Mechanism of Transplanted Hepatocyte Engrafting in Rat of Acute Liver Injury- Interaction of Liver Sinusoidal Endothelial Cell and Transplanted Hepatocyte

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Project title/計畫英文名

Project Number/計畫編號

NSC101-2314-B002-059

Translated Name/計畫中文名

急性肝損傷大鼠接受肝細胞移植後,移植細胞植入機制之研究-肝竇內皮細胞和移植細胞的交互作用

Project Principal Investigator/計畫主持人

CHENG-MAW HO

Funding Organization

National Science and Technology Council

Co-Investigator(s)/共同執行人

李伯皇陳惠玲

Start date/計畫起

01-08-2012

Expected Completion/計畫迄

12-07-2013

摘要：肝細胞移植手術是一項對急性肝衰竭以及代謝性肝疾病的病人有前瞻性的治療選擇。相較於肝臟移植，肝細胞移植手術有侵入性較小的優點。目前臨床肝細胞移植的成功率不如動物實驗一般有效穩定。諸多因素有待改進：細胞來源及品質、純化流程及輸注手術等方面皆會影響最後的成效。輸注手術方面，我們發現臨床細胞輸注入肝門靜脈的速度和大鼠實驗相近皆為107 /ml/min。有時若細胞是輸入脾臟時，速度更快。這引發我們對細胞輸注速度是否會影響細胞移植的成功產生興趣。文獻上有所謂水動力輸入法(hydrodynamic delivery)的理論；主要是說經由快速的輸液將基因或蛋白質打入靜脈可導致肝細胞吸收量的增加。研究指出這種作法會讓肝內竇內皮細胞(sinusoidal endothelial cell) 間的通透性增加而造成實驗觀察到的現象。我們假設類似的現象可能也發生在細胞層次而產生臨床和動物實驗成效不一樣的原因之一。我們發現適當的輸注速度的確在正常大鼠及急性肝損傷大鼠肝臟產生較佳的engraftment。在急性肝損傷大鼠身上更產生較佳的repopulation。我們也發現跟以往論述移植肝細胞並不會馬上進入肝實質而是手術一天後才進入的說法不同處。以適當速度剛打完細胞，即可觀察到移植的肝細胞已離開肝竇血管。移植肝細胞可能藉適當速度輸送經由細胞內 (經由融合的fenestration，和ICAM-1有關)或細胞間(經由adhesion junction disruption, 和VE-cadherin 有關) 而轉離肝竇。這次的研究將著重在肝竇內皮內應力及adhesion 訊息改變的分析。即時的門靜脈壓力監測幫助我們遴選適當的超短時間點，以供檢體收集分析。我們將用免疫組織染色及分子生物學的方法分析肝竇內皮細胞相關應力/adhesion分子及訊息因輸注速度改變而產生的變化。電子顯微鏡檢視可進一步觀察移植的肝細胞是如何在短時間就進入肝實質的。我們希望釐清適當輸注速度對肝竇內皮細胞的分子層次的影響，做出臨床輸注速度的改良建議，並對基本機制進行了解，提出可以改善並穩定臨床細胞移植的成果。
Abstract: Hepatocyte transplantation is a promising alternative to liver transplantation in patients diagnosed with metabolic liver disease or acute liver failure. Excellent results from animal studies cannot, however, fully translated into clinical practice possibly due to several reasons: not so ideal quality of cells transplanted, lack of proper recipient liver preconditioning protocol, and difference of transfusion technique. For hepatocyte transplantation, the infusion rate is around 107 /ml/min, both in human and in rat. Though more cells transplanted in human according to the body weight, the rate of infusion is quite a lot different considering the body size involved. Hydrodynamic gene delivery in gene therapeutic field imply that rapid delivery of fluid containing DNA, RNA or proteins can increase the uptake amounts in liver. We hypothesized that the rate of cell transfusion w the rate of cell transfusion wthe rate of cell transfusion wthe rate of cell transfusion w the rate of cell transfusion w the rate of cell transfusion wthe rate of cell transfusion wthe rate of cell transfusion wthe rate of cell transfusion wthe rate of cell transfusion wthe rate of cell transfusion wthe rate of cell transfusion w the rate of cell transfusion wthe rate of cell transfusion w the rate of cell transfusion wthe rate of cell transfusion wthe rate of cell transfusion wthe rate of cell transfusion wthe rate of cell transfusion w the rate of cell transfusion wthe rate of cell transfusion wthe rate of cell transfusion wouldouldould influence influence influence influence influence influence influence the engraftment of transplanted hepatocytes the engraftment of transplanted hepatocytesthe engraftment of transplanted hepatocytesthe engraftment of transplanted hepatocytesthe engraftment of transplanted hepatocytesthe engraftment of transplanted hepatocytesthe engraftment of transplanted hepatocytes the engraftment of transplanted hepatocytes the engraftment of transplanted hepatocytesthe engraftment of transplanted hepatocytesthe engraftment of transplanted hepatocytesthe engraftment of transplanted hepatocytes the engraftment of transplanted hepatocytesthe engraftment of transplanted hepatocytesthe engraftment of transplanted hepatocytes the engraftment of transplanted hepatocytes the engraftment of transplanted hepatocytesthe engraftment of transplanted hepatocytes the engraftment of transplanted hepatocytes the engraftment of transplanted hepatocytesthe engraftment of transplanted hepatocytesthe engraftment of transplanted hepatocytesthe engraftment of transplanted hepatocytes the engraftment of transplanted hepatocytesthe engraftment of transplanted hepatocytesthe engraftment of transplanted hepatocytesthe engraftment of transplanted hepatocytesthe engraftment of transplanted hepatocytesthe engraftment of transplanted hepatocytes the engraftment of transplanted hepatocytes the engraftment of transplanted hepatocytes. We had found that adequate transfusion rate did provide better early engraftment in normal and acute liver injured (D-gal-induced) rats, the latter of which showed subsequent good repopulation efficiency. We also found that the transplanted hepatocytes were observed outside of the sinusoid immediately after transplantation, in contrast to the previous other research groups. Transmigration through transcellular (fused fenestration, ICAM-1 related) or paracellular (adhesion junction disruption, VE-cadherin related) pathways is hypothesized. Shear-stress and adhesion-mediated transplanted hepatocyte transmigration through the liver sinusoidal endothelial cells are going to be investigated in our experiments. Real-time portal pressure monitoring during intraportal hepatocyte transplantation will be performed to indicate the timing of specimen collection in the ultra-short time period after hepatocyte transplantation. We will examine the changes in liver sinusoidal endothelium specifically focusing on shear-stress and adhesion related signaling modulation with different transfusion rates. Electro-microscopic examination will help capture the transmigration of the transplanted hepatocytes. We hope this experiment can improve the engraftment and repopulation in clinical hepatocyte transplantation by adjusting the rate of transfusion under tolerable hemodynamic disturbance or the manageable variable influenced by the rate and reaching a deeper understanding of basic engraftment mechanism.

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Mechanism of Transplanted Hepatocyte Engrafting in Rat of Acute Liver Injury- Interaction of Liver Sinusoidal Endothelial Cell and Transplanted Hepatocyte

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