CN116712463B - 凝结芽孢杆菌mzy531在制备抗肝癌药物中的应用 - Google Patents
凝结芽孢杆菌mzy531在制备抗肝癌药物中的应用 Download PDFInfo
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Abstract
凝结芽孢杆菌MZY531在制备抗肝癌药物中的应用,涉及抗肝癌药物研发领域。该凝结芽孢杆菌MZY531于2021年6月2日保藏于中国典型培养物保藏中心,保藏编号为:CCTCC NO:M2021662。本发明通过试验证明凝结芽孢杆菌MZY531具有以下功能:诱导小鼠H22肝癌细胞凋亡;抑制小鼠H22肝癌细胞增殖;降低小鼠H22肝癌细胞活力;下调小鼠H22肝癌细胞中PI3K/AKT/mTOR信号通路相关蛋白的表达;上调小鼠H22肝癌细胞中Bax蛋白和Caspase‑3蛋白的表达;抑制小鼠H22肝癌细胞中Bcl‑2蛋白的表达。本发明为凝结芽孢杆菌MZY531作为抗肝癌药物的研究奠定了重要基础。
Description
技术领域
本发明涉及抗肝癌药物研发技术领域,具体涉及凝结芽孢杆菌(Bacillus coagulans)MZY531在制备抗肝癌药物中的应用。
背景技术
凝结芽孢杆菌(Bacillus coagulans)是一类形成孢子、产乳酸的***。作为人们日常生活经常接触的一种重要的益生菌,凝结芽孢杆菌引起了研究者的广泛关注。除了产生乳酸,这种细菌在适当的生长温度和pH值下能够产生孢子,同时具有耐高温特性,可以在胃和肠道的pH值条件下生长。凝结芽孢杆菌通过抗氧化、维持消化道正常菌群、预防肠炎和调节免疫应答等作用促进宿主健康。
目前,临床上多采用化学药物来治疗各类恶性肿瘤。这些化学抗癌药物虽然具有高药效,但是长期使用会造成贫血和免疫力下降等副作用,因此研究安全和对人体健康有益生作用的凝结芽孢杆菌,进一步挖掘其诱导肝癌细胞凋亡的具体机制,对于开发抗肝癌作用的益生菌或其制剂,临床上用来膳食补充辅助治疗肝癌具有重要意义。目前,关于凝结芽孢杆菌对H22肝癌细胞的毒性作用和抗肿瘤作用的研究还未见报道。
发明内容
本发明的目的是提供凝结芽孢杆菌(Bacillus coagulans)MZY531在制备抗肝癌药物中的应用。
本发明为解决技术问题所采用的技术方案如下:
本发明的凝结芽孢杆菌(Bacillus coagulans)MZY531在制备抗肝癌药物中的应用。
作为优选的实施方式,所述凝结芽孢杆菌(Bacillus coagulans)MZY531于2021年6月2日保藏于中国典型培养物保藏中心,保藏编号为:CCTCC NO:M2021662。
作为优选的实施方式,所述凝结芽孢杆菌(Bacillus coagulans)MZY531的功能包括:
(1)诱导小鼠H22肝癌细胞凋亡;
(2)抑制小鼠H22肝癌细胞增殖;
(3)降低小鼠H22肝癌细胞活力;
(4)下调小鼠H22肝癌细胞中PI3K/ AKT/ mTOR信号通路相关蛋白的表达;
(5)上调小鼠H22肝癌细胞中Bax蛋白和Caspase-3蛋白的表达;
(6)抑制小鼠H22肝癌细胞中Bcl-2蛋白的表达。
作为更优选的实施方式,所述小鼠H22肝癌细胞中PI3K/ AKT/ mTOR信号通路相关蛋白包括:PI3K蛋白、p-PI3K蛋白、AKT蛋白、p-AKT蛋白、mTOR蛋白和p-mTOR蛋白。
本发明的有益效果是:
本发明采用凝结芽孢杆菌(Bacillus coagulans)MZY531处理小鼠H22肝癌细胞,通过CCK-8法测定小鼠H22肝癌细胞活力、对凝结芽孢杆菌(Bacillus coagulans)MZY531处理的小鼠H22肝癌细胞进行TUNEL染色、流式细胞术和Western blot方法检测小鼠H22肝癌细胞凋亡,探讨凝结芽孢杆菌(Bacillus coagulans)MZY531对抑制小鼠H22肝癌细胞增殖和诱导小鼠H22肝癌细胞凋亡的治疗作用。上述研究结果显示,凝结芽孢杆菌(Bacillus coagulans)MZY531通过降低小鼠H22肝癌细胞活力、抑制小鼠H22肝癌细胞增殖和增加小鼠H22肝癌细胞凋亡发挥其治疗作用。
