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目的前期制备并鉴定了Tat-E7/pORF-mGM-CSF颗粒疫苗,并在小鼠体内观察到良好的抗肿瘤效果,但其体内具体分子机制不清。本研究拟进一步利用该疫苗免疫小鼠,分析其体内激活T细胞应答的分子机制。方法通过免疫组化检测该纳米颗粒能否将外源基因转入哺乳动物细胞并在动物活体内正确表达;通过CCK-8淋巴细胞增殖实验、细胞内染色及流式细胞术(FCM)、ELISPOT实验、体外特异性细胞毒CTL-LDH释放实验分析疫苗诱导的短期及记忆性免疫应答和其介导的CTL毒性效应。结果当r=2.0时所制备的颗粒为类圆形,大小均匀一致,绝大部分颗粒直径均分布于20~80 nm之间;该纳米颗粒可将mGM-CSF基因导入哺乳动物细胞,并可在体外培养细胞和小鼠体内正确表达。该纳米颗粒疫苗在体外和体内均很大程度上提高了抗原表位特异性的免疫原性,可诱导T淋巴细胞增殖、分化,显著增强抗原特异性CTL应答及其产生的细胞毒效应。结论我们在本研究中设计并合成的Tat-E7/pGM-CSF纳米颗粒疫苗,通过体外免疫学研究证实了其具有良好效果,其机制可能是该颗粒疫苗的免疫可以维持高水平的特异性CD8~+记忆T细胞。
OBJECTIVE To prepare and identify Tat-E7 / pORF-mGM-CSF particle vaccine and to observe the good anti-tumor effect in mice. However, its specific molecular mechanism in vivo is unclear. In this study, the vaccine was further used to immunize mice to analyze the molecular mechanism of T cell activation in vivo. Methods Immunohistochemistry was used to detect whether the nanoparticle was transfected into mammalian cells and correctly expressed in the animal. CCK-8 lymphocyte proliferation assay, intracellular staining and flow cytometry (FCM), ELISPOT In vitro and in vitro cytotoxic CTL-LDH release assays were used to analyze the vaccine-induced short-term and memory immune responses and their CTL toxic effects. Results When r = 2.0, the prepared particles were round and uniform in size. Most of the particles were distributed between 20 and 80 nm in diameter. The nanoparticles could be introduced into mammalian cells by mGM-CSF gene. In vitro cultured cells and mice correctly expressed. The nanoparticle vaccine greatly enhanced antigen epitope specific immunogenicity in vitro and in vivo, induced T lymphocyte proliferation and differentiation, and significantly enhanced the antigen-specific CTL response and the cytotoxic effect thereof. Conclusions The Tat-E7 / pGM-CSF nanoparticle vaccine designed and synthesized in this study has been proved to be effective by in vitro immunological studies. The mechanism may be that the immunization of the particle vaccine can maintain a high level of specific CD8 ~ + Memory T cells.