以人類疾病動物模式探討微環境對幹細胞命運之影響-骨髓間葉幹細胞團塊大小對減緩肝纖維化小鼠之功效及後續之命運

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Title:	以人類疾病動物模式探討微環境對幹細胞命運之影響-骨髓間葉幹細胞團塊大小對減緩肝纖維化小鼠之功效及後續之命運
Other Titles:	Size-Dependent Effect of Mscs Clusters on Migration into Fibrosis Liver of Mice and Subsequent Cell Fate
Authors:	鄭登貴 CHENG TENG-KUEI WIN
Contributors:	東海大學畜產與生物科技學系行政院國家科學委員會
Keywords:	骨髓間葉幹細胞；肝纖維化；轉基因豬；轉基因鼠；細胞移植 Mesenchymal stem cells； EGFP； Transgenic mice； Liver fibrosis； Cell transplantation
Date:	2011
Issue Date:	2014-03-07T03:29:37Z (UTC)
Abstract:	肝纖維化 (liver fibrosis) 為各種慢性肝病進展為肝硬化(cirrhosis)之必經途徑；其成因為肝臟遭受慢性損傷後反覆癒合等反應導致細胞外間質 (extracellular matrix-ECM) 過量堆積形成纖維狀傷疤，逐步惡化將破壞肝組織之結構並影響其功能，最後演變為肝硬化與肝功能衰竭（liver failure），因此探討如何阻止或延緩肝纖維化的發生，將對肝病之治療與後續之研究發展有重要之意義。然而肝臟移植為目前嚴重肝臟疾病患者常採用之治療方式，但其受限於捐贈肝之來源短缺、移植後可能具有免疫排斥反應及多發性併發症等問題，在臨床治療上有其限制；因此如何於惡化至肝硬化之不可逆階段前，予以治療並針對肝臟疾病發展新治療方式，為目前迫切需要解決之重要課題。近來已有研究報告指出，移植骨髓間葉幹細胞 (mesenchymal stem cells, MSCs) 於肝臟受損之動物模式，可有效減緩肝纖維化之病程，然而追蹤所施打之細胞遷移入肝臟之比例仍十分低，因此如若要提升治療之效果或許可藉由提升 MSCs 遷移入損傷肝臟之比例著手，並探討幹細胞遷入損傷組織之後續細胞命運。本研究目標係探討由螢光基因轉殖小鼠及豬所分離之骨髓所分離之間葉幹細胞經預培養為不同大小之細胞團塊後，由脾內注射或肝門靜脈注射移植入肝纖維化小鼠體內，經八週後分析其移植細胞團遷移之效率、改善肝纖維化之功效及移植細胞之命運。本三年期計畫之第一年將以CCL4 處理以建立肝纖維化小鼠模式，並分離螢光基因轉殖小鼠及豬之MSCs，且進一步建立將其條件適化及培養成不同大小之細胞團塊的條件；第二年將移植不同大小之細胞團塊於肝纖維化小鼠，並於八週後評估其移植細胞遷移之效率、肝臟膠原蛋白之含量及血液生化分析；第三年將延續第二年之試驗，並配合laser capture 設備取得植入且遷移至肝纖維化小鼠之細胞，進一步進行組織化學免疫染色、細胞免疫染色及其表現基因之差異性，期能更深入了解其不同大小細胞團塊之移植細胞遷移後之命運。 In chronic liver diseases, liver fibrosis is one of the most common symptoms which is the excessive accumulation of extracellular matrix in the liver, results in destruction of the hepatic tissue architecture by formation of a fibrous scar and in deterioration of the function of liver parenchymal cells, and can subsequently lead to cirrhosis with nodules of partially regenerated hepatocytes surrounded by irregularly distributed fibrous septa. Liver transplantation is the most drastic therapy for cirrhosis with chronic hepatic failure, but shortage of donor organs limits applicability of this therapy. Thus, it is necessary to devise better therapeutic approaches for this disease. Mesenchymal stem cells (MSCs) are defined as plate-adhering, fibroblast-like cells possessing the capacity to self renewal and differentiate into multiple cell types. The unique properties of MSCs are related to easy isolation and high proliferating activity. In previous studies demonstrated that human marrow-derived MSCs under chemically defined conditions can differentiate into functional hepatocyte-like cells. Obviously, MSCs exhibit the potentiality on cell therapy of liver diseases. Recently, transplantation of bone marrow-derived MSCs has been shown to ameliorate liver fibrosis in animal models with only few cells migrated into damaged area. It is reasonable to suppose that liver fibrosis can be better mitigated by improving the migration rate of MSCs to the injured liver and study the fate of migrated cells. In this study, we aim to investigate the effects of cell clusters of MSCs in different sizes when transplanted into portal vein or intra-spleen on migration into fibrosis liver of mice for clarifying the potency of ameliorating liver fibrosis and subsequent cell fate. In the first year, we will establish culture system of cell clusters in different sizes and establish the liver fibrosis mouse model by using CCL4 treatment, as well as isolate bone marrow-derived MSCs from EGFP transgenic mice and optimize condition for cell isolation, purification, culture and maintenance of MSCs from bone marrow of fluorescent transgenic pigs and mice that have been generated from our laboratory. In the second year, transplant cell clusters in different sizes into recipient mice and estimate the efficiency of transplanted cell migration, collagen content assay and biochemical assay of blood after cell transplantation for 8 weeks. In the third year, repeat the experiment as second year to explore cell fate assay of migrated cells in recipient liver by using laser capture associate with immunohistochemistry and immunocytochemistry staining, evaluate therapeutic potency of cell clusters in different sizes.
Relation:	計畫編號：NSC100-2313-B029-002-MY2 研究期間：2011-08~ 2012-10
Appears in Collections:	[畜產與生物科技學系所] 國科會研究報告

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