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目的:构建Egr1介导的人分泌型肿瘤坏死因子相关的凋亡诱导配体(hsTRAIL)重组表达载体pEgr1-hsTRAIL,探讨其对人肺腺癌A549细胞的生长抑制作用。方法:利用基因重组技术构建Egr1介导的hsTRAIL重组载体,PCR、酶切和测序鉴定正确后转染A549细胞,给予6Gy X射线照射。实验分为对照(Control),pEgr1-hsTRAIL、6Gy X射线和pEgr1-hsTRAIL+6Gy X射线组。ELISA法检测各组A549细胞中hsTRAIL的表达,MTT法检测细胞增殖能力,流式细胞术检测细胞周期,TUNEL法检测细胞凋亡变化。结果:成功构建Egr1调控的hsTRAIL重组载体pEgr1-hsTRAIL,质粒转染后经6Gy X射线照射,对照组、6Gy组和pEgr1-hsTRAIL组hsTRAIL蛋白表达随时间延长变化不明显,而pEgr1-hsTRAIL+6Gy组随时间延长hsTRAIL蛋白表达显著增加(P<0.05或P<0.01),8h达到峰值;pEgr1-hsTRAIL组A549细胞增殖能力与对照组比较无明显差异,6 Gy和pEgr1-hsTRAIL+6 Gy组A549细胞增殖能力较对照组显著降低,pEgr1-hsTRAIL+6Gy组降低更明显(P<0.05或P<0.01);与对照组比较,pEgr1-hsTRAIL组A549细胞各期百分比变化不明显,而6Gy和pEgr1-hsTRAIL+6Gy组G0/G1期细胞百分比明显增加(P<0.05),G2/M期细胞百分比明显降低(P<0.05),S期细胞百分比无明显变化;各期细胞百分比在6Gy和pEgr1-hsTRAIL+6Gy组基本一致;与对照组比较,pEgr1-hsTRAIL组A549细胞凋亡百分比无明显变化,而6Gy和pEgr1-hsTRAIL+6Gy组A549细胞凋亡百分比明显增加(P<0.01),其中pEgr1-hsTRAIL+6Gy组增加更明显。结论:成功构建Egr1介导的hsTRAIL重组表达载体pEgr1-hsTRAIL,其能够增加辐射对A549细胞的增殖抑制和凋亡诱导作用,而对细胞周期分布影响不大。
OBJECTIVE: To construct the recombinant plasmid pEgr1-hsTRAIL, which is an inhibitor of human secreting tumor necrosis factor-related apoptosis-inducing ligand (hsTRAIL), and to investigate its inhibitory effect on human lung adenocarcinoma A549 cells. METHODS: Egr1-mediated hsTRAIL recombinant vector was constructed by gene recombination technique. The recombinant plasmid was transfected into A549 cells by PCR, restriction enzyme digestion and sequencing. The cells were irradiated with 6 Gy X-ray. The experiment was divided into control (Control), pEgr1-hsTRAIL, 6Gy X-ray and pEgr1-hsTRAIL + 6Gy X-ray group. The expression of hsTRAIL in each group of A549 cells was detected by ELISA. The cell proliferation was detected by MTT assay. The cell cycle was detected by flow cytometry. The apoptosis was detected by TUNEL assay. Results: The hsTRAIL recombinant plasmid pEgr1-hsTRAIL regulated by Egr1 was constructed successfully. The expression of hsTRAIL protein in the control group, 6Gy group and pEgr1-hsTRAIL group did not change significantly with 6Gy X-ray transfection. However, pEgr1-hsTRAIL + 6Gy The expression of hsTRAIL protein in the group of pEgr1-hsTRAIL was significantly higher than that in the control group (P <0.05 or P <0.01), and reached its peak at 8 h. The proliferation of A549 cells in the pEgr1-hsTRAIL group was not significantly different from that in the control group Compared with the control group, the percentage of A549 cells in the pEgr1-hsTRAIL group did not change significantly, while the expression of 6Gy and pEgr1 in the pEgr1-hsTRAIL + 6Gy group was significantly lower than that in the control group (P <0.05 or P <0.01) The percentage of cells in G0 / G1 phase was significantly increased (P <0.05), the percentage of cells in G2 / M phase was significantly decreased (P <0.05), the percentage of cells in S phase was not changed significantly; the percentage of cells in each phase was 6Gy and pEgr1- hsTRAIL + 6Gy group. The percentage of apoptosis of A549 cells in pEgr1-hsTRAIL group was not significantly different from that in control group, while the percentage of apoptosis in A549 cells in 6Gy and pEgr1-hsTRAIL + 6Gy groups was significantly increased (P <0.01) hsTRAIL + 6Gy group increased more pronounced. CONCLUSION: The recombinant plasmid pEgr1-hsTRAIL mediated by Egr1 can be successfully constructed and can increase the proliferation inhibition and apoptosis induction of A549 cells with little effect on the cell cycle distribution.