以脫細胞化肝臟間質製備之多孔性生物材料應用於受損肝細胞活性恢復之研究

Decellularized Liver Matrix as Substrates for Hepatocyte Rescue in Acute Liver Toxicity

李光閔(Kuang-Min Lee)

指導教授 ： 侯詠德

國立臺灣大學/生物資源暨農學院/生物產業機電工程學研究所/碩士(2018年)

https://doi.org/10.6342/NTU201803378

摘要

愈來愈多學者認為肝硬化的病變是一連串循序漸進的臨床階段，而非單一疾病。一開始慢性肝臟發炎會導致肝細胞壞死，接著若未及時控制則會演變成肝纖維化 (Liver fibrosis)，最終造成肝臟質地變硬而形成肝硬化 (Liver cirrhosis)。然而這樣的病變過程並不可逆，目前的治療方式也僅能停止或延緩肝硬化病變，此外，肝硬化亦長年名列世界成年人口常見死因之一，因此肝硬化的治療在生醫領域一直是研究的重點。
本實驗室既往研究以 D-galactosamine (GaIN) 模擬肝細胞壞死情形，並在體外以肝臟脫細胞化之細胞外間質貼片來培養受損的肝細胞，發現此肝臟細胞外間質貼片有延緩肝細胞受毒害甚至修復受損細胞的效果。因此，本研究提出一新穎的假說，若能將肝臟細胞外間質由肝門靜脈注入纖維化的肝臟來達到肝臟全面性的修復，或許對肝硬化患者而言是一個有效的治療方式。為了證實這樣的概念可行，同時讓肝細胞的體外培養環境更符合實際肝臟情形，本研究首先以大鼠肝臟細胞外間質 (Liver extracellular matrix; LECM) 與 Gelatin-hydroxyphenylpropionic acid (Glt-HPA) 混合成為三次元多孔性生物材料來模擬體內肝臟環境，並將初代大鼠肝細胞培養於其中。接著分別使用含有 GaIN、CHCl3、CCl4 的培養基對肝細胞進行毒化以模擬肝臟受損的情況，最後再以含有肝臟細胞外間質的培養基進行培養，觀察受損肝細胞的生存率與各機能表現，並證實本研究的假說－將肝臟細胞外間質全面性作用於受損肝細胞，能夠使得受損的肝細胞得以修復。
本研究結果得知：(1) 初代大鼠肝細胞在 Glt-HPA:LECM = 4:6 的培養條件下經過三天的培養，肝蛋白 (Albumin) 分泌量較 Glt-HPA:LECM = 5:5 的培養條件提升了約 50 %、較 Glt-HPA (無加入 LECM) 提升了約 20 %；(2) 以 GaIN 毒化處理的肝細胞在 Glt-HPA-LECM (4:6) 的培養條件下，再加入含有 20 mg/mL LECM 的培養基培養五天後，其肝蛋白分泌量較 Negative 組 (GaIN 毒化後的肝細胞以未含 LECM 的培養基培養於 Glt-HPA 中) 提升了 2 倍，同時，其 Lactate dehydrogenase (LDH) 活性也降低至與 Blank 組 (未受毒化的肝細胞以未含 LECM 的培養基培養於 Glt-HPA 中) 相近；(3) 以 CHCl3 毒化處理的肝細胞在 Glt-HPA-LECM (4:6) 的培養條件下，再加入含有 20 mg/mL LECM的培養基培養五天後，其肝蛋白分泌量較 Negative 組 (CHCl3 毒化後的肝細胞以未含 LECM 的培養基培養於 Glt-HPA 中) 提升了 1 倍，同時，其 LDH 活性也降低至 Blank 組 (未受毒化的肝細胞以未含 LECM 的培養基培養於 Glt-HPA 中) 之下；(4) 以 CCl4 毒化處理的肝細胞在 Glt-HPA-LECM (4:6) 的培養條件下，再加入含有 20 mg/mL LECM的培養基培養五天後，其肝蛋白分泌量較 Negative 組 (CCl4 毒化後的肝細胞以未含 LECM 的培養基培養於 Glt-HPA 中) 提升了 12 %，同時，其 LDH 活性也降低至與 Blank 組 (未受毒化的肝細胞以未含 LECM 的培養基培養於 Glt-HPA 中) 相近。本研究證明：(1) 以脫細胞化肝臟間質製備之 Glt-HPA-LECM 可有效提升肝細胞活性及肝蛋白分泌量，為更能模擬肝細胞原生環境之多孔性生物材料；(2) 另外，含有 LECM 的培養基確實有恢復受損肝細胞的活性及延緩毒害的效果。這樣的結果對於未來臨床上肝硬化的治療以及肝組織工程領域中，相信具有非常重要的意義。

