CN115340960A - 猪肠类器官与产肠毒素性大肠杆菌或巨噬细胞共培养体系的构建方法 - Google Patents
猪肠类器官与产肠毒素性大肠杆菌或巨噬细胞共培养体系的构建方法 Download PDFInfo
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- CN115340960A CN115340960A CN202210268503.1A CN202210268503A CN115340960A CN 115340960 A CN115340960 A CN 115340960A CN 202210268503 A CN202210268503 A CN 202210268503A CN 115340960 A CN115340960 A CN 115340960A
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Abstract
本发明属于生物技术领域,公开了一种猪肠类器官与产肠毒素性大肠杆菌或巨噬细胞共培养体系的构建方法,包括如下步骤:步骤1:准备产肠毒素性大肠杆菌或巨噬细胞;步骤2:取仔猪空肠前段组织进行处理得到肠隐窝;步骤3:用稀释后的基质胶重悬肠隐窝,将产肠毒素性大肠杆菌或巨噬细胞、肠隐窝接种于提前温育的孔板上,于孔板中加入类器官生长培养基,培养。本发明的培养基针对于猪肠类器官与产肠毒素性大肠杆菌或巨噬细胞共培养体系的生长特点,选用了多种生长因子成份进行调和、优化了孔板中大肠杆菌和巨噬细胞的浓度,经过不断优化培养基中生长因子的配比,使猪肠类器官与产肠毒素性大肠杆菌、巨噬细胞能够有效地进行接触性共培养。
Description
技术领域
本发明涉及生物技术领域,具体涉及一种猪肠类器官与产肠毒素性大肠杆菌或巨噬细胞共培养体系的构建方法。
背景技术
传统的单层细胞模型和动物模型存在根本性缺陷,单层细胞模型只附着于培养皿上单层生长,它不能模拟正常机体细胞复杂的生存环境(Rahmani et al.,2019)。动物模型虽可模拟体内的生存环境,但其具有培养周期长、费用高等问题,无法满足猪精准营养研究需要(Almeqdadi et al.,2019)。因此近年来研究者们开始关注类器官培养技术,类器官是3D细胞培养,生长在细胞外基质中的干细胞可利用自我分化的能力形成三维腺腔样结构,其能够高度还原肠道上皮等组织结构特异性,更准确地模拟体内的微环境,真实反应细胞异质性与多样性,以及细胞间、细胞与基质间的相互作用(Gjorevski et al.,2016;Lukonin et al.,2020)。
研究表明,肠道干细胞可以在体外分化建立完整的隐窝-绒毛轴(Sato et al.,2009)。通过加入适当的生长因子,肠道干细胞可以在基质胶中存活下来。透过显微镜观察,可以发现许多立体的空心球状结构,它们保留了类似于动物本身的肠道上皮结构,所以被称之为肠道类器官,该培养模型已被证明是在分子水平上研究肠黏膜损伤修复的强大系统(Yu et al.,2017)。
CN201811066368.2公开了一种猪肠道隐窝分离和3D类器官培养的方法,猪肠道3D类器官的培养,所用培养液为含生长因子的DMEM/F12培养液,其配方为:在DMEM/F12培养液中加入100ng/ml的Wnt3a,500ng/ml的R-spondin1,100ng/ml的Noggin,1×的血清替代物B27supplement和N2supplement,1mmol/L n-Acetyl Cysteine,50ng/ml的重组人表皮生长因子(EGF),10μmol/L的SB202190,即P38抑制剂,500nmol/L的LY2157299,即TGFβ抑制剂,10μmol/L的Y27632,即ROCK1抑制剂,10mmol/L的Nicotinamide,100μg/ml的Primocin。
