CN110680830A - 一种溃疡性结肠炎转化动物模型的构建方法 - Google Patents
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Abstract
本发明公开了一种溃疡性结肠炎转化动物模型的构建方法,该方法建立了在DSS急性结肠炎模型中联用艰难梭菌(Clostridium difficile)的炎症转化模型,通过不同的给菌方式,包括腹腔注射、灌胃、灌肠三种手段。该模型优势在于该模型能够显著增加小鼠肠系膜淋巴细胞Th17的比例,并且上调IL‑17的分泌;更接近于临床溃疡性结肠炎患者的病理学特征;小鼠通过灌胃给艰难梭菌的效果最好。
Description
技术领域
本发明涉及动物模型的构建方法,特别涉及一种溃疡性结肠炎转化动物模型的构建方法。
背景技术
艰难梭菌(Clostridium diffficile)是一种孢子形成,革兰氏阳性,厌氧菌,其在1978年被确定为抗生素相关伪膜性结肠炎病因的细菌[Antibiotic-associatedpseudomembranous colitis due to toxin-producing clostridia.N.Engl.J.Med.298,531-534]。尽管过去35年来传染病死亡率总体下降,但美国腹泻病死亡率仍在上升。这一趋势归因于过去十年中艰难梭菌(Clostridium difficile)超强毒株的传播,也包括研究中使用的流行型核糖体027菌株[Binary toxin and death after Clostridium difficileinfection.Emerg.Infect.Dis.17,976-982]。美国疾病控制和预防中心估计仅在美国艰难梭菌每年就会导致近500000例感染和29000例死亡。这些报告和其他报告强调了研究艰难梭菌感染(CDI)和确定降低疾病严重程度的治疗靶点的重要性[Emerging InfectionsProgram C.difficile Surveillance Team(2015).Burden of Clostridium difficileinfection in the United States.N.Engl.J.Med.372,2369-2370]。
溃疡性结肠炎(Ulcerative Colitis,UC):慢性炎症疾病,特点表现为直肠与结肠中的连续黏膜溃疡,起始于直肠,不同程度地扩展,最长可以蔓延到盲肠,其临床表现为腹泻、间断性便血或脓血便、腹痛、呕吐等。但是,其病因至今仍未确定,普遍认为是受到环境、遗传和肠道微生物等因素的影响[Increasing incidence and prevalence of theinflammatory bowel diseases with time,based on systematicreview.Gastroenterology 2012;142:46-54,e42]。其中IBD是CDI的独立危险因素。与非IBD患者相比,IBD患者的CDI更为严重,表现为从入院到CDI的时间缩短、住院时间延长、手术次数增加和住院死亡率增加[Incidence of Clostridium difficile infection ininflammatory bowel disease.Clin.Gastroenterol.Hepatol.5,339-344]。
