CN113151371B - 一种益生菌胞外多糖及其制备方法和抗肿瘤应用 - Google Patents
一种益生菌胞外多糖及其制备方法和抗肿瘤应用 Download PDFInfo
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Abstract
本发明公开了一种益生菌胞外多糖及其制备方法和抗肿瘤应用,该益生菌胞外多糖由植物乳杆菌WLPL09提取得到,植物乳杆菌WLPL09由人类母乳中分离得到,植物乳杆菌WLPL09已保藏于中国典型培养物保藏中心,保藏地址为中国武汉大学,保藏编号为CCTCC M 2021038,保藏日期为2021年1月8日。该益生菌胞外多糖的具体制备方法为:将植物乳杆菌WLPL09发酵过夜后离心,采用乙醇沉淀提取上清液,经纯化和冷冻干燥后得到。本发明公开了一种从人类母乳中分离的植物乳杆菌WLPL09提取出的益生菌胞外多糖,其属于一种新型安全的抗肿瘤活性物质,效果优于现有技术,制备方法简单易行。
Description
技术领域
本发明属于生物学领域,具体涉及一种益生菌胞外多糖及其制备方法和抗肿瘤应用。
背景技术
恶性肿瘤一直是现代医学领域的一大难题,就现在肿瘤治疗的方法——化疗和放疗而言,其两者都能有效的清除肿瘤细胞,但是对正常的细胞也有极大的损伤进而对机体产生副作用,如粒细胞减少性结肠炎、疲劳、消化障碍、贫血等。因此,开发新型有效而低副作用或无副作用的肿瘤制剂是推动肿瘤治疗发展的有效途径。
发明内容
本发明的目的是解决现有技术的不足,提供一种益生菌胞外多糖,其能够用于抗肿瘤药物的制备,进一步能用于抑制黑色素瘤生长药物的制备。具体为:
一种益生菌胞外多糖,该益生菌胞外多糖由植物乳杆菌WLPL09提取得到,该植物乳杆菌WLPL09由人类母乳中分离得到,该植物乳杆菌WLPL09已保藏于中国典型培养物保藏中心,保藏地址为中国武汉大学,保藏编号为CCTCC M 2021038,保藏日期为2021年1月8日。
上述的益生菌胞外多糖实质为一株名为植物乳杆菌WLPL09的菌株所产生的次级代谢产物,其与现有技术相比,在给小鼠灌胃后,明显提高细胞因子IL-2水平,维持和促进小鼠CD8+T淋巴细胞增殖,提高了细胞因子TNF-α水平,从而杀伤肿瘤细胞,参与免疫调节,刺激小鼠脾淋巴细胞增殖,提高免疫力,达到体内抗肿瘤的作用。
因此,上述益生菌胞外多糖能够应用在制备抗肿瘤药物和保健食品中。进一步也能够应用于制备刺激TNF-α、IL-2、IFN-γ、IL-6产生的激动剂,及提高免疫力的作用药物。进一步能够应用于制备抑制皮下黑色素瘤生长药物,如抗肿瘤佐剂。
上述益生菌胞外多糖通过以下制备方法制得:将植物乳杆菌WLPL09发酵过夜后离心,采用乙醇沉淀提取上清液,经纯化和冷冻干燥后得到。
本发明的有益效果为:本发明公开了一种从人类母乳中分离的植物乳杆菌WLPL09提取出的益生菌胞外多糖,其属于一种新型安全的抗肿瘤活性物质,效果优于现有技术,制备方法简单易行。
附图说明
图1所示为B16F10荷瘤小鼠肿瘤生长曲线图;
图2所示为B16F10荷瘤鼠肿瘤重量、抑瘤率及肿瘤取图;
图3所示为EPS对B16F10荷瘤鼠脾淋巴细胞增殖活性的干预示意图;
图4所示为EPS干预后B16F10荷瘤鼠肿瘤组织中CD4+和CD8+T细胞的含量示意图;
图5所示为EPS对B16F10荷瘤鼠肿瘤组织中TNF-α、IFN-γ、P53、IL-1β、Bax、BCL-2、VEGF和Fgf2 mRNA表达量的影响结果图;
图6所示为EPS对B16F10荷瘤鼠血清中TNF-α、IFN-γ、IL-2和IL-6含量的影响结果图;
图7所示为EPS对B16F10荷瘤鼠盲肠内容物中肠道菌群丰度与多样性分析图;
