CN116144530B - 一株植物乳杆菌ay01及其应用 - Google Patents
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Abstract
本发明公开一株植物乳杆菌(Lactobacillus plantarum)AY01,该植物乳杆菌保藏于中国典型培养物保藏中心,保藏编号为CCTCC M 20221517,所述植物乳杆菌具有阻断HT‑29细胞的细胞周期、诱导HT‑29细胞凋亡和抑制HT‑29细胞增殖的特性,其灭活的发酵上清液、菌悬液对结直肠癌细胞HT‑29增殖有较强的抑制作用,实验结果表明,该植物乳杆菌上清液、菌悬液能有效降低小鼠结肠炎相关性结肠直肠癌的敏感性,抑制肿瘤的发生和增殖。本发明的植物乳杆菌在抑制结直肠癌益生菌辅助治疗方面有巨大应用前景。
Description
技术领域
本发明涉及微生物领域,尤其涉及一株性能优良的植物乳杆菌株及其在抑制结直肠癌细胞HT-29增殖、抗结直肠癌益生菌辅助治疗中的应用。
背景技术
结直肠癌(CRC)的高发病率和高死亡率对生命造成严重威胁,研究表明,结直肠癌患者的肠道微生物区系的紊乱与结直肠癌细胞的发生和发展密切相关。与健康人相比,结直肠癌患者肠道微生物群落结构发生了显著变化,导致与结直肠癌相关的致病菌的数量显著增加,如梭杆菌属、肠球菌属和弯曲杆菌属。目前,治疗结直肠癌的主要方法是化疗、放疗和手术,但其毒副作用大限制了临床应用,因此,增强免疫反应和改善肠道菌群是预防结直肠癌的有效策略。
植物乳杆菌属益生菌,其无污染、无残留,同时还可以提高免疫力,增加动物对疾病的抵抗力,已被广泛应用于酸奶发酵和泡菜生产。有研究发现在术后结直肠癌患者中使用益生菌的实验组复发率为10%,而不使用益生菌的对照组复发率为33.3%,也有一些研究分析了乳酸杆菌或其代谢物在CRC辅助治疗中的作用,如萨德吉等人发现热灭活的植物乳杆菌A7、鼠李糖乳杆菌GG和无菌细胞培养上清液对Caco-2和HT-29细胞具有抑制作用。尽管这些结果支持了实验室辅助治疗CRC的可行性,但都没有直接证实实验室辅助治疗CRC的分子机制,因此,有必要进一步研究乳酸菌抑制结直肠癌发生发展的分子机制,筛选出能够阻断HT-29细胞的细胞周期、诱导HT-29细胞凋亡抑制HT-29细胞的增殖的植物乳杆菌,对结直肠癌的预防和抗结直肠癌益生菌辅助治疗具有重要意义。
发明内容
针对上述现有技术中存在的不足,提供一株具有抑制结直肠癌细胞HT-29增殖的植物乳杆菌AY01及其应用。该植物乳杆菌(Lactobacillus plantarum)AY01是从云南传统发酵食品中分离的乳酸菌库中筛选而得,命名为:植物乳杆菌AY01,已于2022年9月27日保藏于中国典型培养物保藏中心(CCTCC,地址:湖北省武汉市武昌区八一路珞珈山17),保藏编号为:CCTCC M 20221517。
本发明采取的技术方案如下:
一株植物乳杆菌(Lactobacillusplantarum)AY01,所述植物乳杆菌AY01保藏于中国典型培养物保藏中心,保藏编号为CCTCC M 20221517。
进一步地,所述植物乳杆菌具有阻断HT-29细胞的细胞周期、诱导HT-29细胞凋亡抑制HT-29细胞增殖的特性。
一株植物乳杆菌的发酵上清液/菌悬液在制备抑制肿瘤或抑制肿瘤细胞增殖产品中的应用。
进一步地,所述肿瘤为结直肠癌;所述肿瘤细胞为结直肠癌细胞HT-29。
本发明的有益效果如下:
本发明的植物乳杆菌AY01是从云南传统发酵食品中分离的乳酸菌库中筛选得到具有肿瘤抑制作用的菌株,通过流式细胞仪检测和转录组检测,该植物乳杆菌具有诱导HT-29细胞凋亡和阻断HT-29细胞的细胞周期抑制肿瘤增殖的特性;小鼠实验结果表明,该菌株发酵的上清液和活菌菌悬液显著降低了小鼠结肠炎相关性结直肠癌的敏感性,为抗结直肠癌益生菌辅助治疗提供理论支持。
附图说明
图1本发明的植物乳杆菌AY01菌落形态图;
图2为本发明的植物乳杆菌AY01系统进化树;
图3为本发明实施例3经浓度为1mg·mL-1的植物乳杆菌培养上清液冻干粉处理后HT-29细胞的生长曲线图;
