CN113817078A - 基于免疫调控具有抗结直肠癌功效的杨黄菌多糖及用途 - Google Patents
基于免疫调控具有抗结直肠癌功效的杨黄菌多糖及用途 Download PDFInfo
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- CN113817078A CN113817078A CN202111223375.0A CN202111223375A CN113817078A CN 113817078 A CN113817078 A CN 113817078A CN 202111223375 A CN202111223375 A CN 202111223375A CN 113817078 A CN113817078 A CN 113817078A
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Abstract
本发明提供一种基于免疫调控具有抗结直肠癌功效的杨黄菌多糖及用途,具有调节免疫功能作用,能够调控肠道菌群物种组成数量、种群丰度及均匀度,具有抑制肿瘤生长及调控T细胞活化相关生化指标作用,可以用于制备治疗结直肠癌药品及保健品。
Description
技术领域
本发明提供一种基于免疫调控具有抗结直肠癌功效的杨黄菌多糖及用途,公开一种杨树桑黄菌多糖及其结构,基于免疫调控具有抗结直肠癌功效,属于医药技术领域。
背景技术
结直肠癌是引发与肿瘤相关死亡的主要癌症之一,早期结直肠癌患者经治疗5年存活率可以达到80%,晚期患者经治疗5年存活率仅为10%。约有5%的结直肠癌患者发病与遗传性疾病(包括Lynch综合症,家族性腺癌性息肉病和MYH相关性息肉病等)相关,约95%的结直肠癌患者疾病的发生无明显遗传相关性,饮食、年龄和生活习惯等因素可能与这一部分结直肠癌的发生发展相关。随着测序技术的发展,肠道菌群在结直肠癌中的作用逐渐被发现,肠道菌群的次级代谢产物中短链脂肪酸能够直接抑制结直肠癌的病理学进展。基于肠道菌群微生物组学开发结直肠癌治疗手段可能是新型有效的结直肠癌治疗策略。
目前结直肠癌的治疗以手术治疗为主,放化疗为辅。随着肿瘤免疫学、分子生物学的发展,免疫治疗已经成为结直肠癌治疗的重要治疗策略之一。基于治疗机制,相关疗法可分为主动免疫和被动免疫两类。特异性主动免疫治疗主要包括全肿瘤细胞疫苗、树突状细胞疫苗以及分子疫苗;非特异性主动免疫治疗主要通过增强免疫系统功能,激活免疫系统对肿瘤细胞的免疫杀伤反应从而实现抗肿瘤的目的。被动免疫治疗:通过引入外源性免疫效应物质,在不刺激机体免疫应答的基础上,发挥免疫治疗作用。随着肿瘤免疫学的发展,免疫治疗有望成为结直肠癌治疗的主要治疗策略之一,在增加患者生存率,提升患者生活质量方面发挥重要作用。
杨树桑黄因其子实体生长在杨树上而得名,是传统菌物药“桑黄”基源物种之一,在我国,杨树桑黄菌主要分布于中国长白山和小兴安岭地区。桑黄是国际公认的生物治癌领域中效率最高的大型真菌,有报道杨树桑黄菌具有与传统桑黄同样的抑制肿瘤的功效。多糖是桑黄发挥功效的主要成分,研究表明杨树桑黄菌具有良好的免疫调节、抗菌、抗氧化和保肝护肝等多种药理活性,但尚未有研究在APC小鼠模型中,对杨树桑黄菌多糖基于免疫调节的抗结直肠癌活性进行研究。
发明内容
本发明公开了一种抗结直肠癌功效的杨树桑黄菌多糖及制备方法,以杨树桑黄菌子实体为原料,通过提取、分离得到一种杨树桑黄菌多糖,对其进行结构表征及基于免疫调控抗结直肠癌活性进行探究。
本发明还进一步提供了杨黄菌多糖抗结直肠癌的医用用途。
本发明提供一种杨树桑黄菌多糖,其特征在于:
分子量为22.5 kDa,主要包括6种单糖:Fuc、Gal、Glc、 Man、 Fru及 GlcUA,其摩尔质量比为24.473: 42.942: 11.892: 19.988: 0.637: 0.068。杨树桑黄菌多糖主要有10个糖苷片段组成, Gal含量比例最高,其中糖苷片段t-Gal(p)、6-Gal(p)及2,6-Gal(p)摩尔比例分别为1.74%,34.50%和18.96%,推测杨树桑黄菌多糖为半乳聚糖。由于2,6-Gal(p),t-Fuc(p),t-Man(p),t-Glc(p),t-Gal(p)存在,推测杨树桑黄菌多糖有分支存在。
本发明提供一种杨树桑黄菌多糖的制备方法,包括以下步骤:
(1)杨树桑黄菌子实体超微粉碎成粉末,过150目筛;
(2)加入质量20倍质量的石油醚进行脱脂,加热回流2小时,回流脱脂两次后,烘干菌粉;
(3)加入质量10-50倍质量的去离子水,于70-90℃水浴中提取2小时,提取两次,合并溶液;
(4)冷却后4000 rpm条件离心20分钟,取上清,旋转蒸发浓缩至1/5-1/10体积,Sevag法去蛋白,旋蒸除去残留的有机试剂后,透析袋透析12-48 h,浓缩至适当浓度;
(5)调节乙醇终浓度至85%(v/v)进行沉淀,4℃条件过夜,离心收集沉淀,进行冻干,即获得杨树桑黄菌粗多糖;
(6)将杨树桑黄菌粗多糖用去离子水溶解,上样于DEAE Sepharose Fast Flow离子交换柱(5 cm×30 cm)层析:在1.0 mL/min的流速下,分别以0、0.1、0.3和0.5 mol/LNaCl进行洗脱,并用洁净、干燥的试管收集,收集量为8 mL/管,将收集到的糖采用硫酸-苯酚法进行检测,绘制出洗脱曲线,将峰值糖进行合并,浓缩,冻干,备用;
(7)将通过DEAE Sepharose Fast Flow离子交换柱获得的杨树桑黄菌多糖冻干粉末溶于0.15 mol/L NaCl,上样于HiLoad 16/600 Superdex 200 prep grade柱:以0.45mol/L NaCl洗脱,流速为0.5 mL / min,将自动收集器设置为体积为2 mL/管,采用苯酚-硫酸法测定多糖含量,收集主峰,旋转蒸发浓缩至1/10体积,经透析除盐,蒸发浓缩,冻干后即获得杨树桑黄菌多糖。
经动物实验证明(详见实施例),本发明所述的基于免疫调控具有抗结直肠癌的杨树桑黄菌多糖,其主要的功效学作用如下:杨树桑黄菌多糖作用后具有抑制APC小鼠结直肠肿瘤发生发展的能力;杨树桑黄菌多糖通过调控APC小鼠肠道菌群组成,直接或间接的限制了小鼠结直肠癌的发生发展;杨树桑黄菌多糖作用后有效地增加APC小鼠脾脏及肿瘤内活化T淋巴细胞的数量;杨树桑黄菌多糖提高APC小鼠脾脏和肿瘤组织内Th1/Th2细胞比例,促进T淋巴细胞介导的杀伤性免疫反应,从而杀伤肿瘤细胞。
本发明的积极效果在于;
提供一种抗结直肠癌活性的杨树桑黄菌多糖,具有调节免疫功能作用,能够调控肠道菌群物种组成数量、种群丰度及均匀度,具有抑制肿瘤生长及调控T细胞活化相关生化指标作用,可以用于制备治疗结直肠癌药品及保健品。
附图说明
图1、杨树桑黄菌粗多糖DEAE Sepharose Fast Flow洗脱曲线;
图2、杨树桑黄菌多糖HiLoad 16/600 Superdex 200 prep grade洗脱曲线;
图3、杨树桑黄菌多糖紫外扫描光谱图;
图4、杨树桑黄菌多糖红外光谱图;
图5、杨树桑黄菌多糖的单糖组分分析图;
图6、杨树桑黄菌多糖绝对分子量及均一性分析色谱图;
图7、杨树桑黄菌多糖NMR的1H谱图;
图8、杨树桑黄菌多糖NMR的13C谱图;
