CN112569336B - 灰肉红菇糖蛋白在制备增强机体免疫及预防和/或治疗肿瘤疾病药物或健康食品中的应用 - Google Patents
灰肉红菇糖蛋白在制备增强机体免疫及预防和/或治疗肿瘤疾病药物或健康食品中的应用 Download PDFInfo
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Abstract
本发明公开了灰肉红菇糖蛋白在制备增强机体免疫及预防和/或治疗肿瘤疾病药物或健康食品中的应用;所述灰肉红菇糖蛋白为灰肉红菇提取物经溶剂沉淀后得到的可溶于水的提取物或级分。本发明首次提出了灰肉红菇糖蛋白在制备增强机体免疫及预防和/或治疗肿瘤疾病药物或健康食品中的应用,发现灰肉红菇提取物中的糖蛋白具有抑制机体肿瘤生长的效果,并能够激活机体被肿瘤所抑制的免疫功能,开发了灰肉红菇糖蛋白的一种新用途。
Description
技术领域
本发明涉及野生菌株应用技术领域,具体涉及灰肉红菇糖蛋白在制备增强机体免疫及预防和/或治疗肿瘤疾病药物或健康食品中的应用。
背景技术
2019年国家癌症中心发布最新统计报告显示:2015年我国恶性肿瘤发病率约为392.9万人,死亡人数约233.8万人,占居民全部死因的23.91%,居全球第一,每天约1万人被确诊为癌症,是威胁我国居民健康的主要公共卫生问题之一。已知癌症的发生是基于自体细胞基因突变,具有强的异质性和遗传性,因此,传统的手术、化疗、放疗等治疗方案在绝大多数情况下不能彻底抑制肿瘤生长,且会导致耐药性,复发后更难治疗。因免疫学和肿瘤学基础研究的深入,人体自身的免疫细胞,尤其是杀伤性T细胞,可以特异性的识别肿瘤抗原和杀伤肿瘤细胞,实现持续性地控制肿瘤生长的效果。但是,在肿瘤患者体内,肿瘤细胞的多种免疫逃逸机制使机体的免疫系统并不能完全控制肿瘤的生长,因此,抑制肿瘤细胞免疫逃逸、恢复机体免疫识别及杀伤清除肿瘤细胞的策略是肿瘤免疫治疗及相应药物开发成功的关键。
食药用菌是传统食药两用大型真菌,根据大量的现代药理研究显示,其提取物具有调节人体免疫力和/或抑制肿瘤的生长功能。在研究基础上,目前已上市多糖类药物有香菇多糖、猪苓多糖、灰树花多糖及云芝糖肽等,其主要作用是通过提高免疫而辅助治疗肿瘤或者增强机体免疫功能。目前世界上已知的大型真菌约14000多种,国内已确认的食用菌有1789种,药用真菌798种,其被研究的仅为少数,因此,从食药用菌中发现和获得具有克服肿瘤患者免疫抑制,实现抑制肿瘤生长的药物具有重要意义。
灰肉红菇(Russula griseocarnosa)是由我国学者王向华首次报道的红菇属新种(Wang XH et al.2009)。灰肉红菇是一种属于红菇属的野生著名食药用菌,目前其不可培养及栽培,主要用其子实体部分,已知灰肉红菇多糖在体外具有抑制肿瘤细胞生长和刺激巨噬细胞分泌细胞因子的作用。灰肉红菇隶属于属担子菌亚门、层菌纲、红菇目、红菇科、红菇属,是一种美味的野生菌根性食用菌,在我国华南梅州地区具有数百年的食用习惯。灰肉红菇的分类学地位确定后,其脂溶性成分的抗氧化功能,杂多糖体外抑制肿瘤细胞生长及体外促进巨噬细胞活性均一一被报道(Chen XH,2010;Liu Y,2017等)。但是,尚未有涉及灰肉红菇糖蛋白抑制机体肿瘤生长和激活肿瘤免疫抑制作用的相关研究报道。
发明内容
为解决上述技术问题,本发明首次提出了灰肉红菇糖蛋白在制备增强机体免疫及预防和/或治疗肿瘤疾病药物或健康食品中的应用,发明人发现灰肉红菇提取物中的糖蛋白具有抑制机体肿瘤生长的效果,并能够激活机体被肿瘤所抑制的免疫功能,属于灰肉红菇糖蛋白的一种新用途。
