CN117801994A - 一株格氏乳杆菌hp2c12及其应用 - Google Patents
一株格氏乳杆菌hp2c12及其应用 Download PDFInfo
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- CN117801994A CN117801994A CN202311845780.5A CN202311845780A CN117801994A CN 117801994 A CN117801994 A CN 117801994A CN 202311845780 A CN202311845780 A CN 202311845780A CN 117801994 A CN117801994 A CN 117801994A
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Abstract
本发明公开了一株格氏乳杆菌为HP2C12及其应用,属于微生物技术领域。该菌株保藏编号为CGMCC No.29261。本发明提供的格氏乳杆菌HP2C12不溶血,对多种常见的抗生素敏感,安全性好,对酸、胆盐耐受力强,对阴道环境适应性强,抗氧化能力强,产乳酸能力强,抑菌抗炎作用效果好,对人子宫颈癌细胞Hela的生长抑制作用和促细胞凋亡作用明显,由此制成的生物制剂在调剂阴道菌群结构和抑制有害菌生长中具有显著的应用价值。
Description
技术领域
本发明属于微生物技术领域,具体涉及一株格氏乳杆菌及其应用。
背景技术
阴道炎是女性常见疾病之一,可由细菌、真菌、病毒等引起,主要发病原因是阴道菌群失调所致。健康女性在内分泌激素的作用下,阴道上皮细胞分泌丰富的糖原,非常有利于兼氧乳酸杆菌的生长,其分泌物抑制了其他致病菌的生长,在阴道内形成了一个正常的生态平衡。当阴道内菌群失调时,过氧化氢等分泌物减少,阴道加德纳菌、大肠埃希氏菌、金黄色葡萄球菌、铜绿假单胞菌等有害菌大量繁殖,导致阴道炎发生。
乳酸菌是一类能利用可发酵碳水化合物产生大量乳酸的细菌的统称,广泛存在于人体肠道、阴道中,对菌群调节、维持菌群平衡具有重要作用。目前已有较多研究证明,乳酸菌是健康女性阴道菌群中的优势菌,它可通过定植于女性阴道,代谢产生乳酸、过氧化氢等产物为阴道提供酸性环境。格氏乳杆菌(Lactobacillus gasseri)属于乳酸菌的一种,可以通过代谢产生抗菌肽和胞外多糖、竞争性占位等方式抑制阴道加德纳菌等阴道致病菌的定植,从而保护女性阴道的健康,维持阴道菌群的稳定。
发明内容
本发明所要解决的技术问题为:提供一株具有多种功能的格氏乳杆菌及其应用。
本发明的技术方案为:一株格氏乳杆菌(Lactobacillus gasseri)HP2C12,于2023年12月07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.29261。
一种含有上所述的格氏乳杆菌(Lactobacillus gasseri)HP2C12或其代谢产物的生物制剂。
上述所述的格氏乳杆菌(Lactobacillus gasseri)HP2C12或生物制剂在制备预防或治疗阴道炎的药物中的应用。
上述所述的格氏乳杆菌(Lactobacillus gasseri)HP2C12或生物制剂在制备抗炎的药物或保健食品中的应用。
上述所述的格氏乳杆菌(Lactobacillus gasseri)HP2C12或生物制剂在制备抗氧化的化妆品或保健食品中的应用。
上述所述的格氏乳杆菌(Lactobacillus gasseri)HP2C12或生物制剂在制备治疗或辅助治疗宫颈癌的药物中的应用。
上述所述的格氏乳杆菌(Lactobacillus gasseri)HP2C12或生物制剂在非疾病治疗目的的抑制阴道加德纳菌、大肠埃希氏菌、鼠伤寒沙门氏菌、金黄色葡萄球菌、铜绿假单胞菌、痢疾杆菌或副伤寒杆菌上的应用。
与现有技术相比,本发明具有以下有益效果:
本发明的格氏乳杆菌HP2C12,不溶血,对多种常见的抗生素敏感,安全性好,对酸、胆盐耐受力强,对阴道环境适应性强,抗氧化能力强,产乳酸能力强,抑菌抗炎作用效果好,对人子宫颈癌细胞Hela的生长抑制作用和促细胞凋亡作用明显,由此制成的生物制剂在调剂阴道菌群结构和抑制有害菌生长中具有显著的应用价值。
保藏信息:
格氏乳杆菌(Lactobacillus gasseri)HP2C12,于2023年12月07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.29261。
