- 學位論文
腸炎沙門氏桿菌感染雞顆粒細胞誘發之基因時序表現研究
A Time-Course Study of Gene Responses in Chicken Granulosa Cells Infected by Salmonella Enteritidis
摘要
食用被腸炎沙門氏桿菌（Salmonella Enteritidis）污染的蛋品已經被認為是人類感染食物媒介沙門氏桿菌症主要原因之一，每年於世界各地引起許多的病例爆發，造成嚴重的經濟損失。而雞顆粒細胞（chicken granulosa cell, CGC）是排卵前濾泡中最靠近卵黃的組織層，為腸炎沙門氏桿菌入侵及增殖偏好的位置。因此本研究利用雞全基因微陣列晶片分析體外培養的雞顆粒細胞感染腸炎沙門氏桿菌後在不同時序的基因表現變化，以了解雞顆粒細胞對於感染的反應。雞顆粒細胞感染腸炎沙門氏桿菌後與未感染腸炎沙門氏桿菌的基因表現比較，共有214個基因表現量顯著地改變(p<0.01)。根據基因於感染後4小時及48小時的表現量變化，分成 1a、1b、1c、2a及2b五個子族群，分別包含70個基因(32.71%)、27個基因(12.62%)、66個基因(30.84%)、41個基因(19.16%)及10個基因(4.67%)。這些改變的基因參與免疫反應、生理調控及訊息傳遞的功能，以及部分目前未知其功能。另外，參與調控細胞激素與生長因子的 JAK-STAT pathway亦被高度活化，其中IL-6、cytokineR、STAT、PI3K四個部分有明顯變化，均屬於JAK-STAT pathway前半部。於微陣列晶片檢測結果表現量上升的基因中，與免疫反應調控相關的TLR15、IL-6、CXCLi1、CXCLi2及K203，其mRNA於兩個時間點均表現量上升，以即時定量反轉錄聚合酶連鎖反應檢測亦得到上升的結果。而與生理反應調控相關的RASD1及HB-EGF以即時定量反轉錄聚合酶連鎖反應與微陣列晶片檢測其表現量均下降。根據本實驗結果得知：雞顆粒細胞感染腸炎沙門氏桿菌後，短時間內即能夠吸引先天性免疫反應的細胞，然而長時間其免疫反應會被抑制。而且雞顆粒細胞細胞生長受到抑制，進而影響濾泡發育及排卵。其中化學激素（chemokine）及旁分泌（paracrine）扮演相當重要的角色。
並列摘要
Consumption of Salmonella Enteritidis (SE)-contaminated eggs has been recognized as one of the important reasons of human food-borne salmonellosis. Chicken granulosa cells (CGC), last tissue layer surrounding the yolk in prevulatory follicles has been demonstrated a preferred site for the Salmonella Enteritidis invasion. To understand how the chicken granulosa cell responds to the infection, a time-course in-vitro study was conducted to identify transcription changes of chicken granulosa cell using chicken whole genome microarrays. The expression of 214 genes of chicken granulosa cell had altered (p<0.01) when compared to those of uninfected cells after the infection. According to the different responses of the altered gene between 4hpi and 48hpi, they are divided into cluster 1a, 1b, 1c, 2a, and 2b those include 70 genes (32.71%), 27 genes (12.62%), 66 genes (30.84%), 41 genes (19.16%), and 10 genes (4.67%) respectively. Among of them, many were related to immune response, physiological process, signal transduction activity, transcription and some of unknown function. We also noted that the JAK-STAT pathway which is essential in regulation of cellular cytokines and growth factors being highly active those are IL-6, cytokineR, STAT,and PI3K in the upstream of this pathway. Among the up-regulated genes, the mRNA levels of TLR15, IL-6, CXCLi1, CXCLi2 and K203 at the two time points from the infected cells were also increased by the validation of real-time RT-PCR (RRT-PCR). However the mRNA expression of RASD1 and HB-EGF decreased according to the microarray and RRT-PCR analyses. These results suggest that during the Salmonella Enteritidis infection, chicken granulosa cell are capable of recruiting cells of innate immune responses in short term. In long term, the growth of chicken granulosa cell is suppressed and then the development and ovulation of follicles is also suppressed. Among of them, the chemokine and paracrine play the important role.