B型肝炎病毒（hepatitis B virus, HBV）是目前已知最小的DNA病毒，具有外套膜（envelope），屬於Hepadnaviridae家族的一種，它的基因體是不完全雙股的DNA，全長約為3.2kb。當被HBV感染之後，一部份的人會變成慢性感染，成為慢性HBV的帶原者，並成為好發肝癌及肝硬化的高危險群。HBV pre-S/S基因可經轉錄及轉譯產生的三種組成病毒外套膜的蛋白，分別稱為大、中、小表面蛋白（LHBs, MHBs, SHBs）。目前已有許多表面抗體的自然突變種自慢性感染者的血清或肝組織分離出來。我們的實驗室過去自兩種毛玻璃狀肝細胞（ground glass hepatocyte, GGH）發現不同的表面基因pre-S區域的中間片段缺失突變種，包涵體型的毛玻璃狀肝細胞（type I GGHs）主要在pre-S1核酸3040-3111的區域有片段缺失突變，稱為deletion I型（S1），其所產生的的中間片段缺失的突變LHBs稱為S1-LHBs；而另一種邊緣型的毛玻璃狀肝細胞（type II GGHs）則是在pre-S2核酸4-57的區域有片段缺失，且伴隨著中表面抗原轉譯起始點序列由ATG變成ATA，稱為deletion II型（S2），其所產生的中間片段缺失突變的LHBs稱做S2-LHBs，並且其MHBs不會表現。這些毛玻璃狀肝細胞中的突變表面蛋白主要聚集在內質網中，在in vitro的實驗也證明了，這些pre-S突變表面蛋白在肝癌細胞株中表現會堆積在內質網並且抑制表面蛋白由細胞分泌出去。本研究在探討這些突變蛋白堆積在內質網中所造成型態學及分子生物學變化。首先利用免疫染色及綠色螢光蛋白融合蛋白(GFP fusion protein)的實驗方法來看其在細胞內表現的位置，結果顯示野生型(wild type, WT)表面蛋白在細胞質中較為均勻分散狀，而S1及S2突變表面蛋白則呈點狀(blot)聚集的特徵，且S1隨時間增加會有較大的包涵體出現。在電子顯微鏡的觀察方面也證實表現這些pre-S突變蛋白造成內質網的增生，且隨之出現內質網被撐大現象而形成空泡狀(vesicle)。轉染pre-S突變蛋白於肝癌細胞株證實了內質網壓力（ER stress）的指標蛋白GRP78及GRP94的表現上升以及細胞內鈣離子濃度增加，以S1所造成的內質網壓力比S2強。但在這些蛋白表現的細胞並沒有看到有死亡的情形出現，且不會抑制MHC class I complex 在細胞膜上的表現。Pre-S突變表面蛋白引起的內質網壓力反應對Hsp60, Hsp70, Hsp90, Erp72及Erp57等伴護蛋白(chaperones)沒有影響。所以pre-S突變蛋白藉由堆積滯留在內質網中造成內質網壓力，可能是藉由釋放內質網內的鈣離子來增加細胞質鈣離子的濃度，造成細胞內ROIs（reactive oxygen intermediates）的增加，本研究證實了pre-S突變表面蛋白確實堆積在內質網中，引起內質網的擴大並經由鈣離子信號造成內質網壓力反應。

　　Hepatitis B virus (HBV), one of the hepadnavirus family, contains a partially double-stranded DNA genome of 3.2 kb. Individuals infected by HBV potentially become chronic carriers that are at a high risk for the development of hepatocellular carcinoma (HCC). HBV encodes three envelope proteins that named large, middle, and small surface protein. Many naturally occurring HBV mutants in sera or liver tissue of patients with chronic hepatitis B have been identified. Previously, two types of HBV pre-S mutant were identified; one contains a deletion in the pre-S1 region and the other contains a deletion in pre-S2 region. Both pre-S1 ad pre-S2 mutant surface proteins are localized in the endoplasmic reticulum (ER) with distinct distribution patterns. This study is designed to investigate the ER stress induction of specific hepatitis B virus pre-S mutant proteins in the cytoplasm. First, we used immunohistochemical staining and GFP fusion protein to monitor the localizations of pre-S mutant proteins in Huh7 cells, and pre-S mutant proteins showed a blot-like distribution pattern. Transmission electron microscope was used to monitor morphological change of ER induced by the accumulation of HBV mutant surface proteins. The expression of pre-S1 and pre-S2 mutant proteins in Huh7 cells resulted in ER proliferation and subsequently ER enlargement which looked like inclusion bodies. ER accumulations of these mutant proteins also activate ER stress signals in Huh7 cells by which ER stress marker GRP78 and GRP94 were significantly upregulated, while other chaperones and ER proteins such as Hsp60, Hsp70, Hsp90, Erp72 and Erp57 remained unchanged. Pre-S mutant proteins induced ER stress also lead to increase cytoplasmic Ca2+ concentration, but did not cause cell death. The ER accumulation effect seems to restrict to the pre-S mutant proteins without any effects on the protein trafficking of other cellular proteins. In fact, we have observed the enhanced expression of MHC class I on the surface of cells with pre-S mutant proteins. In combination of our results, pre-S mutant proteins indeed accumulate in ER, result in ER enlargement, and induce ER stress. These mutant proteins induce ER stress which leads to the release of Ca2+ from the ER and the subsequent production of reactive oxygen intermediates. In conclusion, the clinical observation of ER changes in mutant surface protein-containing hepatocytes in cirrhotic livers could be associated with different biologic functions of pre-S mutant proteins.

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