CN115480062A - 效应cd8+t细胞去分化为cd8+tcm的关键药物靶点筛选及在其药物中的应用 - Google Patents
效应cd8+t细胞去分化为cd8+tcm的关键药物靶点筛选及在其药物中的应用 Download PDFInfo
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Abstract
本发明公开了在严重病毒感染患者中可以促使患者体内的效应CD8+T细胞去分化为CD8+TCM从而提高CD8+TCM比例,重建细胞免疫的关键药物靶点的筛选。本发明通过体外细胞实验筛选8种抑制剂,并通过基因敲降方法验证靶点抑制效果。实验发现趋化因子受体和G蛋白α作用点的抑制可以提高CD8+TCM比例,重建细胞免疫,可用于严重病毒感染的治疗,及相关药物中的应用。
Description
技术领域
本发明属严重病毒感染后可以促使效应CD8+T细胞去分化为CD8+TCM的关键药物靶点筛选的基础研究领域,更具体地说,本发明涉及可以影响严重病毒感染后效应T细胞的能量代谢方式和磷酸化通路,从而促使效应CD8+T细胞去分化为CD8+TCM的药物靶点筛选。
背景技术
病毒感染后,CD8+T细胞会经历扩增、收缩和记忆形成三个阶段。通常使用表面分子KLRG1和CD127(Interleukin-7receptor subunit alpha,IL-7Rα)来表征CD8+T细胞的活化状态,并将活化后的CD8+T细胞分为短活效应细胞(Short-lived effector cells,SLECs,表征为KLRG1hiCD127low)和记忆前体效应细胞(Memory precursor effectorcells,MPECs,表征为KLRG1lowCD127hi)两大类。SLECs能产生大量的细胞毒性分子和细胞因子,大部分在收缩阶段凋亡,而MPECs细胞在清除病原后进一步分化形成记忆细胞。记忆细胞进一步分化为不同的记忆细胞亚群,通常使用表面分子CD45RA、CD45RO、趋化因子受体CCR7和血管L-选择素(CD62L)来表征不同的记忆亚群,具体分为长期记忆T细胞(Centralmemory T cells,TCM,表型为CD45RA-CD45RO+CCR7+CD62L+)、效应记忆性T细胞(Effectormemory T cells,TEM表型为CD45RA-CD45RO+CCR7-CD62L-)和组织特异性T细胞(Tissueresident memory T cells,TRM,表型为CD103+CD69+CD62L-CD27-)。一般情况下TCM主要分布于外周组织免疫器官和淋巴结,当再次受抗原刺激时可迅速分裂增殖和分化。TEM细胞主要存在于非淋巴组织和器官,参与周身循环,可迁移至外周炎症组织发生速发性效应功能。
当持续抗原刺激下CD8+T呈耗竭态(Exhausted T cells,TEX),表现为低水平IL-2、TNF-α、INF-γ,细胞表面表达高水平的PD-1、TAG3、CD244、CD160等抑制分子。有研究表明记忆CD8+T细胞是来自效应T细胞的子集,抑制幼稚相关基因的表达可以逆转效应CD8+T细胞分化为长寿记忆CD8+T细胞。
严重病毒感染机体后,机体会产生一种持续性的免疫应答,这种免疫应答处于一种异常活化状态。研究表明,HIV-1感染机体后,病毒抗原会持续性刺激机体免疫系统处于异常增高的激活状态,具体表现为CD38和HLA-DR表达水平的激增。除此之外CD8+T细胞的细胞毒作用大大增强,过激的细胞毒作用也是HIV-1感染机体后的明显免疫反应。王福生院士对新冠肺炎死亡患者的病理研究结果显示,患者外周血中CD4+T细胞和CD8+T细胞的数量显著减少但CD38和HLA-DR表达水平增高并且CD8+T细胞含有高浓度的细胞毒性颗粒,这表明T细胞的过度活化。还有研究者表明在感染SARS-CoV-2后除了表现出T细胞的过度活化外还检测到T细胞表面的衰竭分子如PD-1表达上升,这表明过度活化的T细胞呈现一种疲惫状态,这种情况在HIV-1感染者中也有相似表达。
