CN113304175A - 丁酸杆菌在制备预防和/或治疗川崎病药物中的应用 - Google Patents
丁酸杆菌在制备预防和/或治疗川崎病药物中的应用 Download PDFInfo
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- CN113304175A CN113304175A CN202110772659.9A CN202110772659A CN113304175A CN 113304175 A CN113304175 A CN 113304175A CN 202110772659 A CN202110772659 A CN 202110772659A CN 113304175 A CN113304175 A CN 113304175A
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Abstract
本发明公开了一种丁酸杆菌在制备预防和/或治疗川崎病药物中的应用,属于医学技术领域。本发明通过实验验证丁酸杆菌通过改善肠道菌群,一方面上调肠紧密连接蛋白Claudin‑1、Jam‑1、Occludin和ZO‑1的表达水平,增强肠屏障功能,减少肠漏,降低IL‑1β和IL‑6炎性因子水平;另一方面增加产短链脂肪酸细菌的数量,提高SCFAs,尤其是丁酸的产量,并上调单羧酸转运体水平,促进SCFAs的吸收,提高机体抗炎能力,以减轻血管炎症,发挥对川崎病的预防及治疗作用。
Description
技术领域
本发明涉及医药领域,特别是涉及一种丁酸杆菌在制备预防和/或治疗川崎病药物中的应用。
背景技术
川崎病(Kawasaki disease,KD)是儿童最常见的获得性心血管疾病,以全身性中小血管炎为主要病理变化。若治疗不及时,川崎病可引发严重的心血管并发症,如冠状动脉瘤、血栓形成等,甚至猝死。所以,提供一种治疗川崎病的药物十分必要。
研究表明,在KD的急性阶段,实验室检查通常显示血象的高炎症状态,如高白细胞计数、C反应蛋白和红细胞沉降率。肠道菌群与多种炎症性疾病具有显著的相关性,其代谢产物短链脂肪酸(short-chain fatty acids,SCFAs)乙酸、丙酸和丁酸均对免疫和炎症反应具有显著的调控作用。其中,丁酸已被证明具有显著的抑炎效应。此外,肠道菌群的失衡将导致肠屏障功能破坏,进而使有害物质进入循环增多。有学者发现KD患儿存在肠道菌群失衡,主要表现为有害菌(如链球菌属,肠球菌属)的增加和有益菌(如乳酸杆菌属)的减少。肠道菌群在KD患者中的变化提示KD的发病机制可能与肠道菌群失衡存在相关性。
丁酸杆菌(Clostridium butyricum),又称酪酸梭菌、酪酸梭状芽孢杆菌、酪酸杆菌、酪酸菌、丁酸梭状芽孢杆菌、丁酸梭菌、丁酸菌,是一种应用广泛的益生菌,具有较强整肠功能,能提高肠道内益生菌的比例,常用于治疗腹泻、肠炎。但是,到目前为止,尚未发现有关丁酸杆菌治疗川崎病的应用。
发明内容
本发明的目的是提供一种丁酸杆菌在制备预防和/或治疗川崎病药物中的应用,以解决上述现有技术存在的问题,提高机体抗炎能力,以减轻血管炎症,这对川崎病的预防及治疗具有重要意义。
为实现上述目的,本发明提供了如下方案:
本发明提供一种丁酸杆菌在制备预防和/或治疗川崎病药物中的应用。
优选的是,所述预防和/或治疗川崎病药物包括预防和/或治疗所述川崎病使用的有效量的丁酸杆菌,所述丁酸杆菌以活细胞形式存在于所述药物中。
优选的是,在所述预防和/或治疗川崎病药物中,所述丁酸杆菌的数目为5×106-6×109CFU/ml。
