CN106947738A - microRNA‑4281的新用途 - Google Patents
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Abstract
本发明提供了microRNA‑4281的新用途,本发明通过microRNA‑4281在体外诱导CD4+初始T细胞向Treg细胞分化,达到在体外制备Treg细胞的目的。本发明发现microRN‑4281能够在体外有效的将初始CD4T细胞诱导扩增为调节性T细胞,并在体外稳定其转录因子表达并促进功能的稳定。
Description
技术领域
本发明涉及microRNA-4281的新用途,具体涉及microRNA-4281在体外诱导或促进CD4+初始T细胞向Treg细胞分化、增强Foxp3基因表达、稳定Treg细胞功能中的用途。
背景技术
调节性T淋巴细胞(Treg细胞)是一群具有免疫抑制功能的CD4+T淋巴细胞亚群,在维持人体免疫耐受和维持自身免疫平衡中起到重要的作用。而FOXP3基因作为转录因子,不仅决定着Treg细胞的分化发育,而且是Treg细胞发挥功能的必要条件。对FOXP3基因的调控对于Treg细胞来说至关重要,而激活FOXP3转录上调的信号研究并不透彻,近几年的研究表明,TCR、CD28、IL-2和TGF-β等信号转导途径与FOXP3基因的表达有联系,然而这些信号转导途径如何发挥调控的作用,是否有其他的信号也影响着FOXP3基因的表达有待进一步的研究和发现。
造血干细胞的移植为很多髓细胞白血病患者提供了治疗的机会,但是移植术后宿主抗移植物病(graft-versus-host disease,GVHD)限制了治疗的发展。基于Treg细胞具有免疫抑制功能,所以Treg细胞的细胞过继免疫治疗在国际上成为了一种新兴有效的治疗和预防急性GVHD的有效手段。将体外诱导的Treg细胞过继回输到移植患者体内,可以有效的提高患者生存率。但是在临床试验当中,该细胞治疗面临了全新的问题:体外诱导Treg细胞数量不足;FOXP3基因的表达不稳定;导致Treg细胞功能性缺失,极大的削弱了细胞治疗的疗效。因此,如何在体外大量诱导扩增Treg细胞,并寻找和验证在体外诱导扩增中可以维持FOXP3稳定性高表达的方法成为了Treg细胞治疗的迫切需要。
发明内容
本发明的目的在于克服现有技术存在的不足之处而提供了microRNA-4281的新用途。
为实现上述目的,所采取的技术方案:一种促进CD4+初始T细胞向Treg细胞分化的试剂盒,所述试剂盒包括microRNA-4281。
本发明提供了一种在体外促进CD4+初始T细胞向Treg细胞分化的试剂盒,,所述试剂盒包括microRNA-4281。
本发明提供了一种增强Foxp3基因表达的试剂盒,所述试剂盒包括microRNA-4281。
本发明提供了一种稳定Treg细胞功能的试剂盒,所述试剂盒包括microRNA-4281。
本发明提供了microRNA-4281在制备诱导或促进CD4+初始T细胞向Treg细胞分化的试剂或药物中的用途。
本发明提供了microRNA-4281在制备在体外诱导或促进CD4+初始T细胞向Treg细胞分化的试剂或药物中的用途。
本发明提供了microRNA-4281在制备增强Foxp3基因表达的试剂或药物中的用途。
本发明提供了microRNA-4281在制备稳定Treg细胞功能的试剂或药物中的用途。
其中,所述Treg细胞为CD4+T细胞。
本发明通过前期专利中报道的一种微小RNA(microRNA,miRNA)全新的基因调节机制,即通过和基因的启动子序列TATA-box基序相互作用,促进和稳定基因的表达,研发了一种全新的调节稳定FOXP3表达的机制。通过miR-4281实现FOXP3基因的稳定性高表达,并极有可能研发出一种GVHD疾病治疗中,提高Treg细胞治疗效果的一种全新的治疗方法,实际解决了Treg细胞在临床细胞治疗过程中所面临的难扩增,扩增后功能不稳定等具体问题,具有广泛的应用前景。
本发明的有益效果在于:
1、本发明利用miR-4281作为核酸类药物,在体外扩增Treg细胞。有非常强的创新性。
2、为我们进一步提高肿瘤细胞治疗提供了强有力的实践基础,具有重要的开发价值和推广意义。通过miR-4281作为核酸类药物,稳定了Treg细胞FOXP3表达,维持了Treg细胞功能的稳定,实际解决了Treg细胞在临床细胞治疗过程中所面临的难扩增,扩增后功能不稳定等具体问题。创新性提出了GVHD疾病治疗中,提高Treg细胞治疗效果的一种全新的治疗方法,具有广泛的应用前景。
附图说明
图1为本发明实施例1中miR-4281特异性的结合FOXP3基因启动子TATA-box基序、促进基因表达的图片;其中,图1中A:双报告系统检测预测的miRNA对FOXP3基因启动子序列的调控作用;B:miR-4281与FOXP3基因启动子TATA-box基序的结合示意图;C:FOXP3基因启动子TATA-box基序的突变影响miRNA对其的转录增强调控;D:miR-4281结合位点的突变影响其对FOXP3基因启动子的转录增强;E:miR-4281的回复突变恢复对Foxp3启动子序列的转录增强效应;
图2为本发明实施例2中miR-4281在体外促进Treg细胞的分化和发育的图片;其中,图2中A、B:miR-4281促进Treg细胞形成;C、D:miR-4281的反义互补链抑制了Treg细胞形成;E:Treg表面相关蛋白的表达增强;
图3为本发明实施例2中miR-4281体外诱导的Treg细胞功能检测图片;其中,图3中A、B、Treg细胞胞内IL-2和IFN-γ的检测;C、Treg细胞体外抑制试验;D、小鼠组织免疫组化HE染色。
具体实施方式
下面结合附图和具体实施例进一步详细说明本发明。除非特别说明,本发明采用的试剂、设备和方法为本技术领域常规市购的试剂、设备和常规使用的方法。
实施例1:miR-4281特异性的结合FOXP3基因启动子TATA-box基序,促进基因表达
(1)将在广州瑞博生物科技公司化学合成五个miRNA的成熟体,与克隆并测序正确的Foxp3启动子序列荧光素酶双报告质粒共转染进293T细胞系,72小时之后收样用双报告系统进行检测FOXP3基因的表达。
(2)对结合区域的Foxp3序列在质粒上进行了突变,突变点在图中用红色标识,将miR-4281和野生型,突变型的Foxp3启动子序列荧光素酶双报告质粒共转染,72小时后收样检测。发现Foxp3结合位点发生突变的质粒,miR-4281对其表达提高的功能消失。
(3)在公司合成了miR-4281的突变型的成熟体,将这些突变型的miR-4281与Foxp3启动子序列荧光素酶双报告质粒共转染,72小时后收样检测。发现序列突变之后的miR-4281对Foxp3启动子序列增强转录效果也同时消失。
(4)将序列对应的Foxp3mt-1启动子序列荧光素酶双报告质粒与miR-4281mt-1微小RNA共转染到293T细胞中,通过荧光素酶双报告系统进行检测,发现miRNA对启动子的增强调控能力恢复。
根据实施例1说明,miR-4281可以促进Foxp3启动子序列的表达,并且调控高度依赖序列的特异性(图1)。
实施例2:miR-4281在体外可以促进Treg细胞的分化和发育
(1)流式细胞分选得到CD4+CD25-的初始CD4T细胞,我们在anti-CD3、anti-CD28激活的CD4+CD25-的初始T细胞中过表达miR-4281,并使用IL-2和TGF-β诱导初始T细胞向Treg细胞分化。过表达miR-4281的48小时之后,我们使用实时定量PCR的方法,对Foxp3的mRNA水平进行了检测。过表达miR-4281的实验组,能检测到mRNA水平显著的提高。
(2)细胞诱导的第五天,我们使用流式细胞技术对Foxp3的蛋白水平进行了检测,同时也检测了CD25+Foxp3+这一类具有Treg细胞表型的细胞亚群形成的情况。结果显示Foxp3的蛋白表达水平显著提高,并且过表达miR-4281的实验组,可以诱导得到更多的CD25+Foxp3+Treg细胞。
