CN114591908B - 一种诱导Tregs得到胚胎抗原特异性iTregs的刺激因子、培养基及方法和应用 - Google Patents
一种诱导Tregs得到胚胎抗原特异性iTregs的刺激因子、培养基及方法和应用 Download PDFInfo
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Abstract
本发明涉及一种诱导Tregs得到胚胎抗原特异性iTregs的刺激因子、培养基及方法和应用,属于细胞体外诱导培养技术领域。本发明提供了诱导Tregs得到胚胎抗原特异性iTregs的刺激因子,所述刺激因子包括雷帕霉素、重组人肿瘤生长因子‑β和绒促性素。本发明所述刺激因子能够诱导得到数量充分,并可根据需要扩增到治疗数量的胚胎抗原特异性iTregs;本发明诱导得到的胚胎抗原特异性iTregs比Tregs具有更强的胚胎抗原特异性、自身稳定性及免疫抑制功能。本发明诱导得到的人外周血胚胎抗原特异性iTregs在维持母胎免疫耐受,抑制母胎免疫排斥方面有广阔的临床应用价值。
Description
技术领域
本发明涉及细胞体外诱导培养技术领域,具体涉及一种诱导Tregs得到胚胎抗原特异性iTregs的刺激因子、培养基及方法和应用。
背景技术
复发性妊娠丢失(Recurrent Pregnancy Loss,RPL)指患者与同一性伴侣连续发生2次及2次以上自然流产,在育龄妇女中发病率约为5%,此类患者再次妊娠失败率高达50%以上。RPL病因复杂,临床上约50%与染色体异常、生殖系统解剖异常、内分泌异常、感染和血栓形成倾向有关,另有50%RPL患者病因不明,称原因不明复发性流产(URSA),被认为与母胎免疫排斥有关,是目前亟待解决的难治性不育症。
自2007年,申请人开始URSA的临床及基础研究,发现:1)URSA患者存在Tregs数量降低,Th17/Treg免疫失衡,提出“URSA患者Th17/Treg免疫失衡”理论[1]。2)相对于既往的破膜检测Foxp3确定Tregs的表达水平,采用CD4+CD25+CD127dim/-作为Tregs的表面标志,发现细胞表面标志CD4+CD25+CD127dim/-的检测结果与Foxp3检测结果相一致,能够简单、高效检测Tregs的表达水平,为目前临床及科研检测所采用,为URSA患者病因学筛查提供了可靠依据[2]。3)URSA患者Tregs调控Th17细胞[3]、巨噬细胞功能异常[4]。4)转输正常孕鼠体外扩增的Tregs能降低rIL-17所致的胚胎丢失[5]。5)种植期Tregs水平、体外倍增的能力以及细胞因子的分泌与种植成败关系密切,反复妊娠失败患者种植期外周血Tregs表达降低[6]。6)Tregs免疫治疗URSA患者,提高了抱婴率[7]。5)TregsPD-1、HLA-G的表达异常与URSA密切相关[8,9]。研究结果均提示与最初的研究假设高度一致,即:Tregs在母胎免疫耐受中发挥“调控师”的作用,而URSA患者Tregs数量、功能异常,导致母胎免疫耐受失调,参与URSA发病。
接触精液或胚胎抗原后,Tregs转化为胚胎抗原特异性iTregs,在孕期发挥更强大的母胎免疫耐受作用。妊娠期间,母体针对胚胎抗原特异性的Tregs大量扩增,其数量及功能的减少与复发性流产、产前子痫及早产及密切相关[10]。
目前尚无研究进行胚胎抗原特异性Tregs的分选、扩增及培养的相关研究。限制因素有几方面:一、母体的胚胎抗原特异性Tregs只有在接触精液或胚胎抗原后才由Tregs转化而来;二、Tregs仅占外周血单个核细胞1%左右,占外周血CD4+T细胞2%~8%,故能够分离出来用于治疗的细胞数量非常少[11];三、Tregs表型多样,分离纯化及扩增过程中,Tregs纯度及稳定性降低[12]。
目前并没有一种来源更充足、表型及功能稳定的胚胎抗原特异性iTregs的制备方法。
参考文献:
1.Wang WJ*,Cui-Fang Hao,Qi-De Lin*,et al.Increased prevalence of Thelper 17(Th17)cells in peripheral blood and decidua in unexplained recurrentspontaneous abortion patients.J Reprod Immunol 2010;84:164-170.