在采用肠隐窝和大肠杆菌进行共培养的过程中,会发现肠隐窝凋亡,导致实验失败。
本案解决的技术问题是:如何实现肠隐窝和大肠杆菌或巨噬细胞共培养,以提高培养成功率。
发明内容
本发明的目的是在于提供一种猪肠类器官与产肠毒素性大肠杆菌或巨噬细胞共培养体系的构建方法,本发明的培养基针对于猪肠类器官与产肠毒素性大肠杆菌或巨噬细胞共培养体系的生长特点,选用了多种生长因子成份进行调和、优化了孔板中大肠杆菌的浓度,经过不断优化培养基中生长因子的配比,使猪肠类器官与产肠毒素性大肠杆菌能够有效地进行接触性共培养。
为实现上述目的,本发明提供如下技术方案:一种猪肠类器官与产肠毒素性大肠杆菌或巨噬细胞共培养体系的构建方法,包括如下步骤:
步骤1:准备产肠毒素性大肠杆菌或巨噬细胞;
步骤2:取仔猪空肠前段组织进行处理得到肠隐窝;
步骤3:采用类器官生长培养基稀释减生长因子的基质胶,用稀释后的基质胶重悬肠隐窝,按照低于1*105个产肠毒素性大肠杆菌/10ul或500-1000个巨噬细胞/10ul、10-20个肠隐窝/10ul接种于提前温育的孔板上,静置,使基质胶凝固,于孔板中加入类器官生长培养基,置于37℃含5%CO2培养箱中,培养,每两天更换一次类器官生长培养基;
所述类器官生长培养基含如下成分:
基础培养基:Advanced DMEM/F12;
L-WRN cells条件性培养基40-50vol%;
100X规格的N2 0.9-1.1倍体积;
100X规格的B27-supplement 0.9-1.1倍体积;
45-55ng/ml EGF;
9-11μM Y27632;
0.45-0.55μM LY2157299;
9-11μM SB202190;
9-11μM CHIR99021;
0.9-1.1mM HEPES;
0.9-1.1mM N-acetylcysteine;
100X规格的Glutamax 0.9-1.1倍体积;
0.9-1.1mM valproic acid。
在上述的猪肠类器官与产肠毒素性大肠杆菌或巨噬细胞共培养体系的构建方法中,含如下成分:含如下成分:
基础培养基:Advanced DMEM/F12;
L-WRN cells条件性培养基40-50vol%;
100X规格的N2 1倍体积;
100X规格的B27-supplement 1倍体积;
50ng/ml EGF;
10μM Y27632;
0.5μM LY2157299;
10μM CHIR99021;
10μM SB202190;
1mM HEPES;
1mM N-acetylcysteine;
100X规格的Glutamax 1倍体积;
1mM valproic acid。
在上述的猪肠类器官与产肠毒素性大肠杆菌或巨噬细胞共培养体系的构建方法中,所述步骤1具体为:
实验前一天准备灭菌的LB液体培养基、LB固体培养基、灭菌ddH2O;
将产肠毒素性大肠杆菌ETEC K88接种至液体培养基中,选取对数期的产肠毒素性大肠杆菌ETEC K88处理细胞,用PBS清洗ETEC K88,然后用DMEM进行稀释。
在上述的猪肠类器官与产肠毒素性大肠杆菌或巨噬细胞共培养体系的构建方法中,所述步骤2具体为:
将仔猪空肠前段组织冲洗后迅速放入含冰冷PBS缓冲液的离心管中;
取平皿,倒入冰冷PBS缓冲液,将肠段转移至大平皿进行清洗,清洗后纵向剪开;
在体视显微镜下用眼科镊剥离肠道浆膜,用镊子夹住肠段在冷PBS缓冲液中彻底冲洗干净;
将含有1%BSA的冷的PBS加入离心管中,用镊子夹住洗净的肠段一端,悬在离心管管口,用手术剪将肠段剪成1-2mm大小的组织块,让这些组织块落入含有1%BSA的冷的PBS中;
用含有1%BSA的冷的PBS润湿移液管,将肠组织块轻轻上下吹打三次;等肠组织块沉底后,吸出上清液,留下的液面刚好没过组织块即可;如此重复直至上清澄清为止;
