CN108588019B - 一种体外诱导初始b细胞分化为调节性b细胞的方法及其培养条件 - Google Patents
一种体外诱导初始b细胞分化为调节性b细胞的方法及其培养条件 Download PDFInfo
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Abstract
本发明公开了一种体外诱导初始B细胞分化为调节性B细胞的方法及其培养条件。本发明的方法是将初始B细胞在诱导培养基中进行诱导培养;所述诱导培养基由RPMI‑1640培养基、胎牛血清、脂多糖、CD40配体、白细胞介素1β、白细胞介素6、白细胞介素4、白细胞介素21和白细胞介素23组成。通过本发明提供的诱导培养基培养初始B细胞,可以得到数量充足的CD19+CD24hiCD27+调节性B细胞。本发明诱导培养基不仅可显著提高初始B细胞分化为CD19+CD24hiCD27+调节性B细胞的诱导分化能力,而且还提高了诱导分化后CD19+CD24hiCD27+调节性B细胞的存活率,具有良好的应用前景。
Description
技术领域
本发明属于生物技术领域,具体涉及一种体外诱导初始B细胞分化为调节性B细胞的方法及其培养条件。
背景技术
胸腔积液(Pleural effusion)是以胸腔内病理性液体积聚为特征的一种常见临床症候。胸膜腔为脏层和壁层胸膜之间的潜在间隙,正常人胸膜腔内有5-15mL液体,在呼吸运动时起润滑作用。任何原因导致胸膜腔内液体产生增多或吸收减少,即可产生胸腔积液。胸腔积液中有大量的免疫细胞如淋巴细胞、巨噬细胞、粒细胞、胸膜间皮细胞等,其中B细胞在胸腔积液中发挥重要的免疫作用。上世纪70年代,科学家们首次描述了一种起免疫抑制作用的B细胞,之后很多年,这类起免疫抑制作用的调节性B细胞(regulatory B,Breg)的功能才被逐渐认识。Breg细胞的分型较为繁杂,目前报道了多种Breg细胞表型:小鼠中,存在T2-MZP(transitional 2marginal-zone precursor)细胞,CD5+CD1dhiB(B10)细胞,MZ(marginal-zone)B细胞,Tim-1+B细胞,CD138+浆细胞和浆母细胞。人类中,CD19+CD24hiCD38hiCD1dhi和CD19+CD24hiCD27+等调节性B细胞表型也被报道。CD19+CD24hiCD27+调节性B细胞通过产生IL-10,产生抑制T淋巴细胞、巨噬细胞和树突状细胞的功能(Rosser EC,Mauri C.Regulatory B cells:origin,phenotype,and function.Immunity 2015;42:607-12.)。
迄今为止,关于Breg细胞发育的研究不多,不同表型Breg细胞之间的关系并不清楚。已知Breg细胞通过分泌IL-10、TGF-β和IL-35影响T细胞的分化。目前有研究报道IL-1β和IL-6可诱导B细胞产生IL-10。但尚无研究报道如何诱导CD19+CD24hiCD27+调节性B细胞的产生。
发明内容
本发明要解决的技术问题是如何诱导CD19+CD24hiCD27+调节性B细胞的产生。
为了解决上述技术问题,本发明首先提供了一种用于诱导初始B细胞分化为CD19+CD24hiCD27+调节性B细胞的培养基。
本发明提供的培养基包括白细胞介素1β、白细胞介素6、白细胞介素4、白细胞介素21和白细胞介素23。
进一步的,本发明提供的培养基由基础培养基、胎牛血清、脂多糖、CD40配体、所述白细胞介素1β、所述白细胞介素6、所述白细胞介素4、所述白细胞介素21和所述白细胞介素23组成。所述基础培养基可为RPMI-1640培养基。
更进一步的,所述胎牛血清在所述培养基中的体积分数为(10~20)%;
所述脂多糖在所述培养基中的浓度为(5~15)μg/ml;
所述CD40配体在所述培养基中的浓度为(0.5~1.5)μg/mL;