本实验室前期研究利用vMIP-II能竞争性抑制HIV与靶细胞上共受体CCR5、CXCR4、CCR3等的结合,以阻止病毒进入靶细胞,研究其具有抗HIV感染的作用,体内实验表明SIV感染初期的食蟹猴体内部分TCRVβ亚家族的表达量有所变化,一些Vβ亚家族有特异性的增生,且增生的TCRVβ亚家族克隆性发生改变,提示其可能为针对病毒的特异性增生。vMIP-Ⅱ对此种Vβ亚家族的表达的增生有促进的作用,说明其对免疫系统的应答有增强的作用,促使特异性应答的免疫细胞的增生。事实上,我们对重组vMIP-II的猴SIV模型和在人AIDS治疗的研究均显示了其具有显著增加病毒感染机体的记忆CD8+T细胞水平作用,对发病期的病毒感染血症具有重要作用。
本实验室前期研究发现在COVID-19的治疗中vMIP-II可以通过促使效应CD8+T细胞去分化为CD8+TCM的方式提高CD8+TCM比例从而发挥治疗效果。通过基因芯片分析,vMIP-II发挥治疗效果可能是通过影响趋化因子受体、磷酸化通路、mTOR通路及甲基化酶Dnmt3a来实现的。为了进一步探索可以促使效应CD8+T细胞去分化为CD8+TCM的关键作用靶点,我们首先验证了严重病毒感染的细胞模型,并通过细胞模型对各作用靶点进行筛选。药物靶点的筛选对开发针对于严重病毒感染的抗病毒药物具有重要意义。
发明内容
本发明通过T细胞表面活化标志物的表达、细胞因子分泌和CD8+TCM的比例对严重病毒感染的细胞模型进行验证。
本发明使用严重病毒感染的细胞模型并将其与各靶点抑制剂共孵育,通过检测效应CD8+T细胞的磷酸化水平、CD8+TCM的比例和细胞的线粒体功能来筛选药物靶点。
本发明通过敲降药物靶点的相关基因,检测其CD8+TCM的比例和细胞的线粒体功能来验证药物靶点。
附图说明
图1为实施例1不同浓度gp120蛋白刺激下的活化T细胞计数。
图2为实施例1不同抗原刺激下的T细胞表面分子表达情况。
图3为实施例1不同抗原刺激下T细胞细胞因子分泌。
图4为实施例1不同抗原刺激下记忆CD8+T细胞和CD8+TCM的百分比。
图5为实施例2使用8种抑制剂后各组中磷酸化蛋白浓度。
图6为实施例2磷酸化通路上靶点抑制剂使用后记忆CD8+T细胞和CD8+TCM的百分比(*p<0.05,n=3)。
图7为实施例2各通路靶点抑制剂使用后记忆CD8+T细胞和CD8+TCM的百分比(*p<0.05,n=3)。
图8为实施例2各通路靶点抑制剂使用后各组线粒体膜电位变化,*p值<0.05,**p值<0.01。
图9为实施例2各通路靶点抑制剂使用后线粒体增殖、自噬基因表达情况。
图10为实施例3细胞内所敲降的基因表达情况。
图11为实施例3敲降靶点基因后记忆CD8+T细胞和CD8+TCM的百分比,*p值<0.05。
图12为实施例3敲降靶点基因后线粒体膜电位变化。
图13为实施例3敲降靶点基因后线粒体增殖、自噬基因表达情况。
具体实施方式
以下结合实施例,对本发明进行进一步详细说明。
实施例1:严重病毒感染细胞模型的验证
材料与方法
病毒、患者和细胞:SARS-CoV-2广州毒株的S蛋白占据其基因组的21563-25384位碱基,对应于1273个氨基酸,编码141.2kDa的蛋白。重组HIV-1的gp120蛋白。HSV-1。人外周血单个核细胞(PBMC)。
实验试剂:抗CD8抗体-APC(allophycocyanin,别藻蓝蛋白)、抗CD4抗体-ECD、单克隆抗体CD45RO-PE-Cy5、单克隆抗体CD62L-APC-H7、单克隆抗体PD-1-PE-Cy5、单克隆抗体Tim-3-APC-H7、单克隆抗体HLA-DR-APC-H7、单克隆抗体CD38-APC、单克隆抗体GNLY-FITC、单克隆抗体PRF-FITC等,均购自Biolegend公司;BD FACS流式细胞仪(美国Bio-Rad公司)等。
