肝細胞在肝臟修復再生過程中之可塑性及治療潛能

Plasticity of Hepatocytes in liver regeneration

陳雅惠(Ya-Hui Chen)

指導教授 ： 張美惠 ； 陳惠玲

國立臺灣大學/醫學院/臨床醫學研究所/博士(2014年)

https://doi.org/10.6342/NTU.2014.03091

摘要

肝細胞是一種具有高度可塑性的細胞。在大部份的肝傷害後，肝臟修復工作主要藉由成熟肝細胞增生來修補受損的區域。然而，在特殊肝損害的情況下，成熟肝細胞亦可轉變為兼具有幹細胞的特性，它們可能經由肝細胞去分化或是轉分化的過程參與肝臟修復，這些過程涉及複雜的信號轉導途徑，調控機制仍不清楚。
關於卵圓幹細胞（Hepatic oval cells, OCs）、小肝細胞樣祖細胞（Small hepatocyte-like progenitor cells, SHPCs）和肝細胞（Hepatocytes）在肝臟修復過程中所扮演的角色及細胞分化源由的關連性仍然備受爭議。SHPCs是一種肝祖細胞（Heaptic progenitor cells），同時具有胚胎肝母細胞（Hepatoblasts）、卵圓幹細胞和成熟肝細胞的表型特徵。為了探討成熟肝細胞和SHPCs細胞分化祖源的關連性，我們應用一個能夠產生50% DPPIV陽性肝細胞的DPPIV chimeric liver的動物實驗模式，大鼠在接受Retrorsine及部分肝切除（Hepatectomy, PH）處理之後，會誘發SHPCs集群的生成，利用DPPIV蛋白表現可以標定追蹤成熟肝細胞的位置以及偵測SHPCs是否具有 DPPIV 蛋白表現。結果在所有的分析樣品內，我們都沒有觀察到任何DPPIV陽性的SHPCs集群。此外，在連續組織切片中使用gamma-glutamyl-transpeptidase（GGT，胚胎肝母細胞的標誌）和glucose-6-phosphatase（G6Pase，成熟肝細胞的標誌）染色進行分析，顯示SHPCs集群是一個包含了不同分化階段肝細胞的異質群體（Heterogeneous population）。在後續連續切片中，使用免疫螢光雙染色分別標示卵圓幹細胞特定標誌（CK-19/OV-6, laminin, EpCAM）和肝細胞的特定標誌（HNF-4α, C/EBPα），也發現卵圓幹細胞的增生和SHPCs集群生成可能是有相關性。4,4’-Methylenedianiline（DAPM）是一種膽道毒素，會破壞膽管週圍細胞進而抑制卵圓幹細胞的活化。在暴露於Retrorsine的大鼠模式中，利用DAPM藥物重複處理全面性抑制卵圓幹細胞的活化，誘發SHPCs集群的生成，亦沒有發現SHPCs集群的產生。另外，將分離出來的DPPIV陽性卵圓幹細胞移植進入已暴露Retrorsine的DPPIV-deficient大鼠肝臟內再進行部分肝切除術後，可以發現有DPPIV陽性SHPCs集群的存在。這些結果強烈顯示，成熟肝細胞無法經由去分化產生SHPCs集群，SHPCs集群可能是由卵圓幹細胞分化而來。
為了探討肝細胞轉分化為膽管上皮細胞（Biliary epithelial cells, BECs）的潛力，在第二部份的研究中，我們使用DAPM和D-galactosamine（DAPM + D-gal）建立急性肝臟及膽道損傷（Acute hepatio-biliary injury）的大鼠實驗模式，進行DPPIV陽性肝細胞移植，利用DPPIV蛋白標記來追蹤移植細胞位置及移植細胞分化狀況。另外，應用DPPIV chimeric liver大鼠模式，分別給予DAPM+D-gal和DAPM+BDL傷害，誘發急性肝臟及膽道損傷和慢性膽道的損傷，探討DPPIV陽性肝細胞在不同肝臟受損情況下如何參與肝臟修復。結果顯示，成熟肝細胞可以逐步轉分化成為BECs。在急性肝臟及膽道損傷進行肝細胞移植的實驗，移植的DPPIV陽性肝細胞會滯留於肝門靜脈內，沿著肝門靜脈增生慢慢排列形成雙層板狀的結構。在雙層板結構內，移植的DPPIV陽性肝細胞會逐漸失去原本肝細胞的特性，慢慢出現膽道（Biliary）特有的基因表現，最後形成具有DPPIV蛋白表現的膽管（Bile duct）。在DPPIV chimeric liver誘發急性肝臟及膽道損傷和慢性膽道損傷的研究，我們觀察到位於肝門靜脈週圍能夠表現膽道特異基因（HNF-1β）的細胞也都具有DPPIV蛋白表現，這些細胞會延續連接到具有DPPIV蛋白表現的膽管。為了進一步確認移植肝細胞和膽管生成的關聯性，應用DPPIV chimer liver動物模式，在誘發急性或慢性膽道損傷之前，先給予動物Retrorsine處理，經由抑制肝細胞的增生阻礙DPPIV陽性BECs的生成，我們沒有發現DPPIV陽性BECs存在。這些結果顯示，在急性和慢性膽道損傷的環境中，成熟肝細胞可能會經由類似膽管板（Ductal plate）結構機制的方式轉化成為BEC進行膽道的再生修復。過程可能會受到Notch信息傳遞所調控。
綜合我們的研究，肝細胞具有很強的可塑性，在不同的肝損傷環境中，可以針對肝臟修復的需求，適時進行細胞轉變參與肝臟修復，有時也會與卵圓幹細胞互相配合來執行肝臟的修復工作。讓我們對於了解肝臟修復再生作用的機制可以更邁前一步。