所述白细胞介素1β在所述培养基中的浓度为(10~30)ng/mL;
所述白细胞介素6在所述培养基中的浓度为(10~30)ng/mL;
所述白细胞介素4在所述培养基中的浓度为(10~30)ng/mL;
所述白细胞介素21在所述培养基中的浓度为(40~60)ng/mL;
所述白细胞介素23在所述培养基中的浓度为(40~60)ng/mL。
在本发明中,所述胎牛血清在所述培养基中的体积分数为10%;
所述脂多糖在所述培养基中的浓度为10μg/ml;
所述CD40配体在所述培养基中的浓度为1μg/mL;
所述白细胞介素1β在所述培养基中的浓度为20ng/mL;
所述白细胞介素6在所述培养基中的浓度为20ng/mL;
所述白细胞介素4在所述培养基中的浓度为20ng/mL;
所述白细胞介素21在所述培养基中的浓度为50ng/mL;
所述白细胞介素23在所述培养基中的浓度为50ng/mL。
上述培养基的pH为7.2-7.4。
为了解决上述技术问题,本发明又提供了上述培养基的新用途。
本发明提供了上述培养基在制备CD19+CD24hiCD27+调节性B细胞中的应用。
本发明还提供了上述培养基在诱导初始B细胞分化为CD19+CD24hiCD27+调节性B细胞中的应用。
本发明还提供了上述培养基在制备诱导初始B细胞分化为CD19+CD24hiCD27+调节性B细胞的产品中的应用。
本发明还提供了上述培养基在提高初始B细胞分化为CD19+CD24hiCD27+调节性B细胞的诱导分化能力中的应用。
本发明还提供了上述培养基在制备提高初始B细胞分化为CD19+CD24hiCD27+调节性B细胞的诱导分化能力的产品中的应用。
本发明还提供了上述培养基在提高诱导分化后CD19+CD24hiCD27+调节性B细胞的存活率中的应用。
本发明还提供了上述培养基在制备提高诱导分化后CD19+CD24hiCD27+调节性B细胞的存活率的产品中的应用。
为了解决上述技术问题,本发明还提供了一种诱导初始B细胞分化为CD19+CD24hiCD27+调节性B细胞的方法。
本发明提供的诱导初始B细胞分化为CD19+CD24hiCD27+调节性B细胞的方法包括如下步骤:在上述培养基中培养初始B细胞,得到CD19+CD24hiCD27+调节性B细胞。
上述方法中,所述培养的条件如下:37℃、5%CO2条件下培养5-7天;所述培养的条件具体可为:37℃、5%CO2条件下培养5天。
上述方法中,所述初始B细胞在上述培养基中的浓度为(1-5)×106细胞/mL;所述初始B细胞在上述培养基中的浓度具体可为1×106细胞/mL。
上述方法中,所述初始B细胞为胸腔积液来源的初始B细胞。
由上述方法制备得到的CD19+CD24hiCD27+调节性B细胞也属于本发明的保护范围。
为了解决上述技术问题,本发明最后提供了一种用于诱导初始B细胞分化为CD19+CD24hiCD27+调节性B细胞的产品。
本发明提供的产品包括上述培养基。
本发明首次提供了一种将初始B细胞分化为CD19+CD24hiCD27+调节性B细胞的方法以及用于将初始B细胞分化为CD19+CD24hiCD27+调节性B细胞的诱导培养基,通过本发明提供的诱导培养基培养初始B细胞,可以得到数量充足的CD19+CD24hiCD27+调节性B细胞。本发明的诱导培养基不仅能够显著提高初始B细胞分化为CD19+CD24hiCD27+调节性B细胞的诱导分化能力,而且还提高了诱导分化后CD19+CD24hiCD27+调节性B细胞的存活率,具有良好的应用前景。
附图说明
图1为胸腔积液患者初始B细胞经不同培养基诱导分化后CD19+CD24hiCD27+调节性B细胞的比例。
图2为胸腔积液患者初始B细胞经本发明的诱导培养基诱导分化后CD19+CD24hiCD27+调节性B细胞的比例。
具体实施方式