分组:SARS-CoV-2组:将实验室保存的S蛋白,调节蛋白浓度为100ng/mL将其与PBMC细胞共孵育12h,作为1组细胞参与后续实验。HIV-1组:将实验室保存的HIV-1的gp120蛋白,调节蛋白浓度为270ng/mL将其与PBMC细胞共孵育12h,作为2组细胞参与后续实验。HSV-1组:将本实验室保存的体外灭活HSV-1病毒刺激PBMC并与PBMC共孵育24h,作为3组细胞参与后续实验。
gp120刺激PBMC活化检测
gp120活化检测分组:共分为I、II、III、IV、V和VI组,每组分别添加0.1ml终浓度为10ng/mL、30ng/mL、90ng/mL、270ng/mL、810ng/mL、2430ng/mL的gp120蛋白溶液和100ng/mL的IL-2,每组设三个复孔。空白对照组中加入等体积RPMI-1640培养基。5%CO2、37℃条件下,进行混合淋巴细胞培养24h。分别取100μl上述培养的细胞于试管中,加入抗人CD8-APC、CD38-PE、HLA-DR-CPCy5单克隆荧光抗体各10μL,震荡器混合均匀,20-25℃下避光放置20到35min。上流式细胞分析仪检测。
严重病毒感染模型验证
根据分组在4个试管中分别放入1.5mL培养的细胞,并将四种单克隆荧光抗体各10μL,包括抗人HLA-DR-CPCy5、CD38-PE、PD-1-PE、Tim-3-APC-H7、GNLY-FITC和PRF-FITC(BDBiosciences),震荡器混合均匀,20-25℃下避光放置20到35min,并在流式细胞仪上分选。
抗原诱导CD8+T细胞的细胞因子检测
将培养的细胞(1.5mL)放入试管中,并添加PMA(50mg/L),离子霉素(750mg/L)和BFA阻滞剂(1x),37℃,5%CO2下孵育6小时。将每组细胞均等地分成五个1.5mL离心管,1500rpm离心5分钟。弃去上清液,并用PBS洗涤细胞。丢弃上清液后,避光将流式细胞仪表面抗体CD8-APC-Cy7添加到细胞中,避光4℃孵育20分钟。然后将细胞用200μL 4%多聚甲醛固定。在黑暗中于4℃固定30分钟后,将每个试管以500rpm离心5分钟,并弃去上清液。加入200μL破膜剂后,将细胞在黑暗中于4℃孵育1h,然后以1500rpm离心5分钟。弃去上清液。将每组的五个试管避光,并将细胞内抗体(包括IFN-γ-APC,TNF-α-APC,IL2-APC,IL4-APC和颗粒酶-APC(BD Biosciences))添加到试管中并进行孵育在4℃下20分钟。流式细胞仪用于检测细胞因子的分泌。
CD8+T细胞亚群检测
根据分组在4个试管中分别放入1.5mL培养的细胞,并将四种单克隆荧光抗体各10μL,包括抗人CD3-PE,CD4-FITC,CD8-APC,CD45RO-PE-Cy5和CD62L-APC将抗人CD3-PE(BDBiosciences)分选后的T细胞中的-H7加入第一个试管中,在流式细胞仪上分选。
统计分析
所有实验均进行3次独立的生物学重复。使用Prism software确定统计显着性。使用两尾t检验确定P值。*P<0.05;**P<0.01。
结果分析
体外gp120刺激PBMC活化检测
将不同浓度的gp120蛋白与PBMC共孵育后,使用HLA-DR与CD38表面分子抗体染色,通过流式细胞仪检测结果如图1,表1所示,空白对照组(不加gp120)HLA-DR与CD38双阳性比例在8%左右。I-VI组中CD38+HLA-DR+的T细胞百分比相对于空白对照组明显升高,且gp120蛋白浓度越高,比例越高,最高的活化比例是第VI组(40.27±3.56%),但活化比例在IV-VI组之间的变化不大说明进入平台期,这就表明抗原过度激活T细胞的最大反应值为270ng/mL gp120。以上结果说明gp120可以明显激活T细胞,且这种激活作用有明显的量效关系。
表1共培养后T细胞激活情况