现有的溃疡性结肠炎(UC)模型常见的动物模型主要为:葡聚糖硫酸钠(DSS)模型、2,4,6-三硝基苯磺酸(TNBS)模型。DSS诱导的结肠炎模型其发病机制DSS是由蔗糖合成的硫酸多糖体,其诱导结肠炎的机制可能与影响DNA合成,通过各种途径造成T细胞、中性粒细胞、巨噬细胞等激活,引起细胞因子表达改变,导致肠上皮屏障破坏有关[Temporal andspatial analysis of clinical and molecular parameters in dextran sodiumsulfate induced colitis.PLoS One,2009,4(6):e6073]。DSS结肠炎表现为浅表性、局灶性炎症和溃疡,全结肠均可发病,炎症主要累及黏膜层和黏膜下层。但是,DSS急性结肠炎模型制作过程中动物死亡率高,停药后8~9d症状自然恢复,影响药物疗效观察,因此适用于发病机制的研究。TNBS结肠炎动物模型发病机制是由乙醇破坏肠黏膜屏障后,TNBS渗入结肠组织,与大分子物质结合形成全抗原,引起肠黏膜的一系列免疫和炎症反应[A Reviewon Chemical-Induced Inflammatory Bowel Disease Models in Rodents.Korean JPhysiol Pharmacol,2014,18(4):279-288]。但是,TNBS模型需严格控制剂量,该模型与Crohn’s病(Crohn’s disease,CD)更为相似,与溃疡性结肠炎相似程度低。目前,针对溃疡性结肠炎的药物研发遇到的主要问题在于,针对免疫反应相关分子和肠道免疫过程的药物靶标发现。随着TNF-α抗体药物在UC中应用的日趋成熟,靶向免疫反应相关分子和肠道免疫过程的药物引发关注,但是其伴随的问题是耐药性的增加。在UC患者的结肠粘膜中IL-17水平较高[Interleukins IL-33 and IL-17/IL-17A in patients with ulcerativecolitis.Hepatogastroenterology,2010,57(104):1442-1444]。这提示IL-17可作为UC的治疗靶点。然而,经典的DSS诱导结肠炎动物模型存在的问题在于小鼠结肠中IL-17的水平并没有显著的增加。因此,需要构建一种新型溃疡性结肠炎转化模型,能够模拟临床溃疡性结肠患者结肠粘膜中IL-17的水平。
发明内容
发明目的:本发明目的是提供一种DSS诱导并联用C.difficile的溃疡性结肠炎转化动物模型。
技术方案:本发明提供一种溃疡性结肠炎转化动物模型的构建方法,先让小鼠饮用葡聚糖硫酸钠(DSS),再通过不同途径给艰难梭菌(Clostridium difficile),之后进行观察和生化指标测定,当出现小鼠结肠中Th1或Th17的数量增加、结肠组织中IL-17的表达增加和炎症因子的表达上调时,则模型构建成功。
进一步地,所述小鼠使用雄性小鼠,相较于雌鼠,雄性性别小鼠受内分泌因素影响较小,体重均一性与造模稳定性更优。
进一步地,所述不同途径为腹腔注射、灌胃或灌肠。通过上述各途径进行菌液感染。
进一步地,所述途径优选为灌胃。
进一步地,所述先让小鼠饮用葡聚糖硫酸钠(DSS)7天,在第8天停止,再给艰难梭菌(Clostridium difficile)。在DSS急性结肠炎模型下,小鼠肠道内Th17细胞数量在造模第三天显著升高,而在肠炎后期Th17细胞数量逐渐降低,这与临床溃疡性结肠炎患者的病理特征不相符,通过在肠炎后期给予艰难梭菌(Clostridium difficile)上调小鼠肠道内Th17的数量,可以更好的模拟临床溃疡性结肠炎的病理特征。在第8天给小鼠艰难梭菌(Clostridium difficile),艰难梭菌(Clostridium difficile)可以根植。
进一步地,所述炎症因子为IL-1β、IL-6或TNF-α。
进一步地,所述模型用于构建DSS合并艰难梭菌(Clostridium difficile)诱导的急性溃疡性结肠炎模型,慢性反复性发作溃疡性结肠炎模型,艰难梭菌(Clostridiumdifficile)感染相关的溃疡性结肠炎模型以及结肠炎癌转化模型。所述模型在急慢性溃疡性结肠炎及结肠炎癌转化相关的治疗药物的临床前药学评价中的用途。
