重组灵芝免疫调节蛋白（rLZ-8）免疫调节及抗癌机制初步研究
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摘要
Ling Zhi-8(LZ-8)是一种从灵芝(Ganoderma lucidium)菌丝体中提取出的真菌免疫调节蛋白。LZ-8具有免疫调节活性,可以促进人外周血淋巴细胞和人外周血单个核细胞以及小鼠脾细胞的增殖,诱导分泌白细胞介素,肿瘤坏死因子和干扰素等细胞因子,以及调节免疫活性细胞黏附分子的表达,并且LZ-8具有抗肿瘤活性。目前其作用机制尚未完全清楚。
本文通过毕赤酵母(Pichia pastoris)表达系统来表达重组灵芝免疫调节蛋白(rLZ-8),经过纯化的rLZ-8用绵羊红细胞凝血实验以及小鼠脾淋巴细胞增殖实验来检测其生理活性,得到了高纯度并且具有稳定生理活性的rLZ-8样品。用异硫氰酸荧光素(FITC)对rLZ-8进行荧光标记,将标记后的FITC-rLZ-8分别与人外周血淋巴细胞(HPBL),小鼠脾细胞,293T细胞,A549细胞进行共培养,利用激光共聚焦显微镜观察rLZ-8在这些细胞中的亚细胞定位,结果发现rLZ-8在有些HPBL的细胞膜上富集,而在有些HPBL细胞的内部富集,仅在小鼠脾细胞的细胞膜上分布,仅在293T与A549细胞内部分布,说明rLZ-8与不同细胞的作用方式和作用位点有所不同。
利用MTT法测定rLZ-8对A549细胞增殖的影响,结果显示rLZ-8能够抑制A549细胞的增殖并且与rLZ-8的剂量和作用时间呈正相关。进一步对与rLZ-8共培养的A549细胞进行衰老相关的[β]-半乳糖苷酶(SA-[β]-Gal)染色实验,结果表明与rLZ-8共培养的A549细胞表现出早熟衰老的特征,说明rLZ-8可能通过诱导A549细胞的早熟衰老来抑制其增殖。
本文表达和纯化了rLZ-8,得到了高纯度并且具有稳定生物活性的rLZ-8样品,为大规模获得rLZ-8从而进一步研究LZ-8的功能和作用机制以及在临床上的应用奠定了基础。并且利用FITC荧光标记技术对rLZ-8在几种细胞中的亚细胞定位做了初步的探索,同时发现rLZ-8对人类肺癌细胞A549的增殖有抑制作用,其原因可能是由于rLZ-8诱导了A549细胞发生早熟衰老,本实验的研究结果,为rLZ-8发挥免疫调节作用以及抗肿瘤活性提供了可能的机制,对rLZ-8在抗肿瘤方面的应用提供了一定的理论支持。
Ling Zhi-8 (LZ-8) is a fungal immunomodulatory protein that is isolated from mycelia of Ganoderma lucidium. In terms of immunomodulatory activity, LZ-8 acts as a mitogen for human peripheral lymphocytes(HPBL), human peripheral blood mononuclear cells and mouse spleen cells, and induces the production of cytokines such as interleukin-2, tumor necrosis factor, interferon-γand so on. LZ-8 also can modifies the expression of adhesion molecules on immunocompetent cells. In addition to its immunomodulatory effects, LZ-8 exhibits anticancer properties. However, its mechanisms of immunomodulatory effects and anticancer activity are still not fully understood now.
In this study, we expressed and purified the recombinant fungal immunomodulatory protein rLZ-8 in Pichia pastoris expression system, and then, used hemagglutination reaction and cell proliferation assay to check the biological activity of rLZ-8. Cells, treated with FITC-rLZ-8(rLZ-8 is linked with FITC), were observed by confocal microscopy scanning laser microscope to detect the cellular localization of rLZ-8. The result shows that rLZ-8 gathers on cell membrane of some HPBLs, and enters into some other HPBLs. But rLZ-8 gathers only on cell membrane of mouse spleen cells, and it only gathers in the cells of A549 and 293T. This means that rLZ-8 may has different action ways and sites to cells. MTT assay was used to determine the effect of rLZ-8 on the proliferation of A549 cells.
The results show that rLZ-8 suppresses the proliferation of A549 cells in a dose and time-dependent manner. Then, we examined the expression of senescence-associated [β]-galactosidase (SA-[β]-Gal) in A549 by staining, the results suggest that rLZ-8 may induce premature senescence in A549, thus it can inhibits the proliferation of A549 cells.
We successfully expressed and purified the recombinant fungal immunomodulatory protein rLZ-8, and investigated the cellular location of rLZ-8 by using FITC. We also found that rLZ-8 can suppresses the proliferation of A549 cells, maybe by trigering premature senescence. These studies would provided some possible mechanisms for immunomodulatory effects and anticancer properties of rLZ-8, and the use of LZ-8 may be a powerful new strategy for chemoprevention and antineoplastic therapy.