*为与阴性对照组相比,P<0.05,**为与对照组相比,P<0.01
严重病毒感染的指标验证
在严重病毒感染时CD4+T细胞和CD8+T细胞的数目减少但细胞呈现一种过度活化状态,具体表现为高比例的CD38和HLA-DR,流式细胞术检测如图2所示在SARS-CoV-2S蛋白组、HIV-1gp120蛋白组中CD38和HLA-DR双阳性的比例(CD4+T细胞3.7%左右,CD8+T细胞33%左右)远高于HSV-1组(CD4+T细胞1.64%左右,CD8+T细胞14.33%)以及空白对照组(CD4+T细胞1.35%左右,CD8+T细胞10.21%)。
细胞毒性增高表现为穿孔素(GNLY)、颗粒溶素(PRF)的表达增多。严重病毒感染时可以观察到CD8+T细胞的高细胞毒作用,流式细胞术检测如图2所示在SARS-CoV-2S蛋白组、HIV-1gp120蛋白组中CD8+T细胞具有高浓度的细胞毒性颗粒,其中穿孔素和颗粒溶素双阳性比例为30%以上。而在HSV-1组中穿孔素和颗粒溶素双阳性比例为14.58%,高于空白对照组的7.45%但远低于其他两组,这表明HSV-1感染也会刺激CD8+T细胞产生细胞毒作用但效力低。
除此之外在严重病毒感染时T细胞表面的耗竭标记物如(PD-1)的表达也有所增强。流式细胞术检测结果如图2所示在SARS-CoV-2S蛋白组、HIV-1gp120蛋白组中CD8+T细胞表面的耗竭分子表达也高于其他两组。通过以上三种指标验证了SARS-CoV-2S蛋白组、HIV-1gp120蛋白组符合严重病毒感染的细胞模型。
抗原蛋白刺激后细胞因子分泌检测
在严重病毒感染时常伴随细胞因子分泌变多,严重的情况可能会造成细胞因子风暴,严重伤害机体功能。对抗原刺激后细胞因子分泌情况的检测结果如图3所示,在SARS-CoV-2S蛋白组、HIV-1gp120蛋白组中细胞因子IFN-γ、IL-4、IL-2(CD4+T细胞分泌)、GNLY(CD8+T细胞)、TNF-α(T细胞分泌)含量明显增高,但在HSV-1组中细胞因子略有上升。
抗原蛋白刺激后CD8+T细胞亚群检测
通过流式细胞术检测SARS-CoV-2S蛋白组、HIV-1gp120蛋白组和空白对照组的CD8+T细胞亚群比例。结果如图所示。相对于空白对照组,SARS-CoV-2S蛋白组、HIV-1gp120蛋白组的记忆CD8+T细胞总体变化不大(图4左)。SARS-CoV-2S蛋白组、HIV-1gp120蛋白组中TCM的比例相较于空白对照组明显下降(图4右),这表明SARS-CoV-2S蛋白、HIV-1gp120蛋白对于CD8+T细胞亚群分化的情况具有一致性。
实施例2:各靶点抑制剂对效应CD8+T细胞分化的影响
材料与方法
实验试剂:鼠γ-疱疹病毒68(MHV-68)编码的病毒性趋化因子结合蛋白—M3、G蛋白α抑制剂抗体、PI3K抑制剂Quercetin、Akt抑制剂MK-2206、p38MAPK抑制剂SB203580、CK2抑制剂Emodin、mTOR抑制剂Rapamycin、DNMT3a抑制剂SGI-1027、FOCUSTM-Phospho RichTM等。
分组:将分离的PBMC细胞以1×107个/mL的细胞浓度调节好,根据本实验室前期研究所发现的8个作用点所在的关键通路分为I、II、III、IV组,分别是效应CD8+T细胞的趋化因子受体(I组)、磷酸化通路G蛋白-PI3K-Akt/G蛋白-PI3K-p38MAPK-CK2(II组)、mTOR信号通路(III组)和甲基化酶DNMT3a(IV组)。每组都分别将PBMC与100ng/mL的SARS-CoV-2S蛋白溶液、靶点抑制剂共孵育。另设空白对照组,空白对照组中不加入靶点抑制剂。
细胞总磷酸化蛋白水平的定量比较