图8所示为EPS干预后B16F10荷瘤鼠盲肠内容物微生物群落组成图。
具体实施方式
以下将结合实施例和附图对本发明的构思及产生的技术效果进行清楚、完整的描述,以充分地理解本发明的目的、方案和效果。
以下实施过程中,植物乳杆菌WLPL09分离自人类健康妇女母乳,为一种具有益生功能的益生菌,已保存于中国典型培养物保藏中心,保藏地址为中国武汉大学,保藏编号为CCTCC M 2021038,保藏日期为2021年1月8日。
此外,本申请的附图中,图1中***表示t检验后,与模型组相比呈显著性差异(P<0.001)。图3中*和#分别表示t检验后,与空白对照组和5-FU组相比呈显著性差异(P<0.05);**和##分别表示t检验后,与空白对照组和5-FU组相比呈显著性差异(P<0.01);***和###分别表示t检验后,与空白对照组和5-Fu组相比呈显著性差异(P<0.001)。图中没有共同字母的组表示相比显著性差异(P<0.05)。
此外,胞外多糖的提取方法是用植物乳杆菌WLPL09发酵过夜后离心去菌体,发酵上清采用乙醇沉法提取后纯化以及冷冻干燥得到纯品多糖。
实施例1:植物乳杆菌WLPL09胞外多糖抑制小鼠黑色素瘤生长实验
1、组别设置:
空白对照组(Control):健康C57BL/6J小鼠,每天灌胃蒸馏水(10mL/kg体重/天),注射生理盐水(10mL/kg体重/天),自由饮食、饮水,持续14天。
模型组(Model):接种B16F10细胞的C57BL/6J小鼠,每天灌胃蒸馏水(10mL/kg体重/天),注射生理盐水(10mL/kg体重/两天),自由饮食、饮水,持续14天。
低剂量EPS组(50mg/kg EPS):接种B16F10细胞的C57BL/6J小鼠,每天灌胃EPS(50mg/kg体重/天),注射生理盐水(10mL/kg体重/两天),自由饮食、饮水,持续14天。
中剂量EPS组(100mg/kg EPS):接种B16F10细胞的C57BL/6J小鼠,每天灌胃EPS(100mg/kg体重/天),注射生理盐水(10mL/kg体重/两天),自由饮食、饮水,持续14天。
高剂量EPS组(200mg/kg EPS):接种B16F10细胞的C57BL/6J小鼠,每天灌胃EPS(200mg/kg体重/天),注射生理盐水(10mL/kg体重/两天),自由饮食、饮水,持续14天。
5-FU组(5-FU):接种B16F10细胞的C57BL/6J小鼠,每天灌胃蒸馏水(10mL/kg体重/天),注射5-氟尿嘧啶(25mg/kg体重/两天),自由饮食、饮水,持续14天。
EPS+5-FU协同组(EPS+5-FU):接种B16F10细胞的C57BL/6J小鼠,每天灌胃EPS(200mg/kg体重/天),注射5-氟尿嘧啶(25mg/kg体重/两天),自由饮食、饮水,持续14天。
2、实验主要步骤:
复苏并活化B16F10细胞,选取生长稳定的B16F10细胞,经胰酶消化,Hanks重悬调整细胞浓度为1×107个/mL。皮下注射。细胞悬液以100μL/只小鼠进行右下腋窝进行注射,操作尽量在1h内完成。注射B16F10细胞24h后对小鼠进行随机分组,按以上分组进行药物干预,期间检测小鼠肿瘤体积变化情况。干预完成后,摘眼球取血备用。断颈法处死小鼠,取其肿瘤组织(拍照)、胸腺、脾脏、肝脏、肾脏进行称重并收集备用。收集小鼠的盲肠内容物,液氮冷冻备用。
肿瘤体积变化曲线如图1所示。结果显示,模型组小鼠皮下肿瘤生长迅速;相较于模型组,不同剂量EPS处理组小鼠肿瘤生长速度明显减缓,且在第15天与模型组出现极显著性差异;5-FU能有效抑制肿瘤的生长,抑制效果优于EPS;EPS与5-FU协同处理组的小鼠肿瘤与5-FU单独处理并未表现出显著性差异。