优选的是,所述预防和/或治疗川崎病药物还包括与所述丁酸杆菌相配伍的其他药类以及药学上可接受的载体和/或辅料。
优选的是,所述其他药类包括与所述丁酸杆菌相配伍,不会使所述丁酸杆菌出现中和、水解以及破坏失效理化反应,同时与丁酸杆菌协同作用后,能提高丁酸杆菌预防和/或治疗川崎病效果的药类。
优选的是,所述预防和/或治疗川崎病药物为药学上可接受的剂型。
优选的是,所述剂型为粉剂、注射液、胶囊、片剂或口服液。
优选的是,所述丁酸杆菌(Clostridium butyricum)于2014年1月22日由中国微生物菌种保藏管理委员会普通微生物中心所保藏,保藏为丁酸杆菌WZ001菌株,保藏编号为CGMCC No.8808,保藏地址为北京市朝阳区北辰西路1号院3号中国科学院生物研究所。
本发明公开了以下技术效果:
本发明提供了丁酸杆菌在制备预防和/或治疗川崎病药物中的应用,发明人研究发现,在川崎病的研究中,丁酸杆菌通过改善肠道菌群,一方面上调肠紧密连接蛋白Claudin-1、Jam-1、Occludin和ZO-1的表达水平,增强肠屏障功能,减少肠漏,降低IL-1β和IL-6炎性因子水平;另一方面增加产SCFAs细菌的数量,增加SCFAs,尤其是丁酸的产量,并上调单羧酸转运体水平,促进SCFAs的吸收,提高机体抗炎能力,以减轻血管炎症,发挥对川崎病的预防和/或治疗作用。因此,本发明通过实验验证丁酸杆菌可用于预防及治疗川崎病,这对于治疗川崎病的新药物的筛选以及川崎病的预防及治疗具有重要的意义。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1是本发明实施例2提供的各组小鼠进行HE染色后的心脏组织冠状动脉形态结构示意图;
图2-1是本发明实施例2提供的各组小鼠血浆中炎症因子IL-1β、TNF-α、IL-6、IL-10和MCP-1的表达水平示意图;*p<0.05,**p<0.01,***p<0.001,ns无统计学意义;
图2-2是本发明实施例2提供的各组小鼠血浆中D-乳酸的表达水平示意图;**p<0.01;
图3-1是本发明实施例2提供的各组小鼠心脏组织冠状动脉CD31/ICAM-1的免疫荧光染色结果示意图;**p<0.01,***p<0.001;
图3-2是本发明实施例2提供的各组小鼠心脏组织冠状动脉周围CD68的免疫荧光染色结果示意图;**p<0.01;
图3-3是本发明实施例2提供的各组小鼠结肠组织中肠紧密连接蛋白Claudin-1、Jam-1、Occludin和ZO-1的免疫荧光染色结果示意图;*p<0.05,**p<0.01,***p<0.001;
图4-1是本发明实施例2提供的,各组小鼠肠道菌群结构变化的非监督多元统计方法PCoA结果示意图;
图4-2是本发明实施例2提供的各组小鼠肠道菌群Venn图;
图4-3是本发明实施例2提供的各组小鼠肠道菌群LEfSe和LDA评分结果图;
图4-4是本发明实施例2提供的各组小鼠肠道菌群组成结果示意图;
图4-5是本发明实施例2提供的各组小鼠产SCFAs菌的相对含量示意图;*p<0.05,**p<0.01,***p<0.001;
图5-1是本发明实施例2提供的各组小鼠结肠紧密连接蛋白Claudin-1、Jam-1、Occludin和ZO-1的表达水平示意图;*p<0.05,**p<0.01,***p<0.001;
图5-2是本发明实施例2提供的各组小鼠结肠单羧酸转运体MCT-1和SMCT-1的表达水平示意图;*p<0.05,***p<0.001;
图6-1是本发明实施例2提供的各组小鼠结肠紧密连接蛋白Claudin-1、Jam-1、Occludin和ZO-1的含量变化示意图;**p<0.01,***p<0.001;
