CN116036285B - 抑制stat6位点泛素化的物质在调控巨噬细胞极化中的应用 - Google Patents
抑制stat6位点泛素化的物质在调控巨噬细胞极化中的应用 Download PDFInfo
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Abstract
本发明涉及生物医药技术领域,具体公开了抑制STAT6位点泛素化的物质在调控巨噬细胞极化中的应用。本发明发现了TRAF3可催化STAT6K450位点泛素化并激发STAT6转录活性,进而促进巨噬细胞向M2表型极化。STAT6K450位点可在巨噬细胞极化功能调控产品中作为靶位点,通过抑制STAT6K450位点泛素化,能够诱导肿瘤相关巨噬细胞从M2向M1表型极化,抑制肿瘤生长,具有良好的潜在实际应用价值。
Description
技术领域
本发明涉及生物医药技术领域,尤其涉及抑制STAT6位点泛素化的物质在调控巨噬细胞极化中的应用。
背景技术
肿瘤相关巨噬细胞(TAM)是乳腺癌等恶性实体肿瘤组织中含量最多的免疫细胞,在炎症反应、病原体防御和损伤修复中发挥重要作用。肿瘤相关巨噬细胞受肿瘤免疫微环境(TME)的影响,可以发生不同的活化,从而形成具有不同分子和功能特征的亚群,主要包括M1型巨噬细胞和M2型巨噬细胞。其中,M1型巨噬细胞能够分泌IL-12、IL-6、NO、ROS和TNFα等促炎因子,发挥杀伤肿瘤细胞的作用,而M2型巨噬细胞会合成和分泌促血管生成因子、趋化因子及基质金属蛋白酶等,抑制效应T细胞的杀伤作用,诱导肿瘤新生血管形成,促进肿瘤细胞的生长和增殖,导致患者预后不良。因此,寻找巨噬细胞极化调控靶点,诱导巨噬细胞从M2型向M1型极化,重塑肿瘤微环境,对恶性肿瘤抗血管新生和免疫治疗具有重要意义。
发明内容
鉴于此,本发明提供抑制STAT6位点泛素化的物质在调控巨噬细胞极化中的应用,通过抑制STAT6 K450位点泛素化,能够诱导肿瘤相关巨噬细胞从M2向M1表型极化,抑制肿瘤生长。
本发明提供一种抑制STAT6位点泛素化的物质在制备巨噬细胞极化功能调控产品中的应用,所述STAT6位点为第450位赖氨酸位点,氨基酸序列如SEQ ID NO.1所示;所述抑制STAT6位点泛素化的物质为基因定点突变载体、抑制TRAF3蛋白活性的物质或降低TRAF3蛋白含量的物质中的至少一种。
TNF受体相关因子3(TRAF3)是一种E3泛素连接酶,参与多种蛋白激酶和磷酸酶的活化调控。发明人通过研究发现,在巨噬细胞条件性TRAF3基因敲除小鼠(TRAF3-MKO)中,肿瘤生长缓慢,肿瘤组织中浸润的M2型巨噬细胞明显减少,新生微血管密度显著降低。同时,发明人通过转录组-蛋白质组技术对野生型(WT)小鼠和TRAF3-MKO型小鼠骨髓来源巨噬细胞(BMDM)进行联合分析,结果显示,TRAF3-MKO型小鼠骨髓来源巨噬细胞中M2型巨噬细胞标志基因(CD206/MRC1、CD36和ABCC3)的mRNA和蛋白质表达均显著下调,而M1型巨噬细胞标志基因(iNOS、FGR和SLC4A7)的表达上调。这说明了,TRAF3在巨噬细胞极化过程中发挥重要作用,其表达缺失可抑制巨噬细胞向M2型极化以及M2型巨噬细胞极化相关的血管新生和肿瘤生长,促进巨噬细胞向M1型极化。
为了明确TRAF3的调控机制,发明人对WT型小鼠和TRAF3-MKO型小鼠原代骨髓来源巨噬细胞总蛋白进行了非标定量蛋白质组学联合泛素化修饰组学分析,结果显示,TRAF3基因缺失引起大量相关蛋白泛素化修饰水平的改变,其中,泛素化水平下调的蛋白质主要富集在IL-4应答信号通路。
信号转导和转录激活因子6(STAT6)(NCBI Entrez Gene:6778;Ensembl:ENSG00000166888)是M2型巨噬细胞诱导剂IL-4和IL-13的共同下游分子,介导M2型巨噬细胞特异性基因的转录激活,其激活或抑制直接影响巨噬细胞M2极化状态和抗肿瘤免疫应答。发明人对IL-4信号途径中的差异泛素化蛋白进行了深入研究,发现STAT6蛋白质多肽链第450位赖氨酸位点(K450)泛素化水平显著下调,随后通过实验表明,过表达TRAF3可通过泛素化修饰底物分子STAT6,促进STAT6转录活性,当K450位点突变后,TRAF3激活STAT6转录作用消失。基于上述研究,本发明提供了将抑制上述STAT6位点泛素化修饰的物质用于制备巨噬细胞极化功能调控产品的技术方案。