關鍵字

脫細胞化 ； 肝臟細胞外間質 ； 明膠 ； 三次元多孔性材料 ； 肝細胞 ； 肝組織工程

並列摘要

More and more scholars believe that the pathological changes of cirrhosis are a series of gradual clinical stages rather than a single disease. In the beginning, chronic hepatitis can lead to hepatocyte necrosis, and then if it is not controlled in time, it will evolve into liver fibrosis, which eventually causes the liver to become hard and form liver cirrhosis. However, such a cirrhosis process is irreversible, and the current treatment can only stop or slow down the damage of the liver. In addition, cirrhosis has long been one of the common causes of death in the adult population of the world, so the treatment of cirrhosis has been studied in the field of biomedicine.
The previous study shows that the decellularized liver matrix-film can promote the recovery of D-galactosamine-induced injured hepatocytes. Therefore, in this study, we proposed a novel hypothesis that the injection of liver extracellular matrix into the fibrotic liver via the hepatic portal vein, may be an effective treatment for patients with cirrhosis. First, we developed a three-dimensional porous biomaterial by mixing liver extracellular matrix (LECM) and gelatin-hydroxyphenylpropionic acid (Glt-HPA). Then we cultured injured hepatocytes (Treated by GaIN, CHCl3, CCl4, repesctively) in the above-mentioned biomaterials with LECM-containing medium, the viability and functionality of injured hepatocytes was determined thereafter.
In the result: (1) After 3 days culture of primary rat hepatocytes in Glt-HPA:LECM = 4:6, the secretion of albumin was about 50% and 20% higher than that in Glt-HPA:LECM = 5:5 and Glt-HPA (without LECM), separately; (2) After 5 days culture, the albumin secretion of the D-galactosamin-induced injury of hepatocytes in Glt-HPA-LECM (4:6) with the LECM-containing medium cultures was 2 times superior to that in Glt-HPA without the LECM-containing medium cultures (Negative). Also, the lactate dehydrogenase (LDH) activity of the GaIN-induced injury of hepatocytes in Glt-HPA-LECM (4:6) with the LECM-containing medium cultures was reduced to be similar to the non-injured hepatocytes in Glt-HPA with normal medium cultures (Blank). (3) After 5 days culture, the albumin secretion of the CHCl3-induced injury of hepatocytes in Glt-HPA-LECM (4:6) with the LECM-containing medium cultures was 1 time superior to Negative condition. Also, the LDH activity of the GaIN-induced injury of hepatocytes in Glt-HPA-LECM (4:6) with the LECM-containing medium cultures was reduced to be lower than Blank. (4) After 5 days culture, the albumin secretion of the CCl4-induced injury of hepatocytes in Glt-HPA-LECM (4:6) with the LECM-containing medium cultures was 12 % superior to Negative condition. Also, the LDH activity of the GaIN-induced injury of hepatocytes in Glt-HPA-LECM (4:6) with the LECM-containing medium cultures was reduced to be similar to Blank.
In summary, Glt-HPA-LECM as a three-dimensional porous substrate showed high potential in increasing cell viability and albumin secretion of the primary hepatocyte. Furthermore, the LECM-containing medium did have the effect of restoring the activity of damaged hepatocytes and delaying the toxicity. Such results are believed to be of great significance for the future treatment of clinical cirrhosis and liver tissue engineering.

並列關鍵字

Decellularization ； Liver extracellular matrix ； Gelatin ； Three-dimensional porous substrate ； Hepatocyte ； Liver tissue engineering

參考文獻

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