本发明的优先方法如下:
步骤一、实验分组与模型建立
选5周龄雄性C57BL/6小鼠,体重18-20g,小鼠适应性喂养一周,按体重随机分组。
组别设置:健康对照组(n=8)、肠炎转化模型组(n=8)
菌液准备:将培养至生长对数期(48h)的C.difficil菌液,6000rpm*5min*4℃,用在厌氧工作站中预还原24h的生理盐水重悬,取100uL菌液进行OD600读数测定细菌数量,调节细菌浓度至2.5*109cfu/ml。
健康对照组:每日自由进饮用水及饲料
DSS肠炎转化模型组:自由进食,自由饮用2%DSS溶液7天,再自由正常饮水3天,并同时经消化道给予C.difficile菌液(灌胃或者直肠给药),每天一次,连续3天。其中,C.difficile菌液具体给药方式有以下两种方式:
①在第8天至第10天,模型组小鼠每日早上8点禁食,早上10点灌胃给予C.difficile菌液0.2ml,2h后恢复进食,平行的,健康对照组小鼠灌胃等体积的生理盐水。
②在第8天至第10天,每天进行灌肠,先用3%水合氯醛按照0.2ml/20g腹腔注射麻醉小鼠,用连有灌肠针的微量注射器抽取0.2ml的菌液,小心的将针头探入小鼠肛门内,深至3-4cm,过程中避免碰伤肠壁,缓缓推入菌液,为确保致敏液与肠道充分作用,将小鼠头朝下。固定在一个45°角倾斜的平面上,静置30min。小鼠苏醒后,将小鼠放回笼中。平行的,健康对照组小鼠以同样方式经直肠给予等体积的生理盐水。
步骤二、收集样本
第11天,禁食4h后,颈椎脱臼处死小鼠,剪开腹腔,收集脾脏,肠系膜淋巴结,置于含2%FBS的PBS中,放在冰上后待流式测定免疫细胞分群;游离结肠,取出肛门上端1cm处至盲肠末端的整个结肠段,沿肠系膜纵轴剖开,用冰预冷PBS反复冲洗干净,再用清洁滤纸吸干水分。宏观检查并进行评分后,剪取结肠组织近端及远端1cm,置于4%多聚甲醛溶液固定,常规石蜡包埋,切片,留作组织病理学检查及免疫组化染色。结肠组织放入-80℃超低温冰箱存放,待用于RT-PCR和ELISA等检测。
步骤三、观察指标
观察并记录给予2%DSS第1天至第10天小鼠每天体重变化,在第11天记录结肠长度。
步骤四、生化指标测定
流式检测脾脏、肠系膜淋巴结淋巴细胞Th1、Th17含量变化;ELISA检测血清、结肠中IL-17的含量;RT-qPCR检测结肠组织中炎症因子(IL-17、IL-1β、IL-6、TNF-α)的表达。
本发明通过DSS诱导的溃疡性结肠炎与C.difficile联用方式,通过不同的给艰难梭菌途径(腹腔注射、灌胃、灌肠),观察小鼠体重变化,结肠长度,结肠组织学损伤,宏观变现;测定淋巴细胞Th17的数量变化,测定结肠组织、血清中IL-17的含量,检测炎症因子的表达情况,摸索一种更接近临床的溃疡性结肠炎模型。
有益效果:本发明的方法可以显著增加肠道免疫系统Th17的比例,同时上调IL-17的含量;并且通过不同的给艰难梭菌途径(腹腔注射、灌胃、灌肠)。该模型方法将DSS诱导的溃疡性结肠炎模型与艰难梭菌感染引发的结肠炎两者联系在一起,建立一种更适合临床溃疡性结肠炎转化的动物模型,能够更有效地模拟临床溃疡性结肠炎患者体内IL-17水平。该模型应用价值在于,基于临床溃疡性结肠炎患者体内IL-17水平显著升高,通过DSS结肠炎和艰难梭菌联用可以显著提高小鼠肠道免疫中Th17的数量,并且结肠组织中IL-17的表达显著增加;同时炎症因子(IL-1β、IL-6、TNF-α)的表达也显著上调。基于实验结果分析,实验的重现性,操作难易等多方面分析,确定通过灌胃的途径给予小鼠艰难梭菌可以构建一种更优的溃疡性结肠炎转化动物模型。
附图说明
图1:不同途径(腹腔注射、灌胃、灌肠)给予小鼠艰难梭菌对DSS模型造模示意图。
