CN116370496A - 胀果甘草多糖在制备治疗和/或预防肝癌的药物中的应用 - Google Patents
胀果甘草多糖在制备治疗和/或预防肝癌的药物中的应用 Download PDFInfo
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Abstract
本发明公开了胀果甘草多糖在制备治疗和/或预防肝癌的药物中的应用,属于疫苗佐剂技术领域。本发明提供的胀果甘草粗多糖GiP及胀果甘草均一多糖GiP‑B1与树突状细胞(DC)协同作用时具有较显著的抑瘤活性。同时,胀果甘草粗多糖GiP及胀果甘草均一多糖GiP‑B1还能够促进正常小鼠骨髓源DC表型的成熟,提高DC的抗原提呈功能。本发明制备的以胀果甘草多糖负载肿瘤抗原的DC疫苗,可以促进DC成熟,上调DC表面标志分子的表达,并对H22肝癌荷瘤小鼠具有一定的免疫治疗作用,胀果甘草多糖可作为DC疫苗佐剂而用于制备治疗和/或预防肝癌的药物。
Description
技术领域
本发明涉及疫苗佐剂技术领域,特别是涉及胀果甘草多糖在制备治疗和/或预防肝癌的药物中的应用。
背景技术
肝癌的发病率居全球恶性肿瘤的第六位,病死率居第三位。肝癌的传统治疗方法有手术、化疗或放疗,尽管是治愈性治疗,但是肿瘤复发率仍然很高。肿瘤免疫治疗主要包括免疫检查点抑制剂、细胞免疫治疗、外泌体免疫治疗和其他新型疗法通过激活个体的免疫系统,选择性地靶向肿瘤抗原,引导免疫细胞杀死肿瘤细胞等途径增强免疫系统预防和对抗肿瘤的能力。
树突状细胞(Dendritic cells,DC)是体内免疫系统中功能最强的抗原呈递细胞,是抗原特异性免疫和耐受性启动的核心,通过主要组织相容性复合体I(MHC-I)和MHC-II分子向先天和适应性免疫系统加工和呈递抗原,并在先天性和适应性免疫系统的连接中发挥关键作用。正常情况下,DC以未成熟状态存在,吞噬抗原能力强,细胞表面共刺激分子表达水平低,在维持机体免疫耐受中起着重要的作用;机体一旦受到致病微生物或其他刺激,DC可以迅速上调其表面共刺激分子的表达,例如CD80、CD86和MHC-II,同时获得迁移能力,将信号从致病微生物携带到淋巴结并激活T细胞。
以DC为基础的疫苗治疗被认为是一种经典的癌症免疫治疗方法,因为它能够激活免疫系统,增强肿瘤特异性细胞毒性反应,杀死肿瘤细胞。DC疫苗是治疗性癌症疫苗,一般从肿瘤患者或健康人外周血中分离出未成熟DC(imDC),经肿瘤抗原刺激成熟,回输患者体内进行治疗,可激活患者体内靶向肿瘤细胞的激活细胞毒性T淋巴细胞(Cytotoxic TLymphocyte,CTL),发挥抗肿瘤作用。近年来,含有抗原的DC疫苗是治疗肿瘤的安全且有前途的疗法,这种基于DC的肿瘤免疫疗法已在乳腺癌、多发性骨髓瘤、前列腺癌、肾细胞癌、恶性黑色素瘤、结直肠癌和非小细胞肺癌等各类肿瘤的临床试验中进行了测试。
然而,随着对免疫治疗的研究不断深入,发现DC疫苗开发中存在的问题是肿瘤抗原的免疫原性较弱,DC在肿瘤组织和外周血中的成熟和功能受到抑制,难以引发机体先天免疫应答。因此,需要添加疫苗佐剂促进DC成熟并增强肿瘤抗原的免疫原性。一些佐剂已被批准用于人用疫苗,包括MF59、AS03、AS04、CpG ODN和AS01,但有效性和安全性问题仍然是阻碍新佐剂开发的挑战,因此需要开发新的安全有效的佐剂。
发明内容
本发明的目的是提供胀果甘草多糖在制备治疗和/或预防肝癌的药物中的应用,以解决上述现有技术存在的问题。
为实现上述目的,本发明提供了如下方案:
本发明提供胀果甘草多糖在制备治疗和/或预防肝癌的药物中的应用。
优选的是,所述药物包括疫苗。