除去上清液,将肠组织块重悬于室温的消化液中,在室温下置于摇床上以40rpm的转速孵育2h,其中消化液的成分为5mMEDTA、1mM二硫苏糖醇和1倍体积的青链霉素;
将组织块通过100um的过滤网进行过滤,将滤液收集于50ml离心管中,将离心管置于冰上;
将滤液进行4℃,300g离心5分钟;弃上清,再重悬于含有1%BSA的冷的PBS中,用含有1%BSA的冷的PBS润湿5mL的移液管,将沉淀轻轻上下吹打三次;
然后进行4℃,200g离心5分钟,弃上清,将肠隐窝沉淀留在管底。
与现有技术相比,本发明的有益效果是:
本发明属于细胞培养技术领域,具体涉及一种猪肠类器官与产肠毒素性大肠杆菌或巨噬细胞共培养体系的构建。所述猪肠类器官与产肠毒素性大肠杆菌或巨噬细胞共培养体系专用培养基由Advanced DMEM/F12;L-WRN cells条件性培养基40-50%,该上清液为富含Wnt3α,R-spondins,Noggin蛋白因子的条件性培养基;1倍体积的N2(100X),1倍体积的B27-supplement(100X),50ng/ml EGF,10μM Y27632,0.5μM LY2157299,10μMCHIR99021,10μM SB202190,1mM HEPES,1mM N-acetylcysteine,1x Glutamax,1mM valproic acid组成。本发明的培养基针对于猪肠类器官与产肠毒素性大肠杆菌或巨噬细胞共培养体系的生长特点,选用了合适浓度的大肠杆菌、多种生长因子成份进行调和,经过不断优化培养基中生长因子的配比,使猪肠类器官与产肠毒素性大肠杆菌能够有效地进行接触性共培养。。
附图说明
图1为本发明的实施例1的产肠毒素性大肠杆菌培养生长曲线图;
图2为本发明的实施例1的肠隐窝分离后的状态图;
图3为本发明的实施例1的猪肠类器官不同发育时间光镜图;
图4为本发明的实施例1的猪肠类器官与ETEC K88接触性共培养光镜图;
图5为本发明的对比例1-5的猪肠类器官与ETEC K88接触性共培养光镜图。
图6猪肠类器官与巨噬细胞3D421接触性共培养光镜图。
具体实施方式
下面将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。
实施例1
参考图1,一种产肠毒素性大肠杆菌培养的方法,至少包括以下步骤:
一、ETEC K88的培养
实验前一天准备灭菌的LB液体培养基、LB固体培养基、灭菌ddH2O。将500ul ETECK88接种至50ml液体培养基中,混匀后分别分装至15ml离心管,每管10ml,37℃200rpm摇菌。
分别于接种后0、2、4、8、12、16、20、24h收样,收取相应时间样品测定OD600并进行涂板测定生长曲线,样品收取后4℃冰箱保存。通过生长曲线确定ETEC K88对数期是在8h.后期选取对数期的ETEC K88处理细胞,用PBS清洗ETEC K88,然后用DMEM进行稀释(图1)。
二、猪肠类器官分离
将仔猪空肠前段组织冲洗后迅速放入含冰冷PBS缓冲液的50ml离心管中;
取15cm大平皿,倒入冰冷PBS缓冲液,将肠段转移至大平皿进行清洗,清洗后纵向剪开;
在体视显微镜下用眼科镊剥离肠道浆膜,用镊子夹住肠段在冷PBS缓冲液中彻底冲洗干净;
将15ml含有1%BSA的冷的PBS加入50ml离心管中,用镊子夹住洗净的肠段一端,悬在50ml离心管管口,用手术剪将肠段剪成1-2mm大小的组织块,让这些组织块落入含有1%BSA的冷的PBS中;
用含有1%BSA的冷的PBS润湿5mL的移液管,将肠组织块轻轻上下吹打三次。等肠组织块沉底后,吸出上清液,留下的液面刚好没过组织块即可;如此重复直至上清澄清为止;
除去上清液,将肠组织块重悬于30ml室温(20-25℃)的消化液中,在室温(20-25℃)下置于摇床上以40rpm的转速孵育2h,其中消化液的成分为5mM EDTA、1mM二硫苏糖醇和1倍体积的青链霉素;