图2:不同途径(腹腔注射、灌胃、灌肠)给予小鼠艰难梭菌对DSS模型小鼠体重(A)和结肠长度(B)影响。*p<0.05,**p<0.01,***p<0.001,vs Control;
图3:不同途径(腹腔注射、灌胃、灌肠)给予小鼠艰难梭菌对DSS模型小鼠结肠近端、结肠远端HE染色分析;
图4:不同途径(腹腔注射、灌胃、灌肠)给予小鼠艰难梭菌对DSS模型小鼠炎症因子的mRNA水平影响。*p<0.05,**p<0.01,***p<0.001,****p<0.0001,vs Vehicle;
图5:不同途径(腹腔注射、灌胃、灌肠)给予小鼠艰难梭菌对DSS模型小鼠脾脏(A)和肠系膜淋巴结(B)淋巴细胞Th1及Th17数量影响。*p<0.05,**p<0.01,***p<0.001,vsDSS;
图6:不同途径(腹腔注射、灌胃、灌肠)给予小鼠艰难梭菌对DSS模型小鼠结肠组织IL-17mRNA水平(A)、血清IL-17含量(B)、结肠组织IL-17(C)的影响。*p<0.05,**p<0.01,***p<0.001,vs DSS。
具体实施方式
实施例1:
本实施例的溃疡性结肠炎转化动物模型的构建方法,包括如下步骤:
一、实验材料
1.实验动物:雄性C57BL/6J小鼠40只、SPF级、鼠龄5wk,体重18-20g,购于上海斯莱克动物中心。
2.菌株:C.difficile ATCC 43255购于American type culture collection(ATCC)
3.主要实验试剂
葡聚糖硫酸钠(DSS)(购于MP Biomedicals公司)、FITC-CD4抗体、PerCP-Cy5.5-CD3e抗体、PE-IFN-γ抗体、Fixation Buffer固定液(均购自Biolegend公司);APC-IL-17A抗体(均购自ebosicence公司);PMA(购于Sigmaaldrich公司)、Ionomycin离子霉素(购于aladdin公司)、含5%FBS 1640培养基(购于Gibco公司)、mouse IL-17A ELISA Kit(购于上海吉泰依科赛公司)、红细胞裂解液(购于碧云天公司)、PBS缓冲液、4%多聚甲醛固定液、Trizol试剂盒(购于Takara公司)、反转录试剂盒(购于诺唯赞公司)、ChamQ SYBR qPCRMaster Mix(购于诺唯赞公司)
二、实验方法
1.艰难梭菌复苏、培养
培养基制备:
称取3.7gBHI培养基粉末、酵母提取物0.5g、刃天青溶液0.4ml、半胱氨酸0.1g,盐溶液4ml,调节Ph至7.2-7.4,煮沸培养基,驱除氧气,121℃高压蒸汽灭菌15min,放入厌氧工作站预还原24h。
1)盐溶液配方:CaCl2 0.2g MgS04 0.2g 加入300ml 去离子水
K2HPO4 1.0g KH2PO4 1.0g NaHCO3 10.0g
NaCl 2.0g 加500ml+200ml 去离子水 并储存在4℃
2)固体培养基:100ml液体培养基中加入2g琼脂粉,混匀;调节Ph至7.2-7.4,煮沸培养基,驱除氧气,121℃高压蒸汽灭菌15min,放入厌氧工作站预还原24h
菌粉活化:在厌氧工作站中,用0.5ml培养基重悬菌粉,轻轻混匀使其溶解;将重悬液转移至含5-6ml培养基的管中培养;菌液培养生长48h,传代。
2.实验分组与模型建立
选雄性C57BL/6J小鼠40只、体重18-20g,小鼠适应性喂养一周,按体重随机分组。组别设置:Con(n=8)、DSS组(n=8)、C.difficile腹腔组(1.0*106cfu/20g,n=8)、C.difficile灌胃组(5.0*108cfu/20g,n=8)、C.difficile灌肠组(5.0*108cfu/20g,n=8)
