摘要
慢性B型肝炎病毒(HBV)感染是造成肝細胞癌的主因，但是我們尚未完全解明HBV引發肝細胞癌的機制。科學家發現HBV產生的HBx可以透過調控細胞內的鈣離子，促進HBV的複製，並造成細胞死亡。科學家還發現HBx轉基因鼠容易得到肝癌，由此研究推論HBx是引發肝細胞癌的重要原因。於是我們研究HBx究竟如何調控細胞內鈣離子訊號，且藉此推敲HBx引發肝細胞癌的機制。我們透過拍攝HepG2和Huh7兩種肝癌細胞的活細胞鈣離子螢光影像進行觀察與分析。透過分析細胞質鈣離子指示劑fura-2訊號，我們發現HBx會提升細胞質鈣離子濃度。除了觀察細胞質鈣離子濃度，我們利用T1ER和4mtD3cpv兩種genetic encoded Ca2+ indicators (GECI)分別測量內質網、粒線體的鈣離子濃度。轉染T1ER的細胞顯示表現HBx會提高內質網鈣離子儲藏量，但HBx對HepG2和Huh7粒線體鈣離子恆定的影響並不一致。我們通過鈣離子指示劑發現HBx會增加細胞質和內質網鈣離子恆定濃度。我們還利用西方墨點法計算肌球蛋白輕鏈2磷酸化比例，肌球蛋白輕鏈2活性會因為鈣離子濃度上升而增加，分析結果顯示不論是單純過量表現HBx或是讓細胞表現完整HBV基因組，肌球蛋白輕鏈2磷酸化程度都上升了，表示HBx引發的細胞鈣離子失衡可能影響癌細胞生理。 我們進一步確認HBx會不會透過改變鈣離子幫浦或鈣離子通道活性使鈣離子濃度增加。我們加入毒胡蘿蔔素(thapsigargin)和EGTA觀察鈣離子流變化，分析結果顯示，HBx除了降低內質網釋出鈣離子的速率，還會減緩鈣離子幫浦將鈣離子送出細胞的能力。使用thapsigargin也令內質網流失鈣離子，於是我們外加氯化鈣引起store-operated calcium entry (SOCE)，發現HBx會讓SOCE活性上升。我們將攜帶完整HBV基因組的質體轉染到細胞內，發現雖然HBx讓HepG2內質網釋放鈣離子的速率及細胞膜排除鈣離子的幫浦活性下降，但是沒有在Huh7細胞看到這些趨勢。值得一提的是，我們在HepG2和Huh7細胞都觀察到表現HBV基因組會讓SOCE活性增加。為了確認上述實驗結果，我們正以慢病毒製作表現GECI或HBx/HBV的細胞株。我們會利用這些研究材料推敲HBx干預胞內鈣離子恆定的機制，也要檢驗這些鈣離子擾動是否會引發肝細胞癌的及影響病情進展。我們的研究將闡釋HBx對鈣離子訊息及恆定的影響，並為B型肝炎治療和預防帶來新的可能性。
並列摘要
Chronic infection of hepatitis B virus (HBV) infection is one of the leading causes of hepatocellular carcinoma (HCC), but how HBV viral proteins cause HCC remains unknown. Previous researches revealed that one of the viral proteins, HBx, induced liver cancer in mouse models. Further investigations implied that HBx might modulate intracellular Ca2+ to promote the replication of HBV viruses and death of hepatocytes. We therefore study how HBx regulates intracellular Ca2+ signaling and its functional significance in HCC. Using live-cell fluorescence Ca2+ imaging on fura-2 loaded HepG2 and Huh7 cells, we noticed that HBx increased cytosolic Ca2+ concentration. Besides cytosolic [Ca2+], we measured [Ca2+] in endoplasmic reticulum (ER) and mitochondria, by applying genetic encoded Ca2+ indicators (GECI), T1ER and 4mtD3cpv, which could reflect [Ca2+] in ER and mitochondria, respectively. T1ER transfected cells showed that HBx increased [Ca2+] inside ER. However, HepG2 and Huh7 showed inconsistent changes of mitochondrial [Ca2+] with HBx expression. So we have found that HBx can increase [Ca2+] in cytosol and ER. Furthermore, western blots showed that Ca2+-dependent phosphorylation of myosin light chain 2 (p-MLC2) increased upon HBx expression, indicating that HBx-induced Ca2+ aberrancy may affect cancer cell physiology. We further examine whether HBx changed Ca2+ channel or pump activities resulting in [Ca2+] increase. With the addition of thapsigargin and EGTA, we found that the HBx-transfected groups had reduced Ca2+ release from ER to cytosol, and decreased Ca2+ pumping from cytosol to the extracellular space. When thapsigargin depleted intra-ER [Ca2+], we administrated CaCl2, allowing cells to replenish Ca2+ through store- operated calcium entry (SOCE). Such experiments demonstrated that HBx upregulated SOCE. In HepG2 but not Huh7 transfected with wild type HBV genome, we also observed decreased Ca2+ flow from ER to cytosol, and downregulated plasma membrane pump activity. Interestingly, both HepG2 and Huh7 expressing HBV genome have increased SOCE activities. To verify the above results, we are making stable cell lines expressing GECI and/or HBx/HBV genome. Using these tools we will elucidate the mechanism how HBx disrupts intracellular Ca2+ homeostasis. We will also investigate whether those Ca2+ changes contribute to the initiation and progression of HCC. Our work will clarify the role of HBx on Ca2+ signaling, thus may shed light on HBV treatment and HCC prevention.