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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。
OBJECTIVE: To evaluate the antitumor efficacy of a combination of interferon alpha (IFN-alpha) and TT-1, an analog of melittin, and to study the antitumor and immunoregulatory mechanisms of the combination. Innovation: In order to enhance the antitumor effect of melittin, our research group modified it and synthesized a new compound TT-1. This study, for the first time, used the combination of the melittin analogue and the immunological cytokine IFN-α and confirmed by experiments that the anti-tumor effect of TT-1 can be enhanced by activating immunomodulation. Methods: The inhibitory effect of TT-1 on proliferation of HepG-2 / Huh7 cells was verified by MTT assay. Then the model of HepG-2 / Huh7 mouse xenografts was established to investigate the antitumor effect of TT-1 + IFN-α in vivo. Natural killer (NK) cells were eliminated by anti-asialo GM- The key role. The effect of TT-1 on MHC I chain-related molecule A (MICA) expression in HepG-2 / Huh7 cells was verified by flow cytometry and enzyme-linked immunosorbent assay (ELISA). Real-time polymerase chain reaction Western blot was used to investigate its mechanism. Cytotoxicity assay was used to investigate whether TT-1 + IFN-α can enhance the specific killing effect of NK cells on HepG-2 / Huh7 cells. Finally, the effect of TT-1 + IFN-α on the expression of MICA and NKG2D in tumor tissue was investigated by immunohistochemistry. Conclusion: MTT assay showed that TT-1 can effectively inhibit the proliferation of HepG-2 / Huh7 cells in vitro. The experimental results of mouse xenograft model showed that the combination of TT-1 + IFN-α could inhibit the growth of HepG-2 / Huh7 transplanted tumor more effectively than TT-1 alone, but the effect was obviously weakened after eliminating NK cells. The antitumor effect of TT-1 + IFN-α is mediated by NK cells. TT-1 can not only upregulate the expression of MICA on the surface of tumor cells, but also decrease the secretion of soluble MICA. Further studies showed that TT-1 can inhibit the expression of MICA from the surface of tumor cells by inhibiting the expression of ADAM 10 Off. Cytotoxicity experiments showed that TT-1 + IFN-α can significantly enhance the killing effect of NK cells on HepG-2 / Huh7 cells. The results of immunohistochemistry showed that the combination of TT-1 + IFN-α could obviously increase the expression of NKG2D on MICA and NK cells on the surface of tumor cells. In conclusion, the combination of TT-1 + IFN-α can achieve significant antitumor effects by enhancing the interaction between MICA and NKG2D.