OSM與LIF蛋白質對HepG2與K562細胞株生長之影響

細胞激素（cytokine）是影響細胞生長、分化與細胞之間相互傳遞訊息的因子，受到刺激後會快速合成。多數細胞素具有多種生物活性，即一種細胞素可以作用於不同細胞，有許多不同的功能，不同的細胞素之間也可能有相同或相似的功能。Oncostatin M（OSM）以及Leukemia inhibitory factor（LIF）都屬於Interleukin-6（IL-6）細胞激素家族（Cytokine family）的一員。LIF可以維持幹細胞的未分化狀態，在其他細胞卻有誘導分化的作用。而OSM可促進白血病細胞分化成類巨噬細胞（macrophage-like cells）。IL-6家族參與許多包括免疫反應、發炎反應、造血作用以及腫瘤發生等的生物反應。而這些反應都是藉由調控細胞的生長、分化以及存活來達成的。我們實驗室利用RT-PCR以及南方點墨法（Southern Blotting）分析許多正常組織、肝癌切片組織與肝癌細胞株HepG2、Huh7及Hep3B中LIF、OSM及其相關受器（Receptor）基因的表現量。我們發現正常的肝組織沒有看到LIF的表現，而OSM在胎兒肝組織中的表現量高出成人肝組織許多，且在胎兒肝組織及肝癌切片組織中其相關受器LIFR、OSMR及gp130的表現量皆有顯著的提高。而受測的肝癌細胞株HepG2、Huh7及Hep3B中皆表現LIF，不表現OSM，同時HepG2與Huh7都會表現相關受器LIFR、OSMR及gp130，而Hep3B則表現微量的LIFR及gp130。隨後我們分析OSM及LIF對肝癌細胞株生長的影響，發現LIF可以抑制HepG2的生長；OSM可以促進Huh7生長，抑制HepG2與Hep3B的生長。由於人類的OSM與LIF可以共享相同的receptor complex，我們在IMPACTTM的系統下製備六種重組蛋白質（OSM、OIO、OIL、LIO、LIL與LIF），並分析他們對肝癌細胞株生長狀態的影響以及其下游訊息傳遞分子ERK的活化。根據MTT assay，HepG2對OSM的感受性最強，生長受到抑制的現象最為明顯。而重组蛋白質亦能在不同程度下誘導細胞ERK的磷酸化。癌細胞的生長受到抑制導因於細胞自戕（apoptosis）、生長停滯（cell cycle arrest）或是細胞分化（differentiation）。倘若能將癌細胞往分化的路徑誘導，使癌細胞分化為正常細胞，則不失為另一種治療癌症的新方向。故我們又選用了K562這種在分化研究上較被廣泛應用的細胞株作為研究的對象。在以流式細胞儀分析細胞週期的數據裡，我們初步排除OSM引導細胞自戕的可能性，並且發現OSM具有調節HepG2與K562細胞週期的作用。接著我們使用K562細胞中的分化負調控基因NDR作為測試細胞是否走向化的標的物（marker）。結果在OSM的處理下K562細胞中NDR蛋白被降解的情況沒有出現，但細胞增生的能力被抑制後，可能會再藉由特定因子的活化後使細胞走向分化，或者提高細胞分化能力，增加分化細胞的比例。

關鍵字

細胞生長；細胞激素

並列摘要

Leukemia Inhibitory factor（LIF）and Oncostatin M（OSM）are members of the interleukin-6（IL-6）cytokine family. And both of them trigger partial overlapping signals through partially shared receptor complexes in human. IL-6 cytokine family is involved in a variety of biological responses. The hematopoiesis, inflammation immune response, and oncogenesis through their effect on cell growth, survival and differentiation are included. In current studies, the gene expression pattern of OSM, LIF and their related receptors in normal tissues and hepatoma cell line is known. OSM was expressed more abundantly in the fetal liver than in adult liver, while the expression of LIF was undetectable in normal livers. However, LIF, but not OSM, was expressed in HCC cell lines Hep3B, Huh7, and HepG2. And their receptors, LIFR, OSMR and gp130, were expressed mainly in fetal liver, Huh7 and HepG2 cell lines. We also found that OSM and LIF could inhibit the growth of HCC and K562 cell lines. In order to enhance the effect of growth inhibition, we try to increase the OSM and LIF concentration on the local surface of the cells by recombine six kinds of recombinant proteins in the IMPACTTM-CN System. They are LIF, OSM, LIL, OIO, LIO and OIL. After prepared these proteins, we first examined their effects on the cell growth and ERK phosphorylation.. Showed that the growth of HepG2 cells about 50%, while 20% of Hep3B cell line have been suppressed by all recombinant proteins. But the biological effects of dimerizative proteins weren’t more dominant than the monomer. We also found that OSM and OIO inhibit the growth of K562 cells, while LIF, LIL, LIO and OIL do not have work. On the contrary, the growth of Huh7 was promoted by OSM, LIO, OIL and OIO. And these proteins can induce the activation of ERK in differential level. We try to figure out what happened during the process of growth inhibition, and use the FACS analysis to examine that. As the results have showed, there was no apoptosis effect in the K562 and HepG2 under the treatment of OSM. But OSM could modulate the cell cycle and may slow down the process through each phase of cell cycle. We also make use of NDR protein which would be down-regulate during the differentiation of K562 cell. And as the result showed, NDR wouldn’t be down-regulate by OSM in our system.

並列關鍵字

OSM ； growth ； K562 ； LIF

參考文獻

1. Peter C. HEINRICH, Iris BEHRMANN, Gerhard MÜLLER-NEWEN, Fred SCHAPER and Lutz GRAEVE. Interleukin-6-type cytokine signaling through the gp130/Jak/STST pathway. Biochem J. 334:297-314（1998）

2. Tracey, K. J., et al. Anti-cachectin/TNF monoclonal antibodies prevent septic shock during lethal bacteraemia. Nature 330:662 - 664（1987）

3. Toshio Hirano, Katsuhiko Ishihara and Masahiko Hibi. Role of STAT3 in mediating the cell growth, differentiation and survival signals relayed through the IL-6 family of cytokine receptor. Oncogene 19:2548-2556（2000）

4. Peter C. HEINRICH, Iris BEHRMANN, Serge HAAN, Heike M. HERMANNS, Gerhard MÜLLER-NEWEN and Fred SCHAPER. Principles of interleukin (IL)-6-type cytokine signaling and its regulation. Biochem. J. 374:1–20（2003）

5. Sutherland GR, Baker E, Hyland VJ, Callen DF, Stahl J, Gough NM. The gene for human leukemia inhibitory factor (LIF) maps to 22q12. Leukemia. 3:9-13.（1989）

國際替代計量

OSM與LIF蛋白質對HepG2與K562細胞株生長之影響

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