将培养的各组细胞以1×107个/mL的浓度调节好在1mL的Phospho-Lysis Buffer中重悬。室温孵育10min后,4℃,14000rpm离心10min,弃沉淀。将蛋白质溶液与0.2体积的10×Phospho-Wash Buffer混合后室温孵育10min,10min后,4℃,14000rpm离心10min,弃沉淀。用1%乙酸滴定磷酸肽溶液至pH<5。用去离子水将适量的10×Phospho-Wash Buffer稀释至1×,用去离子水将适量的5×Phospho-Elution Buffer稀释至1×,用10mL 1×Phospho-Wash Buffer平衡Phospho-Column,使缓冲液在重力作用下通过。将制备的磷酸蛋白溶液上样至色谱柱,并使其在重力作用下通过。收集,并用10mL 1×Phospho-WashBuffer和5mL去离子水洗涤色谱柱,用10mL磷酸洗脱缓冲液洗脱结合的磷酸蛋白。使用BCA法检测磷酸化蛋白浓度。
流式细胞术对磷酸化通路的作用点进行筛选
将培养的PBMC细胞以1×107个/mL的细胞浓度调节好,根据磷酸化通路的5个作用靶点分为五组,分别是G蛋白α(i组),PI3K(ii组),Akt(iii组),p38MAPK(iv组)和CK2(v组)。每组都分别将PBMC与100ng/mL的S蛋白溶液、靶点抑制剂共孵育。另设空白对照组,空白对照组中不加入靶点抑制剂。在六个试管中分别放入1.5mL培养的细胞,并将四种单克隆荧光抗体各10μL,包括抗人CD3-PE,CD4-FITC,CD8-APC,CD45RO-PE-Cy5和CD62L-APC将抗人CD3-PE(BD Biosciences)分选后的T细胞中的-H7加入第一个试管中,在流式细胞仪上分选。
流式细胞术对作用点进行筛选
将培养的PBMC细胞以1×107个/mL的细胞浓度调节好,分别是G蛋白α(i组),PI3K(ii组),趋化因子受体(I组)、mTOR信号通路(III组)和甲基化酶DNMT3a(IV组)。每组都分别将PBMC与100ng/mL的S蛋白溶液、靶点抑制剂共孵育。另设空白对照组,空白对照组中不加入靶点抑制剂。在六个试管中分别放入1.5mL培养的细胞,并将四种单克隆荧光抗体各10μL,包括抗人CD3-PE,CD4-FITC,CD8-APC,CD45RO-PE-Cy5和CD62L-APC将抗人CD3-PE(BDBiosciences)分选后的T细胞中的-H7加入第一个试管中,在流式细胞仪上分选。
线粒体膜电位的测量
根据制造商的说明,使用JC-1kit(上海碧云天公司,中国上海)进行线粒体膜电位的测量。从细胞中除去培养基后,将细胞用PBS洗涤一次。之后,将1mL细胞培养溶液和0.5mLJC-1染色工作溶液添加至细胞。充分摇匀后,将细胞在37℃下孵育20分钟。之后,除去上清液,然后将细胞用稀释的JC-1染色缓冲液(1x)洗涤两次。将细胞培养溶液(2mL)加入到洗涤的细胞中,并在荧光显微镜下观察细胞。
免疫印迹
通过免疫印迹检测各靶点抑制剂组和S蛋白组的CD8+T细胞线粒体相关蛋白表达情况。SDS-PAGE后,我们对每种抗体进行了免疫印迹分析。一抗Anti-G蛋白alpha 12(Abcam,英国剑桥),二抗山羊抗兔IgG-HRP稀释剂,小鼠抗标记单克隆抗体SIRT1,PGC-1a,Parkin,LC3 I/II,PINK1(上海爱比信生物技术公司)。
统计分析
所有实验均进行3次独立的生物学重复。使用Prism software确定统计显着性。使用两尾t检验确定P值。*P<0.05;**P<0.01。
结果分析
细胞内整体磷酸化水平的改变
在SARS-CoV-2S蛋白刺激PBMC后与第二章实验所得到的8个作用靶点抑制剂孵育,使用磷酸化蛋白提取试剂盒提取细胞内总磷酸化蛋白并通过BCA蛋白定量法测蛋白浓度。根据标准蛋白曲线计算出各组的磷酸化蛋白浓度,结果如图5所示相对于空白对照组,实验组细胞内磷酸化蛋白的浓度较低,且其中I组(趋化因子受体)、i组(G蛋白α)和ii组(PI3K)磷酸化水平影响较大。这表明靶点抑制剂的使用改变了细胞内的蛋白磷酸化水平,并且趋化受体、G蛋白α和PI3K位点抑制剂的效果最为明显。
磷酸化通路的作用点筛选