为达到上述目的,本发明提供如下技术方案:
灰肉红菇糖蛋白在制备增强机体免疫力药物或健康食品中的应用。以及,
灰肉红菇糖蛋白在制备预防和/或治疗肿瘤疾病药物或健康食品中的应用。
所述健康食品可以是饮料、非饮料类食品或者食品添加剂等。
优选的,所述灰肉红菇糖蛋白为灰肉红菇提取物经特定溶剂沉淀后得到的可溶于水的提取物或级分;所述提取物或级分可以是溶液、浓缩物或者粉末的形式。
所述级分指利用不同浓度的NaCl洗脱柱层析获得的组分。
优选的,所述灰肉红菇糖蛋白为通过水溶液提取灰肉红菇子实体得到的粗提取物,向粗提取物中加入一种或者多种低级醇而得到的可溶于水的醇沉物。
所述灰肉红菇子实体粗提取物和醇沉物的多糖含量范围为15~60%,蛋白含量大于15~60%。
优选的,所述低级醇选自具有1-3个碳原子的直链或者支链醇。
优选的,所述低级醇包括乙醇、甲醇或者丙酮中的一种或多种。
上述乙醇或者甲醇的浓度为40~90%(v/v),优选浓度为75~85%(v/v),优选乙醇水溶液。
优选的,所述级分为通过NaCl溶液(浓度为0.05~0.8M)对所述醇沉物的水溶液进行离子交换柱(DEAE-Sepharose Fast Flow)层析而得到。
优选的,所述灰肉红菇糖蛋白的制备方法包括如下步骤:
(1)将灰肉红菇的子实体粉碎至40~60目,按灰肉红菇子实体:纯水=1:10~40的质量比加入纯水,提取0.5~10小时(优选1-4小时),提取温度为40~110℃,得到提取物;将所得提取物过滤,收集滤液,再向过滤后的残渣中加入纯水,纯水的加入量为残渣体积的15~25倍,接着提高温度再提取1~4小时,再将所得提取物过滤,重复过滤两次,然后将两次提取后获得的滤液合并,将合并后的滤液在真空下浓缩,获取灰肉红菇提取物原液体积10%~50%的粗提取浓缩液;
(2)向浓缩液中加入1~5倍体积量的含1~3个碳原子的直链或者支链醇,充分混匀,在4℃条件下静置过夜,之后以8000rpm/min的转速离心15~30min,获得沉淀物,并采用冷乙醇将沉淀物洗涤充分,最后将沉淀物冻干,即得所述灰肉红菇糖蛋白。
进一步优选的,步骤(1)中,所述灰肉红菇子实体与纯水的质量比为1:15~25;提取的温度为70~100℃;粗提取浓缩液的体积为灰肉红菇提取物原液体积的15%~30%;步骤(2)中,直链或者支链醇的体积为浓缩液体积的3~4倍;所述直链或者支链醇为乙醇、甲醇、丙酮中的一种(优选无水乙醇)。
上述制备方法中,两步提取和将每次提取后获得的滤液合并可提高提取的效率,此外,在每步提取中检测提取效率发现总提取量的约80~90%是通过两次提取得到的,从而说明两步提取与多于两次提取的多步提取相比具有显著的经济效益。
如果用来制备灰肉红菇提取物的水溶剂的量太少,会导致提取物的溶解性降低从而导致提取效率下降,如果用来制备灰肉红菇提取物的水溶剂的量太多,用于接下来纯化的醇用量会增加,从而引起成本和处理工作量方面的问题,因此,灰肉红菇子实体与纯水的质量比为1:10~40,优选1:15~25。
使用低级醇溶剂获得醇不溶物的步骤是为了取出粗提取物中不必要的杂质如色素、过胶、脂肪酸及小分子脂溶性物质等。因此,如果使用的低级醇的量过多或过少,则不能有效地取出杂质或者造成糖蛋白的损失,因此,低级醇的用量为浓缩液的1~5倍体积量,优选为3~4倍体积量。
步骤(2)中,“将沉淀物洗涤充分”是指将醇沉物经冷乙醇洗涤、离心两次,主要为了控制所得醇沉物中残留的低级醇的含量使其适用作为药物的原材料,同时,“将沉淀物冻干时”,可将醇沉物与10~20倍重量的水共沸1~4次,然后将所得液体经真空冷冻干燥,制备粉末状灰肉红菇提取物。