相对于现有技术,本发明提供的技术方案具有以下优势:
本发明揭示了TRAF3在肿瘤相关巨噬细胞极化过程中的作用,明确了STAT6 K450位点是TRAF3发挥作用的关键位点,首先发现了TRAF3可催化STAT6 K450位点泛素化并激发STAT6转录活性,进而促进巨噬细胞向M2表型极化的机制。本发明提供的技术方案为巨噬细胞极化和功能调控提供了新的方法,以STAT6 K450位点作为巨噬细胞极化功能调控产品的靶位点,通过抑制STAT6 K450位点泛素化,诱导肿瘤相关巨噬细胞从M2向M1表型极化,抑制肿瘤生长,具有良好的潜在实际应用价值,并为代谢疾病的治疗和预防带来新的思路和手段。
可选的,所述基因定点突变载体在所述STAT6位点产生点突变,将第450位赖氨酸定点突变为精氨酸。STAT6 K450位点突变可阻断TRAF3对STAT6的泛素化活化的过程,进而阻断TRAF3诱导的M2型巨噬细胞极化。
可选的,所述降低TRAF3蛋白含量的物质为抑制TRAF3蛋白合成、促进TRAF3蛋白降解、敲低TRAF3基因的物质或敲除TRAF3基因的物质的至少一种。降低TRAF3蛋白含量可抑制STAT6 K450位点泛素化并抑制M2型巨噬细胞极化。
可选的,所述敲低TRAF3基因的物质为抑制TRAF3基因表达的siRNA或shRNA。
可选的,所述敲除TRAF3基因的物质为TRAF3基因编辑系统,所述TRAF3基因编辑系统包括Cas9核酸酶和靶向TRAF3基因的sgRNA。
可选的,所述抑制STAT6位点泛素化的物质通过调控STAT6泛酸化影响IL-4/STAT6信号通路,从而调控巨噬细胞极化。
可选的,所述巨噬细胞极化功能调控产品能够促进巨噬细胞向M1型巨噬细胞极化。
可选的,所述巨噬细胞极化功能调控产品能够抑制巨噬细胞向M2型巨噬细胞极化。
附图说明
为了更清楚地说明本发明实施例中的技术方案,下面将对实施例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一些实施例,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,还可以根据这些附图获得其他的附图。
图1是本发明实施例1提供的Lyz2-cre基因鉴定图;
图2是本发明实施例1提供的TRAF3flox/flox基因鉴定图;
图3是本发明实施例1提供的WT型小鼠和TRAF3MKO型小鼠腹腔巨噬细胞和骨髓来源巨噬细胞中TRAF3蛋白表达水平结果示意图;
图4是本发明实施例1提供的WT型小鼠和TRAF3MKO型小鼠B细胞和T细胞中TRAF3蛋白表达水平结果示意图;
图5是本发明实施例1提供的WT型小鼠和TRAF3MKO型小鼠B16恶性黑色素瘤移植肿瘤模型的肿瘤生长曲线对比图;
图6是本发明实施例1提供的WT型小鼠和TRAF3MKO型小鼠B16恶性黑色素瘤移植肿瘤模型的肿瘤重量分析对比图;
图7是本发明实施例1提供的WT型小鼠和TRAF3MKO型小鼠B16肿瘤组织内M1型巨噬细胞和M2型巨噬细胞流式散点图;
图8是本发明实施例1提供的WT型小鼠和TRAF3MKO型小鼠B16肿瘤组织内M1型巨噬细胞和M2型巨噬细胞数量统计图;
图9是本发明实施例1提供的WT型小鼠和TRAF3MKO型小鼠原代骨髓来源巨噬细胞转录组学差异基因火山图;
图10是本发明实施例1提供的WT型小鼠和TRAF3MKO型小鼠原代骨髓来源巨噬细胞非标定量蛋白质组学分析差异蛋白火山图;
图11是本发明实施例1提供的WT型小鼠和TRAF3MKO型小鼠原代骨髓来源巨噬细胞表达的差异M1/M2标志基因热图;
图12是本发明实施例2提供的WT型小鼠和TRAF3MKO型小鼠原代骨髓来源巨噬细胞非标定量泛素化修饰组学分析差异泛素化修饰蛋白的火山图;
图13是本发明实施例2提供的WT型小鼠和TRAF3MKO型小鼠原代骨髓来源巨噬细胞差异泛素化修饰蛋白的功能富集分析;