2.Shi-Hua Bao,Xi-Peng Wang,Qi-De Lin*,Wen-Juan Wang,Guang-Jie Yin,Li-Hua Qiu.Decidual CD4(+)CD25(+)CD127(dim/-)regulatory T cells in patients withunexplained recurrent spontaneous miscarriage.Eur J Obstet Gynecol ReprodBiol.2011;155:94-98.
3.Wang WJ,Cui-Fang Hao,Qi-De Lin*,et al.The deregulation ofregulatory T cells on interleukin-17-producing T helper cells in patientswith unexplained early recurrent miscarriage.Hum Reprod.2010;25:2591-2596.
4.Wang WJ*,Cui-Fang Hao,Qi-De Lin*.Dysregulation of macrophageactivation by decidual regulatory T cells in unexplained recurrentmiscarriage patients.Journal of Reproductive Immunology.2011;92:97-102.
5.Wang WJ*,Liu FJ,Xin-Liu,et al.Adoptive transfer of pregnancy-induced CD4+CD25+ regulatory T cells reverses the increase in abortion ratecaused by interleukin 17in the CBA/JxBALB/c mouse model.Hum Reprod.2014;29:946-952.
6.Wang WJ*,Liu FJ,Zhang X,et al.Periodic elevation of regulatory Tcells on the day of embryo transfer is associated with better in vitrofertilization outcome.J Reprod Immunol 2017;19:119:49-53.
7.Wang WJ*,Liu XX.Unexplained recurrent spontaneous abortionimmunotherapy using CD4+CD25+CD127dim/-T cells.37th Annual meeting of theAmerican Society for Reproductive Immunology(ASRI)2017.
8.Wenjuan Wang,Nayoung Sung,Alice Gilman-Sachs,Joanne Kwak-Kim.Thelper(Th)cell profiles in pregnancy and recurrent pregnancy losses:Th1/Th2/Th9/Th17/Th22/Tfh cells.Front Immunol 2020;11:2025.
9.Wenjuan Wang,Nayoung Sung,Alice Gilman-Sachs,Joanne Kwak-Kim.Thelper(Th)cell profiles in pregnancy and recurrent pregnancy losses:Th1/Th2/Th9/Th17/Th22/Tfh cells.Front Immunol 2020;11:2025.
10.Rosenblum MD,Way SS,Abbas AK.Regulatory T cell memory.Naturereviews Immunology 2016,16(2):90-101.
11.Gedaly,R.,etal.,mTOR Inhibitor Everolimus in RegulatoryT cellExpansion for Clinical Application in Transplantation.Transplantation,2018.
12.Cheng,Y.,et al.,Categorical Analysis of Human T Cell Heterogeneitywith One-Dimensional Soli-Expression by Nonlinear Stochastic Embedding.JImmunol,2016.196(2):p.924-32.