下述实施例中所使用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材料、试剂等,如无特殊说明,均可从商业途径得到。
下述实施例中的定量试验,均设置三次重复实验,结果取平均值。
下述实施例中的RPMI-1640培养基是GIBCO公司的产品,货号:11875-093;胎牛血清是GIBCO公司的产品,货号:10099-141;脂多糖(LPS)是INVIVOGEN公司的产品,货号:14D09-MM;CD40配体(CD40L)是R&D公司的产品,货号:6420-CL-025;白细胞介素1β(IL-1β)是Peprotech公司的产品,货号:200-01B;白细胞介素6(IL-6)是Peprotech公司的产品,货号:200-06;白细胞介素4(IL-4)是Peprotech公司的产品,货号:200-04;白细胞介素21(IL-21)是Peprotech公司的产品,货号:200-21;白细胞介素23(IL-23)是Peprotech公司的产品,货号:200-23。
实施例1、一种用于体外诱导初始B细胞分化为CD19+CD24hiCD27+调节性B细胞的培养基
本发明提供的用于体外诱导初始B细胞分化为CD19+CD24hiCD27+调节性B细胞的培养基是将RPMI-1640培养基、胎牛血清、脂多糖(LPS)、CD40配体(CD40L)、白细胞介素1β(IL-1β)、白细胞介素6(IL-6)、白细胞介素4(IL-4)、白细胞介素21(IL-21)和白细胞介素23(IL-23)混匀得到的,其中,胎牛血清在培养基中的体积分数为10%,LPS在培养基中的浓度为10μg/mL,CD40L在培养基中的浓度为1μg/mL,IL-1β在培养基中浓度为20ng/mL,IL-6在培养基中浓度为20ng/mL,IL-4在培养基中的浓度为20ng/mL,IL-21在培养基中的浓度为50ng/mL,IL-23在培养基中的浓度为50ng/mL。培养基的pH为7.2-7.4。
实施例2、诱导初始B细胞分化为CD19+CD24hiCD27+调节性B细胞的方法
一、诱导初始B细胞分化为CD19+CD24hiCD27+调节性B细胞的方法
1、经确诊为胸腔积液患者于入院24小时内,通过规范的胸腔穿刺术采集胸腔积液500mL,并向胸腔积液样本中加入肝素抗凝。所有样本立即置于冰盒内转运至实验室。所有胸腔积液患者来源于首都医科大学附属北京朝阳医院,均知情同意。
2、将步骤1中采集的胸腔积液样本在1500rpm,4℃条件下离心10分钟,得到细胞沉淀。
3、向步骤2中获得的细胞沉淀中加入淋巴细胞分离液Ficoll-Hypaque gradientcentrifugation(Pharmacia,Uppsala,Sweden),采用梯度离心法分离单个核细胞,具体步骤参照文献:Determination of Interleukin 27-Producing CD4+and CD8+T Cells forThe Differentiation Between Tuberculous and Malignant Pleural Effusions中的方法。
4、采用美天旎细胞分选试剂盒从步骤3中分离得到的单个核细胞中分选得到初始B细胞,并进行细胞计数。
5、用含10%(体积分数)胎牛血清的RPMI-1640培养基重悬步骤4获得的初始B细胞,按照1×106细胞/mL密度接种于96孔板中;然后每孔分别加入200μL如下32种培养基,在37℃、5%CO2条件下培养5天。32种培养基具体如下:
培养基1:RPMI-1640培养基+胎牛血清(10%)+LPS(10μg/mL)+CD40L(1μg/mL);
培养基2:RPMI-1640培养基+胎牛血清(10%)+LPS(10μg/mL)+CD40L(1μg/mL)+IL-1β;
培养基3:RPMI-1640培养基+胎牛血清(10%)+LPS(10μg/mL)+CD40L(1μg/mL)+IL-4(20ng/mL);
