細菌基因組DNA含有許多未甲基化CpG motifs可活化脊椎動物的免疫系統，對脊椎動物免疫系統而言未甲基化CpG motifs可被視為危險訊號(danger signal) ，但未甲基化CpG motifs對無脊椎動物免疫系統作用的研究目前並不多。黑腹果蠅的血球細胞株Mbn-2 (malignant blood neoplasm-2) 是常用來研究昆蟲體外免疫反應的系統。先前研究顯示在先天性免疫中用來對抗細菌和真菌一般是藉由兩個NF-κB訊號傳導路徑 : Toll和IMD (immune deficiency) pathway，而Toll pathway主要是讓果蠅去對抗真菌和革蘭氏陽性菌的感染。本研究的主要目的為研究藉由E.coli DNA刺激後，果蠅是否經由Toll pathway啟動免疫反應。本研究設計帶有兩個T7 promoters的質體來產生dsRNA以靜默Toll後，經由dsRNA處理Mbn-2細胞後以即時定量PCR分析Toll表現差異，以及其對抗菌胜肽Attacin和Drosomycin表現之影響。可用來推測辨識E.coli DNA時，可能的訊息傳遞路徑。結果顯示轉染dsRNA可抑制Mbn-2細胞內源性的基因表現；當細胞經過dsTOLL干擾24小時，並受到LPS刺激，Toll表現量下降，且抗菌胜肽Drosomycin表現也會下降，由此得知當接收到LPS時，是以Toll pathway來啟動免疫反應；而當接收到E.coli DNA，對抗菌胜肽Attacin和Drosomycin表現無顯著差異，結果顯示目前尚無證據指出是以Toll pathway來啟動免疫反應。 Oligodeoxynucleotides (ODN) containing unmethylated Cytosine phosphorothioate guanine (CpG) motifs can activate innate immune responses in mammals. Unmethylated CpG motifs act as a danger signal in vertebrate. In invertebrate, the effects of unmethylated CpG motif are still not clear. Because NF-κB signal transduction pathways are highly conserved, Drosophila melanogaster provides a good model to study these cascades. Drosophila melanogaster cell line malignant blood neoplasm-2 (Mbn-2) was explored as a model system to study insect immune responses in vitro. Previous studies showed that innate immunity against bacteria and fungi is governed largely by two NF-κB signal transduction pathways, Toll and immune deficiency (IMD). The aim of the study is to investigate whether the stimulation of CpG DNA in Drosophila goes through the Toll pathway. In order to silence Toll, we designed a plasmid with two T7 promoters to product dsRNA (dsTOLL). The expression of Toll in Drosophila cell line Mbn-2 after dsRNA treatment was analyzed by real-time RT-PCR. Furthermore, the expression of antimicrobial peptide Attacin and Drosomycin were also examined to investigate the possible signal transduction pathway of LPS and E.coli DNA recognition.Our results indicated that the transfection of dsTOLL can be used in Drosophila cell line Mbn-2 to disrupt function of endogenous genes in vitro. The dsTOLL decreased the expression of Toll and Drosomycin after treated with LPS. These results showed the stimulation signal from LPS get through the Toll pathway in Drosophila. The expression of Attacin and Drosomycin in dsTOLL treated cells has no significant different after E.coli DNA stimulation. However, there is no evidence to show the recognition of E.coli DNA in Mbn-2 cells signaling by Toll pathway.