图9、杨树桑黄菌多糖NMR的COSY谱图;
图10、杨树桑黄菌多糖NMR的HSQC谱图;
图11、杨树桑黄菌多糖NMR的HMBC谱图;
图12、杨树桑黄菌多糖NMR的NOESY谱图;
图13、杨树桑黄菌多糖对APC小鼠肿瘤组织病理学影响结果图;
图14、小鼠肠道菌群双聚类的属水平物种组成热图;
图15、杨树桑黄菌多糖对APC小鼠血清、脾脏和结肠中IL-12含量影响图;
图16、杨树桑黄菌多糖对APC小鼠血清、脾脏和结肠中TNF-α含量影响图;
图17、杨树桑黄菌多糖对APC小鼠血清、脾脏和结肠中IFN-γ含量影响图;
图18 、杨树桑黄菌多糖对APC小鼠血清、脾脏和结肠中IL-4含量影响图;
图19 、杨树桑黄菌多糖对APC小鼠血清、脾脏和结肠中IL-2含量影响图。
具体实施方式
下面结合实施例对本发明作进一步说明。这些实施例仅是范例性的,并不对本发明的范围构成任何限制。本领域技术人员应该理解的是,在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换,但这些修改和替换均落入本发明的保护范围内。说明书及实施例中采用的化学试剂、层析柱等,如无特殊说明均按常规实验条件下进行操作,或按供应商提供的说明进行操作。
本发明中得到一种新的杨树桑黄菌多糖,以杨树桑黄菌子实体为原料,通过脱脂、热水浸提、醇沉、浓缩、冻干等步骤,获得杨树桑黄菌粗多糖;粗多糖经过DEAE SepharoseFast Flow及HiLoad 16/600 Superdex 200 prep grade分离后获得杨树桑黄菌多糖,通过使用紫外光谱、HPLC、红外光谱、凝胶渗透色谱及核磁共振对杨树桑黄菌多糖进行结构表征。通过动物实验考察其抗结直肠癌活性的作用。
本发明中使用的杨树桑黄菌子实体来自吉林农业大学。
实施例 1 :
(1)杨树桑黄菌子实体超微粉碎成粉末,过150目筛;
(2)加入质量20倍质量的石油醚进行脱脂,加热回流2小时,回流脱脂两次后,烘干菌粉;
(3)加入质量10-50倍质量的去离子水,于70-90℃水浴中提取2小时,提取两次,合并溶液;
(4)冷却后4000 rpm条件离心20分钟,取上清,旋转蒸发浓缩至1/5-1/10体积,Sevag法去蛋白,旋蒸除去残留的有机试剂后,透析袋透析12-48 h,浓缩至适当浓度;
(5)调节乙醇终浓度至85%(v/v)进行沉淀,4℃条件过夜,离心收集沉淀,进行冻干,即获得杨树桑黄菌粗多糖;
(6)将杨树桑黄菌粗多糖用去离子水溶解,上样于DEAE Sepharose Fast Flow离子交换柱(5 cm×30 cm)层析:在1.0 mL/min的流速下,分别以0、0.1、0.3和0.5 mol/LNaCl进行洗脱,并用洁净、干燥的试管收集,收集量为8 mL/管,将收集到的糖采用硫酸-苯酚法进行检测,绘制出洗脱曲线,将峰值糖进行合并,浓缩,冻干,备用;
(7)将通过DEAE Sepharose Fast Flow离子交换柱获得的杨树桑黄菌多糖冻干粉末溶于0.15 mol/L NaCl,上样于HiLoad 16/600 Superdex 200 prep grade柱:以0.45mol/L NaCl洗脱,流速为0.5 mL / min,将自动收集器设置为体积为2 mL/管,采用苯酚-硫酸法测定多糖含量,收集主峰,旋转蒸发浓缩至1/10体积,经透析除盐,蒸发浓缩,冻干后即获得杨树桑黄菌多糖。
测试例1:杨树桑黄菌多糖结构特征分析
(1)试验材料
实施例1获得的杨树桑黄菌多糖;
(2)试验方法及结果
紫外及红外光谱分析:
紫外:适量蒸馏水溶解实施例1中获得的杨树桑黄菌多糖,用超纯水溶解配为0.2mg/mL溶液,于分光光度仪进行200-900 nm全波长扫描记录。空白对照采用超纯水。紫外光谱分析结果如图3所示,实施例1获得的杨树桑黄菌多糖在260/280nm处无吸收峰,说明该多糖中不含蛋白质和核酸。
红外:将实施例1获得的杨树桑黄菌多糖冻干粉与溴化钾(质量比1:200)混合压片,进行红外光谱扫描,空白对照采用KBr粉末压片而成。扫描结果如图4所示,吸收峰在3600-3200 cm-1区间属性为-OH的伸缩振动吸收峰,这个波段的吸收峰为糖类的特征峰。3347.83cm-1处吸收峰峰型宽而强,是O-H的伸缩振动吸收峰,属于糖类的特征峰;在2927.83 cm-1处中等强度的吸收峰归属于C-H伸缩振动;在1633.91 cm-1处吸收峰,归属于C=O伸缩振动;在1346.96 cm-1左右检测到微弱的C-H变形振动峰1078.26 cm-1处弱吸收峰为C-O伸缩振动;在1028.70 cm-1处有吸收峰,归属于O-H变角振动;966.09 cm-1处有吸收峰,为β-糖苷键的特征吸收;814.78 cm-1处的微弱吸收,证明α-糖苷键的存在。
单糖组分分析:
取干净的15 mL EP管,加入8 mL无菌水,依次加入Fuc、Ara、Gal、Glc、Xyl、Man、Fru、Rib、GalUA、GlcUA和ManUA各100 mg,溶解后配制成10 mg/mL的标准液母液。将上述混标母溶液先稀释100倍,制备成100 μg/mL工作液,将取上述溶液按照梯度稀释,装入1.5 mL的EP管中。取干净的色谱瓶,准确称取杨树桑黄菌多糖 5 mg,加入配置好的2 mol/L 三氟乙酸溶液,121℃加热2 h。通氮气,吹干。加入甲醇清洗,再吹干,重复甲醇清洗2-3次。加入无菌水溶解,移入色谱瓶中待测。采用Dionex™ CarboPac™ PA20液相色谱柱,进样量为20μL。流动相A(H2O),流动相B(100 mmol/L NaOH),柱温为30℃,利用电化学检测器对单糖组分进行分析检测。外标法定量,通过配制不同浓度标样来制定标准曲线。根据样品出峰时间计算单糖组成。
结果如图5所示,杨树桑黄菌多糖主要由六种单糖组成,其种类及摩尔比为:Fuc:Gal: Glc: Man: Fru: GlcUA=24.473: 42.942: 11.892: 19.988: 0.637: 0.068。
分子量和均一性分析:配备凝胶排阻色谱柱(Ohpak SB-805 HQ,300×8mm)、差分检测器(Optilab T-rEX)和激光的凝胶色谱-微分-多角度激光光散射系统光散射检测器(DAWN HELEOS 2)用于评估杨树桑黄菌的均匀性和分子量。检测条件为:流动相为0.1 MNaNO3,进样量为100 μL,流速为0.4 mL / min,柱温箱温度为45 ℃。通过结果分析可得实施例1获得的杨树桑黄菌多糖具有均匀的单峰,表明其均一性好,分子量为22.5 kDa,结果如图6。
甲基化及乙酰化分析:
a)准确称取杨树桑黄菌多糖1 mg,加入500 μL DMSO溶解;
b)加入1 mg NaOH,孵育30 min;
c)加入50 μL碘甲烷溶液反应1 h;
d)加入1 mL水和2 mL二氯甲烷;重复水洗3次;
e)吸取下层二氯甲烷相并蒸干;
f)加入100 μL 2M 三氟乙酸,121℃反应90 min;
g)30℃蒸干;
h)加入50 μL 2 M 氨水,50 μL 1 M NaBD4,混匀,室温下反应2.5 h;
i)加入20 μL冰醋酸终止反应,250 μL甲醇洗两次,氮气吹干;
j)加入乙酸酐250 μL,涡旋混匀,100℃反应2.5 h;