本发明还通过检测小鼠H22肝癌细胞中PI3K/ AKT/ mTOR信号通路相关蛋白PI3K、p-PI3K、AKT、p-AKT、mTOR、p-mTOR的表达确定了凝结芽孢杆菌(Bacillus coagulans)MZY531在小鼠H22肝癌细胞中激活的信号通路,结果显示凝结芽孢杆菌(Bacillus coagulans)MZY531可通过下调小鼠H22肝癌细胞中PI3K/ AKT/ mTOR信号通路相关蛋白(PI3K、p-PI3K、AKT、p-AKT、mTOR和p-mTOR)的含量来调控小鼠H22肝癌细胞的凋亡。另外,通过检测小鼠H22肝癌细胞中凋亡信号通路总蛋白和活化蛋白的变化确定了凝结芽孢杆菌(Bacillus coagulans)MZY531诱导小鼠H22肝癌细胞凋亡的可能机制,结果显示,凝结芽孢杆菌(Bacillus coagulans)MZY531上调了Bax蛋白和Caspase-3蛋白的表达,显著抑制了Bcl-2蛋白的表达,通过增加凝结芽孢杆菌(Bacillus coagulans)MZY531的剂量可有效诱小鼠H22肝癌细胞凋亡,且具有显著的剂量依赖性。
本发明为凝结芽孢杆菌(Bacillus coagulans)MZY531作为抗肝癌药物的研究奠定了重要基础。
附图说明
图1为实施例2中凝结芽孢杆菌(Bacillus coagulans)MZY531对小鼠H22肝癌细胞活力的影响。
图2为实施例2中TUNEL法测定各组小鼠H22肝癌细胞在20×视野下的凋亡情况。
图3为实施例2中各组TUNEL染色定量分析结果。
图4为实施例2中凝结芽孢杆菌(Bacillus coagulans)MZY531对小鼠H22肝癌细胞凋亡周期的影响。图3中,Q1-Q4分别代表坏死细胞、晚期凋亡细胞、存活细胞和早期凋亡细胞。
图5为实施例2中凝结芽孢杆菌(Bacillus coagulans)MZY531对小鼠H22肝癌细胞中PI3K/ AKT/ mTOR信号通路相关蛋白表达的电泳图谱。
图6为实施例2中凝结芽孢杆菌(Bacillus coagulans)MZY531对小鼠H22肝癌细胞中PI3K/ AKT/ mTOR信号通路相关蛋白p-PI3K/PI3K的影响。
图7为实施例2中凝结芽孢杆菌(Bacillus coagulans)MZY531对小鼠H22肝癌细胞中PI3K/ AKT/ mTOR信号通路相关蛋白p-AKT/AKT的影响。
图8为实施例2中凝结芽孢杆菌(Bacillus coagulans)MZY531对小鼠H22肝癌细胞中PI3K/ AKT/ mTOR信号通路相关蛋白p-mTOR/mTOR的影响。
图9为实施例2中凝结芽孢杆菌(Bacillus coagulans)MZY531对小鼠H22肝癌细胞中Bax蛋白、Bcl-2蛋白和Caspase-3蛋白表达的电泳图谱。
图10为实施例2中凝结芽孢杆菌(Bacillus coagulans)MZY531对小鼠H22肝癌细胞中Bax蛋白的影响。
图11为实施例2中凝结芽孢杆菌(Bacillus coagulans)MZY531对小鼠H22肝癌细胞中Bcl-2蛋白的影响。
图12为实施例2中凝结芽孢杆菌(Bacillus coagulans)MZY531对小鼠H22肝癌细胞中Caspase-3蛋白的影响。
具体实施方式
下面将结合本发明实施例,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。
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,均为自常规生化试剂商店购买得到的。以下实施例中的定量试验,均设置三次重复实验,结果取平均值。
固体LB培养基:溶剂为水,胰蛋白胨10g/L、酵母浸粉5g/L、氯化钠10g/L、琼脂15g/L;如无特殊说明,pH=7.0。
液体LB培养基;与固体LB培养基的区别仅在于液体LB培养基中不加入琼脂。
液体GPY培养基:葡萄糖20g,胰蛋白胨10g,酵母浸粉10g,氯化钠5g,氯化钙0.7g,氯化锰0.3g,定容至1000 mL蒸馏水,置于高压灭菌锅121℃,灭菌20 min,40℃取出,放入4℃冰箱待用。
胰蛋白胨和酵母浸粉:均购自北京奥博星生物技术有限责任公司。