优选的是,所述疫苗为树突状细胞疫苗;所述胀果甘草多糖为疫苗佐剂。
优选的是,所述胀果甘草多糖为胀果甘草粗多糖GiP或胀果甘草均一多糖GiP-B1中的至少一种。
优选的是,所述胀果甘草的用量为100μg/mL。
优选的是,所述胀果甘草均一多糖GiP-B1的相对分子量为不低于2.0×106Da。
优选的是,所述胀果甘草均一多糖GiP-B1的糖醛酸含量为16.8%。
本发明还提供一种治疗和/或预防肝癌的树突状细胞疫苗,所述疫苗以胀果甘草多糖作为佐剂。
本发明公开了以下技术效果:
本发明提供的胀果甘草粗多糖GiP及胀果甘草均一多糖GiP-B1与DC协同作用时则表现出较显著的抑瘤活性。同时,胀果甘草粗多糖GiP及胀果甘草均一多糖GiP-B1还能够促进正常小鼠骨髓源DC表型的成熟,提高DC的抗原提呈功能。本发明制备的以胀果甘草多糖负载肿瘤抗原的DC疫苗,可以促进DC成熟,上调DC表面标志分子的表达,,并对H22肝癌荷瘤小鼠具有一定的免疫治疗,胀果甘草多糖可作为DC疫苗佐剂而用于制备治疗和/或预防肝癌的药物。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为BMDC培养过程中的形态变化(放大倍数×200);其中,A:第2天;B:第3天;C:第4天;D:第5天;
图2为各组DC疫苗形态(放大倍数×200);其中,A:DC组;B:TNF-α组;C:GiP组;D:GiP-B1组;
图3为胀果甘草多糖佐助的DC疫苗表面标志分子的表达;
图4为免疫治疗期间各组荷瘤小鼠体质量的变化与模型组相比(vs.modelgroup),aP<0.05,bP<0.01;
图5为免疫治疗期间各组荷瘤小鼠瘤体积变化。
具体实施方式
现详细说明本发明的多种示例性实施方式,该详细说明不应认为是对本发明的限制,而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式,并非用于限制本发明。另外,对于本发明中的数值范围,应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明,否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料,但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入,用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时,以本说明书的内容为准。
在不背离本发明的范围或精神的情况下,可对本发明说明书的具体实施方式做多种改进和变化,这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见的。本申请说明书和实施例仅是示例性的。
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等,均为开放性的用语,即意指包含但不限于。
本发明所述技术方案,如未特别说明,均为本领域的常规方案,所用试剂或原料,如未特别说明,均购自商业渠道或是已公开。
实施例1
1、H22小鼠肝癌细胞的培养
1)细胞复苏:将H22小鼠肝癌细胞从液氮罐中取出放入PE手套中,迅速置于37℃恒温水浴锅中,快速摇晃冻存管使其快速溶解(1min左右),待冻存液融化后,快速加入盛有10倍体积的常温完全培养基的离心管中,1000rpm离心5min,弃上清,用2mL完全培养基重悬细胞,移入10cm培养皿中,补足细胞培养液至10mL,置于37℃、5%CO2细胞培养箱中继续培养,2~3天换液一次。
