以real time PCR及Western Blot鑑定肝細胞再生時，基因微陣列監測出免疫相關基因之變遷

Abstract

吾人以前研究顯示，肝臟部分切除 後，除肝細胞再生外，細胞免疫之變遷， 包括CD4、CD8、淋巴球、自然殺手細胞 之增加，血中γ-IFN 之上升，及巨噬細胞 之增加等，均相當明顯，且發生時間在肝 細胞再生後期，是否與再生終結機轉有關 則尚不明瞭。若能研究瞭解肝細胞再生時 免疫相關基因之變遷，對將來以基因治療 控制免疫反應，來促進肝細胞再生、增加 病患存活率必有很大貢獻。 本計劃以重約200 克之Wistar 雄性大 鼠做實驗，以基因微陣列方式監測，並以 real time polymerase chain reaction(RT-PCR) 及Western Blot 鑑定肝細胞再生過程中， 各種免疫相關基因表現在各時段之變遷。 所有大鼠將分別接受約百分之七十及四十 之肝臟部分切除手術，各於術前及術後2、 4、6、12、24、48、72 小時及5、7 天後犧 牲取樣，測定(1)剩餘肝臟之重量比值; (2) 剩餘肝臟之有絲分裂指標; (3)以基因微陣 列尼龍膜晶片、肝細胞mRNA 標號、 hybridization 及影像分析等方法，測定各種 基因表現之變遷，並詳細分析免疫相關基 因群; (4)以RT-PCR 分析剩餘肝臟中，各種 免疫相關基因之mRNA 表現情形，並比較 與Microarray 所監測出免疫相關基因變遷 之一致性; (5) 以Western blot 分析剩餘肝 臟中，免疫相關基因之蛋白質產量，並比 較其與基因及mRNA 變遷之一致性; (6) 比較70%及40%肝臟切除後所有免疫相關 基因、mRNA、蛋白質表現之程度、型態、 時程之差異性。 結果顯示：(1)剩餘肝臟重量比值，切 肝40％後恢復速度較慢，但至術後72 小 時，40％與70％兩組幾乎到達同樣重量； (2)有絲分裂切肝40％後48 小時也出現， 至72 小時逐漸減少，但程度上比70％切肝 後明顯較少；(3)共8 種免疫相關基因在切 肝手術後， 有三種型態明顯的變化； (4)histocompatibility 2, O region alpha locus gene and early B-cell factor gene 在切肝術 後早期明顯上升，表示此2 種基因可能為 肝細胞再生之啟動機轉相關基因； (5)histocompatibility 2, Q region alpha locus gene, immunoglobulin J chain precursor gene, histocompatibility 2, complement component factor gene, and histocompatibility 2, L region locus gene 在切肝術後中期，有明顯下降趨勢，至晚期又有回升現象，表示此4 種基因可能在肝細胞再生之分化機轉，扮 演了down-regulation 的角色；(6) interferon gamma receptor 2 gene and mast cell growth factor gene 在切肝術後晚期明顯上升，表示 此2 種基因可能為肝細胞再生之終結機轉 相關基因；(7)IL-6 及IL-10 兩種基因在 microarry 顯示之基因變化極其相關 m-RNA (RT-PCR 檢測)變化之量、程度及 型態均相似，表示IL-及IL-10 兩種基因在 肝細胞再生機轉中，應扮演較重要的角 色；(8)以Western Blotting 檢測出來之相關 蛋白質量，與基因及m-RNA 量均不符合， 可能需考慮之因素較複雜。本研究結果顯 示，免疫相關基因在肝細胞再生之啟動、 分化、終結等過程之調控機轉，均扮演了 重要之角色。

Cell mediated immune response, including increasing of CD4, CD8 lymphocytes, NK cells, γIFN and macrophage were noted during liver regeneration after partial hepatectomy. Whether immune response is the termination factor for regeneration is not clear. The study about the relationship between immune related gene expressions and liver regeneration could be very important for gene therapy to increase liver regeneration and survival rate. The main purpose of this project was to find out the variation of immune related genes during liver regeneration after partial hepatectomy. Male Wistar rats around 200g were used as subject. Partial hepatectomy around 70% and 40% were be performed and they will be sacrificed at 2, 4, 6, 12, 24, 48, 72 hours and 5, 7days after hepatectomy. We measured: (1) weight of remnant liver; (2) mitotic index; and (3) genomic survey of the expression for many proto-oncogenes by cDNA microarray on nylon membrane, labeling of liver mRNA hybridization and image analysis of the cluster of immune related genes; (4) the mRNA expression of immune related genes by real time polymerase chain reaction (RT-PCR), and compare the compatibility between the genes and the mRNA expression ; (5) the protein product of immune related genes by Western Blot analysis, and compare the compatibility between the genes expressions and the protein products; (6) Compared the difference of all positive immune related gene expression in the variation degree, changing pattern, specific timing and genes subgrouping cluster between 70% and 40% partial hepatectomy. The results were: (1) the remnant liver weight recovered slower, but can reach 90% in 72h after partial hepatectomy; (2) the mitosis of hepatocytes also increased markedly at 48h although not so high as 70% group rats, and also decreased at 72h after partial hepatectomy; (3) there were 8 immune related genes identified with marked changes in 3 different patterns after partial hepatectomy; (4) histocompatibility 2, O region alpha locus gene and early B-cell factor gene showed a marked elevated peak at early PH period, suggesting as initiative genes for regeneration; (5) histocompatibility 2, Q region alpha locus gene, immunoglobulin J chain precursor gene, histocompatibility 2, complement component factor gene, and histocompatibility 2, L region locus gene showed a down-regulated pattern at mid-term PH period, suggest as differentiative genes for regeneration; (6) interferon gamma receptor 2 gene and mast cell growth factor gene showed a late increasing peak suggesting as terminated genes for regeneration; (7) the changing curves of IL-6 and IL-10 genes cloning by microarray and their related m-RNA detected by RT-PCR were similar after 70% vs 40% partial hepatectomy, suggesting IL-6 and IL-10 genes play more important roles in regeneration mechanism; (8) protein products detected by Western Blotting showed no similar changes when compared with microarray and RT-PCR, suggesting protein products were affected by more factors. It is suggested that some immune related genes did play important roles in the mechanism of initiation, differentiation and termination during liver regeneration after partial hepatectomy.