CN104975041A - 一种促进单核细胞向巨噬细胞分化的方法 - Google Patents
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Abstract
本发明公开了一种促进单核细胞向巨噬细胞分化的方法,是通过下调单核细胞内p38IP蛋白水平实现的。本发明利用RNA干扰方法或细胞内信号通路调节单核细胞内p38IP蛋白水平,进而促进单核细胞向巨噬细胞的定向分化,可以增加巨噬细胞的获得率,克服了现有技术中单核细胞诱导向巨噬细胞分化的方法复杂且不理想的技术问题,针对巨噬细胞分化紊乱疾病提出新的研究和治疗思路。
Description
技术领域
本发明涉及生物技术和医学领域,具体地涉及一种促进单核细胞向巨噬细胞分化的方法。
背景技术
p38结合蛋白(p38MAP kinase interacting protein,p38IP)首次发现于酵母双杂交实验中,因其与p38α有相互作用而得名(NCBIAccession#AF093250)。在造血过程中,转录因子和相关的调节蛋白控制细胞生长和分化过程而对正常的血细胞生成过程起到重要作用,而不正常的血细胞生成则可能导致白血病的发生。在探索造血过程中的新型调节因子时,p38IP被发现为CD34抗原阳性造血干细胞的新型转录因子,这些新型调节因子的发现对于了解造血过程和造血过程紊乱的分子机制具有重要意义(1)。在发育生物学的研究方面,p38IP通过结合并激活p38而对钙粘蛋白E-cadherin的下调和上皮间质转化(epithelial tomesenchymal transition,EMT)过程起到重要作用,因而对鼠原肠胚形成过程进行调节。小鼠发育过程中p38IP蛋白的缺陷会导致神经管闭合不全、眼发育及原肠胚形成缺陷等(2)。在癌症发生和形成的研究中,p38IP被发现在前列腺癌病人组织中表达下调(3);此外,p38IP参与人SAGA复合体的组装和激活,酵母中p38IP的同源蛋白yspt20被报道参与SAGA复合体的组装并对其转录活性和结构稳定性都起到重要作用(4-6)。此外p38IP还被报道参与调节ER stress(5)和starvation所引起的Autophagy(7);最近我们的研究表明p38IP能够抑制GCN5的泛素化因此稳定GCN5蛋白,进而调节α-tubulin的乙酰化从而促进细胞周期G2/M期转换(8)。这些研究都说明p38IP是个多功能蛋白,并有可能参与免疫细胞分化的调节作用。
单核巨噬细胞系统(mononuclear phagocyte system,MPS)包括血液中的单核细胞和组织中固定或游走的巨噬细胞,其分化最终起源于造血干细胞。骨髓造血干细胞在各种转录因子和调节蛋白的作用下向多能祖细胞和专能祖细胞分化,其首先在骨髓中分化为髓系祖细胞和淋巴系祖细胞,多能性髓系祖细胞随后向粒细胞--单核细胞前体分化,在这一阶段其表面表达CD16和CD32的细胞经过原单核细胞、前单核细胞阶段后分化发育成单核细胞后由骨髓进入血液循环,随后在组织或浆膜腔中进一步分化成巨噬细胞和树状细胞(9)。当血液中的单核细胞进入组织分化成巨噬细胞后,一般不再返回血液循环;巨噬细胞在组织中虽有增殖潜能,但很少分裂,属于分化成熟细胞,主要通过血液中的单核细胞分化补充(10)。单核巨噬细胞系统作为天然免疫系统的主要组成部分,在机体抵御外源微生物入侵和清除病原体等方面起着重要作用。单核细胞作为免疫效应细胞,在机体受外源入侵时从血液迁移至组织中并产生大量的炎症因子;同时其本身具有吞噬功能,能吞噬细胞和有害分子;此外,单核细胞还可分化成树状细胞或巨噬细胞进一步发挥免疫功能(11,12)。巨噬细胞具有多方面的生物功能,其在天然免疫和适应性免疫的功能主要包括以下几个方面:非特异性免疫防疫,即在机体受到外来病原体入侵激发免疫应答前吞噬清除病原体;清除外来细胞;非特异性免疫监视;递呈抗原;分泌介质IL-1、干扰素及补体(C1,C4,C3,C2,B因子)等细胞因子(13)。此外巨噬细胞在组织发育、修护和维持机体稳定等方面也有重要功能(14-16)。由于单核巨噬细胞系统在机体功能的重要性,使得单核细胞分化成巨噬细胞这一过程尤为重要。而这一过程需要多种转录因子和调节蛋白的参与。巨噬细胞分化的紊乱会导致多种疾病的发生,比如炎症反应,自身免疫性疾病和白血病等。
参考文献
1.Gomes I,etal.(2002)Novel transcription factors in human CD34antigen-positivehematopoietic cells.Blood 100(1):107-119.
