急性肝衰竭和肝細胞移植的研究

Acute liver failure & hepatocyte transplantation

何承懋(Cheng-Maw Ho)

指導教授 ： 李伯皇

共同指導教授 ： 陳惠玲(Hui-Ling Chen)

國立臺灣大學/醫學院/臨床醫學研究所/博士(2016年)

https://doi.org/10.6342/NTU201600315

摘要

背景及目的
急性肝衰竭是一個不常見但卻有高致命風險的症候群。目前的處理大多是根據專家經驗。肝移植是一個黃金標準治療。相較於肝器官移植，肝細胞移植是有發展潛力的另一個選擇。本研究擬針對急性肝衰竭及肝細胞移植的相關題材進行研究，從群體世代的流行病學，個體急性肝衰竭微觀病生理觀察，肝細胞體外功能評估，到最後肝細胞移植臨床前期動物實驗。目前對於體外肝細胞在移植前功能快速評估的工具很少，從肝門靜脈進行肝細胞移植時的輸注速度對移植細胞早期的engraftment 及repopulation的影響也不清楚。因此，吾人欲研究 1) 台灣族群急性肝衰竭的發生率、病因、預後及相關因子探討。2) 急性肝衰竭肝再生及細胞分化的病生理機轉。3) 肝細胞對靛氰綠 (indocyanine green, ICG) 特異性吸收(經由OATP1B1受體)及釋放(面向小膽管的細胞膜)是否可以當作一個快速體外肝細胞功能評估工具。4) 急性肝衰竭大鼠模式下，不同細胞輸注速度對移植肝細胞殖入(engraftment)及繁盛(repopulation)的影響，並試圖以這種臨床可應用的方式改善細胞移植成效 。本論文從四大面向(族群流行病學，橫斷面病生理學，體外細胞學研究，臨床前動物模式轉譯學) 探討相關議題。

研究對象與方法
1)族群研究
從台灣健保資料庫中篩選出在2005年1月至2007年9月因急性肝衰竭相關診斷入院的病人。急性肝衰竭則進一步藉由相關檢驗醫囑、相關處方、住院天數、及沒有先前肝病就醫史等方式確保疾病嚴重度及排除其他可能干擾診斷因子。預後因子再作Cox 迴歸分析。
2)橫斷面病生理學
吾人從急性肝衰竭的病人病肝中，以免疫組織染色法觀察組織中的前驅細胞 (用CK19, EpCAM當標記)，肝細胞(CPS-1, HNF4α)，膽道細胞(HNF1β)，細胞分化(NUMB)及增生(Ki-67)的變化。
3)體外研究
從肝臟移植剩餘肝組織分離出來的人類肝細胞(1 x 106 cells) 在37°C下、分別置於細胞懸浮液和培養基狀態下，加入不同濃度ICG (0-2 mg/ml)浸泡30分鐘。之後將細胞移至不含ICG的培養液中3小時，分別於1, 2, 3小時後收集上清液，測量ICG濃度。細胞存活率用trypan blue 排除法、MTT (mitochondrial dehydrogenase activity) 及 SRB (cell attachment) assays測定。HepG2 細胞用來當作control 比較。
4)動物實驗
以D-galactosamine 誘發Sprague-Dawley 大鼠急性肝衰竭後，從肝門靜脈分別以30, 70或100秒將1 x 107/1 ml 新鮮分離的肝細胞打入肝臟，並於術後2天及7天觀察early engraftment (2天) and repopulation (7天)。

