肝細胞移植應用於急性肝傷害之治療潛能與機制

Abstract

肝細胞移植一直被期待可以取代肝臟移植來治療急性肝衰竭病人。然而，肝細胞移植要能成為可以被信賴的治療方法，前提是移植的肝細胞要能夠在急性肝衰竭的肝臟中有效率的增生。因此，我們主要研究的問題是：(1)移植的肝細胞是否可以在急性肝衰竭受損的肝臟中有效率的增生？如果可以，是透過什麼機轉？(2)肝細胞移植對於急性肝衰竭肝臟的再生過程中被活化的卵圓幹細胞會產生什麼影響？ 為了探討第一個問題，我們使用Retrorsine加上D-galactosamine或單獨使用 D-galactosamine，在雄性的DPPIV-deficient F-344 大鼠誘發急性肝傷害，另外一組則單獨接受retrorsine處理，作為肝細胞移植的接受者。在這些處置中，retrorsine是用來抑制本土肝細胞的增生，而D-galactosamine是用來引發急性肝傷害。這些大鼠在D-galactosamine處置後24小時接受由原生種F-344 大鼠所分離下來的肝細胞移植(1x107/ml)。然後在不同的時間點，對移植肝細胞增生的動力學，肝臟中細胞激素與生長因子基因表現的動力學，肝臟中星狀細胞被活化的動力學，以及matrix metalloproteinase (MMP2)表現的動力學等進行分析。 我們發現，在Retrorsine加上D-galactosamine誘發急性肝傷害組，移植的肝細胞快速的穿過肝竇進入肝臟實質組織中，並且可以進行大量的增生，在第四週時可以取代54.6+7.1%的肝臟實質組織；這些現象在單獨使用 D-galactosamine或單獨接受retrorsine處理組則不會發生。同樣的，在Retrorsine加上D-galactosamine誘發急性肝傷害組，生長因子TGF-α 和HGF的基因表現則顯著的增加，而且持續到肝細胞移植後的第四週。這些生長因子基因表現的動力學和移植肝細胞增生的動力學相一致。肝臟星狀細胞也快速的被啟動活化，其數目顯著的增加直到第四週，然後開始減少；同時間，生長因子基因表現和移植肝細胞的增生也開始減少。MMP2基因表現的動力學，MMP2蛋白質表現的動力學以及在肝臟組織中分佈的位置都和星狀細胞活化反應的動力學以及在肝臟組織中分佈的位置相一致。 為了探討第二個問題，我們使用Retrorsine加上D-galactosamine，在雄性的DPPIV-deficient F-344 大鼠誘發急性肝傷害以及廣泛的卵圓幹細胞活化反應，在D-galactosamine處置後24小時接受由原生種F-344 大鼠所分離下來的肝細胞移植(1x107/ml)。然後在不同的時間點，使用組織化學染色法，免疫組織化學染色法，以及免疫螢光染色法來分析卵圓幹細胞的活化反應及他們分化的命運。 我們發現，在Retrorsine加上D-galactosamine誘發急性肝傷害後，在沒有接受肝細胞移植的時候，卵圓幹細胞很快的在第一天即被啟動活化，進行增生，在第四至五天達到高峰，然後分化成為肝細胞。在接受肝細胞移植後，卵圓幹細胞在第七天開始被啟動增生，在第四週達到高峰。卵圓幹細胞增生動力學和移植肝細胞增生的動力學相一致。卵圓幹細胞增生並以小管狀排列，然後分化成為膽道細胞。我們進一步在Retrorsine加上D-galactosamine處理後的第四天，當卵圓幹細胞活化增生的高峰期，進行肝細胞移植，我們發現，這些已被活化的卵圓幹細胞停止分化為肝細胞，維持小管狀排列，然後分化成為膽道細胞。當我們再度使用Retrorsine將移植肝細胞的增生能力也抑制之後，這些成小管狀排列的卵圓幹細胞又可再度分化成為肝細胞。 總結我們的研究，在急性肝傷害後合併剩餘本土肝細胞的增生能力受損會啟動一系列代償性的肝臟修補機制，這樣的環境提供移植肝細胞進行大量增生的有利條件。此外，肝細胞移植會將本來由卵圓幹細胞主導的肝臟修補機制改變為由移植肝細胞主導的修補機制。而且，肝細胞移植會影響並改變卵圓幹細胞的分化命運。肝細胞移植具有有效治療急性肝衰竭病人的發展性。

