联合使用IFN-α和TT-1通过增强NKG2D和MICA的相互作用达到抗肝癌效果的研究（英文）

发布时间：2018-06-22 20:02

本文选题：TT- + 干扰素α　；参考：《Journal of Zhejiang University-Science B(Biomedicine & Biotechnology)》2017年06期

【摘要】：目的:评估干扰素α(IFN-α)和TT-1(一种蜂毒肽的类似物)联合用药的抗肿瘤效果,并初步研究联合用药的抗肿瘤及免疫调节机制。创新点:为了增强蜂毒肽的抗肿瘤效果,本课题组在其基础上进行改造,合成了一种新的化合物TT-1。该研究第一次将蜂毒肽类似物和免疫细胞因子IFN-α联合使用,并通过实验证实联合用药可以通过激活免疫调节来增强TT-1的抗肿瘤效果。方法:首先通过MTT实验验证TT-1对HepG-2/Huh7细胞的增殖抑制作用。接着建立HepG-2/Huh7小鼠移植瘤模型,考察TT-1+IFN-α的体内抗肿瘤效果;使用anti-asialo GM-1抗体消除自然杀伤(NK)细胞,验证NK细胞在联合用药中的关键作用。使用流式细胞术和酶联免疫吸附法(ELISA)验证TT-1对HepG-2/Huh7细胞MHC I链相关分子A(MICA)表达的影响,并用实时聚合酶联反应(RT-PCR)和蛋白质印迹(Western blot)对其机制进行探究;通过细胞毒性实验考察TT-1+IFN-α是否可以增强NK细胞对HepG-2/Huh7细胞的特异性杀伤作用。最后使用免疫组化的方法考察TT-1+IFN-α联合用药对肿瘤组织中MICA和NKG2D的表达量的影响。结论:MTT实验表明TT-1可以在体外有效地抑制HepG-2/Huh7细胞的增殖。小鼠移植瘤模型实验结果显示TT-1+IFN-α联合用药比TT-1单独给药更能有效地抑制HepG-2/Huh7移植瘤的生长,但是在消除NK细胞之后该效应明显减弱,说明TT-1+IFN-α的抗肿瘤效应是通过NK细胞特异性介导的。TT-1不仅可以上调肿瘤细胞表面MICA的表达量,而且可以减少可溶性MICA的分泌;进一步研究表明,TT-1通过抑制去整合素金属蛋白酶10(ADAM 10)的表达来阻止MICA从肿瘤细胞表面脱落。细胞毒性实验表明,TT-1+IFN-α可以显著增强NK细胞对HepG-2/Huh7细胞的杀伤作用。免疫组化实验结果显示,TT-1+IFN-α联合用药可以明显增加肿瘤组织中肿瘤细胞表面MICA和NK细胞NKG2D的表达量。综上所述,TT-1+IFN-α联合用药可以通过增强MICA和NKG2D的相互作用达到显著的抗肿瘤效果。
[Abstract]:Aim: to evaluate the antitumor effect of interferon 伪 (IFN- 伪) and TT-1 (an analogue of propolis peptide), and to study the mechanism of anti-tumor and immunomodulation. Innovation: in order to enhance the antitumor effect of propolis peptide, a new compound TT-1 was synthesized. In this study, the propolis peptide analogue and IFN- 伪 were used for the first time, and the experimental results showed that the combination therapy could enhance the anti-tumor effect of TT-1 by activating immunomodulation. Methods: MTT assay was used to test the inhibitory effect of TT-1 on the proliferation of HepG-2 / Huh7 cells. Then the transplanted tumor model of HepG-2 / Huh7 mice was established to investigate the anti-tumor effect of TT-1 IFN- 伪 in vivo, and to use anti-asialo GM-1 antibody to eliminate natural killer (NK) cells, to verify the key role of NK cells in combination therapy. Flow cytometry and enzyme-linked immunosorbent assay (Elisa) were used to examine the effect of TT-1 on the expression of MHC I chain associated molecule A (MICA) in HepG-2 / Huh7 cells. The mechanism of TT-1 was investigated by real-time polymerase chain reaction (RT-PCR) and Western blotting (Western blot). To investigate whether TT-1 IFN- 伪 can enhance the specific cytotoxicity of NK cells to HepG-2 / Huh7 cells by cytotoxicity test. Finally, the effects of TT-1 IFN- 伪 on the expression of MICA and NKG2D in tumor tissues were investigated by immunohistochemical method. Conclusion in vitro, TT-1 can effectively inhibit the proliferation of HepG-2 / Huh7 cells. The results of mouse transplanted tumor model showed that TT-1 IFN- 伪 could inhibit the growth of HepG-2 / Huh7 transplanted tumor more effectively than TT-1 alone, but the effect of TT-1 IFN- 伪 on the growth of HepG-2 / Huh7 transplanted tumor was weakened after NK cells were eliminated. The results showed that the anti-tumor effect of TT-1 IFN- 伪 was mediated by NK cells. TT-1 could not only up-regulate the expression of MICA on tumor cells, but also reduce the secretion of soluble MICA. Further studies have shown that TT-1 inhibits the expression of deintegrin metalloproteinase 10 (ADAM10) to prevent MICA from falling off the surface of tumor cells. Cytotoxicity test showed that TT-1 IFN- 伪 could significantly enhance the cytotoxicity of NK cells to HepG-2 / Huh7 cells. The results of immunohistochemistry showed that TT-1 IFN- 伪 could significantly increase the expression of MICA and NKG2D on tumor cells. In conclusion, TT-1 IFN- 伪 can enhance the interaction between MICA and NKG2D to achieve significant antitumor effect.
【作者单位】： Department
【基金】：supported by the Outstanding Young Talent Foundation Project of Science and Technology Department in Jilin Province(No.20170520018JH),China
【分类号】：R96

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