通过本制备方法,可再进一步提高灰肉红菇粗提取物或醇沉物的多糖和糖蛋白的含量(多糖含量范围为15~60%,蛋白含量>15~70%)。
与现有技术相比,本发明具有以下优点:
本发明首次提出了灰肉红菇糖蛋白在制备增强机体免疫及预防和/或治疗肿瘤疾病药物或健康食品中的应用,发现灰肉红菇提取物中的糖蛋白具有抑制机体肿瘤生长的效果,并能够激活机体被肿瘤所抑制的免疫功能,开发了灰肉红菇糖蛋白的一种新用途。
附图说明
图1是模型组与RGP给药组小鼠肿瘤组织大小比较图;
图2是肿瘤组织H&E染色和免疫组织化学染色结果图。
具体实施方式
下面结合实施例对本发明作进一步详细的描述,但本发明的实施方式不限于此。在未作特别说明的情况下,本发明所采用的试剂、设备和方法均为本技术领域常规市购的试剂、设备和常规使用的方法。
实施例1:
1.制备灰肉红菇提取物
1.1用水制备灰肉红菇提取物
1.1.1制备灰肉红菇粗提取物(RG)
用水清洗从广东省梅州市购买的灰肉红菇以除去杂质,将灰肉红菇的子实体粉碎至40目,用10.0L水在100℃下对500g干燥灰肉红菇子实体粉末进行两次热水提取,每次持续2小时,然后对所得提取物进行过滤,收集滤液,并将滤液在真空下浓缩得到25.6g提取物(相对原材料的产率为5.12%),将其命名为RG。
1.1.2制备灰肉红菇醇沉物(RGP)
向获取的25.6g RG中加入0.25L的超纯水,使其溶解,然后缓慢均匀搅拌的同时加入1L无水乙醇,然后放入4℃温箱中,静置过夜。第二天,离心(转速8500rpm,离心温度4℃,离心时间15min),得到沉淀。
将沉淀经冷无水乙醇洗涤两次,再离心去除上清液,获得沉淀。将获得的沉淀用0.1L纯水复溶,并100℃沸腾10min,进行2次。将溶液冷却,过0.8μm滤膜,除去不溶性物质,获得滤液。将滤液经截留分子量大于3500Da的透析袋进行透析,每4-6小时换一次水,重复3-4次,收集透析袋内溶液,将内溶液浓缩至0.1L浓缩液。将浓缩液经冷冻干燥,获得灰肉红菇醇沉物15.05g,并命名为RGP,用于下一实验。
1.1.3级分灰肉红菇提取物
用不同浓度的NaCl溶液对灰肉红菇醇沉物RGP通过阴离子(DEAE-Sepharose FastFlow)交换柱层析进行洗脱,将获得的15.05g RGP分为4个级分。Fr.1为1.42g,Fr.2为1.25g,Fr.3为0.45g,在以下的实验中Fr.1为1.42g,Fr.2为1.25g,Fr.1、Fr.2用作为样本。
1.2结果
对RG、RGP、Fr.1、Fr.2中的多糖和糖蛋白含量进行检测,结果如下表:
实施例2:
2.1灰肉红菇提取物抑制肿瘤生长活性作用的探究实验
2.1.1实验方法:
为了测定实施例1中灰肉红菇提取物的体内抑制肿瘤生长的作用,我们首先以体外肿瘤细胞作为检测对象,探究灰肉红菇提取物对鼠源乳腺癌细胞4T1的影响,同时,在以下步骤中使用申请人实验室已报道的小鼠肿瘤移植瘤动物模型及方法(Xiangmin Li etal.Cell cycle,2019)来进行实验。
1)体外检测样品对小鼠乳腺癌4T1细胞增殖的影响
①小鼠乳腺癌细胞4T1的培养
从液氮储存罐中取出保存的小鼠乳腺癌细胞4T1,进行复苏、活化和培养,培养基为含1%(重量百分比,下同)青霉素、1%链霉素和10%胎牛血清(FBS)的DMEM培养基,培养条件为37℃、5%(浓度)CO2细胞培养箱,培养至对数生长期。
②细胞增殖实验