发明内容
本发明的目的在于提供一种诱导Tregs得到胚胎抗原特异性iTregs的刺激因子、培养基及方法和应用。本发明所述刺激因子诱导获得胚胎抗原特异性iTregs不仅能满足临床应用所需要的细胞数量,还能保持细胞功能的稳定性、免疫抑制功能和特异性,具有纯度高、活性强,稳定性好的效果,安全性及有效性高,为原因不明复发性流产的诊断、治疗提供了新的理论、新方法。
本发明提供了一种诱导Tregs得到胚胎抗原特异性iTregs的刺激因子,所述刺激因子包括雷帕霉素、重组人肿瘤生长因子-β和绒促性素。
优选的是,所述刺激因子中,雷帕霉素、重组人肿瘤生长因子-β和绒促性素的添加量之比为(50~150)μM:(2~10)ng/mL:(250~600)IU/L。
本发明还提供了一种胚胎抗原特异性iTregs的诱导培养基,所述诱导培养基包括培养基成分和上述技术方案所述的刺激因子。
优选的是,所述诱导培养基中,重组人肿瘤生长因子-β的浓度为2~10ng/mL,雷帕霉素的浓度为50~150μM,绒促性素的浓度为250~600IU/L。
优选的是,所述诱导培养基还包括anti-CD3单抗、anti-CD28单抗和rhIL-2。
优选的是,所述诱导培养基中,anti-CD3单抗的浓度为5~15μg/mL,anti-CD28单抗的浓度为1~5μg/mL,rhIL-2的浓度为300~500U/mL。
本发明还提供了基于上述技术方案所述诱导培养基制备胚胎抗原特异性iTregs的方法,包括以下步骤:
将人外周血Tregs置于诱导培养基中进行诱导扩增,得到人外周血胚胎抗原特异性iTregs。
本发明还提供了上述技术方案所述方法制备得到的人外周血胚胎抗原特异性iTregs。
本发明还提供了上述技术方案所述人外周血胚胎抗原特异性iTregs在制备预防和/或治疗复发性流产、产前子痫和/或早产的药物和/或试剂盒中的应用。
优选的是,所述复发性流产包括原因不明复发性流产。
本发明提供了一种诱导Tregs得到胚胎抗原特异性iTregs的刺激因子。本发明所述刺激因子能够诱导得到数量充分,并可根据需要扩增到治疗数量的胚胎抗原特异性iTregs;本发明诱导得到的胚胎抗原特异性iTregs比Tregs具有更强的胚胎抗原特异性、自身稳定性及免疫抑制功能。本发明诱导得到的人外周血胚胎抗原特异性iTregs在维持母胎免疫耐受,抑制母胎免疫排斥方面有广阔的临床应用价值。本发明首次诱导出胚胎抗原特异性的iTregs,胚胎抗原特异性iTregs的高效制备能够为URSA患者的预防及治疗提供一个新的方向。
本发明利用分选出的纯度高的CD4 +CD25 +CD127 dim/-T细胞,通过体外诱导、扩增的方法获得胚胎抗原特异性iTregs,不仅能满足临床应用所需要的细胞数量,还能保持细胞功能的稳定性、免疫抑制功能和特异性,具有纯度高、活性强,稳定性好的效果。本发明通过鼠实验验证了胚胎抗原特异性iTregs的安全性及有效性,为原因不明复发性流产的诊断、治疗提供了新的理论、新方法。
附图说明
图1为本发明提供的本发明所述方法的流程图;
图2为本发明提供的CD4+T细胞、Tregs、扩增iTregs的纯度图;
图3为本发明提供的扩增的iTregs高表达胚胎抗原特异性HLA-G的结果图;
图4为本发明提供的扩增的iTregs高表达胚胎抗原特异性CCR6的结果图;
图5为本发明提供的扩增的iTregs高表达胚胎抗原特异性PD-1的结果图;
图6为本发明提供的胚胎抗原特异性iTregs纯度及活力的结果图;
图7为本发明提供的胚胎抗原特异性iTregs与Tregs的TGF-β、IL-10的表达图;
图8为本发明提供的培养液中加入雷帕霉素对细胞活力的影响结果图;
图9为本发明提供的培养液中加入雷帕霉素对Tregs/iTregs PD-1的影响结果图;