图4为本发明实施例3植物乳杆菌AY01上清液阻断HT-29细胞的细胞周期图;
图5为本发明实施例3植物乳杆菌AY01上清液诱导HT-29细胞的凋亡结果图;
图6为本发明实施例3含和不含植物乳杆菌AY01上清液的HT-29细胞中基因表达水平的差异结果图;
图7为本发明实施例4小鼠结肠组织样品的H&E染色图。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1
植物乳杆菌株AY01的分离筛选、鉴定及保藏
1、从云南石林发酵羊奶中分离筛选所述植物乳杆菌,具体过程:吸取羊奶样品1mL放入5mL灭菌的生理盐水中,震荡混匀后取20uL接种到MRS液体培养基中,37℃静置培养48h,将菌液用生理盐水稀释一定梯度,取100μL直接涂于MRS平板,在37℃培养48h后,挑选形态不同的单菌落,接种至MRS液体培养基中,37℃静置培养48h后,用50%甘油保存至-80℃冰箱备用。
2、菌种形态学鉴定:将筛选得到的菌株进行革兰氏染色,用显微镜的油镜观察细菌的形态及颜色,该菌株为革兰氏阳性菌,短杆状,表面光滑,细密,色白,植物乳杆菌AY01的菌落形态如图1所示。
3、菌株的分子生物学鉴定:将菌株接种于MRS的液体培养基中,放置到37℃恒温培养箱进行活化培养,然后提取菌株的基因组DNA(DNA提取采用试剂盒QIAamp genomic DNAand RNA kits进行),使用细菌16S rRNA基因通用引物27F(5'-AGAGTTTGATCCTGGCTCAG-3')和1492R(5'-TACGACTTAACCCCAATCGC-3')对该离菌株的16S rRNA基因进行PCR扩增。反应体系:2×TaqPCR Master Mix 12.5μL、上下游引物各1μL、DNA模板1μL、ddH2O 9.5μL;反应程序:95℃3min;95℃15s,55℃15s,72℃30s,30个循环;72℃5min;PCR产物送测序公司基因序列测定见序列表,然后将所测的16S rRNA基因序列在NCBI数据库的GenBank中进行Blast比对,并应用MEGA 7.0软件构建系统进化树,构建系统进化树见图2。
实验结果:从云南省石林发酵羊奶中筛选出的菌株,经形态观察、构建系统进化树、16SrRNA基因序列比对,该菌株AY01鉴定为植物乳杆菌。
该菌接种到灭菌的MRS液体培养基中,37℃恒温培养24h,生长良好、呈球形、灰白色菌落如图1所示。该菌株已于2022年9月27日保藏于中国典型培养物保藏中心(CCTCC,地址:湖北省武汉市武昌区八一路珞珈山17),分类命名为植物乳杆菌Lactobacillusplantarum,保藏编号为:CCTCC M 20221517。
实施例2
植物乳杆菌培养上清液冻干粉的制备,具体步骤如下:
(1)以该菌株与灭菌的MRS液体培养基按4‰体积比,将所述菌株20μL接种于5mL灭菌的MRS液体培养基中,37℃恒温培养24h,重复上述操作1次,以获得生长状态良好的菌株。
(2)按照上述(1)进行菌株活化后,把AY01以1×105CFU·mL-1接种于100mL灭菌的MRS液体培养基中,37℃恒温培养72h,培养结束后将菌液在4000rpm,4℃,离心15min,离心后,收集上清液,去除沉淀,使用真空冷冻干燥机将上清液冷冻成冻干粉末。
(3)将步骤(2)的冻干粉末以0.5g·mL-1的浓度溶解在不含胎牛血清(FBS)的RPMI1640培养基中,溶解后,将溶液通过0.22μm滤膜过滤得到上清液,储存在-20℃冰箱中备用。
(4)植物乳杆菌AY01活菌菌悬液制备:将培养好的植物乳杆菌AY01菌液分装至无菌的10mL离心管中,每管装5mL,4000rpm,4℃,离心5min,离心完成后,弃上清,收集沉淀。然后加入5mL无菌生理盐水,用移液枪反复吹打洗涤菌体,4000rpm,4℃,离心5min,离心完成后,弃上清,收集沉淀,用生理盐水反复洗涤三次后,加入300μL无菌生理盐水悬浮菌体,然后将AY01活菌悬浮液转移至新的1.5mL无菌EP管内,每两管菌体转移至一个EP管内,使每个1.5mL EP管内活菌悬浮液的体积为500μL,菌体量为1x109 CFU。