将对数生长期细胞(1×105个细胞/mL,0.5mL)接种到24孔细胞板中,培养基为含有10%(重量百分比,下同)FBS、1%青霉素和链霉素双抗的DMEM培养基,在37℃含有5%(浓度)CO2的培养箱中培养。四小时后,加入配置好的样品(RG、RGP、Fr.1、Fr.2),每个样品分别使用两种浓度(即50ug/mL、100ug/mL),并用样品溶剂做对照,继续培养至48小时,终止实验。采用台盼蓝拒染方法,计算存活细胞数量,并进行统计学分析。
2)体内检测样品对小鼠乳腺癌4T1细胞形成肿瘤的影响
①肿瘤动物模型构建
将上述对数生长期的4T1细胞经消化、计数,调整到细胞密度为5×105cells/mL,将0.1ml细胞接种到BALB/C雌性小鼠(8周龄,体重20±2g)皮下,并随机分配为模型组、测试组(RGP),同时设置正常对照组。
②动物实验及取材
第二天,开始对各组分别给药,给药方式为灌胃给药,测试组(RGP)灌胃灰肉红菇低剂量组(100mg/kg/day)和高剂量组(400mg/kg/day),正常对照组注射阳性紫杉醇,采用腹腔注射,注射剂量为25mg/kg/day,模型组为注射生理盐水,各组连续给药4周。
最后一次给药之后的第二天,牺牲小鼠,取肿瘤,测量肿瘤重量和大小,并将部分肿瘤组织分别置于10%中性福尔马林及液氮中保存备用。
2.1.2实验结果:
体外实验结果
1)样品对小鼠乳腺癌4T1细胞增殖的影响
数据统计分析如表1所示,与对照组相比,样品组(RG、RGP、Fr.1、Fr.2)对小鼠乳腺癌细胞4T1的增殖并不影响,即无直接杀伤细胞的毒作用。
表1-样品对4T1细胞增殖作用影响
体内实验结果
1)给药期间各组小鼠肿瘤生长体积的变化
给药期间,每周测定对照组和给药组小鼠肿瘤生长的体积变化,数据统计分析如表2所示,与模型组相比,给药组显著地抑制小鼠乳腺癌的生长。
表2-与模型组相比各给药组小鼠肿瘤体积的变化
注:肿瘤生长体积变化与模型组相比,*表示p<0.05(差异显著);**表示p<0.01(差异极显著)。
2)小鼠肿瘤重量变化和抑制率
如图1和表3所示,实验结果为实施例1的RGP在剂量为100mg·kg-1·d-1和400mg·kg-1·d-1时,显示出显著的抑制小鼠乳腺癌肿瘤生长的效果。
表3-与模型组相比各给药组小鼠肿瘤重量变化和抑制率
实施例3
灰肉红菇提取物激活肿瘤组织杀伤性T细胞的免疫应答
为了检测灰肉红菇提取物是否激活肿瘤组织的杀伤性T细胞,进而杀伤肿瘤细胞,最终抑制肿瘤生长,本实施例对肿瘤组织的杀伤性T细胞进行检测。
3.1样品组织切片的制备
取实施例2中所获得的肿瘤组织,经10%中性福尔马林固定过夜,再经石蜡包埋,切片和贴片,制备得到肿瘤组织石蜡切片。
3.2肿瘤组织免疫细胞杀伤性T细胞CD8+、辅助性T细胞CD4+和肿瘤细胞表面抑制分子PD-L1表达免疫组织化学染色
实验步骤:
(1)切片常规二甲苯脱蜡,梯度酒精脱水
将石蜡切片在65~68℃烘箱中烤片2h,将石蜡溶解,然后将石蜡切片在二甲苯Ⅰ(浸泡时间15min)、二甲苯Ⅱ(浸泡时间15min)、二甲苯Ⅲ(浸泡时间15min)进行脱蜡。
将脱蜡的切片经100%酒精Ⅰ浸泡5min、100%酒精Ⅱ浸泡5min、90%酒精Ⅰ浸泡5min、90%酒精Ⅱ浸泡5min、80%酒精浸泡5min、75%酒精浸泡5min脱水,之后用纯水清洗3次,每次5min。
(2)灭活内源性抗氧化物酶
将切片放入用甲醇新鲜配置的0.3%过氧化氢中,室温放置15min,之后使用1×PBS缓冲液清洗3次,每次5min。
(3)抗原修复
