CN102755354B - 尖孢镰刀菌提取物及其应用 - Google Patents
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Abstract
本发明公开了尖孢镰刀菌(Fusarium oxysporum)水提取物及其多糖在免疫增强方面的作用与应用，属于生物制药领域。尖孢镰刀菌水提取物及其多糖具有显著免疫增强作用，可促进巨噬细胞增殖、增强巨噬细胞吞噬功能、激活巨噬细胞释放免疫活性因子NO、IL-1α和TNF-α。本发明确定的尖孢镰刀菌水提取物或其多糖免疫增强作用，可用于免疫功能低下疾病及肿瘤辅助治疗。
Description
技术领域
本发明属于生物制药领域，涉及尖孢镰刀菌(Fusarium oxysporum)水提取物及其多糖在免疫增强方面的作用和应用。
背景技术
免疫系统是机体排除病原微生物侵犯，消除衰老、损伤和病变细胞从而维持自身稳定的最重要防御系统。免疫增强药物可以提高免疫系统组织细胞的生物活性，增加机体免疫细胞的生长及抗体的合成，调节机体的免疫功能，维持机体内环境平衡，从而提高机体的抗病力。目前免疫增强药物已广泛应用于抗肿瘤、各种感染性疾病和继发性免疫缺陷疾病的辅助治疗[Hadden，J.W.Immunostimulants.Trends in Pharmacological Sciences，1993，14：169-174]。真菌是重要的天然活性化合物资源库，很多真菌提取物及其多糖具有免疫增强作用。如香菇多糖被证实是一类免疫激活剂，能激活巨噬细胞和淋巴细胞；裂褶菌多糖具有明显的细胞免疫促进作用，对非特异性免疫系统起作用，进而增强机体免疫力，且无毒性被用于治疗白血病和慢性肝炎。因此，开发真菌多糖用于疾病的预防和治疗具有重要的应用价值。
尖孢镰刀菌是一种代表性的根际真菌，也是一种广泛存在的植物内生真菌。目前人们对尖孢镰刀菌的研究主要集中在其对植物致病机理、分子生物学和生物防治方面，最新研究表明：尖孢镰刀菌多糖具有体外抗氧化活性[Li，P.，Luo，C.，Sun，W.，et al.In vitroantioxidant activities of polysaccharides from endophytic fungus Fusarium oxysporum Dzf17.African Journal of Microbiology Research，2011，5：5990-5993]，但国内外至今还没有尖孢镰刀菌水提物及其多糖免疫增强作用的报道。
巨噬细胞是宿主抵抗微生物病原体的首要防线，既是主要抗原呈递细胞，又是免疫效应细胞，可以直接杀伤病原微生物和肿瘤细胞，在机体非特异性免疫中起着重要的作用。活化后的巨噬细胞分泌近百种生物活性物质，如NO、IL-1、TNF-α等，这些免疫活性分子作为细胞间相互作用的内源性信号，对机体的免疫应答起到重要的调节作用[Adams，D.O.and Hamilton，T.A.The cell biology of macrophage activation.Annual Review of Immunology，1984，2：283-318；Aderem，A.and Underhill，D.M.Mechanisms of phagocytosis in macrophages.Annual Review of Immunology，1999，17：593-623]，如IL-1、IL-2能促进T细胞增殖，促进B细胞产生抗体，增强肿瘤特异性杀伤细胞、自然杀伤细胞的杀伤功能；TNF-α对肿瘤细胞具有直接的细胞毒作用和生长抑制作用；NO对肿瘤细胞和微生物病原体则有很强的杀伤作用。因此，具增强巨噬细胞功能的真菌提取物或化合物可以用于免疫功能低下疾病或肿瘤辅助治疗。
发明内容
本发明目的在于尖孢镰刀菌发酵菌丝体水提取物及其多糖的免疫增强作用，可应用于免疫功能低下疾病或肿瘤辅助治疗。
本发明目的是通过如下技术方案实现的：
1、尖孢镰刀菌可从中国普通微生物菌种保藏管理中心(CGMCC)或中国农业微生物菌种保藏中心(ACCC)获得。该真菌形态学特征见图1，该真菌18S rRNA片段，ITS1、5.8S rRNA、ITS2区全序列及28S rRNA序列片断为：