图6-2是本发明实施例2提供的各组小鼠结肠单羧酸转运体MCT-1和SMCT-1的含量变化示意图;*p<0.05,**p<0.01,***p<0.001;
图7是本发明实施例2提供的各组小鼠粪便样品的短链脂肪酸GC-MS定量分析结果示意图;*p<0.05,**p<0.01。
具体实施方式
现详细说明本发明的多种示例性实施方式,该详细说明不应认为是对本发明的限制,而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
本发明实施例提供了丁酸杆菌在制备预防和/或治疗川崎病药物中的应用。
发明人研究发现,在川崎病的研究中,丁酸杆菌通过改善肠道菌群,一方面上调肠紧密连接蛋白Claudin-1、Jam-1、Occludin和ZO-1的表达水平,增强肠屏障功能,减少肠漏,降低IL-1β和IL-6炎性因子水平;另一方面增加产短链脂肪酸(short-chain fatty acids,SCFAs)细菌的数量,增加SCFAs,尤其是丁酸的产量,并上调单羧酸转运体水平,促进SCFAs的吸收,提高机体抗炎能力,以减轻血管炎症,发挥对川崎病的预防和/或治疗作用。可见,本发明通过实验验证了丁酸杆菌可用于治疗川崎病,这对于治疗川崎病具有重要的意义。
具体地,该川崎病药物包括治疗有效量的丁酸杆菌,且丁酸杆菌以活细胞的形式存在。可以理解的是,丁酸杆菌作为该川崎病药物中的活性成分,其以活细胞(即活生物)的形式存在时,能够发挥较佳的治疗川崎病的作用。作为优选,该川崎病药物中,丁酸杆菌的活细胞数目为5×106-6×109CFU/ml,举例来说,可以为5×106CFU/ml、5×108CFU/ml、9×108CFU/ml、1×109CFU/ml、3×109CFU/ml、6×109CFU/ml等,本实验用量为5×108CFU/ml。
进一步地,该川崎病药物还可包括与丁酸杆菌相配伍的其他药类以及药学上可接受的载体和/或辅料。举例来说,此处的“其他药类”指的是与丁酸杆菌相配伍,不会使丁酸杆菌出现中和,水解,破坏失效等理化反应,且与丁酸杆菌协同作用后,能提高丁酸杆菌治疗川崎病效果的药类。而上述的载体可以为本领域常用的药物载体,例如壳聚糖、脂质体、海藻酸、琼脂、纤维蛋白、胶原蛋白以及合成型高分子聚合载体等。上述的辅料为无生理活性,不影响药物制剂活性药物疗效、含量测定和稳定性,且主要目的是方便制剂的制备和临床应用的材料,举例来说,其可以为本领域常用的淀粉、预胶化淀粉、糊精、蔗糖、乳糖、甘露醇、微晶纤维素、硫酸钙、磷酸氢钙、轻质氧化镁、碳酸钙、干淀粉、羟甲基淀粉钠、低取代羟丙基纤维素、泡腾崩解剂、交联聚维酮等。
具体地,该川崎病药物可为药学上可接受的剂型,例如为粉剂、注射液、胶囊、片剂或口服液等。相应地,根据实际的药物剂型,该药物的给药方式可选自吸入、吹入口、鼻给药、经口含化给药、胃肠外或直肠给药、局部椎管给药等。
其中,本发明实施例所述的丁酸杆菌优选为由中国微生物菌种保藏管理委员会普通微生物中心所保藏,保藏为丁酸杆菌WZ001菌株,保藏编号为CGMCC No.8808。利用该丁酸杆菌WZ001菌株,可有效治疗川崎病,降低患者由川崎病导致的死亡率。
进一步可以理解的是,本发明实施例所述的川崎病的受体不仅仅是针对人类,还可针对动物,例如大鼠等。
具体地,本发明实施例针对小鼠由白色念珠菌细胞壁水溶物诱导的川崎病进行了丁酸杆菌治疗实验,为使本发明的目的、技术方案和优点更加清楚,下面将结合附图,以具体实施例的方式,对本发明实施方式作进一步详细的描述。在以下具体实施例中,所涉及的操作未注明条件者,均按照常规条件或者制造商建议的条件进行。所用原料未注明生产厂商及规格者均为可以通过市购获得的常规产品。