图10为本发明提供的培养液中加入雷帕霉素对冷冻细胞的保护作用的结果图;
图11为本发明提供的转输iTregs对CBA/J×BALB/C鼠妊娠的影响结果图。
具体实施方式
本发明提供了一种诱导Tregs(调节性T细胞,Tregs)得到胚胎抗原特异性iTregs的刺激因子,所述刺激因子包括雷帕霉素、重组人肿瘤生长因子-β和绒促性素。其中雷帕霉素维持细胞的稳定性,rhTGF-β促进细胞扩增,HCG诱导细胞转变为抗原特异性Tregs。本发明所述刺激因子能够在体外扩增诱导出足量的、功能稳定的胚胎抗原特异性iTregs(induced Tregs,iTregs)。
在本发明中,所述刺激因子中,雷帕霉素、重组人肿瘤生长因子-β和绒促性素的添加量之比优选为(50~150)μM:(2~10)ng/mL:(250~600)IU/L。在本发明中,雷帕霉素的溶媒优选包括DMEM,绒促性素和TGF-β的溶媒优选包括生理盐水。
本发明还提供了一种胚胎抗原特异性iTregs的诱导培养基,所述诱导培养基包括培养基成分和上述技术方案所述的刺激因子。在本发明中,所述培养基成分优选包括添加有1%青链霉素(100IU/ml青霉素和100μg/ml链霉素)、1%谷氨酰胺酶、10%人AB血清的RPMI 1640完全培养液。
在本发明中,所述诱导培养基中,重组人肿瘤生长因子-β的质量浓度优选为2~10ng/mL,更优选为5ng/mL,雷帕霉素的物质的量浓度优选为50~150μM,更优选为100μM,绒促性素的浓度优选为250~600IU/L,更优选为500IU/L。
在本发明中,所述诱导培养基优选还包括anti-CD3单抗、anti-CD28单抗和rhIL-2。
在本发明中,所述诱导培养基中,anti-CD3单抗的质量浓度优选为5~15μg/mL,更优选为10μg/mL,anti-CD28单抗的质量浓度优选为1~5μg/mL,更优选为2μg/mL,rhIL-2的浓度优选为300~500U/mL,更优选为400U/mL。
本发明还提供了基于上述技术方案所述诱导培养基制备胚胎抗原特异性iTregs的方法,包括以下步骤:
将人外周血Tregs置于诱导培养基中进行诱导扩增,得到人外周血胚胎抗原特异性iTregs。
在本发明中,所述人外周血调节性T细胞优选通过抗体孵育和磁珠分选方法分离获得。本发对所述分离的具体方法没有特殊限定,采用本领域技术人员熟知的常规人外周血调节性T细胞分离方法即可。在本发明中,分离的对象优选为原因不明复发性流产患者。本发明优选先采集原因不明复发性流产患者外周血,分离出外周血单个核细胞,洗涤并计数;然后将洗涤后的外周血单个核细胞中分离出CD4 +CD25 +CD127 dim/-T细胞,即人外周血调节性T细胞Tregs。采集后,外周血优选置于肝素抗凝管中。在本发明中,外周血单个核细胞的分离方法优选包括梯度离心分离法。在本发明中,优选利用磷酸盐缓冲液洗涤所述外周血单个核细胞,洗涤的次数优选为两次。
在本发明中,优选先磁珠分选分离CD4 +T细胞,再磁珠分选分离CD4 +CD25 +CD127 dim/-T细胞。具体的,磁珠分选技术分离CD4 +T细胞的具体步骤优选为:
将所述外周血单个核细胞按比例加分选试剂,即每107细胞加40uL CD4+CD25+CD127-抗生物素抗体、200uL CD4+CD25+CD127-免疫磁珠,震荡均匀,4℃孵育25分钟;再加磷酸盐缓冲液至10mL,1200rpm离心10分钟;离心后去上清,用500uL磷酸盐缓冲液重悬,过LD磁珠分选柱,待阴性细胞完全过柱后用3mL分离缓冲液冲洗柱子3次;将柱子取下,用5mL分离缓冲液将阳性细胞冲洗下来,收集入新的离心管;1200rpm离心5分钟,弃去4mL分离缓冲液,余下1mL缓冲液重悬细胞,取10uL计数,为6.4×106个,占外周血单个核细胞的20%;加分离缓冲液至5mL,1200rpm离心5分钟,弃上清,得CD4 +T细胞。