5′-gtaacaaggt ctccgttggt gaaccagcgg agggatcatt accgagttta caactcccaa 60
acccctgtga acatacctta atgttgcctc ggcggatcag cccgcgcccc gtaaaacggg 120
acggcccgcc agaggaccca aactctaatg tttcttattg taacttctga gtaaaacaaa 180
caaataaatc aaaactttca acaacggatc tcttggttct ggcatcgatg aagaacgcag 240
caaaatgcga taagtaatgt gaattgcaga attcagtgaa tcatcgaatc tttgaacgca 300
cattgcgccc gctggtattc cggcgggcat gcctgttcga gcgtcatttc aaccctcaag 360
cccccgggtt tggtgttggg gatcggctct gcccttctgg gcggtgccgc ccccgaaata 420
cattggcggt ctcgctgcag cctccattgc gtagtagcta acacctcgca actggaacgc 480
ggcgcggcca tgccgtaaaa ccccaacttc tgaatg-3′ 516
所述尖孢镰刀菌(Fusarium oxysporum)在PDA培养基上，菌落白色到浅黄褐色，背面深红色。菌丝多分枝，具分隔，2-4μm宽。小孢子长椭圆形到近柱状，直或略弯曲，6.0-17×2.3-3.3μm，小孢子梗短，柱状，不分枝，8.4-32.3×2.5-3.9μm；大孢子梭状，多数中度弯曲，1-5分隔，顶细胞喙状弯曲，基部具足细胞，23-33.4×3.5-4.1μm。
该真菌通过液体摇瓶培养和发酵罐培养得到发酵物，离心后得到菌丝体。上述液体摇瓶培养和发酵罐培养方法采用本技术领域内常规发酵培养方法。
菌丝体经干燥、粉碎后，采用热水回流提取方法获得提取液，其中优选热水温度为60-140℃，更优选80-100℃。该步骤的目的是获得水提取物，一切本领域可以促使更好的获得上述水提取物的方法都适用于本发明，并且，在提取过程中，采用不同温度下的水进行提取都是可行的。
将上述提取液浓缩干燥后得水提取物。其中，浓缩干燥过程可以采用本技术领域内常规的浓缩干燥方法，如：常压加热浓缩干燥、减压加热浓缩干燥、冷冻干燥等。
上述提取液经过浓缩后，用乙醇进行沉淀、离心得沉淀物，沉淀物干燥后即得粗多糖。上述醇沉过程优选用乙醇进行沉淀。上述过程中乙醇优选60-100％，更优选80-95％，乙醇加入量优选加入0.5-6倍量，更优选2-4倍。该步骤的目的是获得粗多糖，本领域技术人员可知的其他从菌丝体获得粗多糖的方法都可以用来替换本发明制备多糖的方法，均在本发明的保护范围之内。
2、巨噬细胞增殖能力测定(MTT法)
小鼠单核巨噬细胞RAW264.7培养于含10％胎牛血清、1％链霉素/青霉素的DMEM培养基，置37℃、5％CO2恒温培养箱中孵育生长。调整RAW264.7巨噬细胞细胞悬液5×103个/孔接种于96孔培养板内过夜，加入一定浓度的尖孢镰刀菌水提取物或其粗多糖200μL，继续培养24h。MTT法检测细胞活力，结果以加药组与空白对照组的吸光度比值计算巨噬细胞增殖能力。
3、巨噬细胞吞噬能力测定
将RAW 264.7细胞7.5×105个/孔接种于24孔细胞培养板，每孔再加入250μL不同浓度尖孢镰刀菌水提取物或其粗多糖。同体积脂多糖(LPS，400ng/mL)和培养液分别作为阳性对照组和空白对照组，在37℃、5％CO2培养箱里孵育1h后，加入5μL Rainbow荧光颗粒(每孔约10×106个/孔)再避光孵育2h后，弃去培养液，PBS洗涤2次，收集细胞，以流式细胞仪检测吞噬百分数(％)，结果以与溶剂对照组的吞噬率比值表示。
4、巨噬细胞释放免疫活性分子测定
将RAW 264.7细胞6×104/孔接种于96孔细胞培养板内，在37℃、5％CO2培养箱里孵育24h后，每孔加入不同浓度尖孢镰刀菌水提取物或其粗多糖，并以同体积LPS(400ng/mL)或培养液分别作为阳性对照和空白对照，采用Griess法测定巨噬细胞NO释放量，ELISA试剂盒测定细胞培养上清中TNF-α、IL-1α水平，NO释放能力(％)以与LPS阳性对照组吸光度比值计算。