菌液准备:将培养48h的菌液,6000rpm*5min*4℃,用预还原的生理盐水重悬,调节OD600,调节细菌浓度;腹腔注射组艰难梭菌菌液浓度为5.0*106cfu/ml,灌胃组与灌肠组艰难梭菌菌液浓度为2.5*109cfu/ml。
Con组:每日自由进饮用水及饲料
DSS组:自由饮用2%DSS溶液7天,替代饮用水,自由进食饲料,再自由饮水3天。
C.difficile腹腔组(DSS/ip):自由饮用2%DSS溶液7天,替代饮用水,自由进食饲料,再自由饮水3天;在Day8(停止DSS),腹腔注射C.difficile菌液,连续3天。
C.difficile灌胃组(DSS/ig):自由饮用2%DSS溶液7天,替代饮用水,自由进食饲料,再自由饮水3天;在Day8(停止DSS),灌胃C.difficile菌液,连续3天。
C.difficile灌肠组(DSS/pr):自由饮用2%DSS溶液7天,替代饮用水,自由进食饲料,再自由饮水3天;在Day8(停止DSS),灌肠C.difficile菌液,连续3天。
在第8天至第10天,每组小鼠每日早上8点禁食,早上10点给予C.difficile菌液,2h后恢复进食。
灌肠步骤:3%水合氯醛按照0.2ml/20g腹腔注射麻醉小鼠,用连有灌肠针的微量注射器抽取0.2ml的菌液,小心的将针头探入小鼠肛门内,深至3-4cm,过程中避免碰伤肠壁,缓缓推入菌液,为确保致敏液与肠道充分作用,将小鼠头朝下。固定在一个45°角倾斜的平面上,静置30min。小鼠苏醒后,将小鼠放回笼中。
3.收集样本
第11天,禁食4h,采血;颈椎脱臼处死小鼠,剪开腹腔,收集脾脏,肠系膜淋巴结,置于含2%FBS的PBS中,放在冰上后续测流式;游离结肠,取出肛门至盲肠末端的整个肠段,沿肠系膜纵轴剖开,用冰冷PBS反复冲洗干净,再用清洁滤纸吸干水分。宏观检查并进行评分后,剪取结肠组织近端及远端1cm,置于4%多聚甲醛溶液固定,常规石蜡包埋,切片,留作免疫组化染色。结肠组织放入低温冰箱-80℃存放,用于RT-PCR和ELISA实验。
实施例2:观察指标
观察并记录给予2%DSS第1天至第10天小鼠每天体重变化,在第11天记录结肠长度。
实施例3:生化指标测定
1.1流式测定脾脏、肠系膜淋巴结淋巴细胞Th1、Th17比例
1)取出置于2%FBS的PBS中的肠系膜淋巴结和脾脏组织,转移到40um蓝色筛网上,在筛网下方放置无菌六孔板,用2.5ml注射器胶塞分别研磨肠系膜淋巴结和脾脏,并用4ml含2%FBS的PBS冲洗筛网,将滤液转移至10ml无菌离心管中,离心800g*20min*4℃。
2)离心结束后,弃去上清,其中脾脏用1ml红细胞裂解液重悬,冰上裂解10min后离心800g*5min*4℃;淋巴结直接用500ul含2%FBS的PBS重悬,4℃暂存。
3)脾脏离心结束后,弃去上清,用500ul含2%FBS的PBS重悬,4℃暂存,用1ml5%FBS 1640培养基重悬细胞并计数,调整细胞浓度至1*106个/ml,4℃暂存。
4)细胞培养:将细胞浓度调整至2*106个/ml的细胞悬液接种于24孔板中(阴性组,单染组(4组),实验组)。先每孔加入含2*106个细胞的细胞悬液,再用含50ng/ml(80nM)PMA和1ug/ml(1.41uM)Ion的5%FBS 1640培养基定容至800ul,孵育1h后,每孔加入含10ug/ml的BFA的5%FBS 1640培养基200ul,继续孵育3h。
5)细胞表面染色:孵育结束后,收集细胞于1.5ml EP管中,离心弃上清,并用1mlstaining buffer清洗一次,1000g*5min,4℃。再用staining buffer定容至100ul/管(即2*106个/100ul/管),并加入封闭抗体CD16/32,0.2ul/个,4℃孵育10min。封闭结束后,加入FITC-CD4(0.5ul/2*106/100ul)、PerCP-Cy5.5-CD3e(1.25ul/2*106/100ul)4℃避光染色30min。
