CN114540227A - 一种具有抑制肝癌肿瘤生长的副干酪乳杆菌l511及其用途 - Google Patents
一种具有抑制肝癌肿瘤生长的副干酪乳杆菌l511及其用途 Download PDFInfo
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- CN114540227A CN114540227A CN202210158701.2A CN202210158701A CN114540227A CN 114540227 A CN114540227 A CN 114540227A CN 202210158701 A CN202210158701 A CN 202210158701A CN 114540227 A CN114540227 A CN 114540227A
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Abstract
本发明公开了一种具有抑制肝癌肿瘤生长的干酪乳杆菌L511及其用途。一种干酪乳杆菌（Lactobacillus casei）LSC‑L511，于2022年1月14日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC NO.24309。本菌株单菌即具有很强的调节巨噬细胞极化的能力，可显著抑制肝癌的进展，能够通过诱导肝癌细胞铁死亡来增强索拉非尼对肝癌的治疗效果，并且还有缓解结肠炎症，改善肠道菌群结构及短链脂肪酸水平等多种功能和用途。
Description
技术领域
本发明涉及微生物技术领域，涉及一种具有抑制肝癌肿瘤生长的副干酪乳杆菌L511及其用途。
背景技术
肝细胞癌(Hepatocellular carcinoma,HCC)占原发性肝癌病理类型的90％，是男性恶性肿瘤的第二大死因，在我国，每年增加的肝癌患者约占全球新增病例的一半，死亡率也在逐年升高，形势较为严峻。阶段肝癌的治疗方法主要包括手术、药物、放化疗等，但疗效较差，肝癌进展迅速、转移性强导致手术治疗很难实施，即使可以采取手术切除，术后五年的复发率高达70％，五年生存率仅10％，因此，临床迫切需要更有效的治疗策略来延长中晚期肝癌患者的生存时间并改善其生存质量。近年来，大量研究表明肠道微生态失衡与肝癌发生发展密切相关，因此维持肠道菌群稳定对防治肝癌具有重要的意义。肠道和肝脏通过胆汁酸肠肝循环及门静脉循环紧密地联系起来，因此益生菌在治疗肝脏疾病中发挥重要作用。
根据《原发性肝癌诊疗规范(2019年版)》推荐，索拉非尼(sorafenib)作为一种靶向药对于不同国家地区、不同肝病背景的晚期肝癌病人都具有一定的生存获益，因此索拉非尼被推荐作为肝癌的一线治疗用药应用于临床治疗。但根据临床病例统计分析，索拉非尼的客观缓解率仅有5％，且索拉非尼副作用强烈，包括腹泻、体质量下降、手足综合征、皮疹、心肌缺血以及高血压等给，因此提高索拉非尼的疗效、减轻其带来的副作用对临床使用索拉非尼意义重大。有文献报道微生物源性的葡萄球菌超级抗原样蛋白6(SSL6)增强肝细胞癌(HCC)细胞对索拉非尼(SFN)敏感性的潜在机制，为索拉非尼与肠道微生物联合应用提供了理论依据。
在目前已经公开的文献与专利或专利申请中，有少量针对益生菌或益生菌复配、发酵产物抑制肿瘤的专利，但并无真正涉及具有抑制肝癌发生功能的明确益生菌菌株。例如，CN105535650A公开了一种具有抗肿瘤(肝癌细胞株皮下移植)功能的益生菌组合物，组合物涉及到多种益生菌及中药成分，但未明确其中各株菌的肿瘤抑制功能，且所有益生菌均未明确到株，由于在益生功能上同种益生菌不同菌株间差异显著，故该配方并不具有普适性；同样，CN10468665 7A和CN101711775A中的益生菌也未明确到株，且发酵物成分功能不明确。
发明内容
针对现有技术存在的上述问题，本申请提供了一种副干酪乳杆菌及其用途。
本发明的目的可通过以下技术方案实现：
一种副干酪乳杆菌(Lactobacillus paracasei)LSC-L511，于2022年1月14日保藏于中国微生物菌种保藏管理委员会普通微生物中心CGMCC，保藏地址为北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，保藏编号为CGMCC NO.24309。
本发明所述的副干酪乳杆菌L511在制备治疗肝癌的药物中的应用。
由本发明所述的副干酪乳杆菌L511制备的菌剂。
本发明所述的菌剂在制备治疗肝癌的药物中的应用。