附图说明
图1所示为本发明尖孢镰刀菌(Fusarium oxysporum)孢子和分生孢子梗照片。
图中A：小孢子；B、C、D：大孢子；E、F：产小孢子分生孢子梗
具体实施方式
将该真菌通过液体摇瓶培养和发酵罐培养得到发酵物，离心后得到菌丝体。菌丝体经干燥、粉碎后，采用100℃热水回流方法提取1小时，提取液浓缩干燥后得水提取物；或提取液经浓缩后，以4倍量95％乙醇沉淀、离心得沉淀、沉淀干燥后即得粗多糖。分别考察尖孢镰刀菌水提取物和粗多糖对巨噬细胞功能(细胞增殖、吞噬功能及免疫活性因子释放)的影响。
实施例1：
小鼠单核巨噬细胞RAW264.7培养于含10％胎牛血清、1％链霉素/青霉素的DMEM培养基，置37℃、5％CO2恒温培养箱中孵育生长。调整RAW264.7巨噬细胞细胞悬液5×103个/孔接种于96孔培养板内过夜，加入一定浓度的尖孢镰刀菌水提取物或其粗多糖200μL，继续培养24h。MTT法检测细胞活力，结果以加药组与空白对照组的吸光度比值计算巨噬细胞增殖能力。
结果表明：尖孢镰刀菌水提物和多糖在0.5-128μg/mL浓度范围内对巨噬细胞增殖均有显著促进作用，见表1。
表1.尖孢镰刀菌水提取物及其多糖对巨噬细胞RAW264.7增殖能力的影响
数据表示为Mean±SEM，n＝3。与空白对照组比较，*p＜0.05，**p＜0.01。
实施例2：
将RAW 264.7细胞7.5×105个/孔接种于24孔细胞培养板，每孔再加入250μL不同浓度尖孢镰刀菌水提取物或其粗多糖。同体积脂多糖(LPS，400ng/mL)和培养液分别作为阳性对照组和空白对照组，在37℃、5％CO2培养箱里孵育1h后，加入5μL Rainbow荧光颗粒(每孔约10×106个/孔)再避光孵育2h后，弃去培养液，PBS洗涤2次，收集细胞，以流式细胞仪检测吞噬百分数(％)，结果以与溶剂对照组的吞噬率比值表示。
结果表明：尖孢镰刀菌水提取物及其多糖在一定下浓度下可显著促进巨噬细胞吞噬能力。水提物(300μg/mL)提高巨噬细胞吞噬能力为空白组129.04％，多糖(500ng/mL)提高巨噬细胞吞噬能力是空白对照组163.5％，与阳性对照LPS组(400ng/mL)提高巨噬细胞吞噬能力为空白对照组165.6％相当。
实施例3：
将RAW 264.7细胞6×104/孔接种于96孔细胞培养板内，在37℃、5％CO2培养箱里孵育24h后，每孔加入不同浓度尖孢镰刀菌水提取物或其粗多糖，并以同体积LPS(400ng/mL)或培养液分别作为阳性对照和空白对照，采用Griess法测定巨噬细胞NO释放量，ELISA试剂盒测定细胞培养上清中TNF-α、IL-1α水平，NO释放能力(％)以与LPS阳性对照组吸光度比值计算。
结果表明：尖孢镰刀菌水提取物(8-128μg/mL)及其多糖(0.5-128μg/mL)分别能显著(p＜0.05)或极显著(p＜0.01)地促进巨噬细胞释放NO(见表2)；水提取物及其多糖也分别能显著促进巨噬细胞释放免疫活性因子IL-1α和TNF-α(见表3)。
表2.尖孢镰刀菌水提取物及其多糖对巨噬细胞RAW264.7释放NO影响
数据表示为Mean±SEM，n＝3。与空白对照组比较，*p＜0.05，**p＜0.01。
表3.尖孢镰刀菌水提取物及其多糖对巨噬细胞RAW264.7释放细胞因子影响
数据表示为Mean±SEM，n＝3。与空白对照组比较，*p＜0.05，**p＜0.01。
Claims (4)
1.一种尖孢镰刀菌提取物，其特征在于，是将所述尖孢镰刀菌通过液体摇瓶培养和发酵罐培养得到发酵物，离心后得到菌丝体，再经过提取得到的水提取物和/或多糖；
所述菌丝体经干燥、粉碎后，采用热水回流提取，所述热水温度为60-140oC，提取液浓缩干燥后得水提取物；或将所述提取液经浓缩后用乙醇沉淀，离心得沉淀物、沉淀物干燥后即得多糖。
2.权利要求1所述的提取物用于制备免疫增强药物中的应用。
3.权利要求1所述的提取物用于制备治疗免疫功能低下的药物中的应用。
4.权利要求1所述的提取物用于制备肿瘤辅助治疗药物中的应用。
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