附图说明
图1为本发明中的菌株HP2C12的系统发育关系图。
图2为本发明中的格氏乳杆菌HP2C12在MRS琼脂培养基上的菌落形态图。
图3为本发明中格氏乳杆菌HP2C12菌株产乳酸能力测定标准曲线图。
图4为本发明中格氏乳杆菌HP2C12菌株在血平板上的培养结果。
图5为本发明中格氏乳杆菌HP2C12发酵液的抗氧化能力试验中DPPH自由基清除率标准曲线结果。
图6为本发明中格氏乳杆菌HP2C12菌株动物试验正常小鼠(A)、DSS诱导肠炎小鼠(B)和格氏乳杆菌HP2C12试验小鼠(C)结直肠组织切片HE染色。
图7为本发明中格氏乳杆菌HP2C12发酵液对人子宫颈癌细胞Hela的生长抑制和促凋亡作用。其中A:对照组,B:格氏乳杆菌处理组。
具体实施方式
下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的试验材料,如无特殊说明,均为从商业渠道购买得到的。
下述实施例中用到的标准菌株LGG是鼠李糖乳杆菌Lactobacillus rhamnosusATCC 53103。
实验例1格氏乳杆菌的分离和鉴定
1.1材料准备
育龄健康女性的阴道分泌物样本取自四川大学华西第二医院;
16s rRNA通用测序引物27F和1492R由生工生物工程(上海)有限公司合成,序列如下(5’→3’):
27F(SEQ ID No.1):AGAGTTTGATCMTGGCTCAG
1492R(SEQ ID No.2):GGTTACCTTGTTACGACTT
MRS肉汤培养基的配方(每升):酪蛋白酶消化物10.0g,牛肉膏粉10.0g,酵母膏粉4.0g,柠檬酸三铵2.0g,乙酸钠5.0g,硫酸镁0.2g,硫酸锰0.05g,磷酸氢二钾2.0g,葡萄糖20.0g,吐温-80,最终pH为5.7左右。按上述配比称取各组分,加入蒸馏水或去离子水定容1L,分装于锥形瓶,于121℃高压条件灭菌15min即得。
MRS琼脂培养基的配方(每升):蛋白胨10.0g,牛肉膏粉5.0g,酵母膏粉4.0g,葡萄糖20.0g,吐温80 1.0mL,磷酸氢二钾2.0g,乙酸钠5.0g,柠檬酸三铵2.0g,硫酸镁0.2g,硫酸锰0.05g,琼脂15.0g,最终pH为6.2左右。按上述配比称取各组分,加入蒸馏水或去离子水定容1L,分装于锥形瓶,于121℃高压条件灭菌15min即得。
1.2菌株的筛选分离
使用无菌拭子刮取健康女性的阴道分泌物样本,接种于50mL MRS肉汤中,充分振荡混匀,置于37℃恒温摇床培养24h。吸取培养液1mL,采用10倍梯度稀释,分别取20μL,涂布接种于含碳酸钙的MRS琼脂培养基上(碳酸钙2g/100mL),37℃培养24h后,挑取具有较大溶钙圈的单菌落,同样方法连续分离纯化3次。将纯化后的菌株接种于600μL MRS肉汤培养基中,37℃摇床培养18h,加入400μL浓度50%(V/V)的灭菌甘油,于-80℃超低温冰箱中冻存备用。
取冻存菌种20μL,接种于MRS液体培养基进行增殖培养,采用天根细菌基因组DNA提取试剂盒提取菌株DNA,通过采用细菌16S rRNA通用引物27F/1492R以其基因组总DNA为模板进行PCR扩增,得到由1241个碱基对(bp)组成的目的基因序列,序列如SEQ ID No.3所示。
将测序得到的基因序列输入NCBI数据库进行比对,其与GenBank中的标准菌株Lactobacillus gasseri strain LLG-V41相似率达96.39%,可初步鉴定该菌株为格氏乳杆菌(Lactobacillus gasseri)。菌株HP2C12与其它菌株的系统发育关系如图1所示。将该菌株命名为格式乳杆菌(Lactobacillus gasseri)HP2C12,于2023年12月07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.29261。
将格氏乳杆菌HP2C12接种于MRS琼脂培养基上,37℃培养24h后,观察并记录单菌落的形态,格氏乳杆菌HP2C12在MRS琼脂培养基上的菌落形态如图2所示,可见,该菌株生长良好,菌落呈乳白色、圆形凸起、表面光滑。采用革兰氏染色试剂盒对格氏乳杆菌HP2C12进行革兰氏染色,在显微镜下观察并记录染色后的细菌形态,其中,格氏乳杆菌HP2C12在镜检可见菌体呈紫色,符合革兰氏阳性菌的染色特征。
实验例2格氏乳杆菌HP2C12的产乳酸能力测定