2.Zohn IE,etal.(2006)p38and a p38-interactingprotein are critical for downregulationof E-cadherin during mouse gastrulation.Cell 125(5):957-969.
3.SchmidtU,etal.(2005)Quantification ofC13orf19/P38IP mRNA expression byquantitative real-time PCR in patients with urological malignancies.Cancer Lett225(2):253-260.
4.Wang YL,Faiola F,Xu M,Pan S,&Martinez E(2008)Human ATAC Is aGCN5/PCAF-containing acetylase complex with a novel NC2-like histone fold modulethat interacts with the TATA-binding protein.J Biol Chem 283(49):33808-33815.
5.Nagy Z,et al.(2009)The human SPT20-containing SAGA complex plays a direct role inthe regulation of endoplasmic reticulum stress-induced genes.Mol Cell Biol29(6):1649-1660.
6.Marcus GA,Horiuchi J,Silverman N,&Guarente L(1996)ADA5/SPT20links the ADAand SPT genes,which are involved in yeast transcription.Mol Cell Biol16(6):3197-3205.
7.Webber JL&Tooze SA(2010)Coordinated regulation of autophagy by p38alpha MAPKthrough mAtg9and p38IP.EMBOJ29(1):27-40.
8.Liu X,etal.(2013)The p38-interacting protein(p38IP)regulates G2/M progression bypromoting alpha-tubulin acetylation via inhibiting ubiquitination-induced degradationof the acetyltransferase GCN5.J Biol Chem 288(51):36648-36661.
9.ImhofBA&Aurrand-Lions M(2004)Adhesion mechanisms regulating the migration ofmonocytes.Nature reviews.Immunology4(6):432-444.
10.Mosser DM&Edwards JP(2008)Exploringthe full spectrum of macrophage activation.Nature reviews.Immunology 8(12):958-969.
11.Chawla A(2010)Control of macrophage activation and function by PPARs.Circulationresearch 106(10):1559-1569.
12.Heine GH,et al.(2012)Monocyte subpopulations and cardiovascular risk in chronickidney disease.Nature reviews.Nephrology 8(6):362-369.
13.Gordon S(2003)Alternative activation of macrophages.Nature reviews.Immunology3(1):23-35.
14.Naito M(2008)Macrophage differentiation and function in health and disease.PatholInt58(3):143-155.
15.Martinez FO,Helming L,&Gordon S(2009)Alternative activation of macrophages:animmunologic functional perspective.Annual review of immunology 27:451-483.
16.Wynn TA,Chawla A,&Pollard JW(2013)Macrophage biology in development,homeostasis and disease.Nature 496(7446):445-455.