在SARS-CoV-2S蛋白刺激PBMC后与磷酸化通路的5个作用靶点抑制剂孵育,通过流式细胞仪分析,结果如图6所示。相对于空白对照组,五组的记忆CD8+T细胞总数没有明显变化,但是CD8+TCM细胞和效应CD8+T细胞数量的变化明显不同。G蛋白α(i组),PI3K(ii组)中TCM的比例明显上升且两组的结果基本一致,而在Akt(iii组),p38MAPK(iv组)和CK2(v组)中TCM的比例略有上升,增长幅度明显小于G蛋白α(i组),PI3K(ii组)。这表明磷酸化通路中的G蛋白α作用点以及蛋白激酶PI3K对于CD8+T细胞的分化效果更好,后面我们将以G蛋白α和蛋白激酶PI3K作为磷酸化通路上的关键作用点参与与其他通路组的比较。
S蛋白刺激后四组CD8+T细胞亚群检测
在SARS-CoV-2S蛋白刺激PBMC后与趋化因子受体(I组)、G蛋白α(i组)、PI3K(ii组)、mTOR信号通路(III组)和甲基化酶DNMT3a(IV组)四个作用靶点抑制剂孵育,通过流式细胞仪分析,结果如图7所示。相对于空白对照组,五组的记忆CD8+T细胞总数没有明显变化,但是CD8+TCM细胞和效应CD8+T细胞数量的变化明显不同。趋化因子受体(I组)、G蛋白α(i组)和PI3K(ii组)中TCM的比例明显上升,而在mTOR信号通路(III组)和甲基化酶DNMT3a(IV组)中TCM的比例略有上升,增长幅度明显小于趋化因子受体(I组)、G蛋白α(i组)和PI3K(ii组)。
靶点抑制剂对效应CD8+T细胞线粒体功能的影响
在S蛋白刺激PBMC后与趋化因子受体(I组)、G蛋白α(i组)、PI3K(ii组)三个作用靶点抑制剂孵育,检测三组细胞中线粒体膜电位、自噬基因和增殖基因的表达情况。结果如图8、9所示,趋化因子受体组和G蛋白α组绿色荧光的程度更高,而PI3K组也显示绿色荧光,但荧光强度弱于其他两组。说明趋化因子受体组和G蛋白α组膜电位下降更多。此外趋化因子受体组和G蛋白α组线粒体增殖基因SIRT1,PGC-1α的表达被严重抑制,自噬相关基因被激活,而PI3K组的基因表达也有相似的情况但程度不如另外两组。
实施例3:敲降靶点基因对关键药物作用靶点再次验证
材料与方法
实验试剂:Bgl II内切酶、Hind III内切酶、Solution I连接酶、嘌呤霉素、BCA蛋白浓度测定试剂盒等。
敲降靶点基因的PBMC细胞株构建
目的基因shRNA序列设计与合成
敲降趋化因子受体(1组)和GNAT1(2组)的基因。在Genbank中查询各组基因序列,按照shRNA的设计原则对每个基因分别设计shRNA。每条shRNA同时合成四条引物。拿到引物后,一条shRNA一组,取各个shRNA的4条shRNA引物,离心后稀释到100μM,取四条4条shRNA引物各2.5μL加入EP管中,加入ddH2O至50μL,水浴锅100℃加热10min,在锅里自然冷却至室温。
表2 shRNA序列
表达载体的构建
使用Bgl II限制性内切酶和Hind III限制性内切酶将pSuper载体进行双酶切,酶切体系如下。
表3 pSuper载体酶切反应体系
shRNA片段与表达载体的连接
每4条引物完成退火后就会形成一段DNA双链短片段。将pSuper载体与shRNA片段连接,连接体系如下:
表4 pSuper-Vector载体与shRNA片段连接体系
连接产物转化大肠杆菌DH5α感受态细胞
取本实验室冻存的100μL大肠杆菌DH5α感受态细胞于冰上融化,将细胞悬液于1.5mL离心管中,在细胞中加入10μL连接产物,混合均匀,冰浴30min。将上述体系置于水浴锅中42℃水浴,热休克90s,随后将连接产物迅速置于冰上冷却2min。接着立即向上述体系中加入500μL的LB液体培养基,摇匀后置于37℃,200rpm的振荡中培养45-60min,使受体菌活化。取200μL活化后的菌液无菌条件下涂布于Amp抗性的LB固体培养基上,37℃倒置培养过夜。挑取培养基中的单菌落,接种于5mL含Amp的LB液体培养基上,在37℃,200rpm的摇床中培养8h。