k)加入1 mL水静置10 min;
l)加入500 μL二氯甲烷;重复水洗3次;
m)取下层二氯甲烷相,上机检测;
色谱系统采用的是Agilent气象色谱系统(Agilent 7890A;AgilentTechnologies,USA),根据化合物的性质,进样量为1 μL,分流比10:1,载气为高纯氦气;柱温箱的初始温度为140℃保持2.0 min,以3℃/min程序升温至230℃,保持3 min。质谱系统采用的是美国Aiglent公司的四极杆质谱检测系统,配有电子轰击离子源(EI)和MassHunter工作站。采用电子轰击离子源(EI),分析物在全扫描(SCAN)模式下进行检测,质量扫描范围(m/z):30-600。
基于多糖在分析柱上的保留时间确定多糖的单糖组成。通过鉴定二级质谱碎片,获得多糖甲基化后特征性碎片,根据已有的数据库进行比对,进而确认其键合方式。
根据保留时间和复杂碳水化合物研究中心(CCRC)光谱数据库中部分甲基化糖醇乙酸酯(PMAA)的标准数据,杨树桑黄菌多糖主要由8个糖苷片段组成(表1)。 t-Man(p) 和t-Glc(p) 的非还原末端的摩尔比分别达到7.237% 和 26.247%,表明可能存在分支残基。
表1 实施例1获得的杨树桑黄菌多糖的甲基化分析结果
核磁共振(NMR)分析:称取杨树桑黄菌多糖50 mg,将其溶于0.5 mL的D2O中并冻干。随后将冻干粉再溶于0.5 mL的D2O中,并继续冷冻干燥,重复以上过程,以充分交换活泼氢。然后将样品溶于0.5 mL的D2O中,在室温25℃下置于以600 MHz的NMR测定1H NMR谱、13CNMR谱和二维图谱。图7-12分别为杨树桑黄菌多糖核磁共振1H谱图、13C谱图及核磁二维谱图。
氢谱信号主要集中在4.0~5.0ppm之间。如图所示,对氢谱信号进行归属,5.84 ppm处为α-D-Fucp-(1→异头氢信号峰,5.7 ppm处为→2)-β-D-Fucp-(1→异头氢信号峰,5.45ppm处为α-D-Manp-(1→异头氢信号峰,4.59 ppm处为→6)-β-D-Galp-(1→异头氢信号峰,4.5 ppm处为→2,6)-β-D-Galp-(1→异头氢信号峰。1.96 ppm及1.92 ppm分别为α-D-Fucp-(1→及→2)-β-D-Fucp-(1→H-6位。碳谱分析在13C NMR(500MHz, D2O):核磁碳谱信号主要集中在55-110 ppm之间。99.3 ppm处为α-D-Fucp-(1→异头碳信号峰,96.6 ppm处为→2)-β-D-Fucp-(1→异头碳信号峰,100.95 ppm处为α-D-Manp-(1→异头碳信号峰,97.4 ppm处为→6)-β-D-Galp-(1→异头碳信号峰,98.23 ppm处为→2,6)-β-D-Galp-(1→异头碳信号峰。14.19 ppm及14.11 ppm分别为α-D-Fucp-(1→及→2)-β-D-Fucp-(1→C-6位。结合COSY和HSQC对糖苷键其他位移(H-2到H-6,C-2到C-6)进行归属,结果如表2所示。同过HMBC结合NOESY,结果分析糖苷键可能存在的链接方式,从NOESY图谱中看出,→6)-β-D-Galp-(1→异头氢和→2,6)-β-D-Galp-(1→的H-6具有相关信号峰(4.59 ppm/4.18 ppm),可以推测出→6)-β-D-Galp-(1→2,6)-β-D-Galp-(1→的存在;同过HMBC图谱分析,α-D-Fucp-(1→的异头氢和→6)-β-D-Galp-(1→的C-6具有相关信号峰,表明α-D-Fucp-(1→6)-β-D-Galp-(1→的存在。