3、肿瘤抑制率与脏器指数测定
抑瘤率及脏器指数计算公式如下:Tumor inhabit rate(%)=((M-T)/M)*100%,式中M为模型组小鼠瘤体重(g);T为实验组小鼠瘤体重量(g);Organ Index=Worgan/Wbody,式中Worgan为小鼠组织重量(mg);Wbody为小鼠体重(g)。
荷瘤小鼠肿瘤取图(图2C)、重量(图2A)及抑瘤率(图2B)如图2所示。由结果可知,低剂量EPS(50mg/kg)对荷瘤小鼠的肿瘤的生长有轻微的抑制效果,但与Model组无显著性差异;高剂量EPS(200mg/kg)对B16F10肿瘤的抑制率可达42.5%(P<0.05);此外,EPS+5-FU协同处理与5-FU单独处理无显著性差异;肿瘤插图可直观表现出EPS的抗肿瘤效果。
4、脾淋巴细胞增殖实验
每组小鼠随机取三只于生物安全柜中进行解剖,小心取其脾脏,剔除脂肪组织,并用hanks溶液洗去血渍。
将脾脏置于100目尼龙筛网上研磨,用含有血清的RPMI-1640培养基进行冲洗并收集,1,000rpm离心3min。
加入红细胞裂解液,5min后1,000rpm离心5min去上清。
Hanks洗涤两次,用分别含有2μg/mL ConA和5μg/mL LPS的培养基重悬,以1×105个/孔均匀铺至96孔板中培养48h。
CCK-8法检测每个孔细胞的增殖情况,增殖活性计算公式如下:
Proliferation rate(%)=((Atreatment-Ablank)/(Acontrol-Ablank))*100%,式中:Atreatment为各组别肿瘤小鼠的脾淋巴细胞经LPS和ConA刺激所测吸光度;Acontrol为正常小鼠的脾淋巴细胞经LPS和ConA刺激所测吸光度;Ablank为含有LPS和ConA培养基的吸光度。
EPS对B16F10荷瘤小鼠脾脏淋巴细胞增殖的影响如图3所示。结果显示,各组别荷瘤小鼠脾淋巴细胞经ConA和LPS的刺激,高剂量EPS组小鼠脾淋巴细胞的增殖率显著高于空白组与5-FU处理组,200mg/kg EPS处理组小鼠脾淋巴细胞的增殖率可达120.58%(ConA)和169.88%(LPS)。
5、免疫荧光检测CD4+和CD8+T细胞
小鼠的肿瘤组织固定于4%多聚甲醛,送至武汉赛维尔生物科技有限公司(Wuhanservicebio technology CO.,LTD)进行免疫荧光染色,拍照取图分析染色结果。
小鼠肿瘤组织中T细胞(CD4+和CD8+T细胞)聚集情况如图4所示。结果显示,Model组和低、中剂量EPS组(50mg/kg、100mg/kg EPS)和5-FU组小鼠肿瘤组织中仅含有微量的CD4+(红色荧光)和CD8+T(绿色荧光)细胞;相反,200mg/kg EPS和EPS+5-FU处理组小鼠肿瘤组织中富集了大量CD4+T细胞和少量的CD8+T细胞;5-FU组小鼠肿瘤组织细胞核呈现大小不一、核聚等形态学变化,证明5-FU对肿瘤细胞有直接杀伤作用。其中,蓝色为DAPI。
6、qRT-PCR检测抗肿瘤相关基因的表达
提取肿瘤组织的总RNA参照Takara MiniBEST Universal RNA Extraction Kit试剂盒说明书。
参照Takara PrimeScriptTM RT reagent Kit with gDNA Erase试剂盒说明书,将总肿瘤组织RNA反转录为cDNA,并以该cDNA为模板,配制qPCR体系进行检测(TBPremix Ex TaqTM))。