实施例1
1、制备白色念珠菌细胞壁水溶物(CAWS)
①菌株:白色念珠菌菌株ATCC18804(购自宁波明舟生物科技有限公司)。
②白色念珠菌的培养:菌株保存于甘油中,-80℃保存。提取步骤前2天,用一次性接种环接种于沙保氏培养基(C-limiting培养基,青岛海博)中,37℃培养,形成单菌落备用。
③配制5×1L C-limiting培养基,将白色念珠菌接种于加入了200μl生物素溶液的C-limiting培养基中,于270rpm的转速下于27℃摇动扩菌2天。
④加入等体积的乙醇,并放入4℃的冰箱中醇沉过夜。
⑤4℃,9000rmp,15min离心,收集沉淀物,溶解于250ml纯化水中,并将沉淀物在转子充分搅拌2h。
⑥将复合物再次离心,4℃,9000rmp,15min,收集上层可溶水成分,并与等体积的无水乙醇混合,置于4℃冰箱过夜。
⑦将复合物4℃,9000rmp,15min离心,获得沉淀物,用丙酮富集沉淀物,去除丙酮后,于通风橱中风干,得到CAWS。
⑧将获得的CAWS溶解于生理盐水中,配置成0.2g/ml溶液,并在使用前高压灭菌。
2、丁酸杆菌菌液配制
丁酸杆菌WZ001 CGMCC No.8808接种于TPY培养基中,37℃密封厌氧培养。使用时8000rmp,1min离心,用PBS稀释至5×108CFU/ml,灌胃使用。
3、实验分组及处理
将3-4周龄C57BL/6J雄性小鼠随机分为四组(n=10/组),并如下所示进行造模处理:
PBS组:小鼠腹腔注射PBS(4mg/d/只),连续5天。
CAWS组:小鼠腹腔注射CAWS(4mg/d/只),连续5天,进行川崎病小鼠造模。
丁酸杆菌组:小鼠腹腔注射CAWS(4mg/d/只)连续5天进行造模,并且在造模第1天开始每日口服丁酸杆菌(5×108CFU/ml,0.1ml/10g),持续28天。
第28天用戊巴比妥钠麻醉动物,进行眼眶采血,然后用颈椎脱臼处死小鼠。收集升结肠的粪便标本,对结肠和心脏进行解剖、称重,然后具体通过本领域常用的技术对各组小鼠进行相应指标的检测,所得数据进行统计来评价丁酸杆菌对川崎病小鼠的治疗效果。
实施例2
通过检测不同指标评价丁酸杆菌对川崎病小鼠的治疗效果。
(1)通过对实施例1中所提及的各组小鼠心脏组织进行病理切片以及HE染色(hematoxylin-eosin staining,苏木精—伊红染色),来观察各组小鼠冠状动脉的情况,其中,本领域技术人员可以理解的是,上述对小鼠的心脏组织切片进行的HE染色为本领域常用的技术,本发明实施例在此不对其做更具体的限定。
如图1所示,是对各组小鼠的心脏组织冠状动脉进行HE染色后的结果,结果显示PBS组小鼠心脏组织冠状动脉血管内皮光整无肿胀,弹性纤维连续,未见炎症细胞浸润。CAWS组冠状动脉内皮细胞肿胀,血管内皮粗糙、隆起,炎症细胞浸润明显,冠状动脉管腔出现狭窄。丁酸杆菌的干预减轻了冠状动脉炎症,且冠状动脉管腔狭窄的情况也有所改善。
(2)采用双抗体夹心ABC-ELISA法(avidinbiotincomplex-ELISA亲和素-生物素复合ELISA),对实施例1提及的各组小鼠血浆中IL-1β、TNF-α、IL-6、IL-10和MCP-1以及D-乳糖的表达水平进行测试,按照所用试剂盒的说明书如下进行:
①标本稀释液(1%BSA-碳酸盐缓冲液)、洗涤液(0.02mol/L pH=7.4Tris-HCl-Tween20溶液)配制。再将标准品稀释成合适的一系列梯度浓度。
②加样:每孔各加入标准品和胃组织匀浆(按匀浆液:生理盐水=2:3稀释)100μl,将反应板充分混匀后置37℃40min。