表2 实施例1中获得的杨树桑黄菌多糖在氢谱和碳谱中的化学链接
测试例2:杨树桑黄菌多糖抗结直肠癌活性研究
(1)试验材料
药物:生理盐水、实施例1获得的杨树桑黄菌多糖。
动物:吉林大学机构动物伦理委员会批准了动物实验(SY202012012),按照机构指南进行。APC-min(APC)小鼠共30只,SPF级,雄性,8-10周龄,购自江苏集萃药康生物科技有限公司,许可证号:SCXK(苏)2018-0008。小鼠饲养在受控环境中,环境参数:温度23±1°C,湿度50±5%,光照时间每天7:00-19:00。
(2)试验方法及结果
小鼠分组及给药: APC小鼠经适应性喂养后,随机分为2组(n=15),一组小鼠进行500 mg/kg 实施例1获得的杨树桑黄菌多糖灌胃给药,另一组进行生理盐水灌胃给药10 mL/ kg。给药频率每天1次,给药体积为0.2 mL/20 g。整个实验持续10周
样品采集:最后一次给药2 h后,小鼠尾静脉采集血样血液室温静置30 min后3500rpm离心10 min取上清,同样的离心条件再次离心取上清即为小鼠血清样品,-80℃保存备用。取血小鼠安乐死后经过75%乙醇浸泡2 min,随后在无菌条件下收集小鼠盲肠内容物,用细胞冻存管封存,-80°C保存备用。分离小鼠盲肠-结肠-直肠部位组织,用生理盐水去除肠道中的粪便后吸干多余水分,在4%多聚甲醛组织固定液中固定,室温放置保存。
小鼠肠道菌群检测及分析:取用上述收集的盲肠内容物,进行常规微生物组总DNA提取,并进行肠道菌群基因测定,根据基因测序结果,对小鼠肠道菌群改变进行分析,具体内容如下:按照试剂盒说明,每例取500 mg内容物加入到含磁珠的1.5 mL无菌EP管内,依次加入0.7 mL SL1h和0.15 mL增强液,室温震荡5 min后,12000 r/min离心3 min去除沉淀颗粒,上清中再加入0.15 mL SL2冰浴5 min,12000 r/min离心3 min取上清0.7 mL加入粒抑制剂EX,12000 r/min离心1 min取上清,再加入0.25 mL SB缓冲液混匀吸取0.55 mL加入到装有NucleoSpin过滤柱的EP管中,12000 r/min离心1 min,过滤柱转到新管中同样条件再离心后,再转到新的管中加入0.55 mL SB缓冲液同样离心条件离心,过滤柱中依次加入SW1和SW2同样离心条件离心弃掉滤过液,过滤柱再转到新的EP管中加入0.1 mL SE洗脱液室温孵育1 min后同样转速离心1 min取滤过液即为DNA提取物。采用Nanodrop法定量NDA,1.2%琼脂糖凝胶电泳检测DNA质量。
聚合酶链式反应(Polymerase chain reaction,PCR)扩增采用全式金公司的Pfu高保真DNA聚合酶,严格控制循环数,保证样本的扩增条件一致且次数最低。取20 μL磁珠加入到25 μL PCR产物中混合均匀后于磁力架上静置5 min后吸去上清,再加入200 μL 80%乙醇溶液转动PCR管使磁珠移动到另一侧静置5 min,小心吸去上清室温静置至乙醇挥发完全,PCR管中加入25 μL Elution 缓冲液放置于吸附架5 min后取上清于新管中。PCR扩增回收产物用Quant-iT PicoGreen dsDNA Assay Kit进行荧光定量,根据荧光结果对各样本按相应比例进行混合。采用TruSeq Nano DNA LT Library Prep Kit制备测序文库,首先通过End Repair Mix 2切除DNA序列5’端的突出碱基,同时添加磷酸基团、补齐3’端的缺失碱基,在序列5’端添加含Index序列的测序接头,通过AMPure XP Beads磁珠筛选纯化文库体系,纯化后的DNA文库进行PCR扩增,并采用AMPure XP Beads再次纯化文库。