本发明所述的副干酪乳杆菌L511联合索拉非尼在制备治疗肝癌的药物中的应用。
本发明所述的副干酪乳杆菌L511制备的菌剂联合索拉非尼在制备治疗肝癌的药物中的应用。
有益效果：
本菌株单菌即具有很强的调节巨噬细胞极化的能力，可显著抑制肝癌的进展，能够通过诱导肝癌细胞铁死亡来增强索拉非尼对肝癌的治疗效果，并且还有缓解结肠炎症，改善肠道菌群结构及短链脂肪酸水平等多种功能和用途。
附图说明
图1：各组小鼠处死后肝脏情况
图2：各组小鼠肝脏重量(A)、肝脏指数(B)、肝脏结节数(C)、脾脏重量(D)、脾脏指数(E)
图3：各组小鼠肝脏组织HE染色：A为对照组B为本发明菌株干预组C为索拉非尼单独治疗组D为联合治疗组
图4：各组小鼠血清炎症因子表达情况
图5:各组小鼠结直肠长度
图6:各组小鼠结肠组织切片HE染色：A为肝癌模型对照组B为索拉非尼单独治疗组C为索拉非尼与本发明菌株联合治疗组
图7：各组小鼠结直肠组织病理评分
图8：各组小鼠肠组织紧密连接蛋白occludin表达情况
图9：通过流式细胞术检测肝脏组织中M1型巨噬细胞及M2巨噬细胞极化情况
图10：各组小鼠肝脏组织中总铁(A)、二价铁离子(B)、三价铁离子(C)表达情况
图11：小鼠肠道菌群组成(门水平)
具体实施方式
实施例1
副干酪乳杆菌L511的分离筛选：(l)收集酸奶样本，将样品进行梯度稀释后涂布于MRS固体平板上，培养24-48h；(2)选取符合乳酸菌基本形态的单菌落进行平板划线纯化，筛选分离出乳酸菌；(3)将上述单菌落培养于液体MRS培养液中培养24h后提取细菌DNA，进行PCR，并送测序公司进行16s鉴定。
副干酪乳杆菌L511的生理生化特征：革兰阳性杆菌，氧化酶(-)，接触酶(-)，甘油(-)，肌醇(-)，菊糖(-)，赤癣醇(-)，甘露醇(+)，棉子糖(-)，松三糖(-)，山梨醇(+)，D-阿拉伯糖(-)，L-阿拉伯糖(-)，a-甲基_D-甘露糖甙(-)，淀粉(-)，糖原(-)，a-甲基_D-葡萄糖甙(-)，D-核糖(+)，D-木糖(-)，N-乙酰-葡糖胺(+)，木糖醇(-)，龙胆二糖(-)，苦杏仁甙(+)，L-木糖(-)，阿东醇(+)，熊果苷(+)，D-松二糖(+)，D-来苏糖(-)，七叶灵(+)，β-甲基-D-木糖甙(-)，D-半乳糖(+)，水杨苷(+)，D-塔格糖(+)，D-岩藻糖(-)，麦芽糖(+)，D-果糖(+)，麦芽糖(+)，L-岩藻糖(-)，D-甘露糖(+)，乳糖(+)，D-阿拉伯糖醇(-)，L-山梨糖(-)，蜜二糖(-)，L-阿拉伯糖醇(-)，L-鼠李糖(-)，蔗糖(-)，卫茅醇(-)，海藻糖(+)，葡萄糖酸盐(-)，2-酮基-葡萄糖酸盐(-)。
鉴定为副干酪乳杆菌的依据：16S rRNA基因序列测定如SEQ ID NO.1所示。
实施例2
配制MRS培养基：称酪蛋白胨10.0克，牛肉浸取物10.0克，酵母提取液5.0克，葡萄糖5.0克，乙酸钠5.0克，柠檬酸二胺2.0克，吐温80 1.0克，磷酸氢二钾2.0克，七水硫酸镁0.2克，七水硫酸锰0.05克，碳酸钙20.0克，蒸馏水1.0升，pH 6.8。按1：500比例将菌种种于MRS液体培养基中，于37℃厌氧培养箱培养，根据细菌吸光度(OD600)及细菌平板计数，确定细菌浓度。培养至对数期末期，取出菌液，12000rpm/min离心15-20min，用PBS洗涤2-3次后，用PBS将浓度调整至1x109CFU/ml用于小鼠灌胃。
实施例3副干酪乳杆菌L511对小鼠肝癌的缓解作用
取出生15天的雄性C57BL/6小鼠40只，随机分为4组：肝癌模型对照组、本菌株干预组，索拉非尼治疗组及联合治疗组，每组含小鼠10只。
肝癌化学模型小鼠出生14天，按体重腹腔注射DEN试剂，25mg/kg，出生第四周开始每周腹腔注射浓度为20％的CCL4试剂一次，5ul/g，连续注射16周。
造模过程中，化学模型对照组小鼠及索拉非尼组小鼠自出生后第14周开始每两天喂200ulPBS，本菌株干预组及联合治疗组小鼠自出生后第14周开始每两天喂200ul浓度为109CFU/mL的本菌株悬液(PBS配制)，索拉非尼组及联合治疗组自第16周开始灌胃索拉非尼，30mg/Kg，连续灌胃15天。
造模结束后，取小鼠的血清、肝脏、结直肠、脾脏。其中，计算肝癌化学模型小鼠肝脏表面结节个数，统计小鼠肝脏重量、结直肠长度、脾脏重量。同时取结肠组织、肝脏进行石蜡切片操作并进行常规H&E染色。
