CN116606352A - 一种抗菌修复功能的组合物凝胶在妇科疾病中的应用 - Google Patents
一种抗菌修复功能的组合物凝胶在妇科疾病中的应用 Download PDFInfo
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Abstract
本发明涉及一种抗菌修复功能的组合物凝胶在妇科疾病中的应用,具体为一种人源免疫细胞以及细胞裂解蛋白凝胶,包括以下质量浓度的各组分:人源免疫细胞10%‑90%;免疫细胞裂解蛋白1%‑30%;透明质酸钠0.1%~5%;天然海藻糖0.1%‑5%;甘油0.1%~5%;水杨酸0.1%~0.5%:卡波姆0.1%~1%:抗病毒肽0.5%,水余量。该产品可清除阴道内HPV以及HPV阳性的宫颈癌,改善阴道内环境,滋润养护,促进黏膜层再生,加强病人靶向免疫修复,关键在于免疫重建。同时,因最终产品pH值6‑6.5,产品应用于阴道不会对阴道pH以及内环境造成影响,值得在临床推广应用。
Description
技术领域
本发明涉及HPV相关疾病的治疗,具体为一种抗菌修复功能的组合物凝胶在妇科疾病中的应用。
背景技术
宫颈癌是继乳腺癌之后的女性第二大恶性肿瘤,其患病率和病死率都比较高,但是宫颈癌也是目前唯一可以通过医学干预降低患病率和病死率的恶性肿瘤。育龄女性中人乳头状瘤病毒(HPV)感染率较高,高危人乳头状瘤病毒(HPV)持续感染是导致宫颈上皮内瘤变(CIN)和宫颈癌的主要原因。常规进行宫颈癌普查,早期发现宫颈HPV感染,并及时治疗对降低宫颈癌的发病率和病死率非常重要。
清除HPV,免疫重建是关键,HPV感染疾病之所以久治不愈,主要是由于患者体内存在HPV靶向免疫功能缺陷,无法有效地将清除于细胞中的HPV,从而形成长期的病灶。要清除细胞内潜伏的HPV,加强病人靶向免疫修复,关键在于免疫重建。
NKT细胞作为一类新型的免疫调节细胞,其主要特征为T细胞受体(TCR)基因表达的恒定性、CD1d的限制性以及细胞因子产生的迅速、高水平性。NKT细胞的抗原识别与传统的T细胞不同,不能识别由经典的MHCⅠ、Ⅱ类分子提呈的抗原肽,而只识别由细胞表面CD1d分子提呈的脂类、蛋白质抗原,在这些抗原的刺激下NKT细胞被激活,并迅速的产生IL4、IFNγ、IL10、IL13等细胞因子,从而在抗肿瘤、抗感染、抑制自身免疫性疾病及移植免疫中发挥重要的作用。NKT细胞既有增强免疫反应又有抑制免疫反应的双向免疫调节功能,因此与多种疾病如肿瘤、自身免疫性疾病及移植耐受等密切相关。目前研究发现NKT细胞在某些肿瘤患者(如前列腺癌、多发性骨髓瘤等)及自身免疫性疾病患者(如糖尿病、多发性硬化、类风湿性关节炎、特发性血小板减少性紫癜、再生障碍性贫血等)中数目减少,并且在肿瘤患者中NKT细胞分泌IFNγ的能力降低。在小鼠肿瘤模型中NKT细胞的抗肿瘤作用已非常明确。David等在肿瘤患者体内扩增NKT细胞的研究,以及Ishikawa等在晚期肺癌患者的Ⅰ期临床试验表明患者体内应用αGalCer后NKT细胞数目明显增加,从而起到抗肿瘤的作用。现认为NKT细胞抗肿瘤作用的主要机制为:NKT细胞在自身配体及合成配体的刺激下,其表面的IL12R活化,刺激DC细胞分泌大量的IL12,并促使未成熟DC细胞的分化及成熟,而其本身迅速、大量的分泌IFNγ,同时IFNγ作用于NK细胞促使NK细胞亦分泌大量的IFNγ,DC细胞分泌的IL12作用于初始CD4+T细胞,使其向Th1极化,上述因素共同作用而介导了抗肿瘤作用。在小鼠的自身免疫性疾病模型(如Ⅰ型糖尿病、实验性自身脑脊髓膜炎等)中已表明NKT细胞在疾病的发生及治疗中起到了重要的作用,通过应用αGalCer刺激NKT细胞,使其数目增加的同时,极化分泌IL4、IL10等Th2细胞因子而起到治疗自身免疫性疾病的作用。
