)費(fèi)資助項(xiàng)目(穗教高教[2011]34號(hào))
【分類號(hào)】：R96;R94

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相關(guān)期刊論文 前4條

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