重组质粒提取
取适量培养的菌液于离心管中,4℃,13000rpm,离心5min,倒干上清液,收集菌体;向菌体沉淀中加入200μL的S1溶液,涡旋仪振荡使菌体充分裂解;再加入200μL的S2溶液,混匀;加入350μL的S3溶液,剧烈晃动10min-15min,充分混匀后,4℃,13000rpm,离心10min后,取上清;上清液加入吸附柱中,37℃,13000rpm,离心45s,倒弃管内液体;加入300μL漂洗液至吸附柱,13000rpm,离心45s,倒弃管内液体;13000rpm,离心90s,倒弃管内液体,并将吸附柱套于EP管中。在吸附管中部加入50-100μL ddH2O,13000rpm,离心90s,收集EP管中的质粒溶液。
逆转录病毒包装和病毒液的收集
在10cm的细胞培养皿中培养293T细胞;取10μg上述构建的pSuper-shRNA质粒和10μg pCL质粒,添加40μL P3000TM试剂,并补充不含血清和抗生素的DMEM培养基至1.5mL,捶打至混匀使质粒稀释;每个培养皿取50μL LipofectamineTM 6000,补充不含血清和抗生素的DMEM培养基至1.5mL,捶打至混匀使脂质体稀释;将上述制得的质粒稀释溶液和脂质体稀释溶液混合,静置15min后将混合液加入培养皿,培养6h吸除培养基,使用PBS清洗两次。加入10mL含10%FBS的DMEM培养基,培养48h;48h后收集培养皿中的病毒上清液,将上清液过0.45μm的微孔滤膜。接着向培养皿中加入10mL含10%FBS的DMEM培养基,培养24h。24h后收集病毒上清液。
逆转录病毒感染PBMC细胞
将0.5-2×105PBMC细胞种于24孔板中,并加入500μL不含血清的DMEM培养基,保证转染时细胞达到60-70%融合,移液器吸除培养基,每孔加入1mL上述制备的病毒上清液,加入8μg/mL的Polybrene,在37℃,5%CO2的环境下孵育4h,孵育完成后加入2mL含10%血清的DMEM培养基,混匀后培养24h,更换病毒进行二次感染。
嘌呤霉素筛选稳定细胞株
使用嘌呤霉素来筛选可以敲降目的基因的PBMC细胞。分别接种5×105个未处理或者敲降三种目的基因的细胞于3cm中,在培养24h。24h后向每组细胞均加入2μg/mL的嘌呤霉素,培养10天。最后向敲降三种目的基因的细胞加入1μg/mL的嘌呤霉素继续培养两周,获得稳定的细胞株。
Western Blot鉴定基因表达效果
通过免疫印迹检测敲降基因的蛋白表达情况。SDS-PAGE后,我们对每种抗体进行了免疫印迹分析。一抗Anti-G蛋白alpha 12(Abcam,英国剑桥),二抗山羊抗兔IgG-HRP稀释剂,小鼠抗标记单克隆抗体CCR5、CXCR4、CX3CR1(上海爱比信生物技术公司)。
流式细胞术对T细胞亚群进行分析
将敲降基因后的PBMC细胞以1×107个/mL的细胞浓度调节好,分别是G蛋白α组,趋化因子受体组。每组都分别将PBMC与100ng/mL的S蛋白溶液共孵育。另设空白对照组,空白对照组中不加入靶点抑制剂。在3个试管中分别放入1.5mL培养的细胞,并将四种单克隆荧光抗体各10μL,包括抗人CD3-PE,CD4-FITC,CD8-APC,CD45RO-PE-Cy5和CD62L-APC将抗人CD3-PE(BD Biosciences)分选后的T细胞中的-H7加入第一个试管中,在流式细胞仪上分选。
线粒体膜电位的测量
根据制造商的说明,使用JC-1kit(上海碧云天公司,中国上海)进行线粒体膜电位的测量。从细胞中除去培养基后,将细胞用PBS洗涤一次。之后,将1mL细胞培养溶液和0.5mLJC-1染色工作溶液添加至细胞。充分摇匀后,将细胞在37℃下孵育20分钟。之后,除去上清液,然后将细胞用稀释的JC-1染色缓冲液(1x)洗涤两次。将细胞培养溶液(2mL)加入到洗涤的细胞中,并在荧光显微镜下观察细胞。