但是目前,采用NKT细胞来治疗HPV感染的研究还不够多,特别是长效型的凝胶型的药物研究有待于进一步的开发。
发明内容
本发明一方面发现,NKT 细胞既能增强免疫反应又能抑制免疫反应, 从而在抗肿瘤、抗感染、抑制自身免疫性疾病及移植耐受中发挥重要的作用。当HPV病毒侵入人体后,就会启动细胞免疫机制,聚集在感染部位,识别并分析HPV病毒DNA组合,合成相关抗体,同时细胞裂解蛋白富含大量免疫因子,可以促进机体全身及局部免疫细胞的活化和增值,修复破损黏膜屏障,有针对性地消除病原微生物的感染,联合免疫细胞实现增敏增效,1+1大于2的实际效果。病人体内有活性的免疫细胞靶向游走,深入病变的深层和周围有病的组织,以杀灭深部感染及周围的游离病毒和胞内病毒,清除HPV及其感染细胞,纠正损伤细胞功能,矫正病人特异免疫缺损,特异调动病人感染皮肤粘膜细胞修复功能,特异调动病人免疫细胞靶向杀伤功能,对细胞周围微环境进行修复,纠正病理变化,从而阻断宫颈癌等相关疾病的发生。
进一步的,本发明还发现,天然海藻糖具有连接、支持、保水、抗压及保护细胞等物理学作用,而且可以影响细胞的存活,加入海藻糖后,可以使细胞或裂解蛋白在体内微环境下存活更长时间。还可以提供营养促进免疫细胞着床和增殖。
进一步的,发明人通过计算机模拟,通过大批量筛选库的筛选,获得了具有较好病毒活性的抗病毒肽,所述多肽序列如SEQ ID NO:1所示,并且具有较好的抑制HPV病毒的效果,所述多肽具有靶向HPV的E6基因进而和E6特异性的结合形成三维空间结构,以阻碍E6发挥生物学功能。发明人通过研究发现,所述多肽能够抑制宫颈癌CaSki/SiHa细胞中HPV16-E6的表达,抑制活性达到了75.6%和79.6%,体现了较好的抑制效果。由于E6蛋白是一种多功能蛋白,其中最重要的功能是介导p53蛋白的降解,导致p53抑制细胞生长增殖作用减弱甚至消失。因此,已知了该蛋白的功能,能够实现抑制HPV的治疗效果。以此为基础,发明人构建了含有免疫细胞的凝胶。
进一步的,本发明是采用如下技术方案实现的:
一种用于治疗阴道HPV感染的人源免疫细胞或细胞裂解蛋白凝胶,其特征在于:包括以下质量浓度的各组分:
人源免疫细胞10-90%;
免疫细胞裂解蛋白1%-30%;
天然海藻糖0.1%-5%;
透明质酸钠0.1%~5%;
甘油0.1%~5%;
水杨酸0.1%~0.5%;
卡波姆0.1%~1%;
SEQ ID NO:1所示的抗病毒肽0.5%;
水余量。
进一步的,以上人源免疫细胞或细胞裂解蛋白所涉及到的人源免疫细胞可以是跟案列中的人源NK细胞和T细胞,也可以是人源CIK细胞,人源CTL细胞,人源DC细胞,人源TIL细胞,人源CAR-T细胞中的一种或几种。
更进一步的,透明质酸钠也是组合物专利配方的比较重要成分,与海藻糖共同作用在可改善细胞外环境,为细胞生存提供必要的营养支持及环境保证,具备锁水功能,从而促进新生细胞生成及促进细胞新城代谢,加速创面愈合。此外,透明质酸钠还可在体内发挥保水、维持细胞外空间、调节渗透压、润滑的重要功能。
更进一步的,水杨酸:一种有机溶剂,再该配方中用于消炎,防腐和酸碱调节。
更进一步的,卡波姆:是一种非常常用的添加剂,可以降低粘稠度,因为它本身具有一定的疏松性,是能够稀释东西的一种特别强的能力的微酸性的物质。所以说,在制作凝胶类的时候,把卡波姆加进去以后可以降低这些物质的粘稠性,来维持这个产品一个合适的粘度。卡波姆具有一定的消炎、杀菌的作用,因为它本身就是一种天然的药用成分,具有一定的消炎杀菌的作用。
具体的,本发明的上述阴道凝胶的制备方法如下:
先将天然海藻糖、水杨酸、卡波姆、透明质酸钠、甘油溶解到纯水中,搅拌均匀,然后加入制备纯化好的NKT细胞,或者用0.22μm的膜过滤过的人源免疫细胞裂解蛋白,继续搅拌,然后加入SEQ ID NO:1所示的抗病毒肽0.5%,再次轻柔搅拌后用纯水将余量补足,最后将得到的凝胶调节pH值至6.5,即得凝胶成品。
