幽門螺旋桿菌感染胃組織細胞所引發基因差異表現之研究

Abstract

本研究利用mRNA差異表現法(DDRT-PCR)找尋當胃細胞被幽門螺 旋桿菌感染後,其基因表現上的改變。將總RNA從感染與未感染的細胞中萃 取出來之後,以所篩選之cDNA做為探針,用北方墨點法確定是否有差異表現 。總共篩選了43個不同的arbitrary primer後,發現了4個cDNA探針可測定 基因表現差異：04G-1, 04G-2, 01G-1, CPPA-2片段,它們的長度分別 為199bp, 242bp, 228bp, 276bp。在現存的基因資料庫中,這4條cDNA片段 僅CPPA-2與PTI-1的致癌基因有83%的相似性,其他皆無發現相似性的基因, 也因此確定了影響04G-1與04G-2這兩條基因改變的誘導物質是位於幽門螺 旋桿菌的表面。而04G-1的表現在2小時就達到最高點,04G-2卻要到8小時 。04G-1僅在AGS和Hct-15中被影響而表現量增加,04G-2卻在AGS, Hct-15, Raji, HepG2都可被影響而增加。這些結果,顯現當細胞被幽門螺旋桿菌感 染後,會造成基因表現上的改變,也證明了：可以利用mRNA差異表現法來找 尋當細胞被幽門螺旋桿菌感染後所造成基因表現上的改變,若能得到這些 基因之完整序列,並表現出來且研究其特性,相信能更進一步對於幽門螺旋 桿菌的致病原因有更進一步的瞭解。 Changes in genes expression in gastric cells infected with Helicobacterpylori were identified by means of mRNA differential display. Total RNA preparationswere extracted from the Helicobacter pylori infected gastric cells and paired non- infectedcells, and were probed with candidate clones identified after screening up to 43 arbitraryprimers. Among them, four clones, 04G-1, 04G-2, 01G-1, CPPA-2 detected significant changesin gene expression in the infected cells by Northern blot analysis. The four probes are199bp, 242bp, 228bp, 276bp in length, respectively. Database search revealed these nucleotide sequences of these clones share very low identity with any known sequence.And it also identified that the proteins on the Helicobacter pylori's surface are the major inducers caused the change in gene expression using 04G-1 and 04G-2 as probes.When treating gastric cells with Helicobacter pylori lysate for 2, 4, 8, 12, 24 hours, it can find that 04G-1 probed gene was expressed at highest level at 2 hours and 04G-2probed gene was at 8 hours. It also identified that 04G-1 probed gene can be induced onlyin AGS and Hct-15 cells but 04G-2 probed gene can be induced in AGS, Raji, Hct-15, and HepG2 cells respectively. These results indicate that Helicobacter pylori infection can significantly affect gene expression in gastric cells. Furthermore, method of mRNA differential display can be used in pathogenesis studies to identify novel genes in gastric cells in response to insult such as Helicobacter pylori.