6)细胞核内染色:表面染色结束后,离心1000g*5min,弃上清,并用1ml stainingbuffer重悬细胞,清洗一次。清洗结束后,用Fixation Buffer固定细胞,500ul/管,室温避光20min。固定结束后,1000g*5min离心弃上清,并用500ul 1X破膜液重悬固定好的细胞,清洗一次。清洗结束后,弃上清,用100ul 1X破膜液重悬固定破膜后的细胞,并加入相应的荧光标记抗体PE-IFN-γ(1.25ul/2*106/100ul)、APC-IL-17A(0.625ul/2*106/100ul),4℃避光染色30min。染色结束后,1000g*5min,4℃离心弃上清,并用1ml staining buffer重悬细胞,清洗一次。清洗结束后,用200ul staining buffer重悬细胞,流式检测。
流式方案:
FL1:FITC-CD4:0.5ul/2*106/100ul
FL2:PE-IFN-γ:1.25ul/2*106/100ul
FL3:PerCP-Cy5.5-CD3e:1.25ul/2*106/100ul
FL4:APC-IL-17A:0.625ul/2*106/100ul
细胞群:
CD3细胞:CD3+CD4-IFN-γ-IL-17-
CD4细胞:CD3+CD4+IFN-γ-IL-17-
Th17细胞:CD3+CD4+IFN-γ-IL-17+
Th1细胞:CD3+CD4+IFN-γ+IL-17-
1.2结肠近端及远端样本的HE染色
1)石蜡组织块4μm连续切片,组织切片置烤箱60℃30min立即放入二甲苯脱蜡两次,每次10min。
2)无水乙醇(I)10分钟,无水乙醇(II)10min,依次95%、85%、75%梯度酒精各1min,水洗2-3次。
3)苏木素染色3-5min,自来水冲洗2-3次。(吸干水)
4)0.25%氨水返蓝1s,水洗3次。
5)1%伊红染色2min,水洗3次。
6)常规脱水、透明、封片:75%、80%、85%、95%乙醇、无水乙醇II、无水乙醇I脱水,各级2-3min,二甲苯透明两次,各10min,中性树胶封片,镜检。
1.3ELISA法测定血清、结肠组织中IL-17的含量
1.3.1样本的处理
结肠组织上清提取:用冷PBS涮洗结肠,滤纸吸干;在冰板上,纵剖剪取10mg左右的结肠组织,剪碎放置匀浆管中(冰上),每管加入200ulPBS溶液(含PMSF,1∶100),研磨4200-3*15-2两次,每次间歇放冰上静置,离心13000g*10min*4℃,吸取上清至1.5mlEP管,BCA定蛋白浓度:1ul蛋白母液+19ul超纯水即稀释20倍。
血清样本收集:血清7000rpm*10min离心,收集上清。
1.3.2具体操作步骤如下:
1)从冰箱取出试剂盒,室温复温平衡20分钟。
2)配液:用试剂稀释液将标本(结肠上清、血清)稀释一倍(1∶1)
3)分别将标本或不同浓度标准品加入相应孔中(100ul/孔),用封板胶纸封住反应孔,37℃孵箱孵育90分钟。
4)提前30分钟制备生物素化抗体工作液
5)洗板5次,弃去液体,吸水纸上拍干,每孔加满洗涤液,甩去洗涤液,吸水纸上拍干,如此重复洗板4次。
6)除空白孔外,加入生物素化抗体工作液(50ul/孔)。用封板胶纸封住反应孔,37℃孵箱孵育60分钟。
7)提前30分钟制备酶结合物工作液。室温避光放置。
8)洗板5次,弃去液体,吸水纸上拍干,每孔加满洗涤液,甩去洗涤液,吸水纸上拍干,如此重复洗板4次。
9)除空白孔外,加入酶结合物工作液(100ul/孔)。用封板胶纸封住反应孔,37℃孵箱,避光孵育30分钟。
10)洗板5次,弃去液体,吸水纸上拍干,每孔加满洗涤液,甩去洗涤液,吸水纸上拍干,如此重复洗板4次。