③洗板:用洗涤液将反应板充分洗涤5次,向滤纸上印干。
④每孔加入蒸馏水和第一抗体工作液(生物素化单克隆抗体)各50μl(空白除外)。将反应板充分混匀后置37℃20min。并按与步骤③相同的步骤再次进行洗板。
⑤每孔加入酶标抗体工作液100μl,将反应板置37℃10min。并按与步骤③相同的步骤再次进行洗板。
⑥每孔加入底物工作液(TMB溶液)100μl,置37℃暗处反应15min。然后每孔加入100μl终止液(0.1mol/L H2SO4溶液)混匀。
⑦用酶标仪在450nm处测吸光度。根据一系列浓度的标准品吸光度制作标准曲线,然后进行IL-1β、TNF-α、IL-6、IL-10和MCP-1以及D-乳糖表达量的计算。
图2-1是各组小鼠血浆炎症因子IL-1β、TNF-α、IL-6、IL-10和MCP-1的表达水平图,结果显示相较于PBS组,CAWS组IL-1β、TNF-α、IL-6、IL-10和MCP-1水平升高,而经过丁酸杆菌的干预,炎症因子IL-1β和IL-6表达水平下降,IL-10表达水平上升,总体上呈现出炎症减轻的趋势。这表明丁酸杆菌能降低KD小鼠的炎症水平。
血浆中D-乳酸的含量与肠黏膜屏障受损程度成正比。图2-2是各组小鼠血浆中D-乳酸的表达水平图,结果显示,相较于PBS组,CAWS组血浆D-乳酸含量增高,而经过丁酸杆菌的干预,血浆D-乳酸含量出现了下降,可见丁酸杆菌有益于肠黏膜屏障的修复。
(3)对实施例1提及的各组小鼠心脏组织冠状动脉中的CD31/ICAM-1蛋白和CD68蛋白以及结肠组织中的肠紧密连接蛋白Claudin-1、Jam-1、Occludin和ZO-1进行免疫荧光染色。具体操作步骤如下:
①烘片:取出组织切片,置于37℃烘箱中烘30min。
②脱蜡:将组织切片浸泡于二甲苯中,浸泡2次,每次15min。
③水化:依次将组织切片浸泡于梯度浓度下降的乙醇中:100%乙醇浸泡5min,90%乙醇浸泡5min,80%乙醇浸泡5min,70%乙醇浸泡5min,ddH2O浸泡5min。
④用PBS清洗3次,每次5min。
⑤内源性酶去除:将组织切片置于3%H2O2溶液中,室温浸泡15min。
⑥用PBS清洗3次,每次5min。
⑦抗原修复:将组织切片置于枸橼酸溶液中,高压抗原修复2min。
⑧PBS清洗3次,每次5min。
⑨封闭:在组织上滴加5%BSA封闭液,在37℃恒温箱中封闭30min。
⑩孵育一抗:移除组织上的5%BSA封闭液,使用1%BSA溶液按比例(CD31 1:1000;ICAM,CD68,Claudin-1,Jam和Occludin 1:100;ZO-1 1:300;MCT-1和SMCT-1 1:50)配制一抗,滴加覆盖于组织上,放入湿盒,在4℃冰箱中孵育过夜。
图3-1是各组小鼠的心脏组织冠状动脉CD31/ICAM-1的免疫荧光染色结果图。CD31/ICAM-1是一种表达于内皮细胞的粘附因子,当它与其配体LFA-1结合时,会促进炎症细胞在内皮上的滚动、黏附和迁移。如图3-1所示,CD31/ICAM-1在CAWS组小鼠冠状动脉内皮细胞的表达水平明显增加,而丁酸杆菌组冠状动脉内皮细胞上CD31/ICAM-1的表达明显下降。
图3-2是各组小鼠心脏组织冠状动脉周围CD68的免疫荧光染色结果图。冠状动脉周围CD68的荧光染色阳性表明单核-巨噬细胞穿过内皮导致冠状动脉炎症。如图3-2所示,CD68在CAWS组小鼠冠状动脉周围的表达水平明显增加,而丁酸杆菌组冠状动脉周围CD68的表达明显下降。