(3)公司合成的miR-4281的反义互补链inhibitor miR-4281,将其转染至anti-CD3、anti-CD28激活的CD4+CD25-的初始T细胞中,并使用IL-2和TGF-β诱导初始T细胞向Treg细胞分化。结果证明,miR-4281的反义互补链将microRNA抑制后,Foxp3的mRNA和蛋白水平都受到了抑制,并且CD25+Foxp3+Treg细胞形成减少。
(4)对诱导形成的Treg细胞表型进行了检测。分别对Treg相关的表面marker,CD25,CD127,CD103,CTLA4,GITR,PD-1使用流式细胞计数进行了检测,灰色实线代表NC对照组,黑色实验代表miR-4281实验组。流式结果显示,我们诱导得到的Treg,NC对照组和过表达miR-4281的实验组都可以看到比较明显的CTLA-4和GITR的表达,miR-4281有更高的CD25和PD-1表达,以及更低的CD127的表达。
根据实施例2的结果说明,miR-4281在体外可以促进Treg细胞的分化和发育(图2)。
实施例3:miR-4281体外诱导的Treg细胞功能检测。
(1)使用流式细胞技术,破膜对IL-2和IFN-γ的膜内表达进行了检测。灰色实线代表NC对照组,黑色实验代表miR-4281实验组。实验结果表明,过表达miR-4281的实验组,细胞内IL-2和IFN-γ的表达都比对照组降低,其中IFN-γ的表达降低有显著差异。
(2)体外抑制试验中,我们使用来自同一个donor的初始CD4+T细胞和CD8+T细胞进行试验。初始CD4+T细胞按照前文所述的方法,分为对照组合试验组,在有利于Treg细胞分化的条件下体外进行诱导。诱导五天之后,和使用CFSE染色,并且过夜anti-CD3和anti-CD28激活的CD8+T进行共培养。共培养五天之后,使用流式细胞技术对细胞进行检测。在CD8+阳性的门当中检测CFSE的表达。
(3)使用的免疫缺陷型小鼠NOG小鼠。NOG小鼠是由日本实验动物研究所培育的,通过杂交NOD/scid小鼠和γ-链和IL-2受体敲出小鼠成功建立的一个免疫功能严重不全的小鼠品系。我们从健康人的血液中分离得到的PBMC通过尾静脉注射至NOG小鼠体内,实时监测小鼠体重的变化,以确认异种的GVHD模型的建立。GVHD建立之后,我们从尾静脉注射进同个donor来源,经过诱导得到的Treg细胞。在注射Treg细胞10天之后,我们取小鼠的肝脏,肾脏以及肺部进行免疫组化的HE染色。
根据实施例3的结果说明,miR-4281体外诱导得到的Treg细胞功能更加稳定,免疫抑制效果增强(图3)。
最后所应当说明的是,以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制,尽管参照较佳实施例对本发明作了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的实质和范围。
Claims (8)
1.一种促进CD4+初始T细胞向Treg细胞分化的试剂盒,其特征在于,所述试剂盒包括microRNA-4281。
2.一种在体外促进CD4+初始T细胞向Treg细胞分化的试剂盒,其特征在于,所述试剂盒包括microRNA-4281。
3.一种增强Foxp3基因表达的试剂盒,其特征在于,所述试剂盒包括microRNA-4281。
4.一种稳定Treg细胞功能的试剂盒,其特征在于,所述试剂盒包括microRNA-4281。
5.microRNA-4281在制备诱导或促进CD4+初始T细胞向Treg细胞分化的试剂或药物中的用途。
6.microRNA-4281在制备在体外诱导或促进CD4+初始T细胞向Treg细胞分化的试剂或药物中的用途。
7.microRNA-4281在制备增强Foxp3基因表达的试剂或药物中的用途。
8.microRNA-4281在制备稳定Treg细胞功能的试剂或药物中的用途。
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