CN117384842A - 奶牛骨髓源单核巨噬细胞诱导培养方法及应用 - Google Patents
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Abstract
本发明公开了奶牛骨髓源单核巨噬细胞诱导培养方法及应用,涉及生物技术领域。本发明通过在体外诱导分化骨髓中髓系祖细胞来分化成单核巨噬细胞,选择针对单核巨噬细胞特异性抗原受体的单克隆抗体进行细胞鉴定。建立一种获取奶牛骨髓细胞,并能够在体外诱导分化为单核巨噬细胞的方法,为今后从事相关领域研究的科研工作者提供技术支持。
Description
技术领域
本发明涉及生物技术领域,更具体的说是涉及奶牛骨髓源单核巨噬细胞诱导培养方法及应用。
背景技术
巨噬细胞广泛存在于各组织器官中,可参与机体绝大多数免疫相关功能活动,能够执行许多不同的免疫功能。在抵御病原体的过程中,巨噬细胞与其他先天免疫细胞(如中性粒细胞,嗜酸性粒细胞和自然杀伤细胞)发挥着重要作用。巨噬细胞属于细胞免疫,通过吞噬作用和杀死入侵的微生物来消除病原体。此外,巨噬细胞还能通过去除局部病原体和细胞碎片,促进组织修复,诱导伤口愈合。由于组织驻留巨噬细胞难以获得,因此现有技术中有关巨噬细胞的大多数功能研究都是用单核细胞衍生巨噬细胞进行的。M-CSF能够诱导单核细胞分化为巨噬细胞。
现有技术中奶牛巨噬细胞提取方法主要有腹腔巨噬细胞分离法、肺泡灌洗法、外周血分离法。其中奶牛腹腔单核细胞提取过程需打入特定培养基诱导巨噬细胞分化,然后抽出奶牛腹水进行提取,过程困难且造价高;脾脏及肺巨噬细胞提取方法提取的细胞种类太多,且没有办法将巨噬细胞进行纯化,纯度低。近十年间这两种提取方式都已被淘汰。外周血单核细胞需使用试剂盒对血液中的细胞进行提取,但血液中单核细胞的的占比是3%-8%,量较少,不能满足实验的需求。在单核细胞诱导为巨噬细胞的过程中,会导致大部分细胞死亡,剩余细胞不能满足实验需求,无法进行后续实验。
同时现有技术中也有使用月龄三个月以下的犊牛或者未出生胎牛的股骨或者胫骨提取骨髓源细胞,超出2-3月龄后,犊牛股骨或者胫骨中的红骨髓被脂肪组织所代替,颜色变黄,称为黄骨髓,失去造血功能,无法提取单核细胞。而市场上常见的犊牛肉通常产自六个月犊牛,三个月犊牛食用价值不高,并非屠宰场的常规宰杀动物,一般只有患病之后才会宰杀,健康犊牛的新鲜股骨或胫骨获取难度较大,难以适应大规模骨髓源细胞的提取与制备。
现有技术获取的巨噬细胞数量少,提取程序复杂,并且容易受到供体奶牛健康状况的影响。因此,提供一种细胞纯度高、分化程度一致,能够量产的奶牛单核巨噬细胞是本领域技术人员亟需解决的问题。
发明内容
有鉴于此,本发明提供了奶牛骨髓源单核巨噬细胞诱导培养方法及应用。
为了实现上述目的,本发明采用如下技术方案:
奶牛骨髓源单核巨噬细胞诱导培养方法,包括以下步骤:
(1)选取新鲜奶牛肋骨,剔除肋骨周围组织,将肋骨浸泡于75%酒精中10min;
(2)在超净台中将肋骨分段,长度3-5cm,浸泡在RPMI-1640培养基中;使用20mL注射器抽取PBS缓冲溶液刺入奶牛肋骨骨髓腔,反复冲洗直至骨髓腔发白,得冲洗液;
(3)将冲洗液过100μm细胞筛,4℃,1500rpm/min离心8min,弃上清,得细胞沉淀;
加入2-4mL预热至37℃的红细胞裂解液重悬细胞沉淀,裂解10min,4℃,1000rpm/min离心5min,弃上清,得裂解后的细胞沉淀;
裂解后的细胞沉淀即为骨髓造血干细胞;
(4)用预热至37℃的诱导培养基重悬骨髓造血干细胞,均匀接种至无菌细胞培养瓶内,置于含5%体积分数CO2的37℃培养箱中进行培养;
抽取细胞培养瓶中的细胞悬浮液更换新细胞培养瓶,每两个小时一次,重复5次,最后一次取10ul细胞悬液进行计数,以5×106个/孔的密度铺于6孔细胞培养板中;
(5)诱导7天后,即可获得得到成熟的M0型奶牛原代骨髓源巨噬细胞,加入1ug/ml脂多糖诱导,将M0型巨噬细胞诱导为M1型巨噬细胞。
CD11b,CD14是M0型巨噬细胞的表面标记物,CD11b以及CD80通常被视为M1型巨噬细胞特异性标志物,因此使用这些表面标记物对巨噬细胞进行鉴定。