有益效果
本发明涉及阴道HPV感染和HPV阳性的宫颈癌的治疗常用药物组合物,具体为一种人源免疫细胞以及细胞裂解蛋白凝胶,包括以下质量浓度的各组分:人源免疫细胞10%-90%;免疫细胞裂解蛋白1%-30%;透明质酸钠0.1%~5%;天然海藻糖0.1%-5%;甘油0.1%~5%;水杨酸0.1%~0.5%:卡波姆0.1%~1%:抗病毒肽0.5%,水余量。该产品可清除阴道内HPV以及HPV阳性的宫颈癌,改善阴道内环境,滋润养护,促进黏膜层再生,加强病人靶向免疫修复,关键在于免疫重建。同时,因最终产品pH值6-6.5,产品应用于阴道不会对阴道pH以及内环境造成影响,值得在临床推广应用。
附图说明
图1 本发明制备的NKT细胞显微图
图2 本发明的凝胶小鼠抗肿瘤实验结果图
实施方式
本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合,来实现和应用本发明技术。以下实施案例中的方法、设备、材料,如果未进行特别说明,均为本领域常规方法、设备和材料,均可由市场购得。
实施例1人源免疫细胞的制备
具体的,NKT细胞分离及培养步骤如下:
NKT细胞培养
配包被液:7mlD-PBS加入35ugRetronectin、35ugCD16单抗混匀。
包被:将包被液加入T75培养瓶,平放,混匀,4℃,避光,过夜。
1.1采血:取人外周血50ml,加入一支50ml离心管。
1.2离心沉降:800g,20min,室温,上层A为血浆层,下层B为Buffycoat和红细胞。
1.3收集血浆层:轻轻吸出A层液体,56℃,30min。
1.4自体血浆制备:4℃,静置10min,1800rpm/800g,4℃,10min,上清作为自体血浆备用。
1.5淋巴细胞分离液分离:加入PBS到B层至50ml,混匀后缓慢平均加入2支已经添加15ml淋巴分离液的离心管,1800rpm/800g,室温,20min,no-break。
1.6洗涤PBMC:用巴斯德吸管将离心后中间白色的PBMC层吸出,在新的50ml离心管中加入20ml左右PBS清洗,1000rpm,6min→弃上清,再次20mlPBS清洗→1000rpm,6min→弃上清,20ml培养基重悬,混匀,取样,typanblue染色计数→1000rpm,6min弃上清。
1.7配制细胞悬液:用GT-T551H3培养基40ml重悬细胞沉淀,混匀。
1.8清洗培养瓶:取出包被过的培养瓶,弃包被液,6mlPBS洗一次,然后再用6ml培养基洗一次。
1.9接种细胞:40ml细胞悬液加入培养瓶添加IL-15终浓度30ng/ml、IL-21终浓度30ng/ml、自体血浆终浓度5%,血清替代物终浓度5%(美国helios产品)、IL-2终浓度6000U/ml,将接种后的培养瓶置二氧化碳培养箱培养。
DAY4培养至4天均分3瓶继续培养,每瓶补加含IL-2500U/mlGT-T551H3培养基50ml,添IL-21终浓度30ng/ml、加IL-15终浓度30ng/ml,自体血浆终浓度2%
DAY6培养至6天转至培养袋培养,补加含IL-2500U/mlGT-T551H3培养基500ml,添加IL-21终浓度30ng/ml、IL-15终浓度30ng/ml,自体血浆终浓度1%
DAY8培养至8天补加含IL-2500U/mlGT-T551H3培养基500ml,添加IL-21终浓度30ng/ml、IL-15终浓度30ng/ml,自体血浆终浓度1%。
DAY11培养至11天补加含IL-2500U/mlGT-T551H3培养基500ml,添加IL-21终浓度30ng/ml、IL-15终浓度30ng/ml,自体血浆终浓度1%。
DAY14培养至14天补加含IL-2500U/mlGT-T551H3培养基500ml,添加IL-21终浓度30ng/ml、IL-15终浓度30ng/ml,自体血浆终浓度1%。