发明内容
本发明的目的是为了提供一种促进单核细胞向巨噬细胞分化的方法,所述方法可以针对巨噬细胞分化紊乱疾病提出新的研究和治疗思路。
为解决上述技术问题,本发明的技术方案为:
一种促进单核细胞向巨噬细胞分化的方法,是通过下调单核细胞内p38IP蛋白水平实现的。
进一步地,所述下调单核细胞内p38IP蛋白水平可以通过RNA干扰的方法实现。
作为本发明的一种优选的实施方式,所述RNA干扰为shRNA干扰方法。
进一步地,所述shRNA干扰方法中,使用的shRNA序列为:5'-ACACAAGAGCACTGAATCA-3'。
作为本发明的另一种优选的实施方式,所述RNA干扰为siRNA干扰方法,
进一步地,所述siRNA干扰方法中,使用的siRNA序列为:
si-p38IP-1:5'-GCTTGTTATGCAAGAGACT-3',
si-p38IP-2:5'-GCAACAAGCTTTAGAACTA-3'。
作为本发明的另一种优选的实施方式,所述下调单核细胞内p38IP蛋白水平可以通过在所述单核细胞中过表达miR-200b-3p mimics实现。
本发明的优点在于:
通过本发明的方法,只需要下调单核细胞内p38IP蛋白水平,即可实现促进单核细胞向巨噬细胞的定向分化,从而增加巨噬细胞的获得率,克服了现有技术中单核细胞诱导向巨噬细胞分化的方法复杂且不理想的技术问题,针对巨噬细胞分化紊乱疾病提出新的研究和治疗思路。
为了更好地理解和实施,下面结合附图详细说明本发明。
附图说明
图1是PMA诱导U937细胞分化48h后,p38IP蛋白western bolt结果,结果显示,在PMA诱导的U937细胞分化后,p38IP蛋白的表达量有显著降低,而与其相关的p38蛋白表达量则没有太大变化。
图2是PMA诱导U937细胞分化48h后,p38IP mRNA水平的荧光定量PCR检测结果,结果显示,在PMA诱导的U937细胞分化后,p38IP的mRNA水平在分化后也有显著降低。
图3为M-CSF体外诱导人外周血单核细胞分化为巨噬细胞的细胞形态图。结果表明按照本发明方法,M-CSF可以体外诱导人外周血单核细胞分化为巨噬细胞的细胞。
图4为M-CSF体外诱导人外周血单核细胞分化为巨噬细胞的过程中p38IP表达水平的western blot检测。
图5为M-CSF体外诱导人外周血单核细胞分化为巨噬细胞的过程中p38IPmRNA水平的荧光定量PCR检测。结果都表明p38IP在原代单核细胞向巨噬细胞分化过程中表达水平降低。
图6是RNA干扰载体pSUPER.retro.neo+GFP的图谱。
图7是利用shRNA沉默p38IP的U937细胞的western blot结果图,结果显示,p38IP表达被干扰,获得了稳定的p38IP干扰细胞系。
图8是用流式细胞仪检测shRNA干扰p38IP后的U937细胞的结果图。其中,A为流式细胞术的分析结果图;B为PMA刺激后CD11b/CD14阳性细胞占细胞总数的百分比。结果表明,利用shRNA干扰p38IP后的U937细胞中,细胞表面CD11b/CD14阳性细胞比率显著升高。
图9是U937细胞中,利用siRNA针对p38IP不同位点进行干扰的western blot结果图,结果显示,在试验组的U937细胞中p38IP被干扰。
图10是用流式细胞仪检测siRNA针对p38IP不同位点进行干扰U937细胞结果图。其中,A为流式细胞术的分析结果图;B为PMA刺激后CD11b/CD14阳性细胞占细胞总数的百分比。结果表明,利用瞬时siRNA干扰p38IP,可以促进U937在PMA诱导下的分化,其表面CD11b/CD14阳性细胞比率升高,并且这一升高与p38IP干扰效果成正比。
图11是转入miR-200b-3p mimics的U937细胞western blot结果图,结果显示,miR-200b-3p mimics的转入成功的降低了p38IP蛋白的表达水平。
图12是用流式细胞仪检测miR-200b-3p mimics转染的U937细胞表面CD11b/CD14的表达情况,其中,A为流式细胞术的分析结果图;B为CD11b/CD14阳性细胞占细胞总数的百分比。结果显示,当miR-200b-3p过表达后,CD11b/CD14阳性的细胞比率显著升高。