2)磁珠分选技术分离CD4 +CD25 +CD127 dim/-T细胞的具体步骤为:
①在获取的所述CD4+T细胞中,按比例加入分选试剂,即每107细胞加入180uL分离缓冲液、100uL CD25和100uLCD127磁珠,4℃孵育15分钟;
②加磷酸盐缓冲液10mL,1200rpm离心10分钟;离心后弃上清,用500uL分离缓冲液重悬,过MS磁珠分选柱,用3mL分离缓冲液冲洗柱子3次,收集通过柱子的细胞,即为CD4 +CD25 -细胞;
③将柱子取下,用5mL分离缓冲液将阳性细胞冲洗下来,收集入新的离心管,即为CD4 +CD25 +CD127 dim/-T细胞。
得到人外周血调节性T细胞Tregs后,本发明将人外周血调节性T细胞Tregs置于诱导培养基中进行诱导扩增,得到人外周血胚胎抗原特异性iTregs。在本发明中,所述人外周血调节性T细胞Tregs优选置于96孔细胞培养板中进行培养,每个孔优选添加2×105个Tregs(CD4 +CD25 +CD127 dim/-T细胞)。在本发明中,所述诱导的条件优选为37℃、5%CO2;所述诱导优选在培养箱中饱和湿度培养。在本发明中,所述诱导优选每3天换液1次,1周后开始每3天按1:3传代,连续培养3周,得到扩增100倍的人外周血胚胎抗原特异性iTregs。
本发明所述方法不仅能够获得纯度较高的胚胎抗原特异性CD4 +CD25 +CD127 dim/-T细胞(iTregs),还可以通过体外扩增的方法获得临床应用所需要的细胞数量,并保持细胞稳定的功能。
得到人外周血胚胎抗原特异性iTregs后,本发明优选对人外周血胚胎抗原特异性iTregs进行功能、特点鉴定。本发明对功能、特点简单的方法没有特殊限定,采用本领域技术人员公知的常规人外周血胚胎抗原特异性iTregs功能、特点鉴定方法即可。具体的:
1)利用流式细胞技术检测人外周血胚胎抗原特异性iTregs表达特点,检测与妊娠相关的Tregs表面标志的表达(HLA-G,PD-1,CCR6等表达),将胚胎抗原特异性Tregs与Tregs进行比较;
2)将Tregs、胚胎抗原特异性iTregs分别与与效应性T细胞(Teff)以0:1,0.5:1,1:1进行共培养,流式检测iTregs/Tregs的增殖力、抑制力;ELISA检测细胞因子表达,明确胚胎抗原特异性iTregs和Tregs功能的区别。
3)鼠实验确定胚胎抗原特异性iTregs对妊娠的影响。
将胚胎抗原特异性iTregs,通过尾静脉注射的方法,于交配前3天转输至流产鼠模型(CBA/J×BALB/C)体内。对照组为将DMEM于交配前3天转输至流产鼠模型(CBA/J×BALB/C)体内。观察:①胚胎吸收率。②胎鼠发育情况。③仔鼠及胎盘的发育情况。
本发明所获得的胚胎抗原特异性iTregs,具有以下特点:一、可由Tregs或CD4+T细胞诱导而来,细胞来源增多(图2);二、采用HCG诱导扩增,具有特异性的针对胚胎抗原的特性,反应迅速,扩增及免疫调节能力强(图3~图7);三、由雷帕霉素进行培养,iTregs的代谢及功能稳定,能够耐受冷冻、解冻等处理(图8~图11)。
本发明还提供了上述技术方案所述方法制备得到的人外周血胚胎抗原特异性iTregs。
本发明还提供了上述技术方案所述人外周血胚胎抗原特异性iTregs在制备预防和/或治疗复发性流产、产前子痫和/或早产的药物和/或试剂盒中的应用。
在本发明中,所述复发性流产优选包括原因不明复发性流产。
下面结合具体实施例对本发明所述的一种诱导Tregs得到胚胎抗原特异性iTregs的刺激因子、培养基及方法和应用做进一步详细的介绍,本发明的技术方案包括但不限于以下实施例。
实施例1