DAY14收集10ml细胞悬液,送检支原体内毒素,细菌传染病检测,未发现相应的内毒素及传染病。
DAY17回收NKT细胞,细胞是兼具自然杀伤细胞NK的作用及细胞毒T细胞的作用,阳性表达CD3和CD56,同时表达TCRVα24和TCRVβ11。细胞培养过程中的细胞图如图1所示。从图1可以看出,分离得到了外形及活性均较好的NKT细胞。
实施例2 NKT免疫细胞裂解蛋白的制备
a、取实施例1制备的NKT免疫细胞培养上清40ml 4000×g,4℃离心20分钟,去除细胞或细胞碎片(后续d步骤备用),离心后将上清吸入新管中。收集样本至于冰上。
b、取40.0ml处理后的细胞培养上清放入50ml离心管中,12,000×g,4℃离心30min,将上清转移到另一个50ml离心管里,100000×g,离心2h,管底可见白色沉淀,吸去上清,注意不要破坏细胞沉淀。
c、免疫细胞裂解蛋白物重悬
加入100ul 1×PBS,用移液器反复吹打或漩涡混合均匀,彻底溶解沉淀,此重悬液中含有完整的免疫细胞胞外基质蛋白A。
d、NKT免疫细胞裂解蛋白组合制备
把a步骤分离的NKT细胞经过3次反复冻融,形成细胞裂解蛋白B。最后将A和B两者等量混合加入2ml盐水定容。储存在-80℃保存。
实施例3 抗病毒肽及免疫细胞和细胞裂解物的活性鉴定
SEQ ID NO:1的抗病毒肽委托鸿肽生物进行合成,合成后的多肽浓度调整为1mg/mL,纯度为99.97%,备用。
HPV阳性宫颈癌细胞株SiHa购自上海信裕生物技术有限公司;DMEM中加入10%胎牛血清(FBS)和1%青霉素、链霉素混合物。细胞在37℃、5%CO2条件下,放入细胞培养箱中培养。每隔2d更换培养基并在显微镜下观察细胞状态,直至生长到对数期。将对数生长期的C33A细胞接种于96孔板。实验共培养细胞共分为6组:(1)SiHa组,(2)C33A+0.5mg/mL SEQ IDNO:1的抗病毒肽组,(3)SiHa+1mg/mL SEQ ID NO:1的抗病毒肽组,(4)SiHa+NKT细胞组(细胞共培养效靶比为1∶10,SiHa:NKT细胞)+实施例2制备的A+B 5mg/mL,(5)SiHa+NKT细胞组(细胞共培养效靶比为1∶10,SiHa:NKT细胞)+实施例2制备的A+B 5mg/mL+1mg/mL SEQ IDNO:1的抗病毒肽组,(6)阳性对照组:SiHa+ 5-氟尿嘧啶 1mg/mL。培养48h后,去除NKT细胞,然后各孔加入10µL的CCK8工作液,将96孔板置于含5%CO2的37℃培养箱中避光孵育4h。用酶标仪于450nm处测定各孔的吸光度(OD)值。结果如表1所示。
表1 CCK8 法检测各组宫颈癌细胞增殖能力
CCK8法检测共培养后肿瘤细胞增殖能力,结果如表1显示:与SiHa细胞组相比,各实验处理组的细胞增殖能力明显减弱(P<0.01),且从表1可以看出,无论是SEQ ID NO:1的抗病毒肽还是NKT细胞联合A+B裂解蛋白组均可以显著的降低癌细胞的增殖活性,能够有效的抑制HPV阳性的宫颈癌细胞。
实施例4 人源免疫细胞和细胞裂解物凝胶的制备
将实施例1的细胞以及实施例2制备的裂解蛋白按照如下组成进行制备:
人源NKT免疫细胞10%;细胞裂解蛋白A+B 20%;
透明质酸钠2%;天然海藻糖1%;
甘油3%;
水杨酸0.5%;
卡波姆0.5%;
SEQ ID NO:1所示的抗病毒肽0.5%;
水余量。
具体的,先将天然海藻糖、水杨酸、卡波姆、透明质酸钠、甘油溶解到纯水中,搅拌均匀,然后加入制备好的NKT细胞,加入用0.22μm的膜过滤过的人源免疫细胞裂解蛋白A+B,轻柔搅拌,得到均匀体系,然后加入SEQ ID NO:1所示的抗病毒肽0.5%,再次轻柔搅拌后用纯水将余量补足,最后将得到的凝胶调节pH值至6.5,即得凝胶成品。
实施例5 人源免疫细胞和细胞裂解物凝胶无肽对照组的制备
将实施例1的细胞以及实施例2制备的裂解蛋白按照如下组成进行制备:
人源NKT免疫细胞10%;细胞裂解蛋白A+B 20%;
透明质酸钠2%;天然海藻糖1%;
甘油3%;
水杨酸0.5%;
卡波姆0.5%;
水余量。
具体的,先将天然海藻糖、水杨酸、卡波姆、透明质酸钠、甘油溶解到纯水中,搅拌均匀,然后加入制备好的NKT细胞,加入用0.22μm的膜过滤过的人源免疫细胞裂解蛋白A+B,轻柔搅拌,得到均匀体系,用纯水将余量补足,最后将得到的凝胶调节pH值至6.5,即得凝胶成品。
实施例6 多肽对照组的制备
按照如下组成进行制备:
透明质酸钠2%;天然海藻糖1%;
甘油3%;
水杨酸0.5%;
卡波姆0.5%;
SEQ ID NO:1所示的抗病毒肽0.5%;
水余量。
具体的,先将天然海藻糖、水杨酸、卡波姆、透明质酸钠、甘油溶解到纯水中,搅拌均匀,然后加入SEQ ID NO:1所示的抗病毒肽0.5%,搅拌,用纯水将余量补足,最后将得到的凝胶调节pH值至6.5,即得凝胶成品。
实施例7 小鼠动物实验
用6周龄雌性BALB/C小鼠对实施例4-6的水凝胶以及阳性对照组和阴性PBS组进行体内抗肿瘤效力进行评价。0.1ml含1.0×106 SiHa细胞的细胞悬浮液皮下注射于小鼠的右前肢的腋下。2周后,肿瘤的体积达到大约都是150mm3时,将小鼠随机分成5组(每组10只小鼠)。实施例4-6的水凝胶的剂量为20.0mg/kg,于瘤旁仅注射一次。阳性对照组为5-氟尿嘧啶20.0mg/kg。阴性对照组为等体积的PBS。治疗有效性和安全性的评价通过测量28天后的肿瘤体积以及观察小鼠生存状况来实现。肿瘤体积通过下式计算:V=L×W2/2。L、W分别是肿瘤的最大和最小直径。
通过测量肿瘤体积,发现阴性对照组的肿瘤体积为(6352.8±56.7)mm3,而各治疗组的肿瘤体积与阴性对照组相比都显著的缩小,差异及其显著(P<0.01)。并且从图2的结果可以看出,实施例4的凝胶组对应的肿瘤的体积最小(23.4±3.1)mm3,而单独使用细胞和其裂解蛋白以及抗病毒肽的效果都稍逊于实施例4的凝胶组,但是也都比阳性对照组的效果要好,这说明二者组合使用具有较好的协同效果,单独使用也有较好的抗癌效果。
针对实施例4-6的凝胶使用组治疗后进行观察发现正常器官没有明显损害,表明局部治疗没有明显的全身性毒性。从小鼠中收集心脏,肝,脾,肺,肾,并用H&E染色评估。相对与PBS对照组,凝胶治疗组H&E染色无异常变化,这提示在处理后这些器官无明显损害。以上说明了本发明的凝胶的良好安全性。
当对本发明的实施方式进行实施或试验时,可以使用与本说明书中记载的方法和材料类似或等同的任选的方法和材料,本说明书中记载了优选的方法、装置、材料。然而,在记载本发明的材料与方法之前,应当理解为,可以按照常规的实验方法以及最优化的目的来改变本说明书中记载的特定的大小、形状、尺寸、材料、方法、手段等,因此本发明不限于这些。并且应当理解为,本说明书中使用的专业术语仅用于说明特定的类型或实施方式,并不用于限制本发明的范围,本发明的范围仅受添附的权利要求的范围的限制。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (6)
1.一种抗病毒肽,其特征在于其氨基酸序列如SEQ ID NO:1所示,该抗病毒肽能够特异性的抑制含有HPV的宫颈癌的细胞活性。
2.权利要求1所述的抗病毒肽在制备抑制含有HPV的宫颈癌活性的制剂中的用途。
3.一种用于治疗HPV的含有人源免疫细胞和细胞裂解蛋白凝胶,其特征在于:由以下质量浓度的组分组成:
人源免疫细胞10%-90%;细胞裂解蛋白1-30%;
透明质酸钠0.1-5%;天然海藻糖0.1-5%;
甘油0.1%~5%;
水杨酸0.1%~0.5%;
卡波姆0.1%~1%;
SEQ ID NO:1所示的抗病毒肽0.5%;
水余量;