氯化钠、氯化钙、氯化锰和葡萄糖:均购自天津市光复科技发展有限公司。
甲醇:购自天津新通精细化工有限公司。
RPMI-1640培养基、磷酸盐缓冲液(PBS)、胰蛋白酶、小鼠H22肝癌细胞:均购自江苏凯基生物技术股份有限公司。
胎牛血清(FBS):购自浙江天杭生物科技股份有限公司。
BCA试剂盒和组织裂解液(RIPA):均购自北京索莱宝科技有限公司。
蛋白酶抑制剂(PMSF):购自江苏康为世纪生物科技公司。
PAGE凝胶快速制备试剂盒:购自上海雅酶生物科技有限公司。
β-actin、PI3K、p-PI3K、AKT、p-AKT、mTOR和p-mTOR:均购自北京博奥森生物技术有限公司。
Caspase-3和Bax和Bcl-2:均购自GeneTex, Inc。
一、凝结芽孢杆菌菌株的分离
样本取材:传统发酵食品酸菜。
2019年10月,从四川省成都市收集新鲜腌制的老坛酸菜样品放入运输培养基中,迅速置于冰盒内送至实验室进行菌株分离。将酸菜样品置于80℃水浴处理10min,然后进行研磨、梯度稀释后均匀涂于固体LB培养基平板上,50℃培养48h,挑取单菌落接种于固体LB培养基平板上继续培养,经固体LB培养基平板反复划线培养纯化,得到多株纯菌落。纯培养的菌种接种到液体LB培养基培养,然后加入60%甘油,-80℃冰箱保存。将其中1株菌株命名为MZY531。
二、菌株的鉴定
菌株MZY531的生理生化鉴定结果:革兰氏染色阳性,过氧化氢酶试验阴性,联苯胺试验阴性,吲哚试验阴性,乙酰甲基甲醇试验阳性;不水解淀粉,不液化明胶,不产生硫化氢,发酵葡萄糖产酸不产气;不运动的杆菌;在25℃和65℃能够生长,最适生长温度45℃-50℃;适宜pH为5.0-7.0;耐受6.5%NaCl;菌株MZY531在液体LB培养基中呈均匀浑浊生长,久置菌体呈白色沉淀。
16S rDNA序列同源性分析:提取基因组DNA,采用27F(SEQ ID NO:2)和1492R(SEQID NO:3)组成的引物对进行PCR扩增,然后将扩增产物进行测序,16s rDNA序列如序列表的SEQ ID NO:1所示。
将获得的16S rDNA序列通过BLAST程序与GenBank库中已知菌株的序列信息进行同源性比对分析,鉴定待测菌株;以16S rDNA序列同源性大于99%为标准进行属种归类。
根据以上鉴定结果,菌株MZY531被鉴定为凝结芽孢杆菌Bacillus coagulans。
三、菌株的保藏
本发明的一株凝结芽孢杆菌(Bacillus coagulans)MZY531,已于2021年6月2日保藏于中国典型培养物保藏中心,简称CCTCC,地址为:湖北省武汉市武昌区八一路299号武汉大学校内(武汉大学保藏中心),保藏编号为:CCTCC NO:M2021662。
实施例2凝结芽孢杆菌(Bacillus coagulans)MZY531抑制H22肝癌细胞增殖和诱导H22肝癌细胞凋亡作用评价。
一、菌株培养
将冷冻保存的凝结芽孢杆菌(Bacillus coagulans)MZY531接种于液体GPY培养基中,50℃、180rpm摇床孵育20h,3000 rpm、4℃离心10min后收集菌体沉淀,最后将细菌重新悬浮于无菌生理盐水中,调整菌液浓度至1.0×109 CFU/ml,4℃保存备用。
二、细胞培养
将小鼠H22肝癌细胞在95%相对湿度、5%二氧化碳、37℃条件下进行孵育,所用的培养基为RPMI-1640培养基,该RPMI-1640培养基中含有10%胎牛血清和1%(青霉素与链霉素混合物);每1-2天更换一次培养基;在细胞单层覆盖细胞培养板80%的表面后,收集上清培养液,然后用磷酸盐缓冲液(PBS)清洗底部;底部的细胞用胰蛋白酶消化,收集到的液体用于传代培养或实验;在所有的实验中,都使用了处于对数阶段的细胞。
三、细胞活力测试
为验证凝结芽孢杆菌(Bacillus coagulans)MZY531对小鼠H22肝癌细胞增殖和活力的影响,采用CCK-8方法进行实验。将1×104个小鼠H22肝癌细胞接种于96孔板中,按MOI=0、1、10、50、100 CFU/cell分别加入凝结芽孢杆菌(Bacillus coagulans)MZY531干预,以氟尿嘧啶5-FU(100μg/mL)为阳性对照,24h后加入体积比10%的CCK-8溶液,孵育3h,于450nm处检测吸光度。