图3-3是各组小鼠结肠组织中的肠紧密连接蛋白Claudin-1、Jam-1、Occludin和ZO-1的免疫荧光染色结果图,结果显示,相较于PBS组,CAWS组小鼠肠紧密连接蛋白Claudin-1、Jam-1、Occludin和ZO-1的荧光强度出现下降,而经丁酸杆菌处理后上述肠紧密连接蛋白的表达均有所增加。
(4)对实施例1提及的各组小鼠粪便进行16S rRNA高通量测序。操作步骤为采集粪便后立即放入液氮中进行16S rRNA基因测序,送至诺和致源公司的二代测序技术对细菌16S rRNA V3和V4区进行扩增和测序,然后在其分析平台上分析数据。
图4-1为肠道菌群结构变化的非监督多元统计方法PCoA结果图,结果显示,各组之间存在明显的菌群差异。
图4-2为Venn图,结果显示,各组小鼠肠道菌群组成之间存在数量差异。
图4-3为LEfSe和LDA评分结果,结果显示,各组小鼠粪便的微生物区系分布存在差异。
图4-4所示,在门和属的水平上,CAWS组小鼠存在明显的肠道菌群失调,经过丁酸杆菌的干预,肠道菌群的相对丰度显著增加,拟杆菌的相对丰度出现了降低。
图4-5所示,CAWS组小鼠肠道中产生SCFAs的细菌明显减少,而丁酸杆菌组有5种产SCFAs菌增加。
以上结果表明,川崎病模型小鼠存在肠道菌群紊乱的情况,丁酸杆菌能改善小鼠肠道内产SCFAs的菌群减少的情况,改善肠道菌群紊乱。
(5)对实施例1提及的各组小鼠结肠组织中的紧密连接蛋白Claudin-1、Jam-1、Occludin和ZO-1以及单羧酸转运体MCT-1和SMCT-1进行实时荧光定量PCR检测,具体操作步骤如下:
I、RNA提取
①将组织在液氮中磨碎,每50~100mg组织加入1ml TRIzol,用匀浆仪进行匀浆处理。样品体积不应超过TRIzol体积10%。
②每1ml Trizol加入0.2ml氯仿,剧烈振荡15s,室温放置5min。
③2~8℃10000×g离心15min。样品分为三层。RNA主要在水相中,水相体积约为所用Trizol试剂的60%。
④把水相转移到新的EP管中加入等体积的异丙醇,室温放置10min。
⑤2~8℃10000×g离心10min,离心前看不出RNA沉淀,离心后在管侧和管底出现胶状白色沉淀。弃上清进行下一步操作。
⑥用75%冰预冷的乙醇洗涤RNA沉淀。每使用1ml Trizol至少加1ml 75%乙醇。2~8℃不超过7500×g离心5min,弃上清。
⑦超净台中风干大约5~10min。
II、实时定量PCR
①利用核酸蛋白分析仪器检测所提RNA的浓度及纯度。
②按照逆转录试剂盒说明书提示合成样品cDNA。
在反应条件为95℃预变性30s;95℃变性5s;60℃退火30s后采集荧光信号,重复40个循环的条件下以合成的cDNA为模板。按荧光定量PCR试剂盒说明书,在25μl体系内进行PCR扩增。引物序列如表1,并以GAPDH为内参,用所得各炎症因子的Ct计算出其相对表达量。
表1用于扩增靶基因的特异性引物
关于紧密连接蛋白Claudin-1、Jam-1、Occludin和ZO-1的基因表达情况如图5-1所示,相对于PBS组,CAWS组小鼠肠紧密连接蛋白Claudin-1、Jam-1、Occludin和ZO-1的mRNA水平出现下降,而经丁酸杆菌处理后紧密连接蛋白的mRNA水平均有所增加。
肠内SCFAs主要通过肠黏膜上的单羧酸转运体(MCT,SMCT)进入体内发挥作用,图5-2为单羧酸转运体MCT-1和SMCT-1的基因表达情况图,结果显示相对于PBS组,CAWS组小鼠结肠MCT-1和SMCT-1的mRNA水平出现下降,而经丁酸杆菌处理后MCT-1和SMCT-1的mRNA水平均有所增加。