一种人外周血胚胎抗原特异性CD4 +CD25 +CD127 dim/-调节性T细胞的体外分离、扩增及方法(图1为本发明所述方法的流程图),取50mL患者外周血,通过密度梯度仪(流速为1.077g/mL)分离出外周血单个核细胞(PBMC),利用磷酸盐缓冲液(PBS)洗涤两次,计数之后,通过磁珠分选技术分离CD4 +T细胞(纯度为95%~99%),通过抗体孵育及磁珠分选技术分离出CD4 +CD25 +CD127 dim/-T细胞(纯度为90%以上),然后进行扩增CD4 +CD25 +CD127 dim/-T细胞(Tregs)。
具体的体外分离方法如下:
1)采集20例原因不明复发性流产妇女外周静脉血50mL(肝素抗凝,25u/mL血),将静脉血以1:1体积比平铺于淋巴细胞分离液(聚蔗糖-泛影葡胺,d=1.077)上,其中分离液:外周血=1:1.5~2倍体积,室温下离心2000rpm,10分钟,缓慢减速,无制动停转;
2)吸取中间层淋巴细胞层于另外离心管中,离心1200rpm,10分钟,见管底白色沉淀;
3)通过磁珠分选技术分离CD4+CD25+CD127dim/-Treg细胞:
a、弃上清,加PBS至10mL,弹匀,离心1200rpm,10分钟;
b、弃上清,加PBS至10mL,弹匀,计数;
c、离心1200rpm,10分钟,(此时准备LD柱,加PBS冲洗);
d、缓弃上清,弹匀,按比例加入CD4+CD25+CD127-T-cell Biotin-Antibodycocktail Ⅱ(每107细胞加入40uL试剂),混匀,4℃孵育10分钟,每107细胞加入200uL CD4+CD25+CD127-T ceLL Anti-Biotin Microbeads,混匀,4℃孵育15分钟;
e、每107细胞加入PBS1-2mL,离心1200rpm,10分钟;
f、弃上清,弹匀,加PBS混匀(根据细胞量调整),细胞悬液加入LD柱,细胞悬液流完后,加2mLPBS冲洗LD柱,两次;
G、收集流出的液体,即为CD4+细胞,加PBS10mL,细胞计数,离心1200rpm,10分钟;
H、弃上清,弹匀,每107细胞加180uL PBS重悬,100uL CD25和CD127磁珠Ⅱ(磁珠购自美天旎公司)混匀,4℃孵育15分钟(此时准备MS柱);
I、细胞悬液加入PBS至10mL,离心1200rpm,10分钟,弃上清,弹匀,每108细胞加入500uLPBS混合,加至MS柱,液体流尽后,PBS冲洗2-3次,流出的细胞即为CD4+CD25-细胞;
J、将MS柱置于无菌管中,加2mLPBS,迅速推动柱芯,冲出的细胞即为CD4+CD25+CD127dim/-Treg细胞。(4~6)×107PBMC细胞层,可以分离出(1~3)×105个CD4 +CD25 +CD127 dim/-T细胞。
K、检测上述CD4+T细胞、CD4 +CD25 +CD127 dim/-T细胞(Treg)细胞的纯度,检测方法具体如下:
①流式细胞仪(FCM)检测法,取CD4+细胞1×105左右,加入FITC anti-human CD4抗体20uL,室温下避光孵育15分钟;加入2mL PBS,1000rpm,7分钟,弃上清。另设对照组,上机检测,分析CD4+T细胞纯度。
②流式细胞仪(FCM)检测法,取CD4+细胞1×105左右,加入FITC anti-human CD4抗体20uL,PE anti-human CD25抗体20uL,briLLiant vioLet 510anti-CD127抗体20uL,室温下避光孵育15分钟;加入2mL PBS,1000rpm,7分钟,弃上清。另设对照组,上机检测,分析CD4+T细胞和CD4+CD25+CD127dim/-T细胞的纯度。CD4+T细胞、Tregs纯度达到93%以上。
4)体外扩增诱导胚胎抗原特异性iTregs。