进一步的,步骤(2)所述RPMI-1640培养基含有2%体积分数的青-链霉素,所述PBS缓冲溶液含有2%体积分数的青-链霉素;
进一步的,步骤(4)所述诱导培养基的组方以体积分数计为:20%胎牛血清,1.2%谷氨酰胺,2.4%青-链霉素,其余为RPMI 1640培养基;
所述诱导培养基还含有20ng/ml M-CSF。
奶牛骨髓源单核巨噬细胞在免疫学、疾病的发生发展治疗中的应用。
经由上述的技术方案可知,与现有技术相比,本发明使用奶牛肋骨,体外提取奶牛骨髓源单核细胞,并在体外诱导分化骨髓中髓系祖细胞来分化成单核巨噬细胞,可以有效避免巨噬细胞受机体健康情况的影响。本发明针对单核巨噬细胞特异性抗原受体的单克隆抗体进行细胞鉴定,结果表明本发明培养的巨噬细胞不但收获稳定,而且存活效率高,安全可靠。本发明建立了一种简捷、高效、产量多的巨噬细胞提取方法,为今后从事相关领域研究的科研工作者提供技术支持,能够支撑大量巨噬细胞相关科学研究,进而应用到免疫学、疾病的发生发展治疗的研究。
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,下面将对实施例或现有技术描述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,还可以根据提供的附图获得其他的附图。
图1:M0型巨噬细胞形态;
图2:M1型巨噬细胞形态;
图3:普通方法与本方法对于M1型巨噬细胞的区别
图4:免疫荧光鉴定M0型巨噬细胞CD11b;
图5:免疫荧光鉴定M0型巨噬细胞CD14;
图6:免疫荧光鉴定M1型巨噬细胞CD80;
图7:M0型巨噬细胞流式细胞术鉴定;
图8:M1型巨噬细胞流式细胞术鉴定;
图9:PGD2含量及COX-2/H-PGDS含量;
图10:实施例1与对比例1巨噬细胞吞噬杀伤大肠杆菌的能力比较;
图11:加入前列腺素D2(PGD2)后对实施例1巨噬细胞的吞噬杀伤能力的影响。
具体实施方式
下面将结合本发明实施例中的附图,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。
实验材料及器材来源:
试验菌株与细菌悬浮液的制备:内蒙古农业大学兽医学院实验室分离培养的致病性大肠杆菌(E.coli,鉴定证书号:SYS110017)。将细菌在LuriaBertani培养基中培养,200rmp,37℃下培养13小时,将培养物在4℃下以5000g离心5分钟,然后用无菌PBS重悬,在450nm吸光值处将OD调至0.95。
主要试剂:胎牛血清(ExCellBiology,中国上海);M-CSF;RPMI-1640培养基、PBS磷酸盐缓冲溶液购自Hyclone公司;谷氨酰胺(Gln);PGD2 ELISA试剂盒;总RNA试剂盒购自Axygen公司;反转录试剂盒购自Thermo Scientific公司;实时荧光定量PCR试剂盒(FastStart Universal SYBRGreenMaster)购自RocheAppliedScience公司;CD11b/CD14/CD80抗体。
主要仪器设备:超净工作台、37℃恒温CO2细胞培养箱购Thermo公司;高压灭菌锅购自Hirayama公司;电热鼓风干燥箱购自恒丰医疗器械公司;高速低温离心机购自Beckman公司;-80℃超低温冰箱购自海尔公司;-20℃低温冰箱购自美菱公司;电子分析天平购自赛多利斯科学仪器公司;超纯水仪购自Biotech公司;制冰机购自Panasonic;荧光倒置显微镜为日本奥林巴斯产品;BD FACSCalibur流式细胞仪为美国BD公司产品;ELX800酶标仪购自Bio-Tex公司;光学显微镜购自Olympus公司。
实施例1:奶牛骨髓源单核巨噬细胞获取
奶牛骨髓细胞获取
无菌条件下取新鲜奶牛肋骨,剔除肋骨周围的肌肉和结缔组织,将肋骨浸泡于75%酒精中杀菌10min,擦拭后放入超净工作台,横截面将肋骨切成长度3-5cm小段,泡在RPMI-1640培养基(含有2%体积分数的青-链霉素)中,用20mLPBS缓冲溶液(含有2%体积分数的青-链霉素)反复冲洗骨髓直至骨髓腔发白,收集冲洗液;