图14是本发明实施例2提供的WT型小鼠和TRAF3MKO型小鼠原代骨髓来源巨噬细胞白细胞介素4应答通路蛋白泛素化修饰差异倍数(KO/WT log2FC)和P值;
图15是本发明实施例3提供的TRAF3 K450位点位于DNA结合结构域,进化上高度保守;
图16是本发明实施例3提供的过表达TRAF3促进STAT6泛素化水平;
图17是本发明实施例3提供的STAT6 K450位点突变阻断了TRAF3对其转录激活的促进作用;
图18是本发明实施例3提供的STAT6 K450位点突变抑制其磷酸化水平。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述,给出的实施例仅为了阐明本发明,而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南,并不以任何方式构成对本发明的限制。
下述实施例中的实验方法,如无特殊说明,均为常规方法,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。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。
下述实施例中涉及的小鼠实验材料及制备方法如下:
所有小鼠均以C57BL/6N为背景,在河北大学医学综合实验中心SPF动物室无特定病原体的条件下,在受控温度(22℃±2℃)下维持12h光/12h暗周期。除非另有说明,小鼠以标准饮食(#12431,北京科澳协力饲料有限公司)和净化水喂养。所有动物实验均经河北大学实验动物伦理委员会批准。
TRAF3fl/fl小鼠记载于文献“Gardam,S.,Sierro,F.,Basten,A.,Mackay,F.&Brink,R.TRAF2 and TRAF3 signal adapters act cooperatively to control the maturation and survival signals delivered to B cells by the BAFF receptor.Immunity28,391-401,doi:10.1016/j.immuni.2008.01.009(2008).”中。
WT小鼠:野生型C57BL/6N小鼠。
Lyz2-Cre小鼠记载于文献“Clausen,B.E.,Burkhardt,C.,Reith,W.,Renkawit z,R.&Forster,I.Conditional gene targeting in macrophages and granulocytes usingLysMcre mice.Transgenic Res 8,265-277,doi:10.1023/a:1008942828960(1999).”中。
下述实施例中涉及的实验方法如下:
细胞培养:人胚肾293(HEK293)和B16小鼠黑色素瘤细胞来自细胞资源中心(IBMS,CAMS/PUMC,中国北京)。HEK293和B16细胞在Dulbecc o改良Eagle's培养基中培养,培养基中添加10%FBS(Gemini Bio),100U/ml青霉素,100μg/ml链霉素。野生WT或TRAF3MKO小鼠股骨分离的骨髓细胞在添加M-CSF条件培养基的DMEM培养基中培养7天。提取总细胞裂解物用于免疫印迹(IB)分析,提取总RNA用于RT-PCR分析。
小鼠DNA提取:取3周龄小鼠,剪取一小部分鼠尾(约1mm),置于1.5mL离心管中,加入75μL Alkaline裂解液(250μL NaOH 1.0M、4μL Na2EDT A 0.5M和9.746mL三蒸水配制),转移至95℃水浴锅加热30分钟裂解组织,取出离心管,置于4℃冷却5分钟,加入75μL中和试剂(400μL Tris-HCL 1M和9.6mL三蒸水配制),震荡混匀,10000rpm离心3分钟,转移上清到新的1.5mL离心管中,用于后续PCR鉴定。