将2×105Treg细胞培养于加有1%青链霉素,1%谷氨酰胺酶,10%人AB血清,RPMI1640完全培养液的96孔细胞培养板中,每孔中加入游离的anti-CD3单抗(终浓度为10ug/mL)和包被的anti-CD28单抗(终浓度为2ug/mL),400U/mL rhIL-2,100uM的雷帕霉素+5ng/mL rhTGF-β+绒促性素500IU作为刺激因子,置37℃、5%CO2培养箱饱和湿度培养,每3天换液1次,1周后开始每3天按1:3传代,连续培养3周,3周后细胞数量约为原代细胞数的100倍,为胚胎抗原特异性iTregs。
5)检测胚胎抗原特异性iTregs的表达特点及功能。
①流式细胞仪(FCM)检测法检测扩增Tregs纯度及表达特点。取扩增的细胞1×105左右,加入peridinin chlorophyll protein(PerCP)/Cy5.5-conjugated anti-CD3,fluorescein isothiocyanate(FITC)-anti-CD4,PE-conjugated anti-CD25,briLLiantvioLet 510 anti-CD127,briLLiant vioLet 421 anti-CD152(CTLA-4),aLLophycocyaninAPC anti-HLA-G,APC/Fire750 anti-CD279(PD-1),and PE/Cy7 anti-CD357(GITR),(BioLegend,San Diego,CA,USA),室温下避光孵育15分钟;加入2mLPBS,1000rpm,7分钟,弃上清。另设对照组,上机检测,分析胚胎抗原特异性iTregs的纯度及表达特点。扩增的CD4+T细胞,Tregs、iTregs的纯度均>95%(图2)。扩增的iTregs具有胚胎抗原特异性,表现为高表达HLA-G(图3);高表达CCR6(图4);高表达PD-1(图5)。
②流式细胞仪(FCM)检测法检测扩增iTregs的纯度及活力。扩增后的iTregs悬液用10μL台盼蓝染色,镜下计数细胞,并计算活细胞率。图6显示,扩增iTregs的纯度>95%,活力>93%。
③Transwell体系共培养检测扩增iTregs的抑制功能。取原因不明复发性流产患者Tregs、扩增的iTregs各2×105/mL放入Transwell体系上室,取CD4+CD25-T细胞2×105/mL放入下室,共同培养6天。将细胞培养于加有1%青链霉素,1%谷氨酰胺酶,10%热灭活胎牛血清,RPMI 1640完全培养液的24孔细胞培养板中,每孔中加入游离的anti-CD3单抗(终浓度为10ug/mL)和包被的anti-CD28单抗(终浓度为2ug/mL),置37℃、5%CO2培养箱饱和湿度培养,每3天换液1次。流式细胞术检测Tregs、胚胎抗原特异性iTregs细胞因子TGF-β、IL-10的表达,结果见图7,胚胎抗原特异性iTregs TGF-β、IL-10的产生较Tregs明显增加,提示iTregs的免疫功能比Tregs强。
实施例2
按照上述分选方法1)-3),获取CD4+T细胞,Tregs。将Tregs置于不同浓度雷帕霉素中进行培养,其他培养条件不变,观察不同浓度雷帕霉素对于Tregs功能及活性的影响。
①加入不同浓度(0μM,50μM,100μM,200μM)的雷帕霉素,发现100μM的雷帕霉素显著增加Tregs/PD-1、胚胎抗特异性iTregs/PD-1的表达,增强细胞稳定性(结果见图8)。
②比较冷冻、解冻对加入雷帕霉素组与未加雷帕霉素组CD4+T细胞及Tregs活力的影响。加药组CD4+T细胞活力明显增加50.9%±2.9%vs 41.3%±2.4%(P<0.05);加药组Tregs活力明显增加49.9%±2.1%vs 40.9%±3.5%(P<0.05),提示雷帕霉素能增加细胞的稳定性(结果见图9)。