实施例3
1、植物乳杆菌AY01对结直肠癌细胞HT-29的诱导凋亡和阻断周期能力分析
MTT法检测植物乳杆菌培养上清冻干粉对HT-29细胞的抑制能力:将100μL浓度为1×105CFU·mL-1的结直肠癌细胞HT-29细胞悬液加入96孔板,在37℃的5%C02培养箱中培养12h后,向96孔板中加入100μL不同浓度的植物乳杆菌培养冻干粉溶液,使得冻干粉溶液的终浓度分别为250mg·mL-1、125mg·mL-1、10mg·mL-1、5mg·mL-1和1mg·mL-1。同时也选取其他9株植物乳杆菌培养上清冻干粉作为对照实验,5-氟尿嘧啶(5-FU)和RPMI 1640培养基(无血清)用作阳性和阴性对照,每个浓度设置六个生物重复。加入冻干粉溶液的时间为0h,HT-29细胞在37℃的5%C02培养箱中培养12、24、36、48、60、72和96h,然后绘制经不同植物乳杆菌冻干粉培养上清液处理后HT-29细胞的生长曲线如图3。
流式细胞仪检测HT-29细胞周期和凋亡:将1mL浓度为1×106CFU·mL-1的结直肠癌细胞HT-29细胞悬液加入到6孔板中,在37℃含5%的C02的培养箱中培养12h,待细胞完全贴壁后,加入1mL的上述不同浓度的培养上清冻干粉溶液,使得冻干粉溶液的终浓度分别为16mg·mL-1、32mg·mL-1、64mg·mL-1,加入冻干粉溶液的时间为0h,部分HT-29细胞在37℃含5%C02的培养箱中培养24h后取出,用胰蛋白酶消化细胞并分散成单细胞,将细胞离心5min(10000g、4℃),弃去上清液,收集细胞;用预先冷却的无菌PBS洗涤细胞三次,再离心5min(10000g、4℃),向混合物中加入1mL DAPI染料,并通过流式细胞仪(Partec GmbH,Germany)检测,剩余HT-29细胞培养48h后,用胰蛋白酶消化细胞并分散成单细胞,重复上述操作,并通过流式细胞仪检测。
如图3所示,MTT分析显示,当冻干粉溶液的终浓度分别为250mg·mL-1、125mg·mL-1、10mg·mL-1、5mg·mL-1和1mg·mL-1时,植物乳杆菌AY01都对HT-29细胞的生长具有较好的抑制作用,当终浓度较低到1mg·mL-1时,只有AY01和另一株植物乳杆菌JSL8-2能抑制HT-29细胞的增殖,并且植物乳杆菌AY01的抑制效果比其他8株植物乳杆菌的效果更加明显,说明在低浓度下AY01对HT-29细胞就具有较好的抑制效果。
如图4所示,流式细胞仪检测显示,浓度为64mg·mL-1的植物乳杆菌上清液作用48h后,HT-29细胞的细胞周期阻滞在S期,结果表明:浓度为64mg·mL-1的植物乳杆菌AY01培养上清液有效阻断HT-29细胞的细胞周期。
如图5所示,MRS处理24h后,HT-29的活细胞比例为92.96%,凋亡细胞比例仅为6.18%。与MRS相比,5-FU作用24h后,HT-29细胞的凋亡率从6.18%增加到13.06%,表明5-FU在一定程度上也能诱导细胞凋亡。浓度为16mg·mL-1、32mg·mL-1和64mg·mL-1的植物乳杆菌AY01处理HT-29细胞24h后,细胞凋亡率分别为19.71%和25.97%、46.31%,抑制率明显高于阳性对照,细胞凋亡率显著增加,表明随着冻干粉溶液浓度的增加,诱导效果越好。结果表明植物乳杆菌AY01的培养上清液通过细胞周期阻滞和凋亡抑制HT-29细胞的增殖,当用64mg·mL-1植物乳杆菌AY01培养物上清液处理HT-29细胞48h后,达到最佳效果,HT-29细胞周期阻滞在S期,凋亡率达96.73%。
2、利用转录组学分析菌株抑癌机理
HT-29细胞被植物乳杆菌AY01处理后提取总RNA,并对其mRNA进行测序,鉴定出使用植物乳杆菌AY01处理后和处理前显著差异表达的基因,并对这些差异表达基因进行注释和富集分析,从转录组水平上深入研究植物乳杆菌AY01抑制HT-29细胞增殖的分子机理。