關鍵字

肝細胞 ； 肝細胞移植 ； 卵圓幹細胞 ； 小肝細胞樣祖細胞 ； 肝臟再生 ； 細胞分化 ； 轉分化

並列摘要

Hepatocytes are cells with high plasticity. Mature hepatocyte proliferation is usually responsible for liver regeneration after most causes of injury. However, under the special contribution of liver damage, mature hepatocytes can function as facultative stem cells for each other and replenish the inhibited cellular compartment by a process of trans-differentiation, involving complex signaling pathways. Mechanism of its regulation is still unclear.
The potential lineage relationship between hepatic oval cells, small hepatocyte-like progenitor cells (SHPCs), and hepatocytes in liver regeneration is debated. SHPCs are a type of progenitor cells group and express phenotypic characteristics of fetal hepatoblasts, hepatic oval cells, and mature hepatocytes. To test whether mature hepatocytes can give rise to SHPCs, rats with dipeptidyl peptidase IV (DPPIV) chimeric livers, which harbored endogenous DPPIV-deficient hepatocytes and transplanted DPPIV-positive hepatocytes, were subjected to retrorsine treatment followed by partial hepatectomy (PH). DPPIV-positive hepatocytes comprised about half of the DPPIV chimeric liver mass. Tissues from DPPIV chimeric livers after retrorsine/PH treatment showed large numbers of SHPC clusters. None of the SHPC clusters were stained positive for DPPIV in any analyzed samples. Furthermore, serial sections stained for gamma-glutamyl-transpeptidase (GGT, a marker of fetal hepatoblasts) and glucose-6-phosphatase (G6Pase, a marker of mature hepatocytes) showed SHPCs are a heterogeneous population of cells at different stages of differentiation in hepatic progenitor cell and hepatocyte lineage. Using double immunofluorescence staining for markers specific for hepatic oval cells (CK-19/OV-6, Laminin, EpCAM) and hepatocytes (HNF-4α, C/EBPα) in serial sections, we find that a lineage relationship was present within a single oval cell proliferation and between oval cell proliferations and SHPC clusters. Extensive elimination of oval cell response by repeated administration of 4,4’-methylenedianiline (DAPM) to retrorsine-exposed rats impaired the emergence of SHPC clusters. DPPIV-positive hepatic oval cells are transplanted into the DPPIV-deficient rat subjected to retrorsine treatment and followed by partial hepatectomy. The DPPIV-positive SHPC clusters is observed. These findings highly suggest the hepatic oval cells but not mature hepatocytes as the origin of SHPC clusters in retrorsine-exposed rats.
However, we have recently used in vivo lineage tracing technique in rats and resolved the debate on the lineage relationship between mature SHPCs in retrorsine-exposed rats after PH. We have demonstrated that mature hepatocytes do not give rise to SHPCs. Taken together, these studies prompted us to hypothesize that being a terminal differentiated cell type, mature hepatocytes can not convert into BECs in the damaged livers. Therefore, whether hepatocytes can convert into biliary epithelial cells (BECs) during liver injury is much debated. To test this concept, we traced the fate of genetically labeled (DPPIV-positive) hepatocytes in hepatocyte transplantation model following acute hepato-biliary injury induced by DAPM and D-galactosamine (DAPM + D-gal) and in DPPIV-chimeric liver model subjected to acute (DAPM + D-gal) or chronic biliary injury caused by DAPM and bile duct ligation (DAPM + BDL). In both models before biliary injury, BECs are uniformly DPPIV-deficient and DPPIV-deficient hepatocytes are restricted to proliferate by retrorsine. We found that mature hepatocytes underwent a stepwise conversion into BECs. In the hepatocyte transplantation model, DPPIV-positive hepatocytes entrapped periportally proliferated and formed two-layered plates along portal veins. Within the two-layered plates, the hepatocytes gradually lost their identity, proceeded through an intermediate state, acquired a biliary phenotype, and subsequently formed bile ducts along the hilum-to-periphery axis. Additionally, in DPPIV-chimeric liver model, periportally located hepatocytes expressing HNF-1β were exclusively DPPIV-positive and were in continuity to DPPIV-positives bile ducts. Inhibition of hepatocyte proliferation by additional doses of retrorsine in DPPIV-chimeric livers prevented the appearance of DPPIV-positive BECs after biliary injury. Moreover, enriched DPPIV-positive BEC/hepatic oval cell transplantation produced DPPIV-positive BECs or bile ducts in unexpectedly low frequency and in mid-lobular regions. These results together suggest that mature hepatocytes but not contaminating BECs/hepatic oval cells are the sources of periportal DPPIV-positive BECs. These findings highly suggest that mature hepatocytes contribute to the biliary regeneration in the environment of acute and chronic biliary injury through a mechanism similar to fetal development of biliary duct without the need of exogenously genetic or epigenetic manipulation. The Notch signaling pawthay may regulate this response.
Finally, these results showed hepatocytes have a strong ability for regeneration during liver damage in various environment. Hepatocytes can self-replicate proliferation or transdifferentiation to repair the liver.

並列關鍵字

Hepatocytes ； Hepatocyte transplantation ； Hepatic oval cells ； small hepatocyte-like progenitor cells ； Regeneration ； Cell differentiation ； Transdifferentiation

參考文獻

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