将切片放入0.01mol/L柠檬酸盐缓冲液中(保证液面可以将切片完全覆盖),在微波炉中调至中高火加热至沸腾8min,保温7分钟,然后调制中火加热三次,每次6min,之后保温6min,拿出待其降至室温后,将修复后的切片放入PBS缓冲液中清洗3次,每次5min。
(4)血清封闭
37℃下用10%正常山羊血清封闭液将切片孵育30min,倾去,勿洗。
(5)一抗孵育
往切片滴加一抗,放置于湿盒中并于37℃恒温箱中孵育1h,4℃过夜,之后使用1×PBS缓冲液清洗3次,每次5min。
(6)二抗孵育
往切片滴加二抗(山羊抗兔IgG H&L(HRP)),室温孵育50min,之后使用1×PBS缓冲液清洗3次,每次5min。
(7)DAB显色
新鲜配置DAB显色液,在显微镜下滴加DAB显色液到切片上,避光显色,显微镜下控制显色程度,之后使用1×PBS缓冲液或自来水清洗10min,终止显色。
(8)复染
将切片置入苏木精染液中染色5min,用纯水冲洗(切勿将流水对着组织冲洗,以防脱片);然后,放入苏木精透化液染盒中10S分化,冲洗,再放入苏木精返蓝液中10s。
(9)常规脱水,透明,封片
将切片依次放入75%酒精(浸泡时间5min)、85%酒精(浸泡时间5min)、无水乙醇Ⅰ(浸泡时间5min)、无水乙醇Ⅱ(浸泡时间5min)、二甲苯Ⅰ(浸泡时间5min)中脱水至呈透明,再将切片从二甲苯Ⅰ中拿出来稍晾干,最后使用中性树胶封片。
3.3结果
如图2中所示,在400倍视野下,模型组肿瘤组织中检出CD8和CD4微弱,RGP给药组的小鼠肿瘤组织中检出CD8+和CD4+显著增强,表明RGP促进肿瘤微环境的免疫细胞杀伤性T细胞(CD8+)和辅助性T细胞(CD4+)增殖,具有激活免疫细胞的作用。同时,还检测出肿瘤细胞表面免疫抑制因子程序性死亡配体PD-L1的表达显著下调。
因此,RGP具有激活肿瘤组织微环境中的免疫细胞杀伤性T细胞(CD8+)的活性,同时能够抑制肿瘤组织、肿瘤细胞的免疫抑制因子PD-L1的活性。
以上所述,仅为本发明的较佳的具体实施例,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,根据本发明的技术方案及其构思加以等同替换或改变,都应涵盖在本发明的保护范围内。
Claims (1)
1.灰肉红菇糖蛋白(Russula griseocarnosa)在制备治疗乳腺癌疾病药物中的应用,其特征在于,所述应用过程中灰肉红菇糖蛋白的用量为400mg.kg-1.d-1;所述灰肉红菇糖蛋白通过如下方法制备得到:
(1)将灰肉红菇的子实体粉碎至40目,按灰肉红菇子实体:纯水=1:20的质量比加入纯水进行两次热水提取,每次提取2小时,提取温度为100℃,得到提取物;将所得提取物过滤,收集滤液,将滤液在真空下浓缩,获取灰肉红菇提取物原液体积10%~50%的粗提取浓缩液;
(2)向浓缩液中加入4倍体积量的无水乙醇,充分混匀,在4℃条件下静置过夜,之后以8500rpm/min的转速离心15min,获得沉淀物,并采用冷乙醇将沉淀物洗涤充分,离心去除上清液,获得沉淀,将获得的沉淀用纯水复溶,并100℃沸腾10min,进行2次,将溶液冷却,过0.8μm滤膜,除去不溶性物质,获得滤液,将滤液经截留分子量大于3500Da的透析袋进行透析,每4-6小时换一次水,重复3-4次,收集透析袋内溶液,将溶液浓缩后冷冻干燥,获得灰肉红菇醇沉物,即为所述灰肉红菇糖蛋白。
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