(6)对实施例1提及的各组小鼠结肠组织中的紧密连接蛋白Claudin-1、Jam-1、Occludin和ZO-1以及单羧酸转运体MCT-1和SMCT-1进行蛋白质免疫印迹(Western Blot,WB)实验,具体操作步骤如下:
I、组织总蛋白提取
①结肠提总蛋白:取各组结肠组织0.4g,加入400μl组织裂解液(按100:1的比例混合动物组织裂解液和PMSF),然后用组织匀浆器将组织在冰上进行匀浆。将匀浆液放于冰上30min,使脊髓组织充分裂解。然后用低温高速离心机,4℃下,12000rpm离心20min,移液器取上清液。
②Braford法测定蛋白浓度:使用96孔板进行蛋白浓度测定,每孔加入8μl PBS,和2μl待测蛋白液体,最后加入190μl考马斯亮蓝溶液。每个蛋白样品需要做3个复孔,另需要做3个空白孔(10μlPBS溶液,在,加入190μl考马斯亮蓝溶液)进行校准。使用振荡器震荡5min,并用干净的针头戳破气泡。使用酶标仪测定OD值,波长为595nm,根据标准曲线计算出样品蛋白浓度。
③单次上样体积为10μl,动物蛋白上样量为70mg,因而蛋白所需体积为80/c。加入5X loading buffer溶液2μl,再加入纯净水至10μl。配置多次上样量,则将各成分体积都相应翻倍,加完各溶液后混匀,置于100℃沸水中煮10min,室温冷却后,置于-20℃冰箱保存。
II、Western blot检测组织蛋白
①制胶
薄厚板洗净干燥,对齐后垂直放入制胶夹中卡紧。然后垂直卡在制胶架上准备灌胶。选择合适的胶液浓度,依次加入相应制胶试剂(pH=8.8Tris-Hcl缓冲液,pH=6.8Tris-Hcl缓冲液,30%聚丙烯酰胺,ddH2O,10%过硫酸铵,10%SDS),按比例加入TEMED,充分混匀,,快速在两玻璃板的间隙中灌注分离胶,注意避免气泡。同时留出浓缩胶所需的高度(1.5-2.0cm左右)。后立即用加样枪小心的在分离胶上加一层无水乙醇,轻晃使胶线水平,室温静置40分钟待胶完全聚合。当无水乙醇和胶之间有一条折射线时,倒去分离胶上层的无水乙醇,用双蒸水洗涤凝胶顶部2次,除去未聚合的分离胶和残留的无水乙醇,并用滤纸吸干。将配制好的浓缩胶灌满分离胶上层的剩余空间,小心注入避免混入气泡,再小心插入干净的梳子,等待浓缩胶聚合。
②电泳
将凝胶板固定于电泳槽内,加入新的电泳缓冲液,两手分别捏住梳子的两边竖直向上轻轻将其拔出梳子。用加样枪头按预定顺序加蛋白样品,另留左右2孔加入蛋白预染marker。将电泳装置与电源相接,先用80V恒压电泳约1h后,当指示剂进入分离胶或marker清晰地分散后改用120V恒压电泳,当指示剂到达凝胶近下端处时关闭电源,取出胶板。
③转膜
剪下合适大小的0.22μm或0.45μm的PVDF膜,在甲醇中激活30秒左右。把滤纸在预冷的转膜缓冲液中浸泡润湿,将滤纸覆盖于有海绵的黑色转膜夹侧,再放置凝胶,PVDF膜,切记赶除凝胶与PVDF膜之间的气泡,再依次放上浸湿的滤纸和海绵,夹紧夹子。将其按方向放于转膜槽内,倒满转膜液,转膜槽外放碎冰降温,减少转膜过程中产热造成的不良影响。300mA恒流转膜,时间为根据蛋白质量决定。
④封闭
用1×TBST配置5%的脱脂牛奶作为封闭液,涡旋震荡混匀,不能有未凝的牛奶颗粒。转膜结束后,标记膜的正反面后放入封闭液中,室温摇床封闭2小时。
⑤一抗孵育
牛奶封闭结束后,用TBST洗涤3次,每次7分钟,根据所需蛋白质量切出所需位置的PVDF膜,并将对应大小的条带放入相应配置好的一抗中,放4℃低俗摇床孵育过夜,约16-20小时。
⑥二抗孵育
一抗孵育结束后,回收一抗,条带放置孵育盒中,用TBST洗涤3次,每次7分钟,分别加入相应属性的二抗液体中,室温摇床孵育2小时。
⑦曝光