如图6所示,对含植物乳杆菌AY01上清液和不含植物乳杆菌AY01上清液的HT-29细胞中基因表达水平的差异化进行分析,将HT-29细胞的转录组进行分析,通过GO和KEGG富集分析鉴定了在含有和不含植物乳杆菌植物乳杆菌上清液的HT-29细胞中观察到的显著上调和下调的差异基因,根据转录组测序结果计算各个表达基因的RPKM值,有1215个差异表达基因,其中628个基因上调,587个基因下调。GO富集分析结果表明,DEGs主要参与氧化应激反应和细胞增殖调控。在对氧化应激的反应中,上调基因有RCAN1、HMOX1、DUSP1、GCLM、PPP1R15B等共14个,下调基因有TP53、NDUFS2、ERCC2、TXNIP、NAPRT、GSS、PON2、XPA、MT-ND3等共13个。共有25个上调基因如TNFSF9、Bcl-6、Klf4、HIF1A、NFkBIA、JUN和11个下调基因如STAT6、CDCA7和MMP7参与细胞增殖的调节。
上述结果表明:该植物乳杆菌发酵上清液能阻断HT-29细胞的细胞周期、诱导HT-29细胞凋亡抑制HT-29细胞的增殖。
实施例4
该植物乳杆菌上清液降低小鼠对AOM/DSS诱导的结肠炎相关性结肠直肠癌的敏感性、抑制肿瘤的发生和增殖
本实施例实验所用雄性昆明小鼠购自昆明医科大学,购入后饲养在空气经过过滤处理的昆明理工大学动物房内,将所有小鼠随机分装到鼠笼中,每五只小鼠装一笼,动物房内12h昼夜循环,温度控制在25土2℃,相对湿度保持50-70%,房间内低噪音,让其自由采食和饮水,给小鼠饲喂经辐射处理的无菌饲料和经高压蒸汽灭菌的饮用水,并与其他正在进行的动物实验隔离。
将40只小鼠随机分为三组:第一组是正常对照组(小鼠数量n=10),仅灌喂无菌生理盐水,表示为“Control+Vehicle”;第二组是正常造模组,即阴性对照组(n=15),联合使用AOM/DSS诱导CRC,并灌喂无菌生理盐水,表示为“AOM/DSS+Vehicle”;第三组是实验组(n=15),联合使用AOM/DSS诱导CRC,并灌喂AY01活菌菌悬液,表示为“AOM/DSS+AY01”。接受AOM注射的小鼠在第1、4和7周分别接受三轮DSS饮用水,DSS的终浓度分别为2.5%(第一周),2%(第四周)和2%(第七周),每个DSS循环周期持续1周;从腹腔注射AOM的第一天开始,每天通过灌胃的方法向正常对照组小鼠给予500μL生理盐水、向阴性对照组小鼠给予500μL生理盐水、向实验组给予500μL上述植物乳杆菌活菌菌悬液,每天早晚各灌喂1次,持续10周。
实验过程中,对死亡的小鼠进行解剖,取出小鼠结直肠,观察结直肠状态及病变情况;第三个DSS循环结束一周后,使用异氟醚处死所有活着的小鼠,解剖取小鼠结直肠组织,观察结直肠状态及病变情况,并对结直肠做病理切片进行苏木精伊红(Hematoxylin&eosin,H&E)染色和基于Ki-67、PCNA的免疫组化分析。
结果如图7所示,在AOM/DSS+Vehicle组和AOM/DSS+AY01组的小鼠结肠中均观察到高度分化的腺瘤,但是AOM/DSS+Vehicle组的癌变进度远远快于AOM/DSS+AY01组,且在AOM/DSS+Vehicle组中有更多数量的阳性细胞,表明植物乳杆菌AY01上清液能够明显降低小鼠对AOM/DSS诱导的结肠炎相关性结肠直肠癌的敏感性,抑制了肿瘤的发生和增殖。
Claims (3)
1.一株植物乳杆菌(Lactobacillus plantarum)AY01,其特征在于,所述植物乳杆菌AY01保藏于中国典型培养物保藏中心,保藏编号为CCTCC M 20221517。
2.如权利要求1所述的一株植物乳杆菌,其特征在于,所述植物乳杆菌具有阻断HT-29细胞的细胞周期、诱导HT-29细胞凋亡和抑制HT-29细胞增殖的特性。
3.如权利要求1或2所述的一株植物乳杆菌的菌悬液在制备抑制肿瘤或抑制肿瘤细胞增殖产品中的应用,所述肿瘤为结直肠癌;所述肿瘤细胞为结直肠癌细胞HT-29。
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