结果如图1-2所示，与肝癌对照组相比，本发明菌株灌胃的干预组可以显著减少肿瘤的数量，肝脏重量、肝脏指数以及脾脏指数都明显降低；与索拉非尼单独治疗组相比，联合治疗组小鼠肿瘤数量也明显减少，肝脏重量、肝脏指数以及脾脏指数也都明显降低。H&E染色结果如图3所示，从切片中可以明显看出，肝癌对照组小鼠病变严重，相比于对照组小鼠，经本发明菌株灌胃后，肿瘤细胞浸润明显改善，与索拉非尼单独治疗组相比，联合治疗组小鼠肿瘤细胞浸润也明显改善。
实施例4本发明菌株对肝癌小鼠血清中相关炎症因子的调节作用
收集实施例3中的肝癌化学模型小鼠的血液，常温离心，取上清得到小鼠血清，使用Elisa测定血清中IL-12等细胞因子的含量。结果如图4所示，本发明菌株干预组小鼠血清中IL-12、IL-18、TNF-α、IL-1a水平较对肝癌照组显著升高，与索拉非尼单独治疗组相比，联合治疗组小鼠血清中IL-12、IL-18、TNF-α、IL-1a水平也显著升高。
实施例5本发明菌株可减轻索拉非尼治疗带来的肠道炎症副作用
收集实施例3中肝癌化学模型小鼠的结直肠，测量肝癌模型对照组、索拉非尼单独治疗组及索拉非尼本发明菌株联合治疗组小鼠结直肠长度，如图5所示，相较于索拉非尼治疗组，联合治疗组小鼠肠道有增长趋势。
H&E染色结果如图6所示，从切片中可以明显看出，相比于肝癌模型对照组、索拉非尼单独治疗组，经本发明菌株灌胃后，炎症细胞浸润减少，腺体结构有所恢复，症状明显减轻，根据病理评分标准由两人分别独立评分后取均值进行统计，病理评分结果如图7所示，经本发明菌株灌胃后，病理评分降低。
取实施例3中小鼠结直肠组织0.5cm，制作切片，经过脱蜡、抗原修复、封闭、抗体孵育等步骤进行免疫荧光染色，结果如图8所示，联合治疗组肠道紧密连接蛋白Occludin表达较其他两组明显增加。
组织学病理评分标准
实施例6本菌株诱导肝癌小鼠肝脏巨噬细胞极化来调控肿瘤微环境
取实施例3中各组小鼠肝脏组织，经过胶原酶消化后提取单核细胞，通过流式细胞术检测肝癌模型小鼠肝脏中M1、M2型巨噬细胞比例来探究本菌株对巨噬细胞极化的调控作用。结果如图9所示，本菌株可促进肝脏巨噬细胞向M1极化，抑制其向M2极化，进而发挥抑制肿瘤作用。
实施例7本发明菌株促进肝癌细胞铁死亡
取实施例3中小鼠肝脏组织，通过试剂盒检测组织中铁的变化。如图10，相较于肝癌模型对照组、索拉非尼单独治疗组，联合治疗组肿瘤组织内铁离子、亚铁离子及总铁累积增加，促进了肝癌细胞的铁死亡，抑制肝癌的进展。
实施例8本菌株对肝癌小鼠肠道菌群失调的修复作用
取肝癌模型对照组(HCC)、本菌株干预组(LSC)小鼠0.1g粪便，提取参照试剂盒(FastDNA Spin Kit for Soil)说明书，略作改动，具体方法如下所示。将约0.1g粪便加至Lysing Matrix E管中，并加入978μL Sodium Phosphate Buffer和122μL MT Buffer，然后于室温下静置30min；使用Fastprep，设置速度为6.0，设置时间为40，进行破碎；于4℃下以14000×g离心10min后取上清，加入250μL PPS，颠倒混匀；于4℃下以14000×g离心10min，取上清并加入1mL Binding Matrix Suspension，颠倒混匀后，于室温下静置3min，并弃去650μL上清，振荡重悬后，取650μL悬浊液至SPIN Fitter，于4℃下以14000×g离心2min，弃去托管中的液体并重复一次上述步骤；加入500μL SEWS-M(使用前加入100mL无水乙醇，并充分振荡混匀)，于4℃下以14000×g离心1min，弃去托管中的液体，再次以相同条件离心；使用新的集液管，并于室温下静置5min；加入50μL DES，放置在55℃的金属浴中保温5min；然后于4℃下以14000×g离心1min，集液管中即为DNA溶液，将得到的DNA溶液送至二代测序仪测序并分析。结果如图11所示，本菌株的摄入能够显著的增加模型小鼠肠道内厚壁菌门及疣微菌门的丰度。
序列表
<110> 南京医科大学
<120> 一种具有抑制肝癌肿瘤生长的副干酪乳杆菌L511及其用途
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1417
<212> DNA
<213> 副干酪乳杆菌(Lactobacillus paracasei)
<400> 1
ctcgctccct aaaagggtta cgccaccggc ttcgggtgtt acaaactctc atggtgtgac 60
gggcggtgtg tacaaggccc gggaacgtat tcaccgcggc gtgctgatcc gcgattacta 120