将冲洗液用100um细胞筛过滤,1500rpm,4℃,离心8min,弃上清,得细胞沉淀;加入2-4mL预热至37℃的红细胞裂解液重悬细胞沉淀,按照细胞红裂液说明书要求,去除悬液中的红细胞。裂解10min,4℃,1000rpm/min离心5min,弃上清,得裂解后的细胞沉淀(骨髓造血干细胞)。
奶牛骨髓细胞诱导培养及优化
将骨髓造血干细胞用预热至37℃的诱导培养基(20ng/ml M-CSF、20%FBS(胎牛血清)、1.2%谷氨酰胺,2.4%青-链霉素,其余为RPMI-1640培养基)重悬,均匀接种至无菌细胞培养瓶内,置于含5%体积分数CO2的37℃培养箱中进行培养;
抽取细胞培养瓶中的细胞悬浮液更换新细胞培养瓶,每两个小时一次,重复5次,最后一次取10ul细胞悬液进行计数,以5×106个/孔的密度接种于含有诱导培养基的6孔细胞培养板中。细胞放置于培养箱继续培养,在培养至第1、4、7天显微镜下观察细胞形态、细胞计数及诱导情况。
诱导7天后,即可获得得到成熟的M0型奶牛原代骨髓源巨噬细胞(如图1所示),加入1ug/ml脂多糖诱导24h,将M0型巨噬细胞诱导为M1型巨噬细胞(如图2所示);
本发明使用诱导培养基培养单核巨噬细胞,RPMI-1640培养基的基础上加入M-CSF、20%胎牛血清对单核巨噬细胞进行诱导。结果显示,诱导出规则的巨噬细胞。
对比例1:普通巨噬细胞获取(外周血法)
实验动物选择:根据血液中BHBA浓度选出10头健康奶牛和10头临床酮病奶牛。
通过尾静脉穿刺将荷斯坦奶牛血液收集到含有肝素钠作为抗凝剂的抗凝管中。使用牛外周血单核细胞分离试剂盒从抗凝管保存的血液中分离出外周血单核细胞。
外周血单核细胞获取方法:将20mL奶牛全血和试剂盒A液、D液(A:D=3:2)按比例加入50mL无菌离心管中,在800g条件下离心30分钟以获得液体分层。弃去最上层的血浆,吸取第二层的白膜层至15mL无菌离心管中,250g条件下离心10分钟后,弃掉上清液。按细胞体积加入3-4mL红细胞裂解液混匀,在37℃水浴锅中预热2分钟,离心后重复此步骤。然后用细胞洗涤液清洗细胞,室温250g条件下,离心10分钟,得外周血单核细胞;
诱导培养基不添加加M-CSF,其余诱导方法同实施例1;
普通方法与本方法获得的M1型巨噬细胞的区别见图3。
对比例2
奶牛骨髓细胞获取步骤同实施例1;
奶牛骨髓细胞诱导培养及优化步骤中骨髓造血干细胞接种于不含M-CSF(集落刺激因子)的诱导培养基过夜静置培养,第二天取上层细胞离心铺板,使用含有M-CSF的诱导培养基进行培养,以获取纯的单核细胞。
结果发现第二天细胞均沉降,上层细胞量很少,且有一层脂肪膜,而此时的贴壁细胞大部分为树突状细胞等一些杂细胞。无法获得足量单核细胞。
因此为了获得更纯的巨噬细胞,在实施例1中将提取出的细胞每隔2小时更换一次细胞瓶,连续更换5次,去除2小时之内贴住的杂细胞,之后再进行计数铺板。
免疫荧光试验
实验细胞的获取:将实施例1获得的骨髓造血干细胞在免疫荧光小皿诱导培养,方法同实施例1;分别获得M0型巨噬细胞样品,M1型巨噬细胞样品;
弃掉免疫荧光小皿中的骨髓巨噬细胞液,200ul/孔PBST清洗四次。每孔加100ul,4%的多聚甲醛(4℃存放),室温固定10min后,200ul/孔PBST清洗四次。100ul/孔1%PBA室温封闭20min,200ul/孔PBST清洗四次。
分别使用CD11b标记M0型巨噬细胞(如图4所示,左为CD11b,中为DAPI,右为合并),CD14标记M0型巨噬细胞(如图5所示,左为CD14,中为DAPI,右为合并),CD80标记M1型巨噬细胞(如图6所示,左为CD80,中为DAPI,右为合并),4℃湿盒过夜孵育,PBST清洗4次、200μL/孔。在荧光倒置显微镜观察特异性荧光信号。
流式细胞术检测
实验细胞的获取:将实施例1获得的骨髓造血干细胞在6孔细胞培养板中诱导培养,方法同实施例1;分别获得M0型巨噬细胞样品,M1型巨噬细胞样品;
弃掉6孔板中的培养液,使用PBS清洗两次,吸干PBS,加入300ul不含0.25%胰酶置于37℃细胞培养箱中消化5min。加入900ul含体积分数10%胎牛血清的RPMI-1640培养基终止消化,轻轻吹打细胞,使细胞脱落成细胞悬液,转移至1.5ml离心管中,1000rpm离心6min,弃上清。加100ul PBS重悬细胞,1000rpm离心6min,重复三次。