具体实施方式
以下实施例仅用于说明本发明而不用于限制本发明的范围。实施例中未注明具体条件的实验方法,通常按照常规条件,或按照制造厂商所建议的条件。
本发明中使用到的生物材料和试剂:
1.细胞:
U937细胞购自美国模式培养物集存库(ATCC)。U937细胞培养基为RPMI-1640培养基(含10%FBS,100U/ml青霉素及0.1mg/ml链霉素)。稳定干扰细胞系所用培养基另外加入700mg/L(终浓度)G418。细胞培养于37℃、5%CO2细胞培养箱。
2.抗体:
PE Mouse anti-human CD11b/Mac-1(555388)和APC Mouse anti-human CD14(555399)购自BD Pharmingen(San Diego,CA,USA),CD14+microbeads(human)(130-050-201)购自Miltenyi Biotec;Recombinant Human M-CSF(cyt-308)购自Prospec protein specialists。
3.小分子RNA:
siRNA序列及microRNAmimics由广州锐博生物科技公司合成。
siRNA干扰序列:
si-p38IP-1:5'-GCTTGTTATGCAAGAGACT-3'
si-p38IP-2:5'-GCAACAAGCTTTAGAACTA-3'
4.RNA干扰载体:
shRNA干扰序列:Scramble shRNA:5'-CGCTAATTCGACTCGGATA-3'
sh-p38IP:5'-ACACAAGAGCACTGAATCA-3'
本文中使用的RNA干扰载体(RNA interference vector)为pSUPER.retro.neo+GFP,该载体上包含H1启动子、GFP绿色荧光蛋白基因及neo霉素抗性基因,可通过流式细胞仪筛选GFP绿色荧光阳性细胞,或用G418药物筛选含抗性阳性细胞。
U937均为人白血病单核细胞系,研究表明在PMA处理下,这两种细胞会向类巨噬细胞分化,其表现为细胞贴壁性增强,同时细胞表面糖蛋白CD11b和CD14表达量上升,因此在实验中,我们使用这两株细胞系作为研究材料,并以其表面CD11b/CD14阳性细胞比率及细胞形态作为细胞分化的检测指标。
【实施例1】p38IP在单核细胞向巨噬细胞分化过程中表达下调
PMA刺激U937细胞:
1)计数细胞,1000rpm、3min离心收取细胞,PBS洗一次,将上清完全去除;
2)用含10%FBS的RPMI-1640培养基重悬细胞,密度为3×105/ml,0.5ml/孔铺在24孔板内;
3)加入10nM(终浓度)PMA(丙二醇甲醚醋酸酯)刺激细胞,充分混匀后将细胞置于37℃,5%CO2细胞培养箱培养,按照刺激时间点停止刺激,离心后用于后续实验。
对诱导后的细胞进行western bolt和荧光定量PCR检测其p38IP含量,我们观察到,在PMA诱导的U937细胞分化后,p38IP蛋白的表达量有显著降低,而与其相关的p38蛋白表达量则没有太大变化(图1)。同时,我们通过荧光定量PCR检测p38IP在细胞分化前后的mRNA水平,发现p38IP的mRNA水平在分化后也有显著降低(图2)。
M-CSF体外诱导人外周血单核细胞分化巨噬细胞
1.人外周血分离人外周血单核细胞
1)健康人外周血由广州军区总医院馈赠,将人外周血(已加入抗凝剂)与Hanks溶液等体积混合;
2)将15ml Ficoll溶液加入50ml离心管中后,小心用滴管将外周血-Hanks混合液加在Ficoll溶液上层,注意要保持分层界面,加至45ml处即可;
3)离心:2000rpm离心20min。离心后溶液分为3层,在上层和中层分界处有一团白色雾状单个核细胞群,称为白膜层;
4)将吸管小心插入白膜层,吸取白膜层悬液后置于新的50ml离心管中;
5)加入等体积的Hanks溶液后,1500rpm离心15min,去上清;
6)加入25ml Hanks溶液洗涤细胞两次,离心后去上清即可获得PBMC。