其中,人源免疫细胞是分离自人的NKT细胞;细胞裂解蛋白由NKT细胞培养得到的胞外基质和NKT细胞裂解液混合后得到的细胞裂解蛋白。
4.如权利要求3所述的一种用于治疗HPV的含有人源免疫细胞和细胞裂解蛋白凝胶,其特征在于:所述细胞裂解蛋白具体是采用如下方法制备:将NKT免疫细胞培养上清40ml4000×g,4℃离心20分钟,去除细胞或细胞碎片,离心后将上清吸入50ml离心管中,12,000×g,4℃离心30min,将上清再转移到另一个50ml离心管里,100000×g,离心2h,管底可见白色沉淀,吸去上清,注意不要破坏细胞沉淀,加入100μl 1×PBS,用移液器反复吹打或漩涡混合均匀,彻底溶解沉淀,此重悬液中含有完整的免疫细胞胞外基质蛋白A;将分离的NKT细胞经过3次反复冻融,形成细胞裂解蛋白B,最后将A和B两者等量混合加入2ml盐水定容即得细胞裂解蛋白。
5.如权利要求3所述的用于治疗HPV的含有人源免疫细胞和细胞裂解蛋白凝胶,其特征在于:所述凝胶的制备包括以下步骤:
先将天然海藻糖、水杨酸、卡波姆、透明质酸钠、甘油溶解到纯水中,搅拌均匀,然后加入制备好的NKT细胞,加入用0.22μm的膜过滤过的人源免疫细胞裂解蛋白,继续搅拌,得到均匀体系,再加入SEQ ID NO:1所示的抗病毒肽0.5%混合均匀后,然后用纯水将余量补足,搅拌均匀,最后将得到的凝胶调节pH值至6到6.5之间,即得成品。
6.权利要求3-5任一项所述的用于治疗HPV的含有人源免疫细胞和细胞裂解蛋白凝胶在制备用于抑制含有HPV的宫颈癌细胞增殖中的用途。
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Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103119054A (zh) * | 2010-03-26 | 2013-05-22 | 达特茅斯大学理事会 | Vista调节性t细胞介体蛋白、vista结合剂及其用途 |
CN107722115A (zh) * | 2017-11-29 | 2018-02-23 | 吉林大学 | 一种新型重组蜂毒多肽及其制备方法和应用 |
WO2019122050A1 (en) * | 2017-12-22 | 2019-06-27 | Isa Pharmaceuticals B.V. | Methods of immunization |
CN110475571A (zh) * | 2017-04-01 | 2019-11-19 | Avm生物技术有限责任公司 | 细胞免疫疗法前细胞毒性预调理的替代 |
CN116490198A (zh) * | 2020-07-09 | 2023-07-25 | 尤尼根公司 | 用于调节免疫稳态的包含多糖和多酚的基于芦荟的组合物 |
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CN110475571A (zh) * | 2017-04-01 | 2019-11-19 | Avm生物技术有限责任公司 | 细胞免疫疗法前细胞毒性预调理的替代 |
CN107722115A (zh) * | 2017-11-29 | 2018-02-23 | 吉林大学 | 一种新型重组蜂毒多肽及其制备方法和应用 |
WO2019122050A1 (en) * | 2017-12-22 | 2019-06-27 | Isa Pharmaceuticals B.V. | Methods of immunization |
CN116490198A (zh) * | 2020-07-09 | 2023-07-25 | 尤尼根公司 | 用于调节免疫稳态的包含多糖和多酚的基于芦荟的组合物 |
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