結果
1)族群研究方面
從28,078位潛在急性肝衰竭病人中， 篩選出218位條件符合者。發生率為每百萬人年80.2位，而且隨年齡增加而上升。平均年齡是57.9±17.1歲，中位存活時間為171天。最常見的病因是病毒性(45.4%，B型肝炎為主)和酒精/毒藥物(33.0%)。獨立預後因子包括酗酒，惡性腫瘤，每周檢查total bilirubin頻率，敗血症，使用血液透析，以及使用氫離子幫浦阻斷劑 (proton pump inhibitor)。他們的風險比值(HR)及95%信賴區間分別為1.67 (1.01-2.77), 2.90 (1.92-4.37), 1.57 (1.40-1.76), 1.85 (1.20-2.85), 2.12 (1.15-3.9), 0.94 (0.90-0.98)。在存活超過三個月的130病人中，66 人(50.8%)後來追蹤發現有肝硬化的傾向。8 人(3.7%)曾經接受肝移植評估， 只有1位接受移植且存活。
2)橫斷面病生理方面
組織中可觀察到大量肝細胞死亡及顯著的小膽管反應 (ductular reaction)。利用免疫組織染色，可以觀察到不同分化階段的小膽管反應，從早期原始前驅細胞(CK19濃染, 細胞核大細胞小)往外圍成螺旋狀逐漸分化進入肝細胞前期(CK19淡染或消失, 細胞核小細胞大)。 隨著細胞分化進行，這群中間肝細胞(intermediate hepatocyte)也會有NUMB、EpCAM的表現，部分細胞甚至已經出現成熟肝細胞特有與尿素代謝相關的CPS-1表現，但是很少觀察到分裂增生的細胞 (無Ki67表現)。
3)體外研究方面
體外有功能的肝細胞數分鐘內可納入ICG，並可在1~2小時內排除細胞外。排出的ICG在1小時後很快達到一個飽和濃度。ICG濃度超過1.0 mg/ml 對肝細胞有毒性。相較於0.5 mg/ml或對照組，在1.0 mg/ml ICG濃度下，肝細胞有較高粒線體去氫酶活性(0.025 ± 0.0004 v.s 0.019 ± 0.0008 or 0.020 ± 0.002, P < 0.05)。當HepG2細胞浸置在不同ICG濃度(control, 0.5 mg/ml, 1.0 mg/ml)中，上清液白蛋白量呈遞減趨勢(98.9 ± 0.02, 66.6 ± 0.05, 39.1 ± 0.4 ng/ml)，偵測細胞增生的[3H]-thymidine incorporation也呈現相同趨勢。
4)動物實驗方面
不同輸注速度會影響移植肝細胞的engraftment (P = 0.018) 及 repopulation (P = 0.037)，而且有統計上的顯著差異。其中以70秒輸注速度成效較好，移植的肝細胞能夠立即穿越過肝竇內皮血管層，很少累積在門靜脈小管中，也有較顯著的肝功能改善。三組的平均首次門靜脈壓高峰分別是14.8 ± 6.5, 17.7 ± 3.7, 13.6 ± 3.0 mmHg，之間並無統計差異。

結論
台灣族群的急性肝衰竭主要跟病毒感染有關，病人有惡性腫瘤及酗酒者預後較差，使用proton pump inhibitor則有較佳的預後。半數的存活者有肝硬化。急性肝衰竭病肝有明顯的ductular reaction，雖然新生的前期肝細胞已經表現出成熟肝細胞特有的功能，仍不足以成功完成肝臟再生的救援功能，所以肝細胞移植仍能提供急性肝衰竭治療上的實際需求。體外有功能的肝細胞數分鐘內可納入ICG，並可在1~2小時內排除細胞外。再進一步改善後，ICG可發展成快速體外評估肝細胞功能的檢測工具。在急性肝衰竭大鼠模式中，不同肝細胞輸注速度會導致不同早期殖入和繁盛的結果。這些經由動物實驗得到的觀念驗證具有實質臨床意義，可以容易進行臨床的轉譯應用。總體而言，台灣在急性肝衰竭的細胞治療仍有相當多的研究及進步空間。

關鍵字

急性肝衰竭 ； 預後 ； 族群 ； 肝細胞 ； 前驅幹細胞 ； 靛氰綠 (ICG) ； 肝細胞移植 ； 殖入 (engraftment) ； 繁盛 (repopulation)

並列摘要

Background and objective
Acute liver failure (ALF) is uncommon but fatal. Current management is based mostly on clinical experience. Hepatocyte transplantation is a promising alternative to liver transplantation in patients with acute liver failure. The study is to investigate ALF from longitudinal population-scale epidemiological analysis, through individual cross-sectional histopathophysiological observation, ex vivo functional evaluation of hepatocytes, to preclinical animal experiment of hepatocyte transplantation. To this end, we investigated 1) the incidence, etiology, outcomes, and prognostic factors of ALF in Taiwan. 2) pathophysiological expression of regeneration and differentiation in acute failure liver. 3) whether the uptake and release of indocyanine green (ICG) by hepatocytes could be used as a rapid in vitro assay for hepatocyte functional assessment. 4) the impact of the rate of intraportal hepatocyte transplantation on early engraftment and repopulation and to improve the engraftment and repopulation efficiencies of hepatocyte transplantation for treatment of a rat model of acute liver failure in a clinically useful way without preconditioning.