EPS对B16F10荷瘤小鼠肿瘤相关基因表达量的影响如图5所示。结果显示,EPS显著提高了小鼠肿瘤组织中TNF-α、IFN-γ和P53 mRNA的表达量(图5A-C),降低了IL-1βmRNA的表达量;图5E-G结果表明,EPS显著提高了小鼠肿瘤组织中BAX mRNA的表达量,降低了BCL-2mRNA的表达量,整体提高了BAX与BCL-2表达量的比值;VEGF和Fgf2是肿瘤血管生成过程中的重要因子。图5H-I显示,EPS显著降低VEGF和Fgf2 mRNA的表达量。
7、ELISA检测血液细胞因子含量
取出备用的小鼠血清,解冻。后续操作严格按照ELISA试剂盒的操作说明进行实验。
各组小鼠血清中IFN-γ、IL-2、TNF-α、和IL-6含量如图6A-D所示。结果显示,EPS可以显著提高荷瘤小鼠血清中TNF-α、IFN-γ、IL-2和IL-6的含量,且呈现一定的浓度依耐性。200mg/kg EPS处理组小鼠血清中TNF-α(171.94pg/mL)、IFN-γ(259.37pg/mL)、IL-2(34.82pg/mL)和IL-6(73.67pg/mL)的含量显著高于Control组和Model组。
8、16S rDNA扩增子测序
解剖小鼠过程中收集盲肠内容物,标记完成后立即用液氮进行速冻备用。提取盲肠内容物总DNA,经PCR、建库和高通量测序,进一步分析测序数据。具体方法参考华大基因16s rDNA扩增子测序。
荷瘤小鼠肠道菌群多样性与聚类分析结果如图7所示。Shannon指数与Simpson指数(图7A-B)结果显示Model组小鼠与Control组小鼠肠道菌群的多样性基本趋于一致;5-FU组(图中为5.FU)小鼠肠道菌群的多样性低于Control组,且与5-FU+EPS组(图中为EPS.5.FU)小鼠肠道菌群的多样性趋于一致;而200mg/kg EPS处理组小鼠的肠道菌群的多样性高于5-FU组,趋近于近Control组。
Venn图(图7C)结果显示,各组小鼠肠道菌群共同存在的OUT达406,Control、5-FU、Model、EPS+5-FU、和EPS组分别特有32、20、15、10和9个OUT;主成分分析(图7D)结果表明,不同处理组小鼠的肠道菌群在OUT水平存在显著性的差异。
EPS对小鼠肠道微生物群落组成的影响如图8所示(图8A为盲肠内容物菌群在门水平的分布图,n=5;图8B为盲肠内容物菌群在门水平的平均相对度)。结果显示,Control组小鼠肠道内的菌群分布以厚壁菌和拟杆菌为主,并含有低比例的变形菌和疣微菌;相较于Control组,5-FU组(5.FU)小鼠肠道内拟杆菌的相对丰度显著降低,厚壁菌的相对丰度显著升高;此外,EPS处理组小鼠在门水平的肠道菌群组成与Control组更为相似。厚壁菌与拟杆菌的丰度比率结果显示(图8C),Control组小鼠盲肠内容物中厚壁菌与拟杆菌的丰度比率与5-FU组表现出显著性差异,而与EPS组无显著性差异。综上结果,植物乳杆菌WLPL09 EPS在发挥抗肿瘤的同时对小鼠肠道菌群无显著性影响。
以上所述,只是本发明的较佳实施例而已,本发明并不局限于上述实施方式,只要其以相同的手段达到本发明的技术效果,都应属于本发明的保护范围。在本发明的保护范围内其技术方案和/或实施方式可以有各种不同的修改和变化。
Claims (2)
1.一株植物乳杆菌WLPL09,其特征在于,所述植物乳杆菌WLPL09已保藏于中国典型培养物保藏中心,保藏地址为中国武汉大学,保藏编号为CCTCC M 2021038,保藏日期为2021年1月8日。
2.权利要求1所述的植物乳杆菌WLPL09产生的胞外多糖在制备抑制皮下黑色素瘤生长药物中的应用。
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