培养基4:RPMI-1640培养基+胎牛血清(10%)+LPS(10μg/mL)+CD40L(1μg/mL)+IL-6(20ng/mL);
培养基5:RPMI-1640培养基+胎牛血清(10%)+LPS(10μg/mL)+CD40L(1μg/mL)+IL-21(50ng/mL);
培养基6:RPMI-1640培养基+胎牛血清(10%)+LPS(10μg/mL)+CD40L(1μg/mL)+IL-23(50ng/mL);
培养基7:RPMI-1640培养基+胎牛血清(10%)+LPS(10μg/mL)+CD40L(1μg/mL)+IL-1β(20ng/mL)+IL-4(20ng/mL);
培养基8:RPMI-1640培养基+胎牛血清(10%)+LPS(10μg/mL)+CD40L(1μg/mL)+IL-1β(20ng/mL)+IL-6(20ng/mL);
培养基9:RPMI-1640培养基+胎牛血清(10%)+LPS(10μg/mL)+CD40L(1μg/mL)+IL-1β(20ng/mL)+IL-21(50ng/mL);
培养基10:RPMI-1640培养基+胎牛血清(10%)+LPS(10μg/mL)+CD40L(1μg/mL)+IL-1β(20ng/mL)+IL-23(50ng/mL);
培养基11:RPMI-1640培养基+胎牛血清(10%)+LPS(10μg/mL)+CD40L(1μg/mL)+IL-4(20ng/mL)+IL-6(20ng/mL);
培养基12:RPMI-1640培养基+胎牛血清(10%)+LPS(10μg/mL)+CD40L(1μg/mL)+IL-4(20ng/mL)+IL-21(50ng/mL);
培养基13:RPMI-1640培养基+胎牛血清(10%)+LPS(10μg/mL)+CD40L(1μg/mL)+IL-4(20ng/mL)+IL-23(50ng/mL);
培养基14:RPMI-1640培养基+胎牛血清(10%)+LPS(10μg/mL)+CD40L(1μg/mL)+IL-6(20ng/mL)+IL-21(50ng/mL);
培养基15:RPMI-1640培养基+胎牛血清(10%)+LPS(10μg/mL)+CD40L(1μg/mL)+IL-6(20ng/mL)+IL-23(50ng/mL);
培养基16:RPMI-1640培养基+胎牛血清(10%)+LPS(10μg/mL)+CD40L(1μg/mL)+IL-21(50ng/mL)+IL-23(50ng/mL);
培养基17:RPMI-1640培养基+胎牛血清(10%)+LPS(10μg/mL)+CD40L(1μg/mL)+IL-1β(20ng/mL)+IL-4(20ng/mL)+IL-6(20ng/mL);
培养基18:RPMI-1640培养基+胎牛血清(10%)+LPS(10μg/mL)+CD40L(1μg/mL)+IL-1β(20ng/mL)+IL-4(20ng/mL)+IL-21(50ng/mL);
培养基19:RPMI-1640培养基+胎牛血清(10%)+LPS(10μg/mL)+CD40L(1μg/mL)+IL-1β(20ng/mL)+IL-4(20ng/mL)+IL-23(50ng/mL);
培养基20:RPMI-1640培养基+胎牛血清(10%)+LPS(10μg/mL)+CD40L(1μg/mL)+IL-1β(20ng/mL)+IL-6(20ng/mL)+IL-21(50ng/mL);
培养基21:RPMI-1640培养基+胎牛血清(10%)+LPS(10μg/mL)+CD40L(1μg/mL)+IL-1β(20ng/mL)+IL-6(20ng/mL)+IL-23(50ng/mL);
培养基22:RPMI-1640培养基+胎牛血清(10%)+LPS(10μg/mL)+CD40L(1μg/mL)+IL-1β(20ng/mL)+IL-21(50ng/mL)+IL-23(50ng/mL);