11)加入显色底物(包括空白孔)100ul/孔,37℃孵箱,避光孵育15分钟。
12)加入终止液(包括空白孔)100ul/孔,混匀后即刻测量OD450值(10分钟内)
13)计算:计算:根据标准品的浓度及对应的OD值,计算出标准曲线的直线回归方程,再根据样本的OD值,在回归方程上计算出对应的IL-17样品浓度。最终浓度为实际测定浓度乘以稀释倍数。
1.4 RT-PCR技术检测结肠组织内炎症因子mRNA的表达情况:
1.4.1提取结肠组织总RNA
1)从-80℃冰箱中取出结肠样本,置于冰上,在-20℃冰板上展开,从上到下剪下适当组织,置于万分之一天平上称取约10mg(±0.5)组织,在冰板上剪碎。放入匀浆管中(冰上)。
2)匀浆管中加入1ml/管Trizol,打匀浆4000-3*20(2次),至组织被完全打匀。
3)将匀浆管中液体转入1.5ml离心管中,离心12000g*5min*4℃,取上层Trizol800ul至1.5ml EP管中,孵育匀浆样品,室温5min,以保证核蛋白复合体完全分离,加入160ul氯仿(5∶1),用力摇动15s,静置5min,离心12000g*15min*4℃。
4)离心结束后,小心取上层液体300ul+300ul异丙醇,轻轻上下颠倒3-5次,室温静置10min(或4°静置1h),离心12000g*10min*4℃。
5)离心结束后,弃去上清,加入1ml/管75%乙醇(DEPC水),离心12000g*10min*4℃。
6)离心结束后,弃去上清快甩,吸干底部75%乙醇,室温挥发5分钟后置于冰上。
7)每管加入20ul DEPC水复溶,快甩至底部,300rpm混匀5min,快甩,吸取2ul RNA溶液加到Nanodrop仪器上检测RNA浓度。
补的DEPC水量(ul)=5*RNA浓度/0.5-5(只适用于2ul RNA溶液体积的稀释量)
8)从剩下的18ul RNA溶液中吸取5ul加入到200ul EP管中,补加刚才算出的DEPC水量,快甩至底部,涡旋点震混匀,终浓度=0.5mg/ml冰上暂存。剩余RNA母液转移至200ulEP管,-80°保存
9)配置RT溶液(诺维赞,15min)
RT混合液(5X) 2ul
RNA溶液 1ul
DEPC水 7ul
10)逆转录成cDNA,-20℃保存。
11)稀释cDNA:10ul cDNA中加入40ul DEPC水,混匀,稀释5倍。
12)进行PCR:
13)BioRad荧光定量PCR仪,设定PCR反应程序:预变性,95℃90s;95℃10s,60℃30s,72℃30s,40个循环。收集荧光实时定量PCR数据,分析结果。
1.5结果
1、不同的给艰难梭菌途径(腹腔注射、灌胃、灌肠)对DSS诱导结肠炎的影响
给予2%质量体积比(w/v)的DSS溶液(确保水瓶的盖子塞紧,且出水口不堵塞,能流畅出水)自由饮用7天后更换正常饮用水3天建立急性结肠炎小鼠模型。空白对照组始终给予正常饮水。在第八天(即停止给予2%DSS当天),腹腔组(DSS/ip组)腹腔注射0.2ml菌液(1*106cfu/20g)、灌胃组(DSS/ig组)灌胃给予0.2ml菌液(5.0*108cfu/20g)、灌肠组(DSS/pr组)灌肠给予0.2ml菌液(5.0*108cfu/20g),连续给予3天(图1)。每天观察记录小鼠体重变化情况(图2A)。第11天小鼠眼眶取血后,颈椎脱臼处死各组小鼠,收取脾脏、肠系膜淋巴结及结肠标本。测量小鼠结肠长度(图2B)。HE染色分析小鼠结肠组织病理学改变(图3)。RT-qPCR技术考察结肠组织炎症因子(IL-6、TNFa、IL-1β)的变化(图4)。
