以Sf21細胞建立與表現Irisin並探討其對人類神經膠質瘤細胞株U-87 MG之影響

Abstract

中文摘要 運動能幫助預防與減緩許多疾病，像是肥胖、心血管疾病、第二型糖尿病甚至是癌症。然而，對於運動產生諸多益處的生理機制尚未闡明。近年研究發現，運動促使肌肉細胞高量表現膜蛋白FNDC5，隨後，FNDC5的中間部分片段會被截切且釋放到血液循環中，此片段被命名為irisin。Irisin最後作用於白色脂肪細胞促使其進行browning，造成能量型態轉變並增加能量消耗。此外，也有研究發現irisin對於乳癌細胞株與前列腺癌細胞株的增生有抑制的效果，因此，irisin對於癌細胞的抑制效果值得更深入的探討。 在我們先前的研究中發現，以大腸桿菌系統表現的irisin對人類神經膠質瘤細胞株U-87 MG 的細胞增生與侵襲有抑制的效果。此外，有研究指出irisin上帶有的醣基化修飾對於其browning的功能有重要的影響。因此，我們想進一步了解，irisin上的醣基化是否對於抑制U-87 MG也有重要的影響。我們利用昆蟲細胞表現系統表現irisin並探討其對U-87 MG的影響。實驗分成三個部分，首先，我們利用Sf21昆蟲細胞株來表現irisin。在以桿狀病毒感染72小時後，irisin會被外泌至培養液中，接著經由Ni-NTA系統的純化，獲得高純度的irisin。Irisin再經由西方墨點法以FNDC5抗體確認並以質譜儀鑑定，最後並以能截切醣基的酵素加以確認醣基化的存在。第二階段，我們探討irisin對於U-87 MG的影響，發現irisin能抑制細胞增生與侵襲但不影響細胞遷移的能力。第三階段以Sf21表現之irisin與E.coli表現之irisin對於U-87 MG的影響進行比較，發現以Sf21表現之irisin能更早抑制細胞增生，而兩者在細胞遷移與侵襲的影響則沒有明顯的差異，另外，我們也發現，在Sf21表現之irisin上的醣基化比例會影響其抑制細胞增生的效果。綜合以上結果，我們了解到以昆蟲細胞表現之irisin對於U-87 MG的細胞增生與侵襲有抑制效果且其上的醣基化對於抑制細胞增生有影響。

Abstract Exercise help prevent many diseases including obesity, cardiovascular disease and type II diabetes mellitus etc. Besides, it can improve the survival of cancer patients. However, the mechanism of how exercise benefits our health is still unclear. Recently, researchers have found that after exercise, muscle cells elevate the expression of FNDC5, one transmembrane protein. Next, the internal fragment of FNDC5 will be proteolytically cleaved and secreted into circulation and this fragment was named irisin. Irisin will target to white adipocytes and induce white adipocytes browning, which leads to change in energy status and increase in energy expenditure. Moreover, it is reported that irisin has suppressive effects on cell proliferation in breast and prostate cancer cells. Therefore, irisin has attracted more attention on its function to repress cancer cells. In our previous studies, irisin purified from E. coli had inhibitory effects on cell proliferation and invasion in the glioblastoma cell line, U-87 MG. Moreover, glycosylation on irisin was found pivotal for the browning function of irisin. Thus, we wonder if glycosylation on irisin is also vital for suppressive effects on cancer cells as well. Therefore, we used an insect cell expression system to express irisin to further study its effect on U-87 MG cells. There are three parts in this thesis. First, Sf21 insect cells were used to produce recombinant human irisin. After infection of Sf21 cells with baculovirus for 72 hours, irisin was secreted into medium. Afterward, the Ni-NTA purification system was conducted to purify irisin and the identity of irisin was verified by western blotting with -FNDC5 antibody and Mass spectrometry. Furthermore, glycosylation was also confirmed by digestion with glycosidase. Second, the effects of irisin on U-87 MG cells were evaluated by cell proliferation, migration and invasion assays. After irisin treatment, the cell proliferation and invasion were decreased while cell migration remained unaffected. Third, irisin produced in Sf21 cells and E.coli were both used to treat U-87 MG cells. Cell proliferation was suppressed earlier of treatment with irisin from Sf21 cells than treatment with irisin from E.coli. Nevertheless, cell migration and invasion had no difference after cells treated with irisin from these two expression system. Besides, lower glycosylation ratio on irisin from Sf21 cells might lose the repressive effect on cell proliferation of U-87 MG cells. In conclusion, irisin produced in insect cells could inhibit cell proliferation and invasion of U-87 MG cells. Furthermore, the glycosylation on irisin had effect on inhibiting cell proliferation.