研究Huh7細胞株中D型肝炎病毒抗原所誘導的基因及其相關的蛋白

Investigation of HDV antigen induced genes and associated proteins in Huh7 cells

蔡居政(Chu-Cheng Tsai)

指導教授 ： 許國堂

中山醫學大學/醫學院/醫學分子毒理學研究所/碩士(2007年)

https://doi.org/10.6834/CSMU.2007.00085

摘要

D型肝炎病毒 (Hepatitis Delta Virus，簡稱HDV)由一條1.7 kb的單股環狀RNA所構成。HDAg是經由HDV之中的Open Reading Frame (ORF)所轉譯的蛋白，可分為兩種不同的類型，Small-HDAg (S-HDAg)其分子量約為24 kD (195個胺基酸)，主要功能為參與 HDV RNA的複製；Large-HDAg (L-HDAg)其為27 kD (214胺基酸)大小，其功能為參與HDV病毒顆粒的組裝。先前我們實驗室在穩定表現L-HDAg的肝癌細胞株 (Lg stable clone)中發現clusterin的mRNA比不表現L-HDAg的肝癌細胞株要來的高，而我們也進一步證實分泌型clusterin (sCLU)蛋白在Lg stable clone細胞中及分泌到細胞外的表現量比在穩定表現S-HDAg的肝癌細胞 (Sm stable clone)及Huh7中要來的高，且含有較多sCLU的細胞對於adriamycin的感受性變得較低。而L-HDAg會增加細胞中histone H3的acetylation，進而誘導clusterin的表現，這說明了L-HDAg會去調控HATs或是HDACs的活性，可能因此而誘發細胞的癌化。另外我們也發現有將近九成 (7/8)的肝炎病人血清中sCLU較一般體檢病人高，這說明了sCLU似乎與肝炎具有正相關性。
我們實驗室先前用protein A sepharose binding assay和二維膠電泳找到hnRNP H1和S-HDAg有相關，雖然我們之後證實這兩個蛋白並沒有直接結合，但卻也看到在Lg stable clone及Sm stable clone的核蛋白中hnRNP H1表現量較Huh7的核蛋白要多。我們進一步利用了免疫沈澱法和二維膠電泳持續地找出更多在Lg stable clone、Sm stable clone及Huh7核蛋白之間有差異的蛋白，對於以後在HDV的研究上，可以提供一些新的想法以及更多的實驗方向。

關鍵字

D型肝炎病毒 ； D型肝炎病毒抗原 ； 二維電泳

並列摘要

The genome of hepatitis delta virus (HDV) is composed of a circular single-stranded RNA molecule of 1.7 kb. The gene of hepatitis delta antigen (HDAg) consists of two related proteins. The small form is a 24 kD (195 amino acids) of S-HDAg, which is essential for replication of HDV RNA genome. The large form is L-HDAg, which is a 27 kD (214 amino acids), that is essential for particle assembly of HDV RNA. We have found that mRNA level of clusterin of Huh7 cells stablely expressing L-HDAg (Lg stable clone) was higher than Huh7 cells not expressing HDAgs. Our first goal was to examine the regulation of clusterin expression by L-HDAg. Therefore, we demonstrated secretary form of clusterin (sCLU) in Lg stable clone and its medium are higher than Huh7 cells stablely expressing S-HDAg (Sm stable clone) and Huh7 cells. Cells with higher sCLU decrease sensitivity to ADR treatment. Because of L-HDAg enhances histone H3 acetylation and induces expression of clusterin protein, it indicates that L-HDAg regulates the activity of HATs or HDACs to induce clusterin expression. We also found that in about 90% (7/8) of patients with hepatitis, the level of sCLU in the serum was higher than normal control group.
The second goal was to examine the association hnRNP H1 with S-HDAg. Although we demonstrated hnRNP H1 did not bind to S-HDAg directly, the expression level of clusterin in Lg and Sm stable clone nuclear fractions are higher than Huh7 nuclear fraction. To further screen the HDAgs associated proteins, we prepared Huh7 nuclear fraction and Lg or Sm stable clone nuclear fractions for immunoprecipitation and two-dimensional electrophoresis analysis that may provide a new insight for HDV research.

並列關鍵字

HDV ； HDAg ； clusterin ； CLU ； sCLU ； hnRNP H1 ； 2D ； two dimensional electrophoresis

參考文獻

Bergmann KF, Gerin JL (1986) Antigens of hepatitis delta virus in the liver and serum of humans and animals. J Infect Dis 154: 702-706

Bonino F, Brunetto MR, Negro F, Smedile A, Ponzetto A (1991) Hepatitis delta virus, a model of liver cell pathology. Journal of hepatology 13: 260-266

Branch AD, Robertson HD (1984) A replication cycle for viroids and other small infectious RNA's. Science 223: 450-455

Brazas R, Ganem D (1996) A cellular homolog of hepatitis delta antigen: implications for viral replication and evolution. Science (New York, NY 274: 90-94

Brehm A, Nielsen SJ, Miska EA, McCance DJ, Reid JL, Bannister AJ, Kouzarides T (1999) The E7 oncoprotein associates with Mi2 and histone deacetylase activity to promote cell growth. The EMBO journal 18: 2449-2458