用流式染色缓冲液稀释荧光染料标记的CD11b,CD14,CD80抗体。选择一定稀释比例抗体标记细胞。4℃避光旋转孵育45min,抗体标记完成后加入500ul细胞洗液,1200rpm离心6min,重复2-3次(细胞少可适当减少次数)。弃上清,加入500ul 1%多聚甲醛,避光4℃保存。
结果表明通过流式细胞术,激光共聚焦也能检测到巨噬细胞表面特异性抗原标志物(如图7,图8所示)。
巨噬细胞能力检测
取出实施例1诱导出M1型巨噬细胞的细胞培养板,确定细胞数量,按照MOI值5:1将大肠杆菌接种于M1型巨噬细胞,同时设置正常培养的巨噬细胞空白对照组。将细胞置于37℃CO2培养箱培养,收集上清,通过ELISA方法检测PGD2含量、RT-PCR方法检测COX-2/H-PGDS含量(如图9所示)。
当巨噬细胞受到病原微生物刺激,如大肠杆菌感染时,可通过促使其分泌细胞因子进而对病原微生物及其病原相关分子模式(PAMPs)所激活的天然免疫应答进行调控,从而影响炎症过程中各种炎症介质的表达。而PGD2是天然免疫反应发生过程中重要的炎症介质。
通过使用大肠杆菌刺激巨噬细胞后检测PGD2及合成途径中所必需酶的表达,证明本发明提取的巨噬细胞可以支持先天性免疫反应相关研究。
巨噬细胞能力比较
使用MTT方法检测实施例1与对比例1两种M1型巨噬细胞吞噬杀伤大肠杆菌的能力,结果表明,奶牛骨髓源巨噬细胞较外周血单核细胞相比,显著下调了大肠杆菌的存活率(见图10)。
在实施例1所制备的M1型巨噬细胞样品中添加1*10-6M PGD2(前列腺素D2),比较未添加PGD2的M1型巨噬细胞的吞噬杀伤能力,结果表明,添加PGD2后导致细胞的吞噬杀伤能力下降(见图11,左为Dil,中为Hoechst,右为合并)。
对所公开的实施例的上述说明,使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的,本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下,在其它实施例中实现。因此,本发明将不会被限制于本文所示的这些实施例,而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (4)
1.奶牛骨髓源单核巨噬细胞诱导培养方法,其特征在于,包括以下步骤:
(1)选取新鲜奶牛肋骨,剔除肋骨周围组织,将肋骨浸泡于75%酒精中10min;
(2)在超净台中将肋骨分段,长度3-5cm,浸泡在RPMI-1640培养基中;使用20mL注射器抽取PBS缓冲溶液刺入奶牛肋骨骨髓腔,反复冲洗直至骨髓腔发白,得冲洗液;
(3)将冲洗液过100μm细胞筛,4℃,1500rpm/min离心8min,弃上清,得细胞沉淀;
加入2-4mL预热至37℃的红细胞裂解液重悬细胞沉淀,裂解10min,4℃,1000rpm/min离心5min,弃上清,得裂解后的细胞沉淀;
裂解后的细胞沉淀即为骨髓造血干细胞;
(4)用预热至37℃的诱导培养基重悬骨髓造血干细胞,均匀接种至无菌细胞培养瓶内,置于含5%体积分数CO2的37℃培养箱中进行培养;
抽取细胞培养瓶中的细胞悬浮液更换新细胞培养瓶,每两个小时一次,重复5次,最后一次取10ul细胞悬液进行计数,以5×106个/孔的密度铺于6孔细胞培养板中;
(5)诱导7天后,即可获得得到成熟的M0型奶牛原代骨髓源巨噬细胞,加入1ug/ml脂多糖诱导24h,将M0型巨噬细胞诱导为M1型巨噬细胞。
2.根据权利要求1所述的奶牛骨髓源单核巨噬细胞诱导培养方法,其特征在于,步骤(2)所述RPMI-1640培养基含有2%体积分数的青-链霉素,所述PBS缓冲溶液含有2%体积分数的青-链霉素。
3.根据权利要求1所述的奶牛骨髓源单核巨噬细胞诱导培养方法,其特征在于,步骤(4)所述诱导培养基的组方以体积分数计为:20%胎牛血清,1.2%谷氨酰胺,2.4%青-链霉素,其余为RPMI 1640培养基;
所述诱导培养基还含有20ng/ml M-CSF。
4.奶牛骨髓源单核巨噬细胞在免疫学、疾病的发生发展治疗中的应用。
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