Therapeutic Application of Adipose-Derived Stem Cell Transplantation in Rat Model of Acute-On-Chronic Liver Injury

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Project title/計畫英文名

Project Number/計畫編號

MOST106-2314-B002-148-MY2

Translated Name/計畫中文名

應用脂肪幹細胞在急性加慢性肝損傷大鼠治療的研究

Project Principal Investigator/計畫主持人

CHENG-MAW HO

Funding Organization

National Science and Technology Council

Start date/計畫起

01-08-2017

Expected Completion/計畫迄

31-07-2018

摘要：慢性肝病急性發作導致的肝衰竭 (ACLF) 是致死率很高的疾病，需要加護照顧且常要接受肝臟移植才能存活。ACLF 的根本機制仍有待釐清, 轉譯的動物模式有限也不甚完美。吾人為了嘗試細胞治療的可行性，發展了穩定的ACLF 大鼠模式動物。利用可吸收縫線綁總膽管製造肝纖維化，這種肝纖維化損傷術後可持續3-4 週，並注射D-galactosamine 誘發外加的急性肝損傷。人類基質血管部份 (SVF) 是從脂肪組織萃取出來，含有高濃度的初代脂肪間質幹細胞(ADSC)，ADSC 具有免疫調控、分化、血管/組織再生、抗凋亡的潛力，是具有細胞治療潛力的候選細胞。吾人初步測試人類的SVF 細胞和大鼠肝細胞共同移植到ACLF 大鼠肝中，發現表現CD34 的SVF 細胞可消弭肝纖維化並減少小膽管增生現象，不表現CD34 的SVF 細胞反而加重上述種種現象，和SVF 細胞一起移植的肝細胞則無明顯增生反應。進一步釐清這些不同的SVF 細胞作用是有其必要性。因此吾人欲執行CD34+、CD34-、及unsorted 人類SVF細胞單獨移植到ACLF 大鼠病肝中，探討纖維化、免疫發炎反應及小膽管增生相關交互作用及影響。星狀細胞的活化和整體ACLF 的環境變化有很大的關係，因此，體外細胞實驗方面，吾人欲利用LX2 細胞株 (代表活化的星狀細胞) 和SVF 細胞的條件培養液一起培養，觀察並檢驗LX2 細胞的活化、抑制或甚至產生凋亡現象。此外吾人在比對CD34+SVF 細胞和CD34-SVF 細胞的表現標記差異上，發現CD146 在CD34+細胞表現少，CD34-細胞表現多，CD146 表現的間質幹細胞被認為和血管平滑肌分化有關，可溶性CD146 引起的血管增生和肝纖維化惡化有關，CD146 也在肝星狀細胞表現。雖然相關資訊不多，種種跡象顯示CD146 可能是CD34-SVF 細胞移植後造成ACLF 肝纖維化加劇的主因。吾人將進行相關動物實驗(執行CD34-CD146+及CD34- CD146-人類SVF 細胞移植到ACLF 大鼠病肝中)驗證此假說。若上述轉譯實驗群指向一致，吾人將可用最小化操作模式篩選出適合ACLF 病人的SVF 細胞(保留CD34，去掉CD146)，達到臨床應用改善病人預後的最終目的。
Abstract: Acute-on-chronic liver failure (ACLF) is a distinct clinical entity characterized as an acutedeterioration of pre-existing chronic liver disease, usually related to acute insult andassociated with high short-term mortality in need of intensive care and liver transplantationdue to limited liver’s functional reserve. Stromal vascular fraction (SVF) derived fromhuman adipose tissue contains a rich source of primary adiposed derived stem/stromalcells (ADSCs), and ADSCs have great therapeutic potential in cell therapy throughimmunomodulation, differentiation, revascularization, anti-apoptotic and/or tissueregeneration. Our preliminary results of human isolated SVF cells and rat hepatocyteco-transplantation in ACLF rat model showed amelioration of liver fibrosis and ductularreaction by CD34+ (a common marker for diverse early-phase progenitors) SVF cells,aggravation of those histological features by CD34- SVF cells, and without obvious donorhepatocyte proliferation. Further clarified investigation of the roles of CD34+, CD34-,unsorted SVF cells in ACLF rat model will be performed by SVF cell transplantation alone.Additionally, co-culture of LX2 cells (a cell line of activated stellate cells) with conditionedmedium from culture of primary CD34+ or CD34- SVF cells will be performed todemonstrate the in vitro modulating mechanism by SVF cells toward stellate cells (whichplayed central roles in fibrosis and ductular reaction).When we compared the marker expression of CD34+ vs CD34- SVF cells, CD146appeared very high in CD34- and extremely low in CD34+ SVF cells. As CD146 expressionon mesenchymal stem cells is associated with their vascular smooth muscle commitment,soluble CD146-mediated angiogenesis is associated with progression of liver fibrosis andCD146 is actually also expressed in stellate cells, we will further test the hypothesis thatCD146 expression in SVF cells is detrimental in ACLF rat model by transplantation ofCD34- CD146+, CD34- CD146- SVF cells.Through these experiments, we can derive primary SVF cells for potential clinicalapplication in ACLF by positive marker selection (CD34) and negative selection (CD146).We hope these translational findings will improve the outcome of ACLF in the near future.

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Therapeutic Application of Adipose-Derived Stem Cell Transplantation in Rat Model of Acute-On-Chronic Liver Injury

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