Efficient repopulation by transplanted hepatocytes in the severely injured liver is essential for their clinical application in the treatment of acute hepatic failure. We studied here (1) whether and how the transplanted hepatocytes are able to efficiently repopulate the toxin-induced acute injured liver, and (2) the impacts of hepatocyte transplantation on the response and fate of oval cells that are activated to proliferate in acute severe hepatic injury. To address the first issue, we used retrorsine-plus-D- galactosamine (R+D-gal) treatment or D-galactosamine alone (D-gal-alone) to induce acute hepatic injury, or retrorsine-alone in male Dipeptidyl peptidase IV-deficient F-344 rats. In these models, retrorsine was used to inhibit the proliferation of endogenous hepatocytes while D-galactosamine induced acute hepatocyte damage. Wild-type hepatocytes (1x107/ml) were transplanted intraportally 24 hours after D-galactosamine or saline injection. The kinetics of proliferation and repopulation of transplanted cells and the kinetics of cytokine response, hepatic stellate cell (HSC) activation, and matrix metalloproteinase (MMP2) expression were analyzed. We observed that early entry of transplanted hepatocytes into the hepatic plates and massive repopulation of the liver by transplanted hepatocytes occurred in acute hepatic injury induced by R+D-gal treatment but not by D-gal-alone or retrorsine-alone. The expressions of transforming growth factor-α and hepatocyte growth factor genes in the R+D-gal injured liver were significantly up-regulated and prolonged up to 4 weeks after hepatocyte transplantation. The expression kinetics were parallel with the efficient proliferation and repopulation of transplanted hepatocytes, which replaced 54.6+7.1% of damaged parenchyma by four weeks. HSC was activated rapidly, markedly, and prolongedly up-to 4 weeks after hepatocyte transplantation, when the expression of HGF gene and repopulation of transplanted hepatocytes were reduced afterward. Furthermore, the expression kinetics of MMP2 and its specific distribution in the host areas surrounding the expanding clusters of transplanted hepatocytes are consistent with those of activated HSC. To address the second issue, we used retrorsine-plus-D-galactosamine (R+D-gal) treatment to induce acute hepatic injury and to elicit extensive activation of oval cells in male Dipeptidyl-peptidase-IV-deficient F-344 rats. These rats were then randomized to receive wild-type hepatocyte transplantation or vehicle intraportally. The kinetics of oval cell response and their differentiation fate were analyzed using histochemical, immunohistochemical, and immunofluorescent stainings for DPPIV, OV6, CK19, laminin, γ-glutamyl-transpeptidase, and glucose-6-phosphotase. We observed that oval cells were activated early and differentiated into hepatocytes in R+D-gal-treated rats without hepatocyte transplantation. With hepatocyte transplantation, the oval cells were recruited later and continued to proliferate in parallel with the massive proliferation of transplanted hepatocytes. They formed ductules and differentiated into biliary cells. When hepatocytes were transplanted at the day when oval cells were at their peak response, the numerous activated oval cells ceased to differentiate into hepatocytes and remained in ductular form. The ductular oval cells were capable of differentiating into hepatocytes again when the donor hepatocytes were inhibited to proliferate. In conclusion, impaired hepatocyte regeneration after acute severe hepatic injury may initiate serial compensatory repair mechanisms which facilitate the extensive repopulation by transplanted hepatocytes that enter early the hepatic plates. In addition, hepatocyte transplantation changes the mechanism of liver reconstitution from oval cell-mediated to donor hepatocyte-mediated and affects the differentiation fate of host oval cells in acute severe hepatic injury. Hepatocyte transplantation has a great potential in the treatment of patients with acute hepatic failure.