③将CD4+T细胞及Tregs置入含雷帕霉素培养液中培养后进行冷冻、解冻,台盼蓝染色观察CD4+T及Tregs细胞活力。CD4+T细胞活力由82.4%±3.8%下降到50.9%±2.9%(P<0.01);Tregs活力由78.2%±6.7%下降到49.9%±2.1%(P<0.01),提示雷帕霉素能保护冷冻、解冻过程中细胞的活性(结果见图10)。
本部分结果证明了,本发明中加用的雷帕霉素,能稳定体外诱导、扩增过程中iTregs的稳定性,明确了100μM的雷帕霉素的效果最佳。另外,培养液中加入雷帕霉素能增加冷冻、解冻过程中细胞的稳定性。
实施例3
分选正常CBA/J鼠Tregs,按照上述1)-4)步骤进行Tregs分选,诱导及扩增。将胚胎抗原特异性iTregs于交配前3天经尾静脉转输至流产鼠模型CBA/J×BALB/C鼠体内,对照组为DMEM尾静脉输注。发现转输iTregs的CBA/J×BALB/C孕鼠:①胚胎吸收率降低(A,B);②孕鼠体重增加(C);③仔鼠体重增加,胎盘重量增加(D)(结果见图11)。鼠实验证明,转输iTregs能降低流产孕鼠的流产率,为原因不明复发性流产的治疗提供实验依据,为将理论向临床治疗的转化提供理论基础。
本申请通过分选Treg细胞,采用上述1)-4)的方法,对Tregs进行诱导、扩增。本发明的培养条件,具有以下优点:①将Tregs转化为具有胚胎抗原特异性的iTregs,表现为HLA-G的表达增加,CCR6的表达增加,以及PD-1表达的增加,抑制功能增强。②维持诱导、扩增情况下iTregs的稳定性。③提高Tregs/iTregs在冷冻、解冻过程中的稳定性。④采用本方法诱导扩增所得的将胚胎抗原特异性iTregs转输至CBA/J×BALB/C流产鼠体内,能降低胚胎丢失率,增加仔鼠及胎盘重量。
本发明首次应用雷帕霉素,TGF-β和HCG诱导、扩增出胚胎抗原特异性性iTregs,具有功能稳定,免疫调控能力强,能耐受冷冻及解冻的影响,能够减少流产鼠的流产率,增加孕鼠及胎盘的体重,为原因不明复发性流产的诊断、治疗提供了理论及实验依据。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (4)
1.基于胚胎抗原特异性iTregs的诱导培养基制备胚胎抗原特异性iTregs组合物的方法,包括以下步骤:
将人外周血CD4 +CD25 +CD127 dim/-Tregs细胞置于诱导培养基中进行诱导扩增,置37℃、5%CO2、饱和湿度培养,每3天换液1次,1周后开始每3天按1:3传代,连续培养3周,得到胚胎抗原特异性iTregs组合物;
所述诱导培养基包括培养基成分和刺激因子;
所述培养基成分为,加有1%青链霉素,1%谷氨酰胺酶,10%人AB血清的RPMI 1640完全培养液;
所述刺激因子为,5ng/mL重组人肿瘤生长因子-β,100μM雷帕霉素,500IU/L绒促性素,10μg/mL游离的anti-CD3单抗、2μg/mL包被的anti-CD28单抗和400U/mLrhIL-2。
2.权利要求1的方法,所述人外周血CD4 +CD25 +CD127 dim/-Tregs细胞的制备方法包括以下步骤:
人外周血通过流速为1.077g/mL的密度梯度仪分离出外周血单个核细胞(PBMC),利用磷酸盐缓冲液(PBS)洗涤两次,计数之后,通过磁珠分选技术分离CD4 +T细胞,通过抗体孵育及磁珠分选技术分离出CD4 +CD25 +CD127 dim/-Tregs细胞,然后进行扩增。
3.权利要求1或2所述方法制备得到的人外周血胚胎抗原特异性iTregs组合物。
4.权利要求3所述人外周血胚胎抗原特异性iTregs组合物在制备预防和/或治疗原因不明复发性流产的药物和/或试剂盒中的应用。
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