PCR:取上述步骤获得的小鼠DNA进行基因型鉴定。反应体系:2×Taq Master mix15μL、DNA4μL、正负链引物各0.5μL,混匀后放入8联排中准备PCR。提前配置1.5%琼脂糖凝胶(1.5g琼脂糖凝胶粉溶于TAE溶液中,微波炉加热溶解后,加入10μL ExRed配置),加入PCR所得的DNA样品以及DNA maker,150V电泳20分钟。电泳完成后用凝胶成像仪显影DNA条带并拍照,对照DNA maker分析条带并记录小鼠基因型。
流式细胞术:样品中的红细胞用红细胞裂解液(#R1010,Solarbio)裂解。然后将细胞过滤,制成单细胞悬液,在缓冲液(2%BSA)中进行特异性表面抗体染色。将细胞洗涤3次,然后在冰上与抗体孵育,再使用FACS Calibur(BD)进行FACS。所使用的抗体是CD45-FITC(#553080,BD Bioscience)、CD11b-PerCP-Cy5.5(#550993,BD Bioscience)、CD86-APC(#105012,Biol egend)、CD206-APC(#141708,Biolegend)。用FlowJo(Tree Star)软件进行数据分析。
转录组学和蛋白质组学技术:用野生WT和TRAF3MKO小鼠的BMDMs进行总RNA提取,并用BioAnalyzer 2100系统的RNA Nano 6000检测试剂盒(Agilent Technologies,CA,USA)评估RNA完整性。每个样本使用总量为3μg的RNA,并使用UltraTMRNA文库准备试剂盒(美国NEB)用于/>生成测序文库。用Truseq PE聚类试剂盒V3-CBOT-HS(Illumi a)在CBOT聚类生成系统上对索引编码样品进行聚类,并在Illumina HISEQ2500/X平台上对文库制剂进行测序。使用Bowtie V2.2.3将原始转录组读数映射到对照基因组(GRCM39,http://www.ensembl.org/mus_musculus/info/index)。采用cuffquant和cuffnorm(V2.2.1)衡量基因相对表达量,即计算每个样本中基因的每千个碱基的转录每百万映射读取的片段数(Fragments Per Kilobase of exon model per Million mappedfragments,FPKMs)。
GO分析:基因本体论分析(Gene Ontology,GO),目的是判断基因集背后可能涉及的生物学功能。GO分析包括三个部分,分别是生物过程(Biolo gical Process,BP)、细胞组分(Cellular Components,CC)和分子功能(Mol ecular Function,MF)。计算差异基因同GO分类中某(几)个特定的分支的超几何分布关系,GO分析会对每个有差异基因存在的GO返回一个p-value,p<0.05表示差异基因在该GO中出现了富集且具有统计学意义,通过差异基因的GO分析,可以找到富集差异基因的GO分类条目。
质谱分析:WT和TRAF3MKO小鼠的BMDMs使用MG132处理2h后提取总蛋白。细胞在冰上超声3次,并在裂解缓冲液(8M尿素,1%蛋白酶抑制剂,3μM TSA,50mM NaM,2mM EDTA)中裂解。然后用胰蛋白酶消化蛋白质溶液。采用Easy NLC 1000UPLC系统,在Q Exactive TMPlus(Thermo Fisher Scientifial,Waltham,Ma,USA)在线联用串联质谱(MS/MS)对胰蛋白酶肽进行LC-MS/MS分析。使用MaxQuant(V1.6.6.0)对所得的MS/MS数据进行处理。
实时定量RT-PCR(QRT-PCR):用TRIZOL试剂(15596026,Invitroge n,Carlsbad,CA,USA)从BMDMS中分离总RNA。用HiScript III RT Super Mix(Vazyme Biotech,中国南京)进行cDNA合成。采用ChamQ Universal SYBR qPCR Master Mix(Vazyme Biotech,中国南京)进行实时PCR。通过标准曲线法计算目的基因相对于β-actin的表达量来测定基因的表达量。