2.CD14+单核细胞分离
1)计数上述获得的PBMC,300g离心10min,将上清完全去除;
2)每107个细胞加入80ul buffer重悬细胞,并加入20ul CD14Microbeads混合均匀置于2-8℃避光孵育15min;
3)每107个细胞加入1-2ml buffer洗去未结合的Microbeads,离心后每108个细胞用500ul buffer重悬;
4)将LS分离柱置于分离磁场中,预先用3ml buffer润洗柱子;
5)将细胞重悬液加入LS分离柱,重力作用使其缓慢滴下未结合部分,此时结合磁珠部分细胞应吸附于柱子中;
6)用3ml buffer洗涤柱子3次,重力作用使其滴下,洗去多余未结合部分;
7)将分离柱从磁场中取出,置于15ml离心管口,加入5ml buffer后,用针芯将溶液冲入15ml管中,即可获得阳性结合细胞;
8)用培养基洗涤细胞一次,去除缓冲buffer后即可用于后续实验。
3.M-CSF诱导人外周血单核细胞分化:
1)计数细胞,1000rpm、3min离心收取细胞,PBS洗一次,将上清完全去除;
2)用含10%FBS的RPMI-1640培养基重悬细胞,密度为3×105/ml,0.5ml/孔铺在24孔板内;
3)加入50ng/ml(终浓度)M-CSF(巨噬细胞集落刺激因子)诱导细胞分化,充分混匀后将细胞置于37℃,5%CO2细胞培养箱培养相应时间后用于后续实验。
按照上述方法步骤,通过Ficoll梯度离心得到人外周血单核细胞,然后用Miltenyi CD14+Microbeads纯化得到人外周血单核细胞(CD14阳性),并用M-CSF体外诱导其分化为巨噬细胞(如图3)。
通过western blot和荧光定量PCR方法,检测在这一分化过程中p38IP表达水平的变化,发现在原代单核细胞分化过程中p38IP的蛋白水平和mRNA水平持续降低(图4,5),这些结果都表明p38IP在原代单核细胞向巨噬细胞分化过程中表达水平降低。
【实施例2】shRNA干扰p38IP促进单核细胞向巨噬细胞分化
根据p38IP的基因序列设计并合成相应的shRNA干扰序列:
Scramble shRNA:5'-CGCTAATTCGACTCGGATA-3'
sh-p38IP:5'-ACACAAGAGCACTGAATCA-3'
将上述序列与RNA干扰载体pSUPER.retro.neo+GFP连接,所述载体图谱如图6所示。将连接有干扰序列的载体利用Lonza Cell LineKit V转染试剂盒转染至U937细胞,并筛选稳定干扰细胞系,其具体实验操作为:
1)计数细胞,1000rpm、3min离心收取106个U937细胞,PBS洗一次,将上清完全去除;
2)用100ul Cell line nucleofector solution C重悬细胞;
3)加入2ug质粒(或200nM siRNA or miRNAmimics/inhibitors),混合均匀后转移至电转杯中,避免底部有气泡残留;
4)选择相应的电转程序,将电转杯插入电转槽电转;
5)电转完成后迅速将细胞转移至加入正常细胞培养基中的24孔板中培养。
如图7所示,western blot结果显示,转染RNA干扰载体的U937细胞内,p38IP表达被干扰,获得了稳定的p38IP干扰细胞系。
按照实施例1所述方法,用PMA刺激诱导细胞向巨噬细胞分化,并利用流式细胞仪检测细胞分化,其具体步骤为:
1)将刺激处理后的细胞离心收集在1.5ml EP管中,弃上清后用预冷的1×PBS洗一次,弃上后用100ul预冷的1×PBS重悬细胞;
2)每个样品加入5ul PE Mouse anti-human CD11b和5ul APC Mouseanti-human CD14冰上避光染色10min,同时设置unstaining control,isotype control及single staining control;
3)加入1ml 1×PBS洗两次,去除unspecific staining;
4)用200ul 1×PBS重悬后即可用流式细胞仪检测。