将组织块通过100um的过滤网进行过滤,将滤液收集于50ml离心管中,将离心管置于冰上;
将滤液进行4℃,300g离心5分钟。弃上清,再重悬于含有1%BSA的冷的PBS中,用含有1%BSA的冷的PBS润湿5mL的移液管,将沉淀轻轻上下吹打三次;
然后进行4℃,200g离心5分钟,弃上清,将肠隐窝沉淀留在管底(图2)。
三、3D共培养
将减生长因子的matrigel分装到1.5ml离心管中,实验开始前一天放于4℃冰箱中提前融化,并于实验当天取出放在冰上;
实验当天将24孔板放于37℃培养箱中提前加热;
将第(一)步中培养的产肠毒素性大肠杆菌和第(二)步中分离得到的肠隐窝重悬于含10mM HEPES的DMEM/F12中,用含10mM HEPES的DMEM/F12预先润湿的1mL移液管,将沉淀轻轻上下吹打三次;
用含10mM HEPES的DMEM/F12预先润湿的10ul移液管,吸出10ul于血细胞计数仪上,然后在倒置显微镜光镜下进行计数,计数完成后,将类器官进行4℃200g离心3min;
根据2:3的比例用室温的类器官生长培养基稀释减生长因子的Matrigel,根据计数结果,用稀释后的Matrigel重悬隐窝,按照1*10^5个产肠毒素性大肠杆菌/10ul和10-20个隐窝/10ul接种于提前温育的24孔板上;
将接种好的隐窝的24孔板,放于37℃培养箱中静置10-20min,让Matrigel充分凝固。
使用移液枪沿着孔壁加入500ul预热的的类器官生长培养基,置于37℃含5%CO2培养箱中,培养基的配方为:Advanced DMEM/F12;L-WRN cells条件性培养基40-50%,该上清液为富含Wnt3α,R-spondins,Noggin蛋白因子的条件性培养基;1倍体积的N2(100X),1倍体积的B27-supplement(100X),50ng/ml EGF,10μM Y27632,0.5μMLY2157299,10μMCHIR99021,10μM SB202190,1mM HEPES,1mM N-acetylcysteine,1x Glutamax,1mMvalproic acid(图4)。
图3是指采用上述培养基对猪肠类器官进行单独培养的图。图4是指共培养的光镜图。
通过图4可以看到,本发明的共培养体系中有代表隐窝的球状或管状的隐窝,说明共培养体系生长良好,对于产肠毒素性大肠杆菌对于肠道的影响的体系的构建和影响的探讨具有积极意义。
对比例1
与实施例1大体相同,不同的地方在于,接种大肠杆菌的浓度增加一倍,达到了2*10^5个产肠毒素性大肠杆菌/10ul的浓度去接种。
通过图5的对比培养基1的显微照片可以看出,肠隐窝完全凋亡。
通过上述的测试可以得到结论:
高浓度的大肠杆菌会对肠隐窝造成极大的伤害。
低于1*10^5个产肠毒素性大肠杆菌/10ul的浓度的大肠杆菌可以模拟不同浓度情况下的肠道损伤情况。
对比例2
与实施例1大体相同,不同的地方在于,培养基的配方为:Advanced DMEM/F12;L-WRN cells条件性培养基40-50%,该上清液为富含Wnt3α,R-spondins,Noggin蛋白因子的条件性培养基;1倍体积的N2(100X),1倍体积的B27-supplement(100X),10ng/ml EGF,10μMY27632,0.5μM LY2157299,10μM CHIR99021,10μM SB202190。
通过图5的对比培养基2的显微照片可以看出,肠隐窝完全凋亡。
通过该对比例可以发现,EGF的用量对实验影响显著。
对比例3
与实施例1大体相同,不同的地方在于,培养基的配方为:Advanced DMEM/F12;L-WRN cells条件性培养基40-50%,该上清液为富含Wnt3α,R-spondins,Noggin蛋白因子的条件性培养基;1倍体积的N2(100X),1倍体积的B27-supplement(100X),50ng/ml EGF,10μMY27632,0.5μM LY2157299,10μM CHIR99021,1mM HEPES,1mM N-acetylcysteine,1xGlutamax,1mM valproic acid。