上机测序前,采用Agilent High Sensitivity DNA Kit进行质检,确保只有单一的峰之后,再用Quant-iT PicoGreen dsDNA Assay Kit对文库进行定量浓度在2 nM以上,质检后的文库经NaOH变性为单链后上机测序,使用MiSeq Reagent Kit V3测序试剂盒在MiSeq测序仪上进行,目标片段长度选用200~450 bp。
基于原始测序数据,按照index和barcode信息进行文库和样本划分,并去除barcode序列。利用Vsearch软件进行数据序列去噪,并进行运算分类单位(Operationaltaxonomic unit,OTU)聚类分析,根据OTU在不同样本中的分布,评估两组样本的Alpha多样性水平,并计算各样本的距离矩阵,通过聚类手段,结合相应统计学检验方法,比较不同样本组间的beta多样性差异及差异显著性。在物种分类学组成层面,结合相应统计学检验,进一步衡量不同样本组间的物种丰度组成差异,并尝试寻找标志物种。根据物种在各样本中的组成分布,构建关联网络,计算拓扑指数,并尝试找出关键肠道菌群物种。
小鼠结直肠组织病理学检查:获得的APC小鼠结直肠组织固定48 h后,经过50%,65%,80%,95%,100%,100%乙醇溶液的顺序浸泡脱水,然后在二甲苯中进行透明处理,透明后的组织块在65±1℃条件下用融化的石蜡浸泡进行浸蜡包埋。石蜡包埋后的组织切成5 μm厚的切片,在载玻片上展开烘干。再经过透明和脱水的反过程实现组织复水,并进行苏木精-伊红(H&E)染色,在显微镜下观察并拍照记录组织病理学变化。
小鼠生化指标测定:应用用ELISA小鼠血清、脾脏及结肠组织匀浆进行Th1,Th2相关因子含量进行测定。
(3)试验结果
APC小鼠肿瘤组织病理学实验结果:
通过H&E染色对小鼠结直肠肿瘤组织病理学结构进行研究,如图13,未治疗小鼠肿瘤组织发展较大,有明显的空泡出现,细胞形态改变和血管网络生成较为明显。杨树桑黄菌多糖作用后小鼠结直肠中增生组织的发展受到明显的抑制,血管网络生成较少,细胞形态保持原有状态,在病理学层次证实了杨树桑黄菌多糖有抑制肠道组织增生的作用。
杨树桑黄菌多糖对APC小鼠肠道菌群物种组成的影响:表中列出了两组小鼠肠道菌群中从门到种六个水平的微生物分类单元数量,杨树桑黄菌多糖小鼠肠道菌群在微生物分离单元数量上丰度增加。
表3 小鼠肠道菌群各水平微生物分类单元数
小鼠肠道菌群差异性分析:通过在属水平的物种组成热图对菌群物种丰度前20的物种进行双聚类分析,如图14,热图中红色代表该微生物属在样本中相对丰度较高,蓝色代表相对丰度较低。发现差异菌属:Lachnospiraceae_NK4A136_group, Blautia, Muribaculaceae, Parabacteroides, Lactobacillus, Desulfovibrio, Alistipes, Subdoligranulum, Rikenella, Ruminococcaceae_UCG-014, Christensenellaceae_R-7_ group, Odoribacter, [Ruminococcus]_torques_group, Erysipelotrichaceae_UCG- 003, Bifidobacterium, Romboutsia, Rikenellaceae_RC9_gut_group, Ruminiclostridium_9, Faecalibacterium, Dubosiella,该类特征菌属可以作为疾病诊断、治疗的生物标志物。