二抗孵育完毕后,用TBST洗涤3次,每次7分钟,配置1:1的显色发光液,用Bio-Rad化学发光成像仪显色。
关于紧密连接蛋白Claudin-1、Jam-1、Occludin和ZO-1蛋白的表达情况如图6-1所示,相对于PBS组,CAWS组小鼠肠紧密连接蛋白Claudin-1、Jam-1、Occludin和ZO-1的蛋白水平出现下降,而经丁酸杆菌处理后紧密连接蛋白均有所增加。
图6-2是单羧酸转运体MCT-1和SMCT-1蛋白表达情况图,如图所示,可见相对于PBS组,CAWS组小鼠结肠MCT-1和SMCT-1蛋白水平出现下降,而经丁酸杆菌处理后MCT-1和SMCT-1蛋白均有所增加。
(7)对实施例1提及的各组小鼠粪便样品进行短链脂肪酸的GC-MS定量分析,具体操作步骤如下:
取50mg的粪便样品,用100μl的50μg/ml异己酸溶液为内标,加入100μl 15%磷酸,400μl乙醚,70Hz,1min匀浆。随后,样品在4℃下离心10分钟(12,000rpm),将上清液转入小瓶,然后进行GC-MS分析。气相色谱采用Agilent HP-INNOWAX(30m×0.25mm i.d.×0.25μm)毛细管柱(Agilent Technologies,Santa Clara,CA,USA),以1ml/min氦气作为载气。分液进样,进样量为1μl,进样温度为250℃,进样量为10:1。离子源温度为230℃,界面温度为250℃,四极杆温度为250℃。初始柱温为90℃,10℃/min升至120℃,5℃/min升至150℃,最后25℃/min升至250℃,并在此温度下持续2min(总运行时间15min)。探测器采用电子碰撞电离模式(电子能量70eV),采用全扫描和选择性离子监测(SIM)模式。
关于各组小鼠粪便中乙酸,丙酸,丁酸,戊酸,异丁酸和异戊酸的含量如图7所示,结果显示,相对于PBS组,CAWS组所检测的SCFAs含量均出现了降低,而经过丁酸杆菌的治疗,上述降低的SCFAs含量出现了上升。
以上所述的实施例仅是对本发明的优选方式进行描述,并非对本发明的范围进行限定,在不脱离本发明设计精神的前提下,本领域普通技术人员对本发明的技术方案做出的各种变形和改进,均应落入本发明权利要求书确定的保护范围内。
Claims (8)
1.一种丁酸杆菌在制备预防和/或治疗川崎病药物中的应用。
2.根据权利要求1所述的应用,其特征在于,所述预防和/或治疗川崎病药物包括预防和/或治疗所述川崎病使用的有效量的丁酸杆菌,所述丁酸杆菌以活细胞形式存在于所述药物中。
3.根据权利要求1或2所述的应用,其特征在于,在所述预防和/或治疗川崎病药物中,所述丁酸杆菌的数目为5×106-6×109CFU/ml。
4.根据权利要求1所述的应用,其特征在于,所述预防和/或治疗川崎病药物还包括与所述丁酸杆菌相配伍的其他药类以及药学上可接受的载体和/或辅料。
5.根据权利要求4所述的应用,其特征在于,所述其他药类包括与所述丁酸杆菌相配伍,不会使所述丁酸杆菌出现中和、水解以及破坏失效理化反应,同时与丁酸杆菌协同作用后,能提高丁酸杆菌预防和/或治疗川崎病效果的药类。
6.根据权利要求1所述的应用,其特征在于,所述预防和/或治疗川崎病药物为药学上可接受的剂型。
7.根据权利要求6所述的应用,其特征在于,所述剂型为粉剂、注射液、胶囊、片剂或口服液。
8.根据权利要求1所述的应用,其特征在于,所述丁酸杆菌由中国微生物菌种保藏管理委员会普通微生物中心所保藏,保藏为丁酸杆菌WZ001菌株,保藏编号为CGMCC No.8808。
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