培养基23:RPMI-1640培养基+胎牛血清(10%)+LPS(10μg/mL)+CD40L(1μg/mL)+IL-4(20ng/mL)+IL-6(20ng/mL)+IL-21(50ng/mL);
培养基24:RPMI-1640培养基+胎牛血清(10%)+LPS(10μg/mL)+CD40L(1μg/mL)+IL-4(20ng/mL)+IL-6(20ng/mL)+IL-23(50ng/mL);
培养基25:RPMI-1640培养基+胎牛血清(10%)+LPS(10μg/mL)+CD40L(1μg/mL)+IL-4(20ng/mL)+IL-21(50ng/mL)+IL-23(50ng/mL);
培养基26:RPMI-1640培养基+胎牛血清(10%)+LPS(10μg/mL)+CD40L(1μg/mL)+IL-6(20ng/mL)+IL-21(50ng/mL)+IL-23(50ng/mL);
培养基27:RPMI-1640培养基+胎牛血清(10%)+LPS(10μg/mL)+CD40L(1μg/mL)+IL-1β(20ng/mL)+IL-4(20ng/mL)+IL-6(20ng/mL)+IL-21(50ng/mL);
培养基28:RPMI-1640培养基+胎牛血清(10%)+LPS(10μg/mL)+CD40L(1μg/mL)+IL-1β(20ng/mL)+IL-4(20ng/mL)+IL-6(20ng/mL)+IL-23(50ng/mL);
培养基29:RPMI-1640培养基+胎牛血清(10%)+LPS(10μg/mL)+CD40L(1μg/mL)+IL-1β(20ng/mL)+IL-6(20ng/mL)+IL-21(50ng/mL)+IL-23(50ng/mL);
培养基30:RPMI-1640培养基+胎牛血清(10%)+LPS(10μg/mL)+CD40L(1μg/mL)+IL-4(20ng/mL)+IL-6(20ng/mL)+IL-21(50ng/mL)+IL-23(50ng/mL);
培养基31:RPMI-1640培养基+胎牛血清(10%)+LPS(10μg/mL)+CD40L(1μg/mL)+IL-1β(20ng/mL)+IL-4(20ng/mL)+IL-21(50ng/mL)+IL-23(50ng/mL);
培养基32:RPMI-1640培养基+胎牛血清(10%)+LPS(10μg/mL)+CD40L(1μg/mL)+IL-1β(20ng/mL)+IL-4(20ng/mL)+IL-6(20ng/mL)+IL-21(50ng/mL)+IL-23(50ng/mL)。
二、CD19+CD24hiCD27+调节性B细胞的表型鉴定
培养5天后,分别检测上述各个培养体系中CD19+CD24hiCD27+调节性B细胞所占的比例。具体步骤如下:将培养第5天的各个培养体系中的细胞,用PBS重悬离心后,用流式抗体These human Abs included anti–CD19(BD Biosciences,Franklin Lakes,NJ,货号:561295)、anti–CD24(Ebioscience公司,货号:25-0247-42)、anti–CD27(BD Biosciences,货号558664)和anti-IgD(BD Biosciences,货号555778)及同型对照抗体(BDBiosciences,Franklin Lakes,NJ)进行染色15分钟后,用PBS清洗后,加入4%多聚甲醛固定,用BD流式仪进行分析,通过与同型对照的比较进行圈门,从而确定CD19+CD24hiCD27+调节性B细胞所占的比例。