参见图1,结果表明,凝结芽孢杆菌(Bacillus coagulans)MZY531显著抑制了小鼠H22肝癌细胞的生长,且呈剂量依赖性。随着凝结芽孢杆菌(Bacillus coagulans)MZY531浓度的增加,小鼠H22肝癌细胞的活力逐渐下降。以MOI = 100 CFU/cell浓度处理小鼠H22肝癌细胞24h后,细胞存活率达到52.74±1.36%。经计算凝结芽孢杆菌(Bacillus coagulans)MZY531的半抑制浓度值为MOI = 107 CFU/cell。
四、TUNEL染色分析实验
采用TUNEL染色分析研究凝结芽孢杆菌(Bacillus coagulans)MZY531对于小鼠H22肝癌细胞的影响。
将小鼠H22肝癌细胞以每孔2×105个细胞的数量接种于预先放置爬片的6孔板中,并用凝结芽孢杆菌(Bacillus coagulans)MZY531(MOI=0、50、100 CFU/cell)和氟尿嘧啶5-FU(100μg/mL)分别干预24h,磷酸盐缓冲液(PBS)清洗爬片三次后,用4%多聚甲醛固定细胞30min;固定后用磷酸盐缓冲液(PBS)清洗细胞3次,加50μL-100μL的0.3% Triton x-100破膜工作液,室温孵育20min,磷酸盐缓冲液(PBS)清洗3次;爬片甩干后滴加TUNEL试剂盒内的缓冲液Buffer覆盖细胞,常温孵育10min;取TUNEL试剂盒内TDT酶、dUTP和Buffer(TDT酶、dUTP和Buffer的体积比为1:5:50)的混合液覆盖细胞,置37℃恒温培养箱孵育2h;磷酸盐缓冲液(PBS)清洗3次,每次5min,去除磷酸盐缓冲液(PBS)并滴加DAPI(4',6-二脒基-2-苯基吲哚)染液复染,避光室温孵育10min;于荧光显微镜下观察并采集图像。参见图2(图2中的SpOrange为CY3荧光素标记),DAPI紫外激发波长330nm-380nm,发射波长420nm,发蓝光;CY3激发波长510nm-560nm,发射波长590nm,发红光;DAPI染活细胞的细胞核在紫外的激发下为蓝色,CY3荧光素标记凋亡细胞且细胞核为红色;ImageJ软件统计TUNEL阳性细胞数。
观察TUNEL染色结果,TUNEL阳性细胞的细胞核被染成红色,TUNEL阳性细胞数量可反应出其细胞凋亡程度。参见图3,随着MOI的增加,小鼠H22肝癌细胞数量逐渐降低,TUNEL阳性细胞数量增加,结果表明,凝结芽孢杆菌(Bacillus coagulans)MZY531可诱导小鼠H22肝癌细胞凋亡,抑制小鼠H22肝癌细胞增殖。
五、流式细胞仪检测细胞凋亡
将小鼠H22肝癌细胞以单孔2×105个细胞的数量接种于6孔板中;以凝结芽孢杆菌(Bacillus coagulans)MZY531(MOI = 0、50、100 CFU/cell)和氟尿嘧啶5-FU(100μg/mL)分别干预24 h后,用不含EDTA的胰蛋白酶消化细胞,离心(4℃,1000×g,2 min);计数1×105的小鼠H22肝癌细胞悬浮于100μL的1×combined buffer,用Annexin V / PI solution染色,在37℃下孵育15 min;采用流式细胞术分析在530 nm(FITC通道)和617nm(FITC通道)下488 nm激光激发的荧光发射光谱。
参见图4,流式细胞术结果显示,凝结芽孢杆菌(Bacillus coagulans)MZY531可诱导小鼠H22肝癌细胞凋亡;凝结芽孢杆菌(Bacillus coagulans)MZY531(MOI=50CFU/cell和100 CFU/cell)处理24 h后小鼠H22肝癌细胞的凋亡率分别为25.53%和42.39%,分别显著高于对照组(5.34%),且MOI=100 CFU/cell组与阳性组5-FU(47.59%)较为接近。
六、蛋白质印迹