杨树桑黄菌多糖对APC小鼠血清、脾脏和结直肠肿瘤中免疫相关蛋白的影响:
IL-12能够促进T淋巴细胞增殖和Th1细胞的分化,参与促进杀伤性T细胞介导的免疫杀伤反应过程,与未治疗的APC小鼠相比,杨树桑黄菌多糖作用后显著提升了小鼠血清和结肠组织中IL-12含量(图15)。TNF-α作为肿瘤坏死因子调控效应性T淋巴细胞的增殖分化,如图(图16)杨树桑黄菌多糖作用后提升了APC小鼠TNF-α含量。
杨树桑黄菌多糖作用后,APC小鼠血清、脾脏和肿瘤组织中IFN-γ(图17)呈现升高趋势,而IL-4(图18)呈现降低趋势。表明杨树桑黄菌多糖作用后T淋巴细胞总数增多,且Th1/Th2细胞比例增大,整体向促进免疫反应一侧倾斜。如(图19),杨树桑黄菌多糖作用后IL-2蛋白含量升高,进而促进了T淋巴细胞的活化水平及肿瘤杀伤活性。
Claims (5)
1.一种杨树桑黄菌多糖,其特征在于:
分子量为22.5 kDa,主要包括6种单糖:Fuc、Gal、Glc、 Man、 Fru及 GlcUA,其摩尔质量比为24.473: 42.942: 11.892: 19.988: 0.637: 0.068。
2.根据权利要求1所述的一种杨树桑黄菌多糖,其特征在于:
杨树桑黄菌多糖主要有10个糖苷片段组成,其中糖苷片段t-Gal(p)、6-Gal(p)及2,6-Gal(p)摩尔比例分别为1.74%,34.50%和18.96%。
3.根据权利要求1或2所述的一种杨树桑黄菌多糖的制备方法,包括以下步骤:
(1)杨树桑黄菌子实体超微粉碎成粉末,过150目筛;
(2)加入质量20倍质量的石油醚进行脱脂,加热回流2小时,回流脱脂两次后,烘干菌粉;
(3)加入质量10-50倍质量的去离子水,于70-90℃水浴中提取2小时,提取两次,合并溶液;
(4)冷却后4000 rpm条件离心20分钟,取上清,旋转蒸发浓缩至1/5-1/10体积,Sevag法去蛋白,旋蒸除去残留的有机试剂后,透析袋透析12-48 h,浓缩至适当浓度;
(5)调节乙醇终浓度至85%(v/v)进行沉淀,4℃条件过夜,离心收集沉淀,进行冻干,即获得杨树桑黄菌粗多糖;
(6)将杨树桑黄菌粗多糖用去离子水溶解,上样于DEAE Sepharose Fast Flow离子交换柱(5 cm×30 cm)层析:在1.0 mL/min的流速下,分别以0、0.1、0.3和0.5 mol/L NaCl进行洗脱,并用洁净、干燥的试管收集,收集量为8 mL/管,将收集到的糖采用硫酸-苯酚法进行检测,绘制出洗脱曲线,将峰值糖进行合并,浓缩,冻干,备用;
(7)将通过DEAE Sepharose Fast Flow离子交换柱获得的杨树桑黄菌多糖冻干粉末溶于0.15 mol/L NaCl,上样于HiLoad 16/600 Superdex 200 prep grade柱:以0.45 mol/LNaCl洗脱,流速为0.5 mL / min,将自动收集器设置为体积为2 mL/管,采用苯酚-硫酸法测定多糖含量,收集主峰,旋转蒸发浓缩至1/10体积,经透析除盐,蒸发浓缩,冻干后即获得杨树桑黄菌多糖。
4.根据权利要求1或2所述的一种杨树桑黄菌多糖在治疗结直肠癌药物中的用途。
5.一种药物制剂以本发明所述的具有抗结直肠癌活性的杨树桑黄菌多糖为活性成分,同时含有一种或多种药学上可接受的载体物质和/或辅剂。
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