Bergant Martina, Meden Luka, Repnik Urska, Sojar Valentin, Stanisavljević Dragoje, Jeras Matjaz
Tissue Typing Center, Blood Transfusion Centre of Slovenia, Ljubljana, Slajmerjeva 6, SI-1000 Ljubljana, Slovenia.
Immunobiology. 2006;211(3):179-89. doi: 10.1016/j.imbio.2005.08.008. Epub 2006 Feb 7.
Recent research indicates that dendritic cells transfected with RNA-encoded tumour-associated antigens (TAA) can generate potent anti-tumour immune responses in vitro and in vivo. RNA is an important source of TAA, but its relatively unstable nature, in addition to often limited availability of tumour tissue, may represent a considerable obstacle for its use. Our first goal was to establish an efficient protocol for the preparation of high quality total RNA from tumour samples. This should then be used as such or be pre-amplified for DC transfection. Therefore native total RNA was prepared from stabilised tissue samples obtained from liver metastases of colon cancer using either solution- or silicagel-based protocols for RNA isolation. The first isolation protocol yielded higher amounts of total RNA, but with lower purity as compared to the second one. No degradation of RNA was observed regardless of the protocol used. Subsequently, we focused on the amplification of mRNA. The fidelity of the amplified mRNA was confirmed by RT-PCR for glyceraldehyde-3-phosphate-dehydrogenase (GADPH) and carcinoembryonic antigen (CEA) coding sequences. We found no differences in the induction of CEA-specific CTL responses between native and amplified RNA-transfected DCs. Additionally, we tested the induction of CTL responses and found that DCs transfected with amplified mRNA originating from either tumour tissue or a cell line were able to induce strong anti-tumour CTL responses in vitro. They were comparable to those induced by native total RNA-transfected DCs. Our results therefore indicate that the amplified mRNA is equivalent to the native one in the induction of anti-tumour CTL responses and can be used for generation of RNA-transfected DCs.
最近的研究表明，用RNA编码的肿瘤相关抗原（TAA）转染的树突状细胞可在体外和体内产生强大的抗肿瘤免疫反应。RNA是TAA的重要来源，但其相对不稳定的性质，加上肿瘤组织的可用性往往有限，可能是其应用的一个重大障碍。我们的首要目标是建立一种从肿瘤样本中制备高质量总RNA的有效方案。然后应直接使用该总RNA或对其进行预扩增以用于树突状细胞转染。因此，使用基于溶液或硅胶的RNA分离方案，从结肠癌肝转移获得的稳定组织样本中制备天然总RNA。与第二种方案相比，第一种分离方案产生的总RNA量更高，但纯度较低。无论使用哪种方案，均未观察到RNA降解。随后，我们专注于mRNA的扩增。通过逆转录聚合酶链反应（RT-PCR）检测甘油醛-3-磷酸脱氢酶（GADPH）和癌胚抗原（CEA）编码序列，证实了扩增mRNA的保真度。我们发现，天然RNA和扩增RNA转染的树突状细胞在诱导CEA特异性细胞毒性T淋巴细胞（CTL）反应方面没有差异。此外，我们测试了CTL反应的诱导情况，发现用源自肿瘤组织或细胞系的扩增mRNA转染的树突状细胞能够在体外诱导强烈的抗肿瘤CTL反应。它们与天然总RNA转染的树突状细胞诱导的反应相当。因此，我们的结果表明，扩增的mRNA在诱导抗肿瘤CTL反应方面与天然mRNA相当，可用于生成RNA转染的树突状细胞。