本文通过毕赤酵母(Pichia pastoris)表达系统来表达重组灵芝免疫调节蛋白(rLZ-8),经过纯化的rLZ-8用绵羊红细胞凝血实验以及小鼠脾淋巴细胞增殖实验来检测其生理活性,得到了高纯度并且具有稳定生理活性的rLZ-8样品。用异硫氰酸荧光素(FITC)对rLZ-8进行荧光标记,将标记后的FITC-rLZ-8分别与人外周血淋巴细胞(HPBL),小鼠脾细胞,293T细胞,A549细胞进行共培养,利用激光共聚焦显微镜观察rLZ-8在这些细胞中的亚细胞定位,结果发现rLZ-8在有些HPBL的细胞膜上富集,而在有些HPBL细胞的内部富集,仅在小鼠脾细胞的细胞膜上分布,仅在293T与A549细胞内部分布,说明rLZ-8与不同细胞的作用方式和作用位点有所不同。
利用MTT法测定rLZ-8对A549细胞增殖的影响,结果显示rLZ-8能够抑制A549细胞的增殖并且与rLZ-8的剂量和作用时间呈正相关。进一步对与rLZ-8共培养的A549细胞进行衰老相关的[β]-半乳糖苷酶(SA-[β]-Gal)染色实验,结果表明与rLZ-8共培养的A549细胞表现出早熟衰老的特征,说明rLZ-8可能通过诱导A549细胞的早熟衰老来抑制其增殖。
本文表达和纯化了rLZ-8,得到了高纯度并且具有稳定生物活性的rLZ-8样品,为大规模获得rLZ-8从而进一步研究LZ-8的功能和作用机制以及在临床上的应用奠定了基础。并且利用FITC荧光标记技术对rLZ-8在几种细胞中的亚细胞定位做了初步的探索,同时发现rLZ-8对人类肺癌细胞A549的增殖有抑制作用,其原因可能是由于rLZ-8诱导了A549细胞发生早熟衰老,本实验的研究结果,为rLZ-8发挥免疫调节作用以及抗肿瘤活性提供了可能的机制,对rLZ-8在抗肿瘤方面的应用提供了一定的理论支持。
Ling Zhi-8 (LZ-8) is a fungal immunomodulatory protein that is isolated from mycelia of Ganoderma lucidium. In terms of immunomodulatory activity, LZ-8 acts as a mitogen for human peripheral lymphocytes(HPBL), human peripheral blood mononuclear cells and mouse spleen cells, and induces the production of cytokines such as interleukin-2, tumor necrosis factor, interferon-γand so on. LZ-8 also can modifies the expression of adhesion molecules on immunocompetent cells. In addition to its immunomodulatory effects, LZ-8 exhibits anticancer properties. However, its mechanisms of immunomodulatory effects and anticancer activity are still not fully understood now.
In this study, we expressed and purified the recombinant fungal immunomodulatory protein rLZ-8 in Pichia pastoris expression system, and then, used hemagglutination reaction and cell proliferation assay to check the biological activity of rLZ-8. Cells, treated with FITC-rLZ-8(rLZ-8 is linked with FITC), were observed by confocal microscopy scanning laser microscope to detect the cellular localization of rLZ-8. The result shows that rLZ-8 gathers on cell membrane of some HPBLs, and enters into some other HPBLs. But rLZ-8 gathers only on cell membrane of mouse spleen cells, and it only gathers in the cells of A549 and 293T. This means that rLZ-8 may has different action ways and sites to cells. MTT assay was used to determine the effect of rLZ-8 on the proliferation of A549 cells.
The results show that rLZ-8 suppresses the proliferation of A549 cells in a dose and time-dependent manner. Then, we examined the expression of senescence-associated [β]-galactosidase (SA-[β]-Gal) in A549 by staining, the results suggest that rLZ-8 may induce premature senescence in A549, thus it can inhibits the proliferation of A549 cells.
We successfully expressed and purified the recombinant fungal immunomodulatory protein rLZ-8, and investigated the cellular location of rLZ-8 by using FITC. We also found that rLZ-8 can suppresses the proliferation of A549 cells, maybe by trigering premature senescence. These studies would provided some possible mechanisms for immunomodulatory effects and anticancer properties of rLZ-8, and the use of LZ-8 may be a powerful new strategy for chemoprevention and antineoplastic therapy.
引文
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[2]Kino K, Yamashita A, Yamaoka K, et al. Isolation and characterization of a new immunomodulatory protein, Ling Zhi-8 (LZ-8), from Ganoderma lucidium[J].The Journal of Biological Chemistry, 1989, 264(1): 472-478.
[3]Ko J L, Hsu C I, Lin R H, et al. A new fungal immunomodulatory protein, FIP-fve isolated from the edible mushroom, Flammulina velutipes and its complete amino acid sequence[J]. Eur J Biochem, 1995, 228: 244-249.
[4]Hsu H C, Hsu C I, Lin R H, et al. Fip-vvo, a new fungal immunomodulatory protein isolated from Vovariella volvacea[J]. Biochem J, 1997, 332(2): 557-565.