2)细胞换液:H22细胞为悬浮细胞,用移液枪吸取培养液轻轻吹打混匀细胞,收集细胞悬液于无菌离心管中,1200rpm离心5min,弃上清,加入新鲜的完全培养基,吹打混匀细胞,移入10cm培养皿中,置于37℃、5% CO2细胞培养箱中继续培养。2~3天换液一次。
2、H22肿瘤细胞裂解液的制备
收集对数生长期的H22细胞,1200rpm离心5min,弃上清,调整细胞悬液密度为1×107个/mL,移入5mL冻存管中,放入液氮罐中30min,取出置于37℃水浴中溶解5min,重复上述步骤,反复冻融5次得到H22细胞裂解液。将H22肿瘤细胞裂解液1500rpm,离心5min,收集上清液,过0.22μm微孔滤器,放入液氮中保存备用。将过滤后的H22细胞裂解液取适量,用BCA试剂盒测定蛋白含量。
经BCA法测得肿瘤细胞裂解液的蛋白浓度为11.33mg/mL,使用25μg/mL的TCL负载DC,制备DC疫苗。
3、小鼠骨髓源DC的分离培养分离
C57BL/6雄性小鼠股骨及胫骨于装有RPMI 1640基础培养液的培养皿中,剪去股骨与胫骨的两端,用1mL注射器抽取无血清RPMI1640基础培养液,针头分别从骨两端插入骨髓腔,反复冲洗出骨髓至培养皿中,直至骨髓腔完全变白;用1mL移液枪吹打收集骨髓悬液,用70μm无菌细胞滤网过滤,转移到无菌离心管中,1200rpm离心5min,弃上清后再加3mL RPMI1640重悬,1200rpm离心5min,弃上清,用小鼠骨髓源树突状细胞完全培养基重悬后,转移于25cm2培养瓶中,37℃、5%CO2细胞培养箱中进行培养,48h后,全量换液,去除未贴壁的细胞继续培养;培养至第4天,进行半量换液;第五天,即为未成熟的小鼠骨髓源树突状细胞。
4、肝癌树突状细胞疫苗的制备
培养至第5天的BMDC,计数后按每孔5×105个细胞铺于六孔板中,依次设置为Control、DC、TNF-α、GiP和GiP-B1组,其中Control组不加TCL和佐剂;DC组只加TCL,不加佐剂;TNF-α组加TCL和佐剂细胞因子TNF-α;GiP组加TCL和佐剂胀果甘草粗多糖GiP;GiP-B1组加TCL和佐剂胀果甘草均一多糖GiP-B1。故除Control组外,每孔加入H22肝癌细胞裂解液,使终浓度为25μg/mL,放入37℃、含5%CO2细胞培养箱中培养12h。除Control和DC组外,TNF-α、GiP、GiP-B1组分别加入rmTNF-α(20ng/mL)、GiP(100μg/mL)、GiP-B1(100μg/mL)。继续培养48h后即可获得负载肿瘤抗原的DC疫苗,同时用荧光倒置显微镜观察并记录各组DC疫苗形态。
BMDC培养的第一天,培养瓶中大多细胞呈圆形,继续静置培养,不移动细胞;第二天全量换液,弃去不贴壁细胞,镜下观察只有少量、单个的DC,偶见集落生长的DC;第三天,悬浮细胞逐渐增多,有短小“毛刺”样突起的DC增多,DC集落增多;第四天,镜下观察见大量的悬浮细胞,DC数量增多,呈典型的集落生长状态;第五天,大多集落生长细胞有短小“毛刺”样突起,为典型的imDC。imDC经抗原致敏、佐剂刺激为mDC,与imDC相比,mDC“毛刺”样突起明显变长,TNF-α、GiP和GiP-B1组与Control、DC组相比较,突起更长,各组DC疫苗呈集落生长,为典型的mDC的形态(如图1、2所示)。
5、流式细胞术检测DC疫苗的成熟程度
