論文名稱:

口腔細胞中皰疹病毒進入調控因子的表現及單純皰疹病毒感染後對口腔細胞之影響

 

Expression of herpesvirus entry mediators in oral cells and cellular changes of oral cells of herpes simplex virus infection

研究生:

鄭宜芸  Yi-Yun Cheng

 

(以作者名查詢陽明大學館藏系統)

 

(以作者名查詢全國圖書書目資訊網)

指導教授:

洪善鈴  Shan-Ling Hung

        學位類別:

碩士

        學校名稱:

國立陽明大學

系所名稱:

口腔生物研究所

            學號:

g880h01

          學年度:

89

          語文別:

中文

          出版年:

90

關鍵字:

單純皰疹病毒  herpes simplex virus

 

皰疹病毒進入調控因子  viral entry mediators

 

角化上皮細胞  oral keratinocytes

 

牙齦纖維母細胞  gingival fibroblasts

 

細胞激素  cytokine

全文說明:

(全文限校內瀏覽)

 

電子全文

        論文頁數:

82

摘要:

摘要    單純皰疹病毒(herpes simplex virusHSV)感染人體後可能引起齦口炎(gingivostomatitis)、唇皰疹(herpes labialis)、角膜性結膜炎(keratoconjunctivitis)、無菌性腦膜炎(aseptic meningitis)及生殖器皰疹(genital herpes)等疾病。HSV可依其血清型分為HSV-1HSV-2HSV先藉由病毒蛋白C(glycoprotein CgC)gB與細胞膜上的肝燐脂類物質(heparan sulfate proteoglycan)結合,再藉由gD與細胞膜上接受器(receptor)作用,並藉由gBgH-gL的協同作用而侵入細胞。數種細胞膜上接受器可以調控病毒進入細胞,包括:皰疹病毒進入調控因子(herpesvirus entry mediator) HveAHveBHveCHveD3-O-sulfated heparan sulfateHSV-1主要會造成口咽之感染,然而探討HSV進入口腔細胞機轉及口腔細胞受HSV感染後細胞激素分泌之改變的研究較缺乏。本論文比較HSV-1病毒進入初代培養的口腔角化細胞(keratinocyteKC)及牙齦纖維母細胞(gingival fibroblastGF)之能力,在HSV-1感染口腔細胞的過程中,KC受病毒感染後造成的細胞病理性變化比GF明顯,且病毒感染KC後產生的病毒量比GF多。以反轉錄脢連鎖反應(reverse transcriptase-polymerase chain reactionRT-PCR)探討KCGF二種口腔細胞中調控因子HveAHveBHveC mRNA的表現情形,同時以免疫化學染色法鑑定HveAHveC蛋白質在初代培養的KCGF及牙齦組織切片的表現。結果顯示,初代培養的KCGF皆有調控因子HveAHveBHveC mRNA的表現;此外,比較mRNA及蛋白質的表現,HveAKCGF的差別不大,但HveCKC的表現比GF來的明顯。牙齦組織切片染色結果得知,上皮細胞明顯表現HveAHveC二種調控因子,結締組織中纖維母細胞有微弱的表現。此外,利用酵素連結免疫吸附定量法(enzyme-linked immunosorbent assayELISA)偵測病毒感染KCGF後,腫瘤壞死因子 (tumor necrosis factor TNF-)間白素6 (interleukin-6IL-6)及間白素8 (interleukin-8IL-8)等細胞激素分泌之變化。結果得知,TNF-KCGF的分泌量不明顯,且不受病毒感染所影響;而隨著病毒感染時間增加,GF分泌IL-6的量隨著增加,但KC不管是否有病毒感染,皆未分泌IL-6;另外IL-8KCGF受病毒感染後的分泌量皆受抑制,明顯比未受病毒感染的細胞低。由本論文實驗得知,口腔細胞中確實有調控病毒進入因子可調控病毒進入口腔細胞,且在KCGF間具有差異性。對於病毒感染與口腔細胞之間的交互作用引起細胞激素調控的影響也有差異性存在。

   
 

Abstract    Herpes simplex virus (HSV) can cause several human diseases, such as gingivostomatitis, herpes labialis, keratoconjunctivitis, aseptic meningitis and genital herpes. HSV can be divided into two serotypes: HSV-1 and HSV-2. HSV initially binds heparan sulfate proteoglycan on the cell surface via glycoprotein C (gC) or glycoprotein B (gB). Viral gD then interacts with receptors on the membrane, cooperates with gBz gH-gL and penetrates into cells. These specific receptors, which regulate viral entry, are herpesvirus entry mediatorsA (HveA), HveB, HveC, HveD and 3-O-sulfated heparan sulfate. HSV-1 mainly infects oropharyngeal mucosa. However, there are little studies on HSV entry mechanisms and the changes of cytokine secretion in oral cells after HSV infection. This study compared HSV-1 entry into primary cultured keratinocytes (KC) and gingival fibroblasts (GF). The results showed that the pathologic changes of KC shown are more obvious than GF after HSV-1 infection. The viral titer produced by infected KC was higher than by infected GF. The mRNA expression of HveA, HveB and HveC was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in KC and GF, and the protein expression of HveA and HveC in KC and GF or in gingival tissue sections was examined by immunochemical analysis. The results showed that the mRNA of HveA, HveB and HveC was expressed in KC and GF. Expression of HveA was similar in KC and GF. Expression of HveC in KC was higher than in GF. The staining of HveA and HveC was pronounced in the epidermis, but was weak in fibroblasts in gingival tissues. The possible changes of tumor necrosis factor- (TNF-), interleukin-6 (IL-6) and interleukin-8 (IL-8) in infected KC and GF were also examined by enzyme-linked immunosorbent assay (ELISA). The data showed that TNF- secretion was not obvious in KC and GF. Expression of TNF- in KC and GF was not affected by viral infection. Secretion of IL-6 was enhanced as infection time increased in GF, but IL-6 expression was not detected in KC whether cells were infected or not. On the other hand, secretion of IL-8 in KC and GF was inhibited after infection when compared to mock-infected cells. In conclusion, the results suggest that several cellular receptors were expressed in oral cells. Difference between KC and GF were observed. Differences in cytokine production in KC and GF after HSV-1 infection were demonstrated.

   

       發表著作

Hung S-L, Cheng Y-Y, Wang Y-H, Chang K-W, Chen Y-T. Expression and roles of herpesvirus entry mediators A and C in cells of oral origin. Oral Microbiology and Immunology 2002; 17: 215-223

Chen Y-T, Wang Y-H, Cheng Y-Y, Hung S-L. Direct binding of herpes simplex virus type 1 virions to complement C3. Viral Immunology 2003; 16(3): 347-355

Teng Y-C, Chen Y-T, Cheng Y-Y, Hung S-L. Requirements for the upregulation of interleukin-6 by herpes simplex virus-infected gingival fibroblasts. Viral Immunology 2005; 18(1): 170-178

Hung S-L, Cheng Y-Y, Peng J-L, Chang L-Y, Liu T-Y, Chen Y-T. Inhibitory effects of areca nut extracts on phagocytosis of Actinobacillus actinomycetemcomitans ATCC 33384 by neutrophils. Journal of Periodontology 2005; 76(3): 373-379

Hung S-L, Lee Y-Y, Liu T-Y, Peng J-L, Cheng Y-Y, Chen Y-T*. Modulation of phagocytosis, chemotaxis and adhesion of neutrophils by areca nut extracts. Journal of Periodontology 2006; 77(4): 579-585.