不同的给艰难梭菌途径(腹腔注射、灌胃、灌肠)对溃疡性结肠炎的造模结果见图1、2、3。实验结果如下,相比于空白对照组,模型组及不同的给艰难梭菌途径(腹腔注射、灌胃、灌肠)组小鼠体重和结肠长度显著降低。同时,通过HE染色分析结肠组织损伤(图3),可以看出,相比于对照组,模型组及不同的给艰难梭菌途径(腹腔注射、灌胃、灌肠)组结肠近端都存在严重的结肠组织损伤,炎症细胞浸润增加;同样在结肠远端也可以看出,模型组及不同的给艰难梭菌途径(腹腔注射、灌胃、灌肠)组的结肠近端也存在严重的组织损伤,炎症因子浸润和上皮细胞增生增加,其中灌胃组和灌肠组更为显著。通过RT-qPCR可以看出,相比于空白对照组,模型组及不同的给艰难梭菌途径(腹腔注射、灌胃、灌肠)组IL-6、TNFa的mRNA水平显著上调,但是在IL-1β的mRNA水平上,仅有灌胃组显著增加。因此,从病理炎症角度考察,可能灌胃给菌造模是更优的溃疡性结肠炎模型。
2、在肠炎下不同的给艰难梭菌途径(腹腔注射、灌胃、灌肠)对肠道免疫Th17淋巴细胞的作用
接下来想探索不同的给艰难梭菌途径(腹腔注射、灌胃、灌肠)对Th17及IL-17的作用。通过流式细胞技术考察脾脏(图5A)和肠系膜淋巴结(图5B)中Th1、Th17数量的变化。在脾脏淋巴细胞中,相比于模型组,在给菌三组中Th1与Th17没有显著增加;虽然在肠系膜淋巴结淋巴细胞中相比于造模组,在给菌三组中Th1没有显著上调,但是在给菌三组中Th17显著上调,其中灌胃组和灌肠组效果最显著。另外,还通过结肠组织RT-qPCR分析(图6A),得到结肠组织中灌胃组和灌肠组的IL-17的转录水平显著增加;在血清和结肠组织中ELISA分析(图6B、图6C)可以看出,在血清上在给菌三组中IL-17都有显著增加,而在结肠组织中,仅有灌胃组和灌肠组的IL-17含量显著增加。
综上所述,对于优化溃疡性结肠炎模型,通过对不同的给艰难梭菌途径(腹腔注射、灌胃、灌肠)病理炎症角度和生化分析,灌胃和灌肠的方式给小鼠艰难梭菌(C.difficile)是更好的选择。但是,基于实验结果的分析,模型的重现性和稳定性,小鼠灌肠给菌操作过程的繁琐,认为灌胃给予小鼠艰难梭菌是构建DSS结肠炎联用艰难梭菌溃疡性结肠炎模型更优途径。
Claims (8)
1.一种溃疡性结肠炎转化动物模型的构建方法,其特征在于:先让小鼠饮用葡聚糖硫酸钠(DSS),再通过不同途径给艰难梭菌(Clostridium difficile),之后进行观察和生化指标测定,当出现小鼠结肠中Th1或Th17的数量增加、结肠组织中IL-17的表达增加和炎症因子的表达上调时,则模型构建成功。
2.根据权利要求1所述的溃疡性结肠炎转化动物模型的构建方法,其特征在于:所述小鼠使用雄性小鼠。
3.根据权利要求1所述的溃疡性结肠炎转化动物模型的构建方法,其特征在于:所述不同途径为腹腔注射、灌胃或灌肠。
4.根据权利要求1所述的溃疡性结肠炎转化动物模型的构建方法,其特征在于:所述途径为灌胃。
5.根据权利要求1所述的溃疡性结肠炎转化动物模型的构建方法,其特征在于:所述先让小鼠饮用葡聚糖硫酸钠(DSS)7天,在第8天停止,再给艰难梭菌(Clostridiumdifficile)。
6.根据权利要求1所述的溃疡性结肠炎转化动物模型的构建方法,其特征在于:所述炎症因子为IL-1β、IL-6或TNF-α。
7.根据权利要求1-6任一项所述的溃疡性结肠炎转化动物模型的构建方法,其特征在于:所述模型用于构建DSS合并艰难梭菌(Clostridium difficile)诱导的急性溃疡性结肠炎模型,慢性反复性发作溃疡性结肠炎模型,艰难梭菌(Clostridium difficile)感染相关的溃疡性结肠炎模型以及结肠炎癌转化模型。
8.根据权利要求1-6任一项所述的溃疡性结肠炎转化动物模型的构建方法,其特征在于:所述模型在急慢性溃疡性结肠炎及结肠炎癌转化相关的治疗药物的临床前药学评价中的用途。
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