结果如图1所示。初始B细胞经过培养基1-32培养5天后,培养体系中CD19+CD24hiCD27+调节性B细胞的比例分别为3.8%、6.3%、6.9%、5.8%、4.5%、5.3%、11.5%、6.9%、5.7%、6.0%、9.4%、7.8%、7.1%、5.7%、8.3%、6.0%、7.0%、7.2%、9.6%、9.8%、11.5%、9.0%、10.7%、12.2%、9.4%、11.2%、14.3%、16.8%、19.2%、19.5%、18.4%和23.5%。上述结果表明:初始B细胞经过培养基1-32培养5天,在培养基32(实施例1中的培养基)的培养体系中CD19+CD24hiCD27+调节性B细胞的比例最高,达到23.5%(图2),说明该培养基可提高初始B细胞分化为CD19+CD24hiCD27+调节性B细胞的诱导分化能力及诱导分化后CD19+CD24hiCD27+调节性B细胞的存活率,可用于制备或生产CD19+CD24hiCD27+调节性B细胞。
Claims (13)
1.一种用于诱导初始B细胞分化为CD19+CD24hiCD27+调节性B细胞的培养基,所述培养基由基础培养基、胎牛血清、脂多糖、CD40配体、白细胞介素1β、白细胞介素6、白细胞介素4、白细胞介素21和白细胞介素23组成。
2.根据权利要求1所述的培养基,其特征在于:所述基础培养基为RPMI-1640培养基。
3.根据权利要求1或2所述的培养基,其特征在于:
所述胎牛血清在所述培养基中的体积分数为(10~20)%;
所述脂多糖在所述培养基中的浓度为(5~15)μg/ml;
所述CD40配体在所述培养基中的浓度为(0.5~1.5)μg/mL;
所述白细胞介素1β在所述培养基中的浓度为(10~30)ng/mL;
所述白细胞介素6在所述培养基中的浓度为(10~30)ng/mL;
所述白细胞介素4在所述培养基中的浓度为(10~30)ng/mL;
所述白细胞介素21在所述培养基中的浓度为(40~60)ng/mL;
所述白细胞介素23在所述培养基中的浓度为(40~60)ng/mL。
4.根据权利要求3所述的培养基,其特征在于:
所述胎牛血清在所述培养基中的体积分数为10%;
所述脂多糖在所述培养基中的浓度为10μg/ml;
所述CD40配体在所述培养基中的浓度为1μg/mL;
所述白细胞介素1β在所述培养基中的浓度为20ng/mL;
所述白细胞介素6在所述培养基中的浓度为20ng/mL;
所述白细胞介素4在所述培养基中的浓度为20ng/mL;
所述白细胞介素21在所述培养基中的浓度为50ng/mL;
所述白细胞介素23在所述培养基中的浓度为50ng/mL。
5.权利要求1-4任一所述的培养基在制备CD19+CD24hiCD27+调节性B细胞中的应用。
6.权利要求1-4任一所述的培养基在诱导初始B细胞分化为CD19+CD24hiCD27+调节性B细胞中的应用。
7.权利要求1-4任一所述的培养基在制备诱导初始B细胞分化为CD19+CD24hiCD27+调节性B细胞的产品中的应用。
8.权利要求1-4任一所述的培养基在提高诱导分化后CD19+CD24hiCD27+调节性B细胞的存活率中的应用。
9.权利要求1-4任一所述的培养基在制备提高诱导分化后CD19+CD24hiCD27+调节性B细胞的存活率的产品中的应用。
10.一种诱导初始B细胞分化为CD19+CD24hiCD27+调节性B细胞的方法,包括如下步骤:在权利要求1-4任一所述的培养基中培养初始B细胞,得到CD19+CD24hiCD27+调节性B细胞。
11.根据权利要求10所述的方法,其特征在于:所述培养的条件如下:37℃、5% CO2条件下培养5-7天。
12.根据权利要求10或11所述的方法,其特征在于:所述初始B细胞在权利要求1-4任一所述的培养基中的浓度为(1-5)×106细胞/mL。
13.一种用于诱导初始B细胞分化为CD19+CD24hiCD27+调节性B细胞的产品,其包括权利要求1-4任一所述的培养基。
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