Materials and methods
1) For population study, patients with the admission diagnosis of ALF between January 2005 and September 2007 were identified from the Longitudinal Health Insurance Database of Taiwan. ALF was further confirmed by disease severity based on laboratory orders, prescriptions, and duration of hospital stay, and acute onset without prior liver disease. Prognostic factors were identified using Cox regression analysis.
2) For microscopic cross-sectional observational study, a human explant liver from acute HBV infection was examined for immunohistochemical expression of progenitors [marker: CK19, epithelial cell adhesion molecule (EpCAM)], differentiation [NUMB (an inhibitor of the Notch pathway), carbamoyl phosphate synthetase 1 (CPS-1, urea cycle enzyme), HNF4α, and HNF1β], and proliferation (Ki-67).
3) For in vitro study, human hepatocytes (1 x 106 cells) isolated from unused donor livers were incubated at 37°C for 30 min with ICG (0-2 mg/ml) in both cell suspension and on collagen-coated culture plates. Cells were then incubated in medium without ICG for 3 h with supernatants collected at 1, 2, and 3 h for measurement of ICG release. Viability of cells was determined by trypan blue exclusion, MTT (mitochondrial dehydrogenase activity) and SRB (cell attachment) assays. HepG2 cells were also used as comparison.
4) For animal study, acute hepatic injury was induced in Sprague-Dawley rats with D-galactosamine. Hepatocytes (1 x 107/ml) were infused intraportally over 30, 70, or 100 seconds to study early engraftment (2 days) and repopulation (7 days).

Results
1) For population study, during the study period, 218 eligible cases were identified from 28,078 potential eligible ALF patients. The incidence was 80.2 per million person-years in average and increased with age. The mean age was 57.9±17.1 years and median survival was 171 days. The most common etiologies were viral (45.4%, mainly hepatitis B virus) and alcohol/toxin (33.0%). Independent prognostic factors included alcohol consumption (HR 1.67 [1.01-2.77]), malignancy (HR 2.90 [1.92-4.37]), frequency of check-ups per week for total bilirubin (HR 1.57 [1.40-1.76]), sepsis (HR 1.85 [1.20-2.85]), and use of hemodialysis/hemofiltration (HR 2.12 [1.15-3.9]) and proton pump inhibitor (HR 0.94 [0.90-0.98]). Among the 130 patients who survived ≥90 days, 66 (50.8%) were complicated by liver cirrhosis. Eight (3.7%) were referred for liver transplantation evaluation, but only one received transplantation and survived.
2) For cross-sectional study, histological examination of the explant liver showed submassive necrosis and prominent ductular reaction. The road of hepatocyte differentiation was nicely shown from the bipotential progenitor cells (thick stained, small cell size, high nuclear-cytoplasm ratio) and gradually spirally spreading outward to form daughter intermediate hepatocytes (light stained, larger cell size, lower nuclear-cytoplasm ratio). These differentiating cells did not proliferate actively, and express EpCAM and transition of NUMB and CPS-1.
3) For in vitro study, ICG was taken up and secreted by hepatocytes with the release reaching a plateau level soon after 1 hour. Concentrations of ICG above 1.0 mg/ml, had toxic effects on hepatocytes. Hepatocytes incubated with 1.0 mg/ml ICG had higher mitochondrial dehydrogenase activity compared to 0.5 mg/ml ICG or control (0.025 ± 0.0004 v.s 0.019 ± 0.0008 or 0.020 ± 0.002, P < 0.05). Incubation of HepG2 cells with ICG reduced albumin production (98.9 ± 0.02, 66.6 ± 0.05, 39.1 ± 0.4 ng/ml for control, 0.5 mg/ml, and 1.0 mg/ml ICG respectively) and also decreased [3H]-thymidine incorporation in a dose-response manner.
4) For animal study, three groups had significant difference in hepatocyte engraftment (P = 0.018) and repopulation efficiencies (P = 0.037) and infusion over 70 seconds produced superior outcomes. After the 70-second infusion, the transplanted cells immediately transmigrated the sinusoidal endothelial layer and rarely accumulated in the portal venules, with improved liver function significantly. The mean first peak pressures, without significant difference, were 14.8 ± 6.5, 17.7 ± 3.7, and 13.6 ± 3.0 mmHg in the 30, 70, and 100-second groups, respectively.

Conclusion
ALF in Taiwan is mainly due to viral infection. Patients with malignancy and alcohol exposure have worst prognosis. The use of proton pump inhibitor is associated with improved survival. Half of the ALF survivors have liver cirrhosis. Prominent ductular reaction with at-least partially functional hepatocyte differentiation did not guarantee successful regeneration in acute liver failure and there is demand left for hepatocyte transplantation. With further refinement of ICG could be used to develop a rapid assay for assessment of the function of isolated human hepatocytes. Differential hepatocyte transfusion rate contribute to accelerated early engraftment and repopulation in rats with acute liver
injury. These proof-of-concept findings are of clinical significance because they are easy to translate into practice. Further studies are needed for improvement of hepatocyte transplantation for ALF in Taiwan, albeit some problems solved.

並列關鍵字

acute liver failure ； prognosis ； population ； hepatocyte ； progenitor cell ； indocyanine green (ICG) ； hepatocyte transplantation ； engraftment ； repopulation

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