收集上述各组DC疫苗的悬浮及半贴壁细胞于15mL离心管中,离心,弃上清。细胞沉淀用1mL预冷的PBS缓冲液清洗两遍,之后用100μL PBS重悬细胞,加入抗小鼠的FITC-CD11c、FITC-CD80、PE-CD86和PE-MHC-Ⅱ流式抗体各2μL,4℃下避光孵育30min,反应完成后用1mL PBS洗去未结合的抗体,最后500μL PBS重悬,细胞重悬液过300目尼龙筛网于流式管中,使之成为单细胞悬液并防止细胞团块阻塞流式检测仪管路。每个流式管中细胞数量不得低于1×106个,用流式细胞仪检测相应抗体的表达,检测结果如表1所示。
注:与空白对照组相比,*表示差异显著P<0.05,**表示差异极显著P<0.01;与DC组相比,▲表示差异显著P<0.05,▲▲表示差异极显著P<0.01。
各组DC培养至第8天,流式细胞仪检测DC的细胞表面标志分子的表达。CD11c为鉴定DC纯度的标志分子;CD80、CD86和MHC-II是鉴定DC成熟的表面标志分子。CD80、CD86可促进共刺激信号以激活T细胞反应;MHC-II将来自外源性蛋白质的肽呈递给CD4+T细胞。由各组DC疫苗CD80、CD86和MHC-II的表达水平与Control组相比(如表1所示),差异有统计学意义(P<0.05),说明负载肝癌细胞抗原能够刺激DC成熟;与DC组相比,差异具有统计学意义(P<0.05),说明佐剂胀果甘草多糖能有效刺激DC成熟。胀果甘草粗多糖GiP与均一多糖GiP-B1比较,CD80、CD86和MHC-II的表达水平GiP-B1高于GiP,其中两组比较MHC-II的差异有统计学意义(P<0.05);CD80、CD86的差异无统计学意义(P>0.05)。
实施例2
1胀果甘草多糖佐助的DC疫苗对H22肝癌荷瘤小鼠的免疫治疗作用
1.1H22肝癌荷瘤小鼠模型的建立及分组
收集对数生长期的H22细胞,用PBS重悬调至细胞密度为1×107个·mL-1,取0.2mL细胞悬液注射于健康KM雌性小鼠腹腔,注射5天后,无菌条件下抽取腹水,调整浓度至1×107个·mL-1,注射细胞悬液于适应饲养7d后的小鼠右侧腋部皮下,第7天接种部位有肿瘤出现即为造模成功,按肿瘤体积,随机分为6组,为模型组、阳性组、DC组、TNF-α组、GiP组、GiP-B1组。
1.2胀果甘草多糖佐助的DC疫苗治疗H22肝癌荷瘤小鼠
取上述制备好的DC疫苗,经腹腔注射给药,给各组荷瘤小鼠注射相应的DCs疫苗200μL(5×105个),模型组注射PBS,阳性组注射奥沙利铂(10mg·kg-1),H22细胞接种后第7天开始治疗,每隔7天治疗一次,共治疗2次。
1.3胀果甘草多糖佐助的DC疫苗对H22肝癌荷瘤生长状态、体质量、瘤重和脏器指数的影响
自初次给药起,观察小鼠的进食进水量、活跃程度、毛发等情况。每2天测量肿瘤体积和小鼠体重,末次给药5天后禁食12h,称量各组小鼠体质量后,摘眼球采血,颈椎脱臼法处死小鼠,剥离小鼠腋下肿瘤,剖取脾脏、胸腺及肝脏组织,立即称质量记录,并计算抑瘤率、各脏器指数。按下面公式计算:
肿瘤体积=A×B2×0.5(A:肿瘤长径,B:肿瘤短径)
抑瘤率%=(模型组平均瘤重-给药组平均瘤重)/模型组平均瘤重×100%
肝脏指数=肝脏质量(mg)/小鼠体重(g);
脾脏指数=脾脏质量(mg)/小鼠体重(g);
胸腺指数=胸腺质量(mg)/小鼠体重(g)。
1.4胀果甘草多糖佐助的DC疫苗对H22肝癌荷瘤小鼠血清中细胞因子的影响
各组小鼠经摘眼球采血,室温静置4h后3000rpm离心10min,收集血清。按Elisa试剂盒使用说明书分别测定IL-12(P70)、IFN-γ、IL-4和IL-10细胞因子的含量。
1.5统计学分析
数据结果以均值标准差表示,数据比较采用单因素方差分析(One-wayANOVA)或独立样本t检验,P<0.05为差异有统计学意义。FCM检测结果采用Flowjo10.8.1软件进行分析,免疫组化结果采用Fiji软件进行分析。
2结果