乳酸具有重要的生理功能,能调节阴道菌群,从而改善阴道功能、降低阴道环境的pH值、抑制阴道内病原菌生长、提高机体免疫力等。乳酸在乳酸脱氢酶的作用下生成丙酮酸,将NAD+还原成NADH和H+,生成紫色物质,经分光光度计测量570nm处的特征吸收峰,以此定量测定乳杆菌代谢产生的乳酸含量。
2.1材料准备
仪器设备:天平、研钵/匀浆器、离心机、可见分光光度计、微量玻璃比色皿、恒温水浴锅、乙醇和蒸馏水。
试剂及溶液配制:采用乳酸含量测定试剂盒(北京索莱宝科技有限公司),各试剂如表1所示。试剂二:液体置于试剂瓶内EP管中。临用前按试剂二(V)∶蒸馏水(V)=10μL∶450μL的比例配制试剂二溶液,现用现配;试剂四:临用前每瓶加入3mL蒸馏水混匀,可分装后-20℃保存,避免反复冻融;标准品:临用前加入1.04mL蒸馏水配成100μmol/mL的标准溶液。
表1试剂名称、规格和保存条件
2.2标准曲线的绘制
仪器准备:分光光度计预热30min以上,波长调至570nm,分光光度计用乙醇调零。
标准液的稀释:将100μmol/mL的标准溶液用蒸馏水稀释为2.5、1.25、0.625、0.3125、0.15625、0.078μmol/mL的标准溶液待测。各试剂使用如表2所示。
表2测定管及试剂用量
标准曲线的绘制:以各标准溶液浓度为X轴,以其对应的吸光值(ΔA标准)为y轴,绘制标准曲线,得到标准方程:y=kx+b,将ΔA测定带入公式中计算求得的x值(μmol/mL)为乳酸产量,标准曲线如图3所示。
2.3发酵液乳酸含量测定
将实验例1中鉴定保存的格氏乳杆菌HP2C12菌种活化培养后,接种于MRS肉汤培养基,37℃培养24h,4℃、12000g离心10min后取上清液保存备用。取100μL发酵上清液,加入1mL提取液一,4℃12000g离心10min,取0.8mL上清液,再加入0.15mL提取液二,12000g离心10min后取上清液,在酶标仪570nm处测定OD值。
其中,乳酸含量的计算公式如下:
乳酸含量(μmol/mL)=X×(V上清+V提取液二)÷[V液体×V上清÷(V提取液一+V液体)]。
式中的V上清:加入的上清液体积,0.8mL;V提取液二:加入提取液二的体积,0.15mL;V提取液一:加入的提取液一,1mL;V液体:液体样本体积,0.1mL。
格氏乳杆菌HP2C12发酵液中的乳酸含量测定结果如表3所示,发酵液中乳酸浓度为104.72±1.83μmol/mL,显著高于其它菌株,表明格氏乳杆菌HP2C12产乳酸能力强,具有很好的开发利用价值。
表3格氏乳杆菌HP2C12发酵液乳酸含量(μmol/mL)
实验例3格氏乳杆菌HP2C12的溶血性和抗生素敏感性测定
采用纸片琼脂扩散法检测待测菌株对常见抗生素的敏感程度。将格氏乳杆菌HP2C12菌株活化培养,菌液浓度调整至1×106CFU/mL,用无菌棉签将菌液均匀地涂抹于MRS培养基平板表面,室温放置10min后放入药敏纸片,37℃培养24h后,用游标卡尺测量各药敏纸片周围的抑菌圈直径,每种抗生素重复3次,试验结果参照美国临床实验室标准委员会(NCCLS)标准判断菌株的药物敏感性,结果以敏感(S)、中介(I)和耐药(R)表示。格氏乳杆菌HP2C12对6种抗生素的敏感性测定结果见下表4,可知,格氏乳杆菌HP2C12对测试的四环素、氨苄西林、克林霉素、克拉霉素、头孢曲松和氯霉素5种抗生素表现为敏感(S),对头孢曲松抗生素表现为中介。
表4格氏乳杆菌HP2C12对6种抗生素的敏感性
有的细菌在生长过程中,可能产生溶血素,使红细胞破裂溶解,影响人体健康。在血平板上生长时,菌落周围可观察到透明或半透明的溶血环,许多细菌的致病性都与溶血特性相关。溶血情况主要分为α溶血(菌落周围出现草绿色的溶血环)、β溶血(菌落周围出现清晰透明的溶血环)、γ溶血(菌落周围无溶血环)。因此,溶血试验常用来进行细菌的安全鉴定。将HP2C12待测菌株使用接种环在MRS琼脂平板划线后,挑选单菌落,在血平板培养基上划线,培养24h后,观察溶血情况,三次重复。格氏乳杆菌HP2C12菌株在血平板上的培养结果如图4所示,菌落周围没有出现溶血现象,为γ溶血,表明其具备安全性。
以上结果可证明格氏乳杆菌HP2C12不具有溶血性,对常见抗生素敏感,具备良好的安全性。
实验例4格氏乳杆菌HP2C12的耐酸耐盐能力评估
4.1耐酸性和耐胆盐性评估:
将待测菌株格氏乳杆菌HP2C12在MRS琼脂平板上进行复苏并纯化3代,并调节菌液初始浓度达到1×106CFU/mL。使用盐酸和猪胆盐分别配制酸度(pH=3.0)及胆盐浓度(0.3%)的MRS肉汤培养基。将1mL待测菌株植物乳杆菌HP2C12菌液接种于pH 3.0的肉汤培养基中,37℃培养18h。培养完毕,取20μL菌液涂布于MRS琼脂平板培养基,设置3个重复,于37℃培养18h。观察培养后MRS平板表面是否有菌落生长。同样,将1mL待测菌株格氏乳杆菌HP2C12菌液接种于胆盐浓度为0.3%的MRS肉汤培养基,37℃培养18h后,涂平板,37℃培养24h,查看菌落生长情况。
根据耐酸性和耐胆盐性试验结果可知,格氏乳杆菌HP2C12在pH 3.0或胆盐浓度为0.3%的MRS肉汤培养基中处理18h后仍可在MRS琼脂平板上长出正常菌落,说明格氏乳杆菌HP2C12具有较强的耐酸和耐胆盐特性。
4.2模拟胃液、肠液耐受性试验:
模拟胃液和肠液购自上海源叶生物科技有限公司;人工胃液模拟液成分为稀盐酸、胃蛋白酶和氯化钠,最终为2.5;人工肠液模拟液成分为磷酸二氢钾和胰蛋白酶,最终pH为6.8。将格氏乳杆菌HP2C12菌株复苏并活化,取1mL浓度为1×108CFU/mL菌液加人9mL模拟人工胃液中,并进行10倍梯度稀释,吸取20μL涂平板观察是否存在活菌,作为耐受人工胃液的初始活菌状态;将加入活菌的模拟胃液于37℃培养3h,再次涂平板观察是否存在活菌,作为耐受人工胃液的最终活菌状态。同理,将1mL浓度为1×108CFU/mL格氏乳杆菌HP2C12发酵液加入9mL模拟肠液中,并进行活菌观察,37℃培养6h后再次观察是否存在活菌。最终状态存在活菌即为通过模拟胃液、肠液耐受实验。
根据模拟胃液、肠液耐受性试验结果可知,进一步证明格氏乳杆菌HP2C12菌株耐酸耐盐能力强,对阴道环境适应能力强,有利于进一步开发利用。
实验例5格氏乳杆菌HP2C12菌株对常见肠道致病菌的抑制能力
阴道中的加德纳杆菌、大肠埃希氏菌和沙门氏菌等致病菌的大量增殖,引起阴道菌群失调,是阴道炎的重要原因之一。将大肠埃希氏菌(Escherichia coli CMCCB 44102)、金黄色葡萄球菌(Staphylococcus aureus CMCCB 50094)、鼠伤寒沙门氏菌(Salmonellatyphimurium ATCC 14028)、铜绿假单胞菌(Pseudomonas aeruginosa CMCCB 10104)、痢疾杆菌(Shigella Castellani CMCCB 51105)、副伤寒杆菌(paratyphoidfever CMCCB50094)和阴道加德纳菌(Gardnerella vaginalis BNCC 354890)分别接种于其固体培养基,复苏并纯化3次,将活化好的致病菌接种于液体培养基中,并调节菌液浓度达到1×108CFU/mL。吸取上述致病菌和液体培养基的混合液60μL加至600mL灭菌后暂未凝固的固体培养基内(温度冷却至40℃左右),充分混匀后每皿约20mL分装至培养皿中。待培养基冷却凝固后,使用直径6mm的打孔器在平板上打孔,制成致病菌琼脂板,每板对应一株菌,进行三次重复。将复苏后的待测菌株的菌液浓度调至1×108CFU/mL。吸取50μL的待测菌菌液加至上述致病菌琼脂板孔内,于37℃培养24h。培养后,使用游标卡尺测量打孔点周围的抑菌圈直径大小(mm)并记录。将标准菌株LGG作为对照菌株,与待测菌株同时进行以上实验操作。
格氏乳杆菌HP2C12的抑菌活性评估测定结果见下表5所示,可知,格氏乳杆菌HP2C12菌株的发酵液对大肠埃希氏菌、金黄色葡萄球菌、鼠伤寒沙门氏菌、铜绿假单胞菌、痢疾杆菌、副伤寒杆菌或和阴道加德纳菌等致病菌的生长具有较强的抑制活性,总体的抑菌效果优于LGG标准菌株,说明由格氏乳杆菌HP2C12制备的生防菌剂可用于抑制上述7种致病菌的生长繁殖。
表5格氏乳杆菌HP2C12菌株的抑菌活性评估(直径:mm)
实例6格氏乳杆菌HP2C12发酵液的抗氧化能力
抗氧化剂被定义为任何以较低浓度就可有效抑制自由基氧化反应的物质,它可抵消自由基对人体细胞的氧化攻击。其作用机理为直接作用在自由基或间接消耗易产生自由基的物质,防止其发生进一步反应。抗氧化的能力越强,机体越健康,越不易产生炎症反应。越来越多的研究显示抗氧化是预防衰老的重要步骤,因为自由基或氧化剂会将细胞和组织分解,影响代谢功能,并会引起不同的健康问题。如果能够消除过多的氧化自由基,对于许多自由基引起的及老化相关疾病都能够预防。
6.1发酵液的制备:
将格氏乳杆菌HP2C12冻存菌种复苏培养24h后,按2%接种量接种于MRS液体培养基中,在37℃条件下培养24h,菌液在4℃、4000r/min条件下离心15min,上清液经0.22μm滤膜过滤得到发酵上清液冻存备用。
