TLR9信号通路介导缺血性脑损伤的研究进展

炎症反应是缺血性脑损伤的重要机制之一,过度的炎症免疫反应会加剧脑损伤的程度。作为先天免疫系统的重要组成部分,Toll样受体9(Toll like receptor 9,TLR9)通过识别其特异性的配体Cp G-DNA,从而激活先天免疫系统,释放大量前炎症细胞因子,参与缺血性脑损伤的炎症反应过程。近年来,有学者提出将TLR9作为一个新的缺血预处理靶点,即通过启动TLR9信号转导通路增加体内炎症细胞因子的水平来对抗缺血损伤。本文通过对近年TLR9信号通路介导的炎症反应与缺血性脑损伤相关研究进展作一综述,为寻求临床安全高效的缺血性脑损伤防治措施提供理论依据。     

DNA-PKcs, but not TLR9, is required for activation of Akt by CpG-DNA

CpG-DNA and its related synthetic CpG oligodeoxynucleotides (CpG-ODNs) play an important role in immune cell survival. It has been suggested that Akt is one of the CpG-DNA-responsive serine/threonine kinases; however, the target protein of CpG-DNA that leads to Akt activation has not been elucidated. Here, we report that ex vivo stimulation of bone marrow-derived macrophages (BMDMs) from mice lacking the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs) results in defective phosphorylation and activation of Akt by CpG-DNA. Unexpectedly, loss of the Toll-like receptor 9 has a minimal effect on Akt activation in response to CpG-DNA. Further in vitro analysis using purified DNA-PK and recombinant Akt proteins reveals that DNA-PK directly induces phosphorylation and activation of Akt. In addition, in BMDMs, DNA-PKcs associates with Akt upon CpG-DNA stimulation and triggers transient nuclear translocation of Akt. Thus, our findings establish a novel role for DNA-PKcs in CpG-DNA signaling and define a CpG-DNA/DNA-PKcs/Akt pathway.     

Expression of human β-defensin-2 gene induced by CpG-DNA in human B cells

Defensins have a broad range of antimicrobial activity against bacteria, fungi, and viruses. The expression of human β-defensin-2 (hBD-2) is prevalently observed in epithelial cells and is induced by bacterial infection. Here, we have shown that the expression of the hBD-2 gene and release of hBD-2 protein into the medium is up-regulated in response to CpG-DNA in human B cell line RPMI 8226. The induction of hBD-2 was dependent on CG sequence and phosphorothioate backbone-modification. This was also confirmed in primary human lymphocytes. To shed light on the molecular mechanism involved in hBD-2 induction by CpG-DNA, we examined the contribution of the NF-κB signaling pathway in RPMI 8226 cells. Suppression of MyD88 function and inhibition of NF-κB nuclear localization blocked hBD-2 induction. The NF-κB pathway inhibitors also abolished hBD-2 induction. These results may contribute to a better understanding on the therapeutic effects of CpG-DNA against infectious diseases.     

CpG-DNA活性的影响因素

细菌DNA疫苗的发现引发了对免疫刺激序列CpG-DNA的研究。CpG-DNA被细胞吞噬后与胞内受体结合,引发信号转导,产生免疫应答,从而发挥抗病毒、抗肿瘤免疫作用。针对该过程中影响CpG-DNA活性的诸多因素,从核心结构、骨架结构及信号转导等进行综述,希望对合理有效的DNA疫苗的构建有所帮助。     

TLR9的特性及其介导的TLR9/CpG-DNA免疫信号通路

TLRs在天然免疫应答中发挥着重要作用,构成了机体抗感染的第一道防线,是沟通天然免疫和获得性免疫的桥梁。其中TLR9已经被证实能够识别细菌DNA中免疫刺激序列CpG,从而激活哺乳动物细胞的天然免疫机制,这一发现具有重要的生物学意义与应用价值。它不仅进一步推进了外源DNA激活哺乳动物抗感染天然免疫的进一步研究,更为CpG-DNA应用于抗感染、肿瘤和免疫缺陷疾病等的治疗提供新的思路,为改进DNA疫苗效果提供非常有益的帮助。     

Bioluminescence-Based Tumor Quantification Method for Monitoring Tumor Progression and Treatment Effects in Mouse Lymphoma Models

Although bioluminescence imaging (BLI) shows promise for monitoring tumor burden in animal models of cancer, these analyses remain mostly qualitative. Here we describe a method for bioluminescence imaging to obtain a semi-quantitative analysis of tumor burden and treatment response. This method is based on the calculation of a luminoscore, a value that allows comparisons of two animals from the same or different experiments. Current BLI instruments enable the calculation of this luminoscore, which relies mainly on the acquisition conditions (back and front acquisitions) and the drawing of the region of interest (manual markup around the mouse). Using two previously described mouse lymphoma models based on cell engraftment, we show that the luminoscore method can serve as a noninvasive way to verify successful tumor cell inoculation, monitor tumor burden, and evaluate the effects of in situ cancer treatment (CpG-DNA). Finally, we show that this method suits different experimental designs. We suggest that this method be used for early estimates of treatment response in preclinical small-animal studies.