免疫印迹:收集细胞,用RIPA缓冲液(Solarbio,北京)裂解。细胞裂解物进行IB和coIP测定。简单地说,细胞裂解物通过SDS-PAGE分离并转移到PVDF膜上。用5%脱脂乳封闭膜,孵育特异性一抗。辣根过氧化物酶结合二抗与特异性一抗结合,用ECL检测膜。
泛素化分析:细胞用MG132预处理2h,然后用含有6M尿素和蛋白酶抑制剂的RIPA裂解缓冲液裂解。用免疫沉淀法(IP)在4℃下用指示抗体1小时分离指示蛋白,然后用proteinA-琼脂糖(Santa Cruz Biotechnology,Santa Cruz,CA,USA)在4℃下孵育过夜。然后将蛋白A-琼脂糖-抗原-抗体复合物颗粒洗涤5次,用抗泛素抗体IB检测泛素化蛋白。
定量泛素组学分析:WT和TRAF3MKO小鼠来源的BMDMs用MG132预处理2h。用含8M尿素、1%蛋白酶抑制剂、3μM TSA、50mM NAM和2mM EDTA的裂解缓冲液对细胞进行超声裂解,然后用胰蛋白酶消化蛋白质溶液。将溶于NETN缓冲液(100mM NaCl,1mM EDTA,50mM Tris-HCl,0.5%NP-40,pH 8.0)的胰蛋白酶肽与预洗的抗泛素抗体珠(浙江杭州PTM Bio,China)在4℃下孵育过夜,以富集泛素修饰肽。抗泛素抗体珠洗涤,结合肽用0.1%三氟乙酸洗脱。对洗脱后的肽进行LC-MS/MS分析。
肿瘤模型:利用6-8周龄、年龄和性别匹配的WT和TRAF3MKO小鼠,皮下注射B16黑色素瘤细胞(5×105)。肿瘤体积按体积=1/2×a×b2的公式计算,其中(a)和(b)分别代表肿瘤的最大直径和最小直径。按河北大学动物伦理与福利委员会(AEWC)批准的方案,于注射后第21天处死小鼠。对移植瘤进行解剖、称重和拍照。
肿瘤浸润免疫细胞分析:取肿瘤组织,在含10%FBS的RPMI1640培养基中研磨,然后用DNASEI(0.2mg/mL,DN25,Sigma-Aldrich,St.Louis,MO,USA)和胶原酶IV(0.5mg/mL,V900893,Sigma-Aldrich,St.Louis,MO,USA)在37℃下处理30min后将细胞悬液用70微米的滤膜过滤,加入红细胞裂解液静置3min,并在PBS中洗涤两次,进行流式细胞术分析。
双荧光素酶报告基因分析:将HEK293细胞接种于24孔板中,在转染ST AT6荧光素酶质粒p4×Stat6-Luc2P,对照报告质粒pRL-TK和相应基因表达质粒后,用Lipofectamine2000试剂(Invitrogen,Carlsbad,CA,USA)转染HEK293细胞。24小时后,细胞裂解并使用双荧光素酶报告分析系统(Promeg a,Madison,WI,USA)进行荧光素酶分析。特异启动子活性表示为Firefly荧光素酶与Renilla荧光素酶的比值。
统计分析:数据以均值±SD表示,所有提供的数据都是至少三个独立重复的代表性结果。用GraphPad Prism 8软件进行统计分析。根据被比较组的数量,采用Two-tailedunpaired Student’s t tests、Two-way ANOVA分析或者Tukey'smultiple comparisons检验。P<0.05认为差异有统计学意义。
实施例1敲除巨噬细胞中TRAF3对巨噬细胞极化的影响
1、构建TRAF3MKO基因敲除小鼠
将TRAF3fl/fl小鼠与Lyz2-Cre小鼠杂交,经基因型鉴定在杂交后代中选取的含有flop序列且不含有Lyz2-Cre基因的小鼠及其后代。
结果如图1-4所示,在小鼠腹腔巨噬细胞(PEM)和骨髓来源巨噬细胞(BMDM)中,TRAF3MKO小鼠巨噬细胞TRAF3表达缺失;在WT小鼠和TRAF3MKO小鼠的B细胞和T细胞中,TRAF3表达无显著差异(图4),成功构建了TRAF3flox/floxLyz2-Cre+(TRAF3MKO)小鼠。
2、TRAF3基因缺失对小鼠B16-OVA恶性黑色素瘤生长的影响
对WT小鼠和TRAF3MKO小鼠注射小鼠B16-OVA恶性黑色素瘤细胞,从接种后第4天开始每隔2天测量肿瘤体积。接种后21天观察肿瘤情况和测量肿瘤重量,荷瘤小鼠肿瘤组织消化后进行流式细胞分析,对CD45+CD11b+CD86+CD45+M1型巨噬细胞和CD11b+CD206+M2型巨噬细胞进行统计。