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[6]Hsiao Y M, Huang Y L, Tang S C, et al. Effect of a fungal immunomodulatory protein from Ganoderma tsugae on cell cycle and interferon-gamma production through phosphatidylinositol 3-kinase signal pathway[J]. Process Biochemistry, 2008, 43: 423-430.
[7]Ko J L, Lin S J, Hsu C I, et al. Molecular cloning and expression of a Fungal Immunomodulatory Protein, FIP-fve, from Flammulina velutipes[J]. J Formos Med Assoc, 1997, 96: 517-524.
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[2]Kino K, Yamashita A, Yamaoka K, et al. Isolation and characterization of a new immunomodulatory protein, Ling Zhi-8 (LZ-8), from Ganoderma lucidium[J].The Journal of Biological Chemistry, 1989, 264(1): 472-478.
[3]Ko J L, Hsu C I, Lin R H, et al. A new fungal immunomodulatory protein, FIP-fve isolated from the edible mushroom, Flammulina velutipes and its complete amino acid sequence[J]. Eur J Biochem, 1995, 228: 244-249.
[4]Hsu H C, Hsu C I, Lin R H, et al. Fip-vvo, a new fungal immunomodulatory protein isolated from Vovariella volvacea[J]. Biochem J, 1997, 332(2): 557-565.
[5]Lin W H, Huang C H, Hsu C I, et al. Dimerization of the N-terminal amphipathicα-helix domain of the fungal immunomodulatory protein from Ganoderma tsugae (Fip-gts) defined by a yeast two-hybrid system and site-directed mutagenesis[J]. The Journal of Biological Chemistry, 1997, 272(32): 20044-20048.
[6]Hsiao Y M, Huang Y L, Tang S C, et al. Effect of a fungal immunomodulatory protein from Ganoderma tsugae on cell cycle and interferon-gamma production through phosphatidylinositol 3-kinase signal pathway[J]. Process Biochemistry, 2008, 43: 423-430.
[7]Ko J L, Lin S J, Hsu C I, et al. Molecular cloning and expression of a Fungal Immunomodulatory Protein, FIP-fve, from Flammulina velutipes[J]. J Formos Med Assoc, 1997, 96: 517-524.
[8]Murasugi A, Tanaka S, Komiyama Naoki, et al. Molecular cloning of a cDNA and a gene encoding an immunomodulatory protein, Ling Zhi-8, from a fungus, Ganoderma lucidium[J]. The Journal of Biological Chemistry, 1991, 266(4): 2486-2493.
[9]Paaventhan P, Joseph J S, Seow S V, et al. A 1.7 ? structure of fve, a member of the new fungal immunomodulatory protein family[J]. J Mol Biol, 2003, 332: 461-470.
[10]Seow S V, Kuo I C, Paaventhan P, et al. Crystallization and preliminary X-ray crystallographic studies on the fungal immunomodulatory protein Fve from the golden needle mushroom (Flammulina velutipes) [J]. Acta Crystallog sect, 2003, 59(9): 1487-1489.
[11]Huang L, Sun F, Liang C Y, et al. Crystal structure of LZ-8 from the medicinal fungus Ganoderma lucidium[J]. Proteins, 2008, 75(2): 524-527.
[12]常团结,朱祯.植物凝集素及其在抗虫植物基因工程中的应用[J].遗传HEREDITAS(beijing), 2002, 24(4): 493-500.
[13]林景卫,孙非,张韧,等.真菌免疫调节蛋白的研究进展[J].中国免疫学杂志,2005, 6: 477-450.
[14]Wang P H, Hsu C I, Tang S C, et al. Fungal immunomodulatory protein from Flammulina velutipesinduces interferon-γproduction through p38 mitogen-activated protein kinase signaling pathway[J]. J Agric Food Chem, 2004, 52: 2721-2725.
[15]孔祥辉,孙宇峰,任永春,等.金针菇免疫调节蛋白的研发和应用[J].生物技术, 2006, 16(4): 84-88.
[16]Hsieh K Y, Hsu C I, Lin J Y, et al. Oral administration of an edible-mushroom-derived protein inhibits the development of food-allergic reactions in mice[J]. Clin Exp Allergy, 2003, 33(11): 1595-1602.
[17]Liu Y H, Kao M C, Lai Y L, et al. Efficacy of local nasal immunotherapy for Dp2-induced airway inflammation in mice: Using Dp2 peptide and fungal immunomodulatory peptide[J]. J Allergy Clin Immunol, 2003, 112(2): 301-310.
[18]Kino K, Mizunoto K, Sone T, et al. An immunomodulatory protein, Ling Zhi-8 (LZ-8) prevents insulitis in nonobese diabetic mice[J]. Diabetologia, 1990, 33(12): 713-718.
[19]Van der Hem L G, Van der Vliet J A, Bocken C F M, et al. Ling Zhi-8: studies of a new immunomodulating agent[J]. Transplantation, 1995, 60(5): 438-443.
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