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。我們初步利用肝細胞株(clone 9)當移植的肝細胞，以不同的速度 (10s, 30s, 和100s) 從門靜脈輸入細胞到雌性Sprague-Dawley大鼠的肝臟中並觀察術後24 和48h 觀察細胞進入基質(liver parenchyma)存活的情況，利用綠螢光蛋白的表現標記，追蹤移植肝細胞的早期進駐的程度；我們發現以30秒輸注細胞可產生最佳的engraftment。肝竇內皮血管內應力改變是這次實驗的唯一變數。應力的變化會誘發VEGFR2及往核內位移，並形成VEGFR2、VE-Cadherin,及βcatenin複合體；進而引發訊息傳遞。血管內皮產生誘導的訊號(VEGFR2-Id1-HGF/Wnt 2)也被發現和肝細胞增生及肝臟再生有關。進一步我們希望利用D-galactosamine引發急性肝損傷模式的F344 DPPIV-/-大鼠，接受不同速度(10s, 30s, 和100s) wild type 肝細胞輸注後，觀察肝竇內皮細胞受應力的影響並和engraftment及之後的repopulation差異做相關性比較。以便釐清輸注速度對細胞移植的影響程度，做出臨床輸注速度的改良建議，並對基本機制進行了解。
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 hepatocytes the engraftment of transplanted hepatocytesthe engraftment of transplanted hepatocytesthe engraftment of transplanted hepatocytes the engraftment of transplanted hepatocytesthe engraftment of transplanted hepatocytes the engraftment of transplanted hepatocytesthe engraftment of transplanted hepatocytes the engraftment of transplanted hepatocytes the 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 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 hepatocytes the engraftment of transplanted hepatocytes the engraftment of transplanted hepatocytes the engraftment of transplanted hepatocytes. We used hepatocyte cell line (clone 9) as the hepatocytes to be transplanted into portal vein of female Sprague-Dawley adult rats in rates over 10s, 30s, and 100s. Green fluorescent protein expression was used to evaluate the degrees of early engraftment of transplanted clone 9 cells. We found that transfusion over 30s would provide the best early engraftment. Shear stress in the liver sinusoidal endothelial cells was the only one variable changed in our experiments. Shear stress causes the induction and translocation of VEGFR-2 to the nucleus in bovine endothelial cells. In addition, it promotes the formation of a complex comprising VEGFR-2, VE-cadherin and β-catenin and triggers subsequent signaling pathways. Inductive angiocrine signals (VEGFR2-Id1-hepatocyte growth factor (HGF) and Wnt2) from sinusoidal endothelium are required for hepatocyte proliferation and liver regeneration. We, therefore, hope to further extend these findings to D-gal-induced acute liver injury in F344 DPPIV-/- rat model and apply to cell transplantation by transfusing wild type hepatocyte in different speeds (10s, 30s, and 100s). We will examine the changes in liver sinusoidal endothelium specifically focusing on shear stress related signaling modulation, and correlate with the engraftment and repopulation. We hope this experiment can improve the engraftment and repopulation in clinical hepatocyte transplantation by simply increasing the rate of transfusion under tolerable hemodynamic disturbance and reaching a deeper understanding of basic engraftment mechanism.