6.2试剂与仪器:
总抗氧化能力(T-AOC)检测试剂盒(A015-1-2)、DPPH自由基清除能力试剂盒(A153-1-1)和抑制与产生超氧阴离子自由基测定试剂盒(比色法A052-1-1)均由南京建成生物工程研究所生产。紫外可见分光光度计(UV752N型),上海佑科仪器仪表有限公司生产;发酵液抗氧化能力测定由成都里来生物科技有限公司完成。对照菌株为LGG标准菌株。
6.3总抗氧化能力测定:
总抗氧化能力(Total antioxidant capacity,T-AOC)是指各种抗氧化物质构成的总抗氧化水平。研究抗氧化可以有效克服其所带来的危害,所以抗氧化被保健品、化妆品企业列为主要的研发方向之一,也是市场最重要的功能性诉求之一。许多抗氧化物质能使Fe3+还原成Fe2+,后者可与菲啉类物质形成稳固的络合物,通过比色可测出其抗氧化能力的高低。使用波长520nm,1cm光径,双蒸水调零,测定吸光度值。步骤按照总抗氧化能力(T-AOC)检测试剂盒说明书进行。格氏乳杆菌HP2C12发酵液的抗氧化能力测定结果见下表6,可知,格氏乳杆菌HP2C12发酵液的总抗氧化能力为12.29±0.63U/mL,接近对照菌株LGG,说明HP2C12发酵液的总抗氧化能力较强。
6.4DPPH自由基清除能力:
DPPH又称1,1-二苯基-2-三硝基苯肼,是一种很稳定的氮中心的自由基。由于DPPH自由基有单电子,在517nm处有一强吸收,其醇溶液呈紫色,当有自由基清除剂存在时,由于与其单电子配对而使其吸收逐渐消失,呈现的颜色越浅,因此,可以对样本中DPPH清除能力进行定量分析。按照试剂盒说明书将标准品粉剂一支加无水甲醇2mL溶解,即为0.5mg/mL(Trolox)标准应用液,再用无水甲醇分别稀释成5μg/mL、10μg/mL、15μg/mL、20μg/mL、25μg/mL,使用波长517nm,1cm光径,无水乙醇调零,测定各管吸光度,制作标准曲线,得到DPPH自由基清除率标准曲线如图5所示。
DPPH自由基清除率(%)=(1-(A测定-A对照)÷A空白)×100%
用从标准曲线算得的相当于抗氧化剂Trolox的量来表示样本的DPPH自由基清除能力。发酵液样品DPPH自由基清除能力(μg Trolox/mL)=代入标准曲线得相当于Trolox的浓度×稀释倍数。格氏乳杆菌HP2C12发酵液的DPPH自由基清除能力测定结果见下表6,可知,格氏乳杆菌HP2C12发酵液的DPPH自由基清除能力42.81±0.88μg Trolox/mL,高于对照菌株LGG,表明HP2C12发酵液对DPPH自由基清除能力较强。
6.5抗超氧阴离子能力:
超氧阴离子自由基作为生物体代谢过程中产生的一种自由基,可攻击生物大分子,如脂质、蛋白质、核酸和聚不饱和脂肪酸等,使其交链或者断裂,引起细胞结构和功能的破坏,与机体衰老和病变有很密切的关系。本实验模拟机体中黄嘌呤与黄嘌呤氧化酶反应系统,产生超氧阴离子自由基,加入电子传递物质及Gress氏显色剂,使反应体系呈现紫红色,用分光光度计测其吸光度,当被测样本中含有超氧阴离子自由基抑制剂时,则比色时测定管的吸光度低于对照管的吸光度,通过以维生素C做标准,可计算出被检物品对超氧阴离子自由基的抑制能力。测定时用1cm光径比色杯,双蒸水调零,波长550nm处比色。
抗超氧阴离子能力(U/L)=(A对照-A测定)/(A对照-A标准)×C标准×1000×N
在反应系统中,每升发酵液在37℃反应40分钟所抑制的超氧阴离子自由基相当于1mg的维生素C所抑制的超氧阴离子自由基的变化值为一个活力单位。格氏乳杆菌HP2C12发酵液的抗超氧阴离子能力测定结果见下表6,可知,格氏乳杆菌HP2C12发酵液对超氧阴离子自由基的抑制能力为277.74±5.36U/L,接近对照菌株LGG,表明该菌对超氧阴离子自由基的抑制能力较强。
6.6抑制羟自由基能力:
羟基自由基是一种重要的活性氧,从分子式上看是由氢氧根(OH-)失去一个电子形成。它十分活泼,可以氧化氨基酸、糖类、核酸、蛋白质和脂类等物质,不及时清除会对机体的细胞造成破坏,加快机体衰老,甚至会导致肿瘤的产生。因此,抑制羟自由基对维持机体正常生理活动、减缓衰老具有重要的作用。
本实验通过Fenton反应对HP2C12发酵液抑制羟自由基的能力进行探究。过氧化氢的量和Fenton反应产生的OH-量成正比,当给予电子受体后,用griess试剂显色,形成红色物质,其呈色与OH-的多少成正比关系。在550nm下使用分光光度计测定吸光度值。实验步骤参照试剂盒完成。