2.1胀果甘草多糖佐助的DC疫苗对H22肝癌荷瘤小鼠生长状态和体质量的影响
如图4所示,小鼠荷瘤后,各组小鼠活动状态、精神、进食与接种前几乎相同;第7天,各组小鼠腋下可见直径约10mm左右的肿瘤组织。给药第三天后,阳性对照组小鼠明显可见呼吸急促、精神状态较差,体重下降(P<0.05);与模型组相比,TNF-α、GiP、GiP-B1和DC组小鼠饮食、精神状态方面较好,与皮下荷瘤、给药前相比,无明显变化,体重均增长。
2.2胀果甘草多糖佐助的DC疫苗对H22肝癌荷瘤小鼠瘤重、瘤体积变化以及抑瘤率的影响
由表2及图5可见,与模型组相比,阳性对照组、TNF-α和GiP-B1组小鼠肿瘤体积均减小,差异具有统计学意义(P<0.05);与模型组相比,阳性对照组、TNF-α、DC和GiP-B1组瘤重均减小,差异具有统计学意义(P<0.05);提示各组DC疫苗对H22荷瘤小鼠肿瘤的生长有抑制作用。
注(note):与模型组相比(vs.model group),aP<0.05,bP<0.01。
2.3胀果甘草多糖佐助的DC疫苗对H22肝癌荷瘤小鼠脏器指数的影响
由表3可见,与模型组相比,TNF-α、DC、GiP和GiP-B1组胸腺指数均明显升高,差异有统计学意义(P<0.05);与模型组相比,阳性组、TNF-α、DC和GiP组脾脏指数均明显升高,差异有统计学意义(P<0.05);与模型组相比较,阳性组、DC和GiP组肝脏指数均明显升高,差异具有统计学意义(P<0.05)。甘草多糖佐助的DC疫苗升高胸腺指数和脾脏指数,该结果提示胀果甘草多糖佐助的DC疫苗具有改善机体免疫功能的作用。
注(note):与模型组相比(vs.model group),aP<0.05,bP<0.01。
3.4胀果甘草多糖佐助的DC疫苗对H22肝癌荷瘤小鼠血清中细胞因子含量的影响
由表4可见,与模型组相比较,阳性组、DC、GiP和GiP-B1组IL-12(P70)、IFN-γ含量均明显升高,差异具有统计学意义(P<0.05);各组IL-10含量均下降,差异具有统计学意义(P<0.01);各组IL-4含量均下降,其中GiP和GiP-B1组,差异具有统计学意义(P<0.05)。该结果提示胀果甘草多糖佐助的DC疫苗可调节荷瘤小鼠Th1/Th2失衡向以Th1介导的细胞免疫偏移,增强机体的细胞免疫功能,通过细胞免疫途径发挥抗肿瘤作用。
注(note):与模型组相比(vs.model group),aP<0.05,bP<0.01。
以上所述的实施例仅是对本发明的优选方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。
Claims (8)
1.胀果甘草多糖在制备治疗和/或预防肝癌的药物中的应用。
2.根据权利要求1所述的应用,其特征在于,所述药物包括疫苗。
3.根据权利要求2所述的应用,其特征在于,所述疫苗为树突状细胞疫苗;所述胀果甘草多糖为疫苗佐剂。
4.根据权利要求1所述的应用,其特征在于,所述胀果甘草多糖为胀果甘草粗多糖GiP或胀果甘草均一多糖GiP-B1中的至少一种。
5.根据权利要求4所述的应用,其特征在于,所述胀果甘草的用量为100μg/mL。
6.根据权利要求4所述的应用,其特征在于,所述胀果甘草均一多糖GiP-B1的相对分子量为不低于2.0×106Da。
7.根据权利要求6所述的应用,其特征在于,所述胀果甘草均一多糖GiP-B1的糖醛酸含量为16.8%。
8.一种治疗和/或预防肝癌的树突状细胞疫苗,其特征在于,所述疫苗以胀果甘草多糖作为佐剂。
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