结果如图5-8所示,TRAF3MKO小鼠肿瘤生长速度显著低于WT小鼠。
3、小鼠原代骨髓来源巨噬细胞中TRAF3基因缺失对巨噬细胞极化标志基因表达的影响
通过转录组学和蛋白质组学技术,对WT小鼠和TRAF3MKO小鼠骨髓来源巨噬细胞进行联合分析。
结果如图9-11所示,与WT小鼠骨髓来源巨噬细胞相比,TRAF3MKO小鼠骨髓来源巨噬细胞中M2型巨噬细胞标志基因(CD206/MRC1、CD36、ABCC3)的mRNA和蛋白质表达均显著下调,而M1型巨噬细胞标志基因(iNOS、FGR、SLC4A7)的表达上调。该结果证实了,TRAF3基因缺失抑制M2型巨噬细胞极化和肿瘤生长。
实施例2小鼠巨噬细胞中蛋白质泛素化水平变化
对WT小鼠和TRAF3MKO小鼠原代骨髓来源巨噬细胞总蛋白进行非标定量蛋白质组学联合泛素化修饰组学分析,WT组和TRAF3MKO组之间具有显著差异的泛素化修饰位点共1421个(991个位点泛素化水平下调,430个位点上调),涉及的蛋白质共1060种(709种蛋白质泛素化水平下调,351种上调)。对这些差异泛素化修饰蛋白进行GO富集分析。
结果如图12-14所示,TRAF3基因缺失引起的泛素化水平下调的蛋白质主要富集在IL-4应答信号通路。上述研究结果表明,在巨噬细胞中,TRAF3通过泛素化修饰调控IL-4通路蛋白,可能在IL-4信号途径的激活或抑制过程发挥重要作用。
实施例3TRAF3对STAT6转录活性的影响
对上述检测到的IL-4信号途径中的差异泛素化蛋白进行了深入分析和比对。结果显示,IL-4信号通路关键激酶STAT6的两个赖氨酸位点(K450和K129)均可检测到泛素化修饰,且在TRAF3基因敲除的骨髓来源巨噬细胞中,STAT6分子K450和K129位点泛素化水平显著下调,其中K450位点位于DN A结合结构域,进化上高度保守。
应用His-STAT6、Flag-TRAF3和HA-Ubiqutin表达质粒共转染HEK293细胞,收集细胞裂解液,利用免疫沉淀法进行泛素化分析,结果显示,过表达TRAF3促进STAT6泛素化水平。
通过构建STAT6野生型和突变型(WT、K129R和K450R)表达载体,与STAT6启动子报告基因质粒(p4×STAT6-luc2P)和pRL-TK质粒共转染HEK293细胞,收集细胞裂解液,利用免疫沉淀法进行泛素化分析,结果显示,过表达TRAF3促进STAT6泛素化水平。
HEK293细胞中共转染p4×STAT6-luc2P、pRL-TK、HA-TRAF3和Flag-STAT6 WT、Flag-STAT6 K129R、Flag-STAT6 K450R,加入IL-4刺激细胞24小时,收集细胞,用双荧光素酶报告基因检测系统,分析STAT6特异性靶向的启动子报告基因转录活性。
结果如图15-18所示,过表达TRAF3可促进野生型STAT6转录活性,当K450位点突变后,TRAF3激活STAT6转录作用消失。
以上实施例1~实施例3的结果说明,基因定点突变载体、抑制TRAF3蛋白活性的物质或降低TRAF3蛋白含量的物质能够抑制STAT6位点泛素化,可用于制备巨噬细胞极化功能调控产品。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
Claims (2)
1.抑制STAT6位点泛素化的物质在制备调控巨噬细胞极化以抑制肿瘤生长的产品中的应用,其特征在于,所述STAT6位点为第450位赖氨酸位点,STAT6的氨基酸序列如SEQIDNO.1所示;
所述抑制STAT6位点泛素化的物质为基因定点突变载体或敲除TRAF3基因的物质中的至少一种;
所述基因定点突变载体在所述STAT6位点产生点突变,将第450位赖氨酸定点突变为精氨酸。
2.如权利要求1所述的应用,其特征在于,所述敲除TRAF3基因的物质为TRAF3基因编辑系统,所述TRAF3基因编辑系统包括Cas9核酸酶和靶向TRAF3基因的sgRNA。
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