抑制羟自由基能力(U/mL)=(A对照-A测定)/(A标准-A空白)×C标准×1/V样×样本测试前稀释倍数(N倍)
格氏乳杆菌HP2C12发酵液的抑制羟自由基能力测定结果见下表6,可知,格氏乳杆菌HP2C12发酵液对羟自由基的抑制能力为4198.47±10.42U/mL,抗氧化效果接近对照菌株LGG,表明该菌对羟自由基的抑制能力较强。
表6格氏乳杆菌HP2C12发酵液的抗氧化能力
综上可知,HP2C12发酵液的抗氧化能力、抑制羟自由基能力、自由基清除能力和抗超氧阴离子较强,在抗炎类的生防制剂领域具有潜在的应用价值。
实验例7格氏乳杆菌HP2C12对小鼠炎症模型的预防作用
7.1实验方法
DSS水溶液:用葡聚糖硫酸钠盐(DSS)配制浓度为4%(w/v)的水溶液。
菌悬液的制备:将益生菌在MRS肉汤培养基培养24h后,在-4℃、6000r/min条件下离心5min,弃上清液。使用无菌PBS缓冲液重悬菌体,调节菌液浓度为5×109CFU/mL,冰箱保存备用。
实验动物:由成都达硕实验动物有限公司提供4周龄SPF级雄性昆明小鼠27只,随机分成9笼,每笼3只,适应性饲养后,随机分为3组,每组3笼,包括空白对照组(CK)、DSS模型组、益生菌(格氏乳杆菌HP2C12)处理组,实验处理如表7所示。
表7DSS诱导小鼠结肠炎实验
血清炎症因子检测:在实验结束时,每组分别随机处死3只小鼠,进行眼眶取血,血液经离心机3000r/min离心15min,获得血清,使用ELISA试剂盒测定血清的炎症因子(IL-1β、IL-6、IL-8和TNF-α),步骤按照试剂盒说明书进行。
脏器指数、结直肠长度和组织切片:在建模期结束后,每组处死并解剖3只小鼠,迅速取其脾脏及肝脏,用生理盐水冲洗,滤纸吸干后称量,并用直尺测量结直肠长度,浸泡于4%多聚甲醛固定液24h,切片后对组织进行HE染色观察组织情况。
脏器指数=脾脏或肝脏质量(g)/小鼠体重(g)×100%
7.2结果分析
7.2.1血液炎症因子检测:实验结束时,测定TNF-α、IL-1β、IL-6和IL-8四个炎症因子指标,结果显示,4个炎症因子都显著低于DSS模型组,且接近空白对照组。说明格氏乳杆菌HP2C12菌株对DSS诱导的小鼠炎症有明显的缓解作用(表8)。
表8各组小鼠血清中各炎症因子浓度变化(单位:pg/mL)
与CK组比较,*p<0.05,**p<0.01;与DSS组比较,#p<0.05,##p<0.01
7.2.2脏器指数、结直肠长度和组织切片:解剖发现DSS处理会引起脾脏指数增加,结直肠缩短(表9),饲喂格氏乳杆菌HP2C12组结直肠长度接近于空白对照组。在显微镜下可观察到结直肠炎症反应(图6),DSS组小鼠的结肠组织黏膜固有层全层坏死,炎性细胞浸润程度明显,且存在严重的纤维组织增生情况。而预防性饲喂格氏乳杆菌HP2C12的小鼠的结肠组织炎性浸润程度低,黏膜固有层坏死程度大大减轻,较DSS组而言,症状明显减轻。进一步表明格氏乳杆菌HP2C12可减缓小鼠炎症症状。
表9小鼠脏器指数及结直肠长度变化
实验例8格氏乳杆菌HP2C12对人子宫颈癌细胞生长抑制和促凋亡作用
8.1格氏乳杆菌HP2C12对人子宫颈癌细胞Hela的粘附性试验
8.1.1实验方法
将格氏乳杆菌HP2C12冻存菌种复苏培养后接种于MRS肉汤培养基中,37℃恒温培养24h。培养完毕后置于-4℃、5000r/min条件下离心10min,并用无菌PBS缓冲液多次洗涤。调整菌悬液浓度至1×106CFU/mL后备用。复苏人子宫颈癌细胞Hela,将其接种至六孔细胞培养皿,添加DMEM完全培养基并置于37℃、5% CO2中培养,两天更换一次培养液。当细胞贴壁状态达80%时,使用0.25%胰酶-EDTA进行消化,并传代培养。培养完毕后用细胞血球计数板对其进行计数,并将细胞浓度调整至5×106个/mL。将1mL细胞悬浮液加至培养孔中,置于培养箱中培养。待培养板中的细胞长至单层,弃掉DMEM培养液,用无菌PBS将每孔冲洗3次。将1mL已制备好的菌悬液加入细胞孔,轻微晃动细胞培养板,吸取孔中少量菌液用于平板计数,其结果作为菌悬液中的初始活菌数。将细胞板置于37℃孵育2h,弃去培养基并用无菌PBS缓冲液洗涤3次。使用0.7mL 0.25%胰蛋白酶-EDTA消化细胞10min,待细胞完全脱落后加入0.3mL DMEM培养液终止消化,收集黏附实验结束后的培养液进行平板计数,该结果作为黏附活菌数。粘附率(%)=结束期乳酸菌数目/初始乳酸菌接种数目×100%
