siRNA靶向抑制4-1BBL基因表达在HL-60细胞对淋巴细胞增殖和凋亡的影响
Effect of siRNA targeting inhibition of 4-1BBL gene expression in HL-60 tumor cells on lymphocytic proliferation and apoptosis
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摘要: 目的：观察4-1BB配体（4-1BBL）-siRNA对HL-60细胞细胞因子分泌的影响；对比观察4-1BBL基因被干扰前后，HL-60细胞培养上清液对淋巴细胞增殖、凋亡诱导作用的影响；探讨肿瘤细胞的免疫逃逸机制。方法：采用化学合成法人工合成靶向人4-1BBL基因的siRNA，脂质体转染法转染HL-60细胞；半定量RT-PCR和流式细胞术检测干扰前后HL-60细胞4-1BBL-mRNA和表面蛋白的表达水平；ELISA法检测4-1BBL干扰前后HL-60细胞培养上清液转化生长因子-β（TGF-β）、血管内皮生长因子（VEGF）分泌水平。密度梯度离心法分离外周血单个核细胞，收集4-1BBL干扰后的HL-60细胞培养上清液与体外分离的人淋巴细胞共培养，MTT法检测机体淋巴细胞增殖水平；流式细胞术检测淋巴细胞凋亡比率的变化。结果：靶向4-1BBL-siRNA转染HL-60后，4-1BBL-mRNA和蛋白的表达水平明显降低（P<0.01）； ELISA结果显示，4-1BBL基因被干扰后，HL-60细胞培养上清液中TGF-β和VEGF分泌水平明显降低（P<0.01和P<0.05）。与干扰前比较，干扰后的HL-60细胞培养上清液对机体淋巴细胞的增殖抑制和诱导凋亡作用均下降（P<0.05）。结论：HL-60细胞高表达的4-1BBL信号具有反向调节作用，通过促进其分泌TGF-β、VEGF，使肿瘤细胞培养上清液对淋巴细胞活性的抑制作用增强并促进淋巴细胞凋亡，可能是肿瘤细胞逃避宿主免疫监视的机制之一。Abstract: Objective:To observe the effects of 4-1BBL siRNA on the cytokine production of HL-60 cells,compare the effects of culture supernatant of HL-60 cells on lymphocyte proliferation and inducing apoptosis between before and after interfering 4-1BBL,and explore the immune escape mechanism of tumor cells.Methods:The siRNA targeting 4-1BBL gene was designed and synthesized in vitro,then transfected into HL-60 cells using Lipofectamine.The expression levels of 4-1BBL mRNA and protein in HL-60 cells were detected using RT-PCR and flow cytometry,respectively.The production levels of TGF-β and VEGF in culture supernatant of HL-60 cells were determined by enzyme linked immunosorbent assay(ELISA).Peripheral blood mononuclear cells(PBMC) were collected by density gradient centrifugation,and co-cultured with HL-60 cells culture supernatant in vitro.The proliferation and apoptosis rates of lymphocytes were determined by MTT assay and flow cytometry,respectively.Results:After the siRNA targeting 4-1BBL gene was transfected into HL-60 cells,the expression levels of 4-1BBL gene and protein significantly decreased(P<0.01).The results of ELISA showed that the secretion levels of TGF-β and VEGF in culture supernatant of HL-60 cells significantly decreased after the 4-1BBL gene expression was interfered(P<0.01 and P<0.05).Compared with before interfering,the effects of the culture supernatant of HL-60 cells on inhibiting the proliferation and inducing apoptosis of lymphocyte decreased after interfering(P<0.05).Conclusions:The high expression of 4-1BBL in HL-60 cells has a reversely regulatory function.The production of TGF-β and VEGF in HL-60 cells can strengthen the inhibiting effect of tumor cell culture supernatant on lymphocyte activity,and promote the lymphocytes apoptosis,which can be an important mechanism of tumor cells escaping from the immune surveillance of tumor-bearing host.
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Key words:
- tumor immunity /
- 4-1BB ligand /
- siRNA /
- HL-60 cells /
- proliferation /
- apoptosis /
- immunity escape
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