gcgattccga cttcgtgtag gcgagttgca gcctacagtc cgaactgaga atggctttaa 180
gagattagct tgaccgcggt ctcgcaactc gttgtaccat ccattgtagc acgtgtgtag 240
cccaggtcat aggggcatga tgatttgacg tcatccccac ctccccgttt gtcaccggca 300
gtcttactag agtgcccaac taaatgctgg caactagtca taagggttgc gctcgttgcg 360
ggacttaacc caacatctca cgacacgagc tgacgacaac catgcaccac ctgtcatttt 420
cccccgaagg ggaaacctga tctctcaggt gatcaagatg tcaagacctg gtaaggttct 480
tcgcgttgct tcgaattaaa ccacatgctc caccgcttgt gcggcccccg tcaattcctt 540
tgagtttcaa ccttgcggtc gtactcccca ggcggaatgc ttaatgcgtt agctcgcggc 600
actgaagggc gaaaccctcc aacacctagc attcatcgtt tacggcatgg actaccaggg 660
tatctaatcc tgttcgctac ccatgctttc gagcctcagc gtcagttaca gaccagacag 720
ccgccttcgc cactggtgtt cttccatata tctacgcatt tcaccgctac acatggagtt 780
ccactgtcct cttctgcact caagtttccc agtttccgat gcgcttcctc ggttaagccg 840
agggctttca catcagactt aaaaaaccgc ctgcgctcgc tttacgccca ataaatccgg 900
ataacgcttc cacctacgta ttaccgcggc tgctggcacg tagttagccg tggctttctg 960
gttggatacc gtcacgccga caacagttac tctgccgacc attcttctcc aacaacagag 1020
ttttacgacc cgaaagcctt cttcactcac gcggcgttgc tccatcagac ttgcgtccat 1080
tgtggaagat tccctactgc tgcctcccgt aggagtttgg gccgtgtctc agtcccaatg 1140
tggccgatca acctctcagt tcggctacgt atcatcgcct tggtgagcca ttacctcacc 1200
aactagctaa tacgccgcgg gtccatccaa agcgatagct tacgccatct ttcagccaag 1260
aaccatgcgg ttcttggatc tatgcggtat tagcatctgt ttccaatgtt atcccccact 1320
taagggcagg ttacccacgt gttactcacc cgtccgccac tcgttccatg ttgaatctcg 1380
gtgcaagcac cgatcatcaa cgagaactcg ttcgact 1417
Claims (6)
1.一株具有抑制肝癌肿瘤生长的干酪乳杆菌(Lactobacillus casei)L511，于2022年1月14日保藏于中国微生物菌种保藏管理委员会普通微生物中心CGMCC，保藏地址为北京市朝阳区北辰西路1号院3号中国科学院微生物研究所，保藏编号为CGMCC NO.24309。
2.权利要求1所述的干酪乳杆菌L511在制备治疗肝癌的药物中的应用。
3.由权利要求1所述的干酪乳杆菌L511制备的菌剂。
4.权利要求3所述的菌剂在制备治疗肝癌的药物中的应用。
5.权利要求1所述的干酪乳杆菌L511联合索拉非尼在制备治疗肝癌的药物中的应用。
6.权利要求3所述的干酪乳杆菌L511制备的菌剂联合索拉非尼在制备治疗肝癌的药物中的应用。
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