8.1.2结果分析
结果如表10,格氏乳杆菌HP2C12对人子宫颈癌细胞Hela的粘附率为10.88%。
表10格氏乳杆菌HP2C12对人子宫颈癌细胞Hela的粘附率试验结果(%)
8.2格氏乳杆菌HP2C12对人子宫颈癌细胞Hela生长抑制试验
8.2.1实验方法
细胞传代:提前将PBS、RPMI培养基和胰蛋白酶进行37℃水浴预热,将CO2培养箱中培养的Hela培养皿取出,在超净工作台进行后续操作。首先将培养皿中的培养基吸走,往培养皿中加入1ml PBS洗一次,吸走后加入1ml胰酶,轻轻混匀,将培养皿放入培养箱中消化2min,直至在光学显微镜下观察细胞变圆。消化完成后,将胰酶吸出。再加入1ml培养基,用移液枪吹打使细胞悬浮,并转移至1.5ml EP管,离心3min,1200rpm/min。离心完成后吸走上清液,往细胞沉淀中加入300μl培养基,吹打混匀,吸取150μl到装有4ml培养基的培养皿中,轻轻混匀,放入CO2培养箱中继续培养,48h后可进行下一步实验。
细胞铺板:按照细胞传代的方法操作至离心,弃上清液,用1ml PRMI培养基混匀细胞,接着用血细胞计数板进行计数,将细胞悬浮液稀释至3×104CFU/ml。往96孔板每个孔中加入100μl细胞悬浮液,空白组只加培养基,加完后放入CO2培养箱培养,24h后待细胞贴壁生长良好时,可进行下一步实验。
加样处理:先用无血清的培养基按照1:100体积比对格氏乳杆菌HP2C12发酵上清液进行稀释。将96孔板里的培养液洗干净,再向孔中加入对应稀释后的格氏乳杆菌HP2C12发酵上清液,三个重复,培养72h。乳杆菌MRS液体培养基作为阴性对照。
CCK-8检测细胞增殖情况:提前按照CCK-8:RPMI基本培养基按照体积比1:9的比例混合均匀,将给药处理72h的96孔板里的100μl混合液吸出,向每个孔中加入100μl混合后的CCK-8溶液,继续放入培养箱中培养1.5h,用酶标仪测OD450值。
计算细胞抑制率:细胞抑制率=[(Ac-As)/(Ac-Ab)]×100%。As:实验孔,细胞、CCK-8溶液和格氏乳杆菌HP2C12发酵上清液混合液吸光度;Ac:对照组,细胞、CCK-8溶液和MRS液体培养基的吸光度;Ab:空白孔,CCK-8溶液的吸光度。
8.2.2结果分析
表11格氏乳杆菌HP2C12发酵上清液对人宫颈癌细胞的生长抑制作用(%)
测定结果如表11所示,1%格氏乳杆菌HP2C12发酵上清液,培养72小时,对人子宫颈癌细胞的生长抑制作用为13.91%,抑制生长作用效果明显。
8.3HP2C12发酵上清液对人子宫颈癌细胞的促凋亡作用
8.3.1材料与方法
细胞培养:将对数生长期的Hela细胞消化离心后均匀铺入96孔板中,计数保证每孔2000个细胞。培养12小时待细胞贴壁稳定后加入HP2C12发酵上清液2%,每个处理设置5个重复,培养48小时后进行蛋白质提取。
蛋白提取:将培养好的细胞用PBS洗两遍后,尽可能地吸掉细胞表面所有残余的PBS。将细胞置于冰浴中,加入含有蛋白酶抑制剂Cocktail的RIPA裂解液(蛋白酶抑制剂按照1:100的比例稀释),将细胞全部刮到1.5ml无菌离心管中,置于冰浴中裂解30min。12000r/min 4℃离心10min,收集上清液到新的1.5ml无菌离心管中,即为所提取的蛋白样品。该蛋白样品可以直接进行蛋白定量分析或者保存在-80℃冰箱中备用。对于定量分析之后的蛋白样品,可加入蛋白缓冲液,置于金属浴中98℃煮10min,使蛋白变性,瞬时离心后保存于-20℃冰箱备用。
蛋白定量:用碧云天蛋白定量试剂盒进行定量分析。将蛋白样品稀释20倍加入到标记好的96孔板中。按照BCA试剂A与B的比例为50:1配制BCA工作液,充分混匀,每孔加入200μl BCA工作液后,摇晃混匀30s,盖住各样品孔,放置在37℃的恒温培养箱中30min。在562nm处测定吸光值,根据各蛋白样品的校正吸光值,在标准曲线的线性范围内读出各样品的蛋白浓度,根据样品体积和稀释度计算原来样品中的蛋白量。
8.3.2实验结果
(1)HP2C12发酵上清液对细胞粘附因子β-连环蛋白的影响