免疫印迹
通过免疫印迹检测各靶点抑制剂组和S蛋白组的CD8+T细胞线粒体相关蛋白表达情况。SDS-PAGE后,我们对每种抗体进行了免疫印迹分析。一抗Anti-G蛋白alpha 12(Abcam,英国剑桥),二抗山羊抗兔IgG-HRP稀释剂,小鼠抗标记单克隆抗体SIRT1,PGC-1a,Parkin,LC3 I/II,PINK1(上海爱比信生物技术公司)。
统计分析
所有实验均进行3次独立的生物学重复。使用Prism software确定统计显着性。使用两尾t检验确定P值。*P<0.05;**P<0.01。
结果分析
敲降基因的Western Blot检测
如图10所示使用qRT-PCR检测到PBMC细胞中GNAT1、CXCR4、CCR5、CX3CR1的表达水平与空白对照组相比显著降低,这表明我们成功构建了敲降GNAT1、CXCR4、CCR5、CX3CR1基因的细胞。
敲降基因后T细胞亚群分析
在完成趋化因子受体和G蛋白α的基因敲降后,通过流式细胞仪分析,结果如图11所示。相对于空白对照组,趋化因子受体、G蛋白α组的记忆CD8+T细胞总数没有明显变化,但是CD8+TCM细胞变化明显不同。趋化因子受体、G蛋白α组中TCM的比例明显上升
敲降基因后效应CD8+T细胞线粒体功能检测
S蛋白刺激敲降基因后的PBMC,检测三组细胞中线粒体膜电位、自噬基因和增殖基因的表达情况。结果如图12、13所示,在敲降趋化因子相关基因和GNAT1后细胞呈绿色荧光,说明敲降后细胞的膜电位降低。Western Blot检测细胞中线粒体的增殖基因SIRT1,PGC-1α的表达水平,发现敲降趋化因子相关受体和GNAT1后SIRT1,PGC-1α的表达被严重抑制。这表明,GNAT1的敲降会影响线粒体的增殖基因的表达水平。同时发现敲降趋化因子相关受体和GNAT1后LC3-II,LC3-I,PINK1,Parkin的表达大量增加条带明显。这表明,敲降GNAT1会影响线粒体的自噬基因的表达水平。
Claims (6)
1.一个可以诱导严重病毒感染患者体内效应CD8+T细胞去分化为CD8+TCM的关键药物靶点,其特征在于,抑制其关键作用靶点可以改变细胞的能量代谢方式、磷酸化通路功能从而诱导效应CD8+T细胞去分化为CD8+TCM,提高CD8+TCM的比例,重建细胞免疫。
2.权利要求1所述的严重病毒感染包括但不限于HIV、SARS-CoV-2、流感病毒、柯萨奇病毒和肝炎病毒。
3.权利要求1所述可以诱导效应CD8+T细胞去分化为CD8+TCM的关键药物靶点的适用细胞模型,其特征在于,病毒感染后T细胞高度活化、CD8+T细胞高细胞毒性及T细胞表面耗竭分子表达上升。
4.权利要求1所述可以诱导严重病毒感染患者体内效应CD8+T细胞去分化为CD8+TCM的关键药物靶点的验证方法,其特征在于,使用不同作用点抑制剂对靶点抑制后通过磷酸化水平、CD8+TCM的比例及线粒体功能进行筛选。
5.权利要求1所述可以诱导严重病毒感染患者体内效应CD8+T细胞去分化为CD8+TCM的关键药物靶点的验证方法,其特征在于,敲降权利要求3所筛选的位点基因通过CD8+TCM的比例及线粒体功能进行筛选。
6.权利要求1所述可以诱导严重病毒感染患者体内效应CD8+T细胞去分化为CD8+TCM的关键药物靶点,其特征在于,其抑制剂在病毒感染中如病毒性肺炎、病毒性心肌炎、病毒性肝炎、病毒性脑炎以及HIV/艾滋病等疾病中细胞免疫功能重建中药物的应用。
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CN116211860B (zh) * | 2023-05-10 | 2023-08-22 | 细胞生态海河实验室 | Ck2抑制剂cx4945在制备肿瘤治疗中防止免疫细胞耗竭药物应用、抑制剂及联合物 |
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