取对数生长期的小鼠H22肝癌细胞以每孔3×105个细胞的数量接种于6孔板中,用凝结芽孢杆菌(Bacillus coagulans)MZY531(MOI=0、50、100 CFU/cell)和氟尿嘧啶5-FU(100μg/mL)处理;培养24 h后,加入组织裂解液(RIPA)、蛋白酶抑制剂(PMSF)和磷酸酶抑制剂(PI)的混合物100μL裂解细胞,其中,组织裂解液(RIPA)、蛋白酶抑制剂(PMSF)和磷酸酶抑制剂(PI)的体积比为100:1:1,提取蛋白,并在冰浴中裂解60 min,随后采用低温离心机离心(10000 rpm,4℃,10 min),最后收集上清液待用,BCA法测定蛋白浓度。根据BCA试剂盒说明书的指导方法,选用96孔板的方法利用酶标仪测定各组织样品在570 nm处的吸光度,并根据标准曲线计算各组织样品的蛋白浓度;SDS-PAGE电泳并转移到PVDF膜上,使用以下单克隆抗体的一抗稀释液进行Western blot分析:PI3K、p-PI3K、AKT、p-AKT、mTOR、p-mTOR以及Bcl-2相关死亡启动子Bax、caspase-3和Bcl-2,用结合辣根过氧化物酶的抗兔二抗在37℃孵育1 h,使用化学发光成像分析仪(Image Quant LAS 4000)对目的条带进行量化,β-肌动蛋白(β-actin)作为加载对照。
为了确定凝结芽孢杆菌(Bacillus coagulans)MZY531在小鼠H22肝癌细胞中激活的信号通路,本发明检测了小鼠H22肝癌细胞中PI3K/ AKT/ mTOR信号通路相关蛋白PI3K、p-PI3K、AKT、p-AKT、mTOR、p-mTOR的表达。与对照组相比,凝结芽孢杆菌(Bacillus coagulans)MZY531处理组的磷酸化蛋白p-PI3K、p-AKT和p-mTOR的表达显著降低(图5)。小鼠H22肝癌细胞中的PI3K和AKT的磷酸化水平也同样降低(图6-图8),这些结果表明凝结芽孢杆菌(Bacillus coagulans)MZY531可通过下调小鼠H22肝癌细胞中PI3K/ AKT/ mTOR信号通路相关蛋白(PI3K、p-PI3K、AKT、p-AKT和p-mTOR)的含量来调控小鼠H22肝癌细胞的凋亡。
为了研究凝结芽孢杆菌(Bacillus coagulans)MZY531诱导小鼠H22肝癌细胞凋亡的可能机制,本发明检测了小鼠H22肝癌细胞中凋亡信号通路总蛋白和活化蛋白的变化。参见图9-图12,结果显示,凝结芽孢杆菌(Bacillus coagulans)MZY531上调了Bax蛋白和Caspase-3蛋白的表达,显著抑制了Bcl-2蛋白的表达;此外,低剂量的凝结芽孢杆菌(Bacillus coagulans)MZY531可促进小鼠H22肝癌细胞的凋亡,但其作用不是特别明显;增加凝结芽孢杆菌(Bacillus coagulans)MZY531的剂量可有效诱小鼠H22肝癌细胞凋亡,且具有显著的剂量依赖性。
本发明公开了凝结芽孢杆菌MZY531在制备抗肝癌药物中的应用,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。本发明的产品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产品进行改动或适当变更与组合,来实现和应用本发明技术。
Claims (3)
1.凝结芽孢杆菌(Bacillus coagulans)MZY531在制备抗肝癌药物中的应用,其特征在于,所述凝结芽孢杆菌(Bacillus coagulans)MZY531于2021年6月2日保藏于中国典型培养物保藏中心,保藏编号为:CCTCC NO:M2021662。
2.根据权利要求1所述的应用,其特征在于,所述凝结芽孢杆菌(Bacillus coagulans)MZY531的功能为:
(1)诱导小鼠H22肝癌细胞凋亡;
(2)抑制小鼠H22肝癌细胞增殖;
(3)降低小鼠H22肝癌细胞活力;
(4)下调小鼠H22肝癌细胞中PI3K/AKT/mTOR信号通路相关蛋白的表达;
(5)上调小鼠H22肝癌细胞中Bax蛋白和Caspase-3蛋白的表达;
(6)抑制小鼠H22肝癌细胞中Bcl-2蛋白的表达。
3.根据权利要求2所述的应用,其特征在于,所述小鼠H22肝癌细胞中PI3K/AKT/mTOR信号通路相关蛋白为:PI3K蛋白、p-PI3K蛋白、AKT蛋白、p-AKT蛋白、mTOR蛋白和p-mTOR蛋白。
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