癌细胞扩散的特征是细胞间无序的相互作用和细胞粘附。β-连环蛋白(β-catenin)是一种多功能蛋白,是重要的细胞粘附分子,参与细胞生长与修复,在肿瘤发生与转移的过程中起着重要作用。几乎所有宫颈癌中Wnt/β-catenin信号通路的突变最终都会导致β-catenin的积累。因此,β-连环蛋白的含量被作为临床上评判宫颈癌发展及预后的辅助因素,β-连环蛋白的核积累可能是宫颈癌侵袭和预后不良等恶性相关的标志物。结果显示,在子宫颈癌(Hela)培养液中加入2%的HP2C12发酵上清液,培养48h后,可明显观察到HP2C12发酵液处理组癌细胞发生凋亡,并伴有自噬小泡生成,如图7。β-连环蛋白(β-catenin)含量大大降低,仅为不加发酵液的对照的34.05%。说明HP2C12发酵上清液可降低细胞粘附因子β-连环蛋白的表达,从而降低癌细胞的粘连与扩散。
表12HP2C12发酵上清液对结肠癌细胞β-连环蛋白含量的影响(灰度值)
(2)HP2C12发酵上清液对细胞凋亡抑制基因蛋白含量的影响
髓样细胞白血病1(Mcl-1)是抗凋亡bcl2家族成员之一,MCL1的过度表达或扩增常见于多种癌症类型,因此被认为是最相关的癌蛋白之一。结果表明,培养基中加入2%HP2C12发酵上清液,使癌细胞MCL-1蛋白总量降低40.9%(表13),说明HP2C12发酵液具有促癌细胞凋亡活性。研究表明,mcl1基因亚型1在肿瘤组织及相应旁组织中的mRNA和蛋白质水平表达均高于正常组,mcl1亚型1和亚型2与肿瘤恶性程度呈正相关,可以作为临床评估子宫颈癌恶性程度有效的诊断指标之一。
B细胞淋巴瘤/白血病-2基因(bcl-2)也具有明显的抑制细胞凋亡的作用,可以抑制由多种细胞毒素所引起的细胞死亡,与多种癌症密切相关,该基因的过度表达能增强细胞对大多数细胞毒素的抵抗性。实验结果表明,细胞培养基中加入2% HP2C12发酵上清液,使癌细胞Bcl-2蛋白总量下降77.9%(表13),说明发酵上清液具有促癌细胞凋亡活性。
表13HP2C12发酵上清液对结肠癌细胞凋亡抑制基因蛋白含量的影响(灰度值)
(3)HP2C12发酵上清液对细胞凋亡因子蛋白含量的影响
P62蛋白为癌基因蛋白,其转录调控的靶基因参与细胞周期的调控、自噬、细胞增殖、细胞凋亡及永生化等过程,在诸多肿瘤的发生中发挥重要作用。P62基因可帮助机体更好地识别和抵御外源抗原,从而减轻感染带来的危害。P62蛋白还可以通过促进选择性自噬,来防止遗传毒性和致癌突变的积累,从而发挥抑癌作用。当P62基因与抗原相结合时,它会引发抗原特异性的T细胞和B细胞功能。T细胞会分泌特异性Cytokine来杀死外来病原体,而B细胞会分泌特异性抗体来抑制病原体的复制。因此,P62基因能够结合抗原,促进免疫应答,帮助人体抵御外来的抗原,从而减少病毒感染或细菌侵染所造成的伤害。结果表明,细胞培养基中加入2% HP2C12发酵上清液,能使癌细胞P62蛋白总量大大增加,约为对照的2.38倍(表14),说明HP2C12发酵上清液具有促进子宫颈癌细胞凋亡的能力。
表14HP2C12发酵上清液对宫颈癌细胞促凋亡因子蛋白的影响(灰度值)
综上所述,格氏乳杆菌HP2C12菌株对宫颈癌细胞粘附性好,其发酵上清液具有明显的抑制宫颈癌细胞生长的作用,能降低与粘连扩散有关的β-连环蛋白含量,能使代表凋亡抑制基因的MCL-1、Bcl-2蛋白含量下降,能使促代表自噬的P62蛋白含量增加,从多个方面说明格氏乳杆菌HP2C12具有促进宫颈癌细胞凋亡的能力,有广阔的应用前景。
Claims (7)
1.一株格氏乳杆菌(Lactobacillus gasseri)HP2C12,保藏编号为CGMCC No.29261。
2.含有权利要求1所述的格氏乳杆菌(Lactobacillus gasseri)HP2C12或其代谢产物的生物制剂。
3.权利要求1所述的格氏乳杆菌(Lactobacillus gasseri)HP2C12或权利要求2所述的生物制剂在制备预防或治疗阴道炎的药物中的应用。
4.权利要求1所述的格氏乳杆菌(Lactobacillus gasseri)HP2C12或权利要求2所述的生物制剂在制备抗炎的药物或保健食品中的应用。
5.权利要求1所述的格氏乳杆菌(Lactobacillus gasseri)HP2C12或权利要求2所述的生物制剂在制备抗氧化的化妆品或保健食品中的应用。
6.权利要求1所述的格氏乳杆菌(Lactobacillus gasseri)HP2C12或权利要求2所述的生物制剂在制备治疗或辅助治疗宫颈癌的药物中的应用。
7.权利要求1所述的格氏乳杆菌(Lactobacillus gasseri)HP2C12或权利要求2所述的生物制剂在非疾病治疗目的的抑制阴道加德纳菌、大肠埃希氏菌、鼠伤寒沙门氏菌、金黄色葡萄球菌、铜绿假单胞菌、痢疾杆菌或副伤寒杆菌上的应用。
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