图8是用流式细胞仪检测shRNA干扰p38IP后的U937细胞的结果图。其中,A为流式细胞术的分析结果图;B为PMA刺激后CD11b/CD14阳性细胞占细胞总数的百分比。结果表明,利用shRNA干扰p38IP后的U937细胞中,细胞表面CD11b/CD14阳性细胞比率显著升高。
【实施例3】siRNA干扰p38IP促进单核细胞向巨噬细胞分化
针对p38IP不同位点,合成干扰的特异性siRNA
si-p38IP-1:5'-GCTTGTTATGCAAGAGACT-3',
si-p38IP-2:5'-GCAACAAGCTTTAGAACTA-3'。
按照按照实施例1、2的方法,在U937细胞中瞬时干扰p38IP(图9),检测p38IP瞬时干扰对细胞分化的影响。结果表明:瞬时干扰p38IP促进U937在PMA诱导下的分化,其表面CD11b/CD14阳性细胞比率升高,并且这一升高与p38IP干扰效果成正比,p38IP表达被干扰越彻底,细胞分化越完全(图10A,B)。
【实施例4】microRNA-200b-3p作用下p38IP表达降低,促进单核细胞向巨噬细胞分化
在研究中,我们发现p38IP在分化过程中受miR-200b-3p的调节,我们在U937细胞中过表达miR-200b-3p mimics(Gene ID:406984),发现miR-200b-3pmimics的转入成功的降低了p38IP蛋白的表达水平(图7),通过流式细胞仪检测miR-200b-3p mimics转染两天后的U937细胞表面CD11b/CD14的表达情况,发现当miR-200b-3p过表达后CD11b/CD14阳性的细胞比率显著升高(图12),这说明miR-200b-3p过表达能通过降低p38IP的表达来促进单核细胞分化。
综上所述,p38IP蛋白在单核细胞向巨噬细胞分化过程中下调,p38IP蛋白的下调会促进单核细胞向巨噬细胞分化;通过shRNA、siRNA和microRNA作用导致的p38IP的下调都会促进单核细胞向巨噬细胞分化。本发明的方法,通过下调单核细胞内p38IP蛋白水平,实现促进单核细胞向巨噬细胞的定向分化,从而增加巨噬细胞的获得率,克服了现有技术中单核细胞诱导向巨噬细胞分化的方法复杂且不理想的技术问题,针对巨噬细胞分化紊乱疾病提出新的研究和治疗思路。
以上仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,凡在本发明的精神和原则之内所做的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (8)
1.一种促进单核细胞向巨噬细胞分化的方法,其特征在于:是通过下调单核细胞内p38IP蛋白水平实现的。
2.根据权利要求1所述的促进单核细胞向巨噬细胞分化的方法,其特征在于:所述下调单核细胞内p38IP蛋白水平可以通过RNA干扰的方法实现。
3.根据权利要求2所述的促进单核细胞向巨噬细胞分化的方法,其特征在于:所述RNA干扰为shRNA干扰方法。
4.根据权利要求3所述的促进单核细胞向巨噬细胞分化的方法,其特征在于:所述shRNA干扰方法中,使用的shRNA序列为:
sh-p38IP:5'-ACACAAGAGCACTGAATCA-3'。
5.根据权利要求2所述的促进单核细胞向巨噬细胞分化的方法,其特征在于:所述RNA干扰为siRNA干扰方法。
6.根据权利要求5所述的促进单核细胞向巨噬细胞分化的方法,其特征在于:所述siRNA干扰方法中,使用的siRNA序列为:
si-p38IP-1:5'-GCTTGTTATGCAAGAGACT-3'。
7.根据权利要求5所述的促进单核细胞向巨噬细胞分化的方法,其特征在于:所述siRNA干扰方法中,使用的siRNA序列为:
si-p38IP-2:5'-GCAACAAGCTTTAGAACTA-3'。
8.根据权利要求1所述的促进单核细胞向巨噬细胞分化的方法,其特征在于:所述下调单核细胞内p38IP蛋白水平可以通过在所述单核细胞中过表达miR-200b-3p mimics实现。
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