通过图5的对比培养基3的显微照片可以看出,肠隐窝生长不及实施例1。
通过实验结果可以看出,SB202190对于隐窝细胞在共培养状态下的生长是有利的。
对比例4
与实施例1大体相同,不同的地方在于,培养基的配方为:Advanced DMEM/F12;L-WRN cells条件性培养基40-50%,该上清液为富含Wnt3α,R-spondins,Noggin蛋白因子的条件性培养基;1倍体积的N2(100X),1倍体积的B27-supplement(100X),50ng/ml EGF,10μMY27632,0.5μM LY2157299,10μM SB202190,1mM HEPES,1mM N-acetylcysteine,1xGlutamax,1mM valproic acid。
通过图5的对比培养基4的显微照片可以看出,肠隐窝生长不及实施例1。
通过实验结果可以看出,CHIR99021对于隐窝细胞在共培养状态下的生长是有利的。
对比例5
与实施例1大体相同,不同的地方在于,培养基的配方为:Advanced DMEM/F12;L-WRN cells条件性培养基40-50%,该上清液为富含Wnt3α,R-spondins,Noggin蛋白因子的条件性培养基;1倍体积的N2(100X),1倍体积的B27-supplement(100X),10ng/ml EGF,0.5μM LY2157299。
通过图5的对比培养基5的显微照片可以看出,肠隐窝完全凋亡。
通过实验结果可以看出,在抑制剂明显缺失且EGF用量明显降低的情况下,肠隐窝完全凋亡。
实施例2
一、猪巨噬细胞培养
3D421猪巨噬细胞用完全培养液配置:取450mLDMEM高糖培养基,加入50mL胎牛血清(FBS)和1x青链霉素,充分混匀后4℃保存,使用前37℃预热;
3D421猪巨噬细胞培养:完全培养液于37±0.5℃、5%CO2及饱和湿度的细胞培养箱内培养至猪巨噬细胞融合度达到80%-95%进行传代,传代时除去原培养基,加入新鲜培养基轻柔吹打,收集细胞,以1:4或1:5比例进行传代;
二、猪肠类器官分离参考实施例1。
三、巨噬细胞和肠道类器官共培养
将减生长因子的matrigel分装到1.5ml离心管中,实验开始前一天放于4℃冰箱中提前融化,并于实验当天取出放在冰上;
实验当天将24孔板放于37℃培养箱中提前加热;
将第(一)步中培养的巨噬细胞和第(二)步中分离得到的肠隐窝重悬于含10mMHEPES的DMEM/F12中,用含10mM HEPES的DMEM/F12预先润湿的1mL移液管,将沉淀轻轻上下吹打三次;
用含10mM HEPES的DMEM/F12预先润湿的10ul移液管,吸出10ul于血细胞计数仪上,然后在倒置显微镜光镜下进行计数,计数完成后,将类器官进行4℃200g离心3min;
根据2:3的比例用室温的类器官生长培养基稀释减生长因子的Matrigel,根据计数结果,用稀释后的Matrigel重悬隐窝,按照500-1000个3D421巨噬细胞/10ul和10-20个隐窝/10ul接种于提前温育的24孔板上;
将接种好的隐窝的24孔板,放于37℃培养箱中静置10-20min,让Matrigel充分凝固。
使用移液枪沿着孔壁加入500ul预热的的类器官生长培养基,置于37℃含5%CO2培养箱中,培养基的配方同实施例1。
每两天更换一次类器官生长培养基,可用于后续实验研究,共培养结果可参考图6。
需要说明的是:经过额外验证,我们发现,CN201811066368.2所记载的完全培养基也能用于巨噬细胞和肠隐窝的共培养,效果近似。
Claims (4)
1.一种猪肠类器官与产肠毒素性大肠杆菌或巨噬细胞共培养体系的构建方法,其特征在于,包括如下步骤:
步骤1:准备产肠毒素性大肠杆菌或巨噬细胞;
步骤2:取仔猪空肠前段组织进行处理得到肠隐窝;
步骤3:采用类器官生长培养基稀释减生长因子的基质胶,用稀释后的基质胶重悬肠隐窝,按照低于1*105个产肠毒素性大肠杆菌/10ul或500-1000个巨噬细胞/10ul、10-20个肠隐窝/10ul接种于提前温育的孔板上,静置,使基质胶凝固,于孔板中加入类器官生长培养基,置于37℃含5%CO2培养箱中,培养,每两天更换一次类器官生长培养基;
所述类器官生长培养基含如下成分:
基础培养基:Advanced DMEM/F12;
L-WRN cells条件性培养基40-50vol%;
100X规格的N2 0.9-1.1倍体积;
100X规格的B27-supplement 0.9-1.1倍体积;
45-55ng/ml EGF;
9-11μM Y27632;
0.45-0.55μM LY2157299;
9-11μM SB202190;
9-11μM CHIR99021;
0.9-1.1mM HEPES;
0.9-1.1mM N-acetylcysteine;
100X规格的Glutamax 0.9-1.1倍体积;
0.9-1.1mM valproic acid。
2.根据权利要求1所述的猪肠类器官与产肠毒素性大肠杆菌或巨噬细胞共培养体系的构建方法,其特征在于,含如下成分:
基础培养基:Advanced DMEM/F12;
L-WRN cells条件性培养基40-50vol%;
100X规格的N2 1倍体积;
100X规格的B27-supplement 1倍体积;
50ng/ml EGF;
10μM Y27632;
0.5μM LY2157299;
10μM CHIR99021;
10μM SB202190;
1mM HEPES;
1mM N-acetylcysteine;
100X规格的Glutamax 1倍体积;
1mM valproic acid。
3.根据权利要求1所述的猪肠类器官与产肠毒素性大肠杆菌或巨噬细胞共培养体系的构建方法,其特征在于,所述步骤1具体为:
实验前一天准备灭菌的LB液体培养基、LB固体培养基、灭菌ddH2O;
将产肠毒素性大肠杆菌ETEC K88接种至液体培养基中,选取对数期的产肠毒素性大肠杆菌ETEC K88处理细胞,用PBS清洗ETEC K88,然后用DMEM进行稀释。
4.根据权利要求1所述的猪肠类器官与产肠毒素性大肠杆菌或巨噬细胞共培养体系的构建方法,其特征在于,所述步骤2具体为:
将仔猪空肠前段组织冲洗后迅速放入含冰冷PBS缓冲液的离心管中;
取平皿,倒入冰冷PBS缓冲液,将肠段转移至大平皿进行清洗,清洗后纵向剪开;
在体视显微镜下用眼科镊剥离肠道浆膜,用镊子夹住肠段在冷PBS缓冲液中彻底冲洗干净;
将含有1%BSA的冷的PBS加入离心管中,用镊子夹住洗净的肠段一端,悬在离心管管口,用手术剪将肠段剪成1-2mm大小的组织块,让这些组织块落入含有1%BSA的冷的PBS中;
用含有1%BSA的冷的PBS润湿移液管,将肠组织块轻轻上下吹打三次;等肠组织块沉底后,吸出上清液,留下的液面刚好没过组织块即可;如此重复直至上清澄清为止;
除去上清液,将肠组织块重悬于室温的消化液中,在室温下置于摇床上以40rpm的转速孵育2h,其中消化液的成分为5mM EDTA、1mM二硫苏糖醇和1倍体积的青链霉素;
将组织块通过100um的过滤网进行过滤,将滤液收集于50ml离心管中,将离心管置于冰上;
将滤液进行4℃,300g离心5分钟;弃上清,再重悬于含有1%BSA的冷的PBS中,用含有1%BSA的冷的PBS润湿5mL的移液管,将沉淀轻轻上下吹打三次;
然后进行4℃,200g离心5分钟,弃上清,将肠隐窝沉淀留在管底。
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