CN115518158A - mTOR抑制剂体外诱导树突状细胞及其应用 - Google Patents
mTOR抑制剂体外诱导树突状细胞及其应用 Download PDFInfo
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Abstract
本申请公开了一种mTOR抑制剂或mTOR抑制剂体外诱导树突状细胞在制备抗输血相关急性肺损伤预防/治疗药物中的用途。本申请还公开了一种体外筛选抗输血相关急性肺损伤预防/治疗药物的方法,包括以下步骤:以mTOR作为药物作用对象,挑选出mTOR抑制剂作为抗输血相关急性肺损伤预防/治疗候选初筛药物。本申请还公开了mTOR信号通路作为靶标在筛选抗输血相关急性肺损伤预防/治疗药物中的用途。本申请还公开了mTOR抑制剂在制备抗输血相关急性肺损伤预防/治疗药物中的用途。本申请还公开了一种抗输血相关急性肺损伤预防/治疗药物,包括有效量的mTOR抑制剂、mTOR抑制剂体外诱导树突状细胞和药学上可接受的载体或辅料。
Description
技术领域
本说明书涉及免疫细胞领域,特别涉及一种mTOR抑制剂体外诱导树突状细胞及其应用。
背景技术
输血相关急性肺损伤(transfusion related acute lung injury,TRALI),临床上属于间接性急性肺损伤,在输血6小时内(迟发性72小时内),突发双肺渗透水肿,肺气体交换功能急剧下降,呼吸频速,进行性低血氧,影像学双肺渗出性病变,以及其他血流动力学改变,如低血压和发烧。主要症状急性呼吸窘迫症(acute respiratory distresssyndrome,ARDS),是近年来导致输血相关死亡率的主要输血后并发症,死亡率占比高达53%。
目前的主要观点认为,70-80%的输血相关急性肺损伤的发病与输注血液中的白细胞抗体相关,因此国际输血协会提出减少使用已育女性献血者血浆(富含抗白细胞抗体),以期减少发病率。然而,在美国血库和输血协会执行减少使用已育女性血液的策略后,仍不能排除急性肺损伤的发生。临床数据表明,除抗白细胞抗体因素外,输注陈旧红细胞,储存后的血小板,血浆等均与输注后急性肺损伤的发病率呈正相关。并且,库存血液成分中的生物活性脂,免疫复合物,微粒,可溶性CD40配体,老化的细胞碎片,异基因抗体等等,多种成分能诱发TRALI。因此,单纯依靠舍弃已育女性的血液成分,并不能从根本上控制输血后急性肺损伤的发生。同时,国内血液资源的严重紧缺的现状,也让这样的控制手段难以执行。临床上尚无特异性措施治疗TRALI,只能通过吸氧或机械通气改善症状,因此急需找到科兴的防治策略,降低该病对患者的危害。
发明内容
本申请的目的,为目前尚无有效治疗手段的TRALI,提供一种安全稳定有效的细胞治疗手段,为TRALI的防控提供一种有效的干预方案。
本申请提供一种mTOR抑制剂或mTOR抑制剂体外诱导树突状细胞在制备抗输血相关急性肺损伤预防/治疗药物中的用途。
本申请还提供一种体外筛选抗输血相关急性肺损伤预防/治疗药物的方法,包括以下步骤:以mTOR作为药物作用对象,挑选出mTOR抑制剂作为抗输血相关急性肺损伤预防/治疗候选初筛药物。
本申请还提供mTOR信号通路作为靶标在筛选抗输血相关急性肺损伤预防/治疗药物中的用途。
本申请还提供mTOR抑制剂在制备抗输血相关急性肺损伤治疗预防/药物中的用途。
本申请还提供一种抗输血相关急性肺损伤预防/治疗药物,包括有效量的mTOR抑制剂、mTOR抑制剂体外诱导树突状细胞和药学上可接受的载体或辅料。
本说明书实施例带来的有益效果包括但不限于:(1)DC是一种高效安全,且需要量相对较少的细胞治疗策略。(2)针对DC在炎性环境下稳定性不足的缺点,我们利用mTOR抑制剂,已经是临床级药用小分子,制备的tDC,在炎症环境刺激后,能依然稳定保持耐受功能特质,解决了DC在体内复杂炎性环境下稳定性的重要技术难点。(3)利用适宜浓度mTOR抑制剂对树突状细胞进行短时处理,将最大可能性的保证了树突状细胞的存活率和活性,以保证细胞治疗的效果。(4)通过调控mTOR信号靶点来诱导临床级tDC,并对TRALI高风险的患者进行输注干预的过继细胞治疗,能有效降低TRALI死亡率,是一种更安全,便捷、经济、高效、易操作且稳定的技术手段。
附图说明
本申请将以示例性实施例的方式进一步说明,这些示例性实施例将通过附图进行详细描述。这些实施例并非限制性的,其中:
图1为根据本申请一些实施例所示的mTOR-tDC控制TRALI疾病小鼠的死亡率以及胸部浸润的示意图;
图2为根据本申请一些实施例所示的各组小鼠肺脏组织病理检测的染色图;
图3为根据本申请一些实施例所示的各组小鼠肺脏组织免疫组化检测的染色图。
具体实施方式
为了更清楚地说明本说明书实施例的技术方案,下面将对实施例描述中所需要使用的附图作简单的介绍。显而易见地,下面描述中的附图仅仅是本说明书的一些示例或实施例,对于本领域的普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图将本说明书应用于其它类似情景。除非从语言环境中显而易见或另做说明,图中相同标号代表相同结构或操作。
如本说明书和权利要求书中所示,除非上下文明确提示例外情形,“一”、“一个”、“一种”和/或“该”等词并非特指单数,也可包括复数。一般说来,术语“包括”与“包含”仅提示包括已明确标识的步骤和元素,而这些步骤和元素不构成一个排它性的罗列,方法或者设备也可能包含其它的步骤或元素。
本说明书中使用了流程图用来说明根据本说明书的实施例的系统所执行的操作。应当理解的是,前面或后面操作不一定按照顺序来精确地执行。相反,可以按照倒序或同时处理各个步骤。同时,也可以将其他操作添加到这些过程中,或从这些过程移除某一步或数步操作。
后疫情时代,病毒或细菌感染或许将成为常态,输血作为医学治疗中的重要手段,面对的将不是单纯的基础疾病和输血不良反应,而是更为复杂的患者基础状态。输血引发急性肺损伤的核心因素,除了大量输血时遭遇供者血液中的炎性物质之外,还取决于被输注患者的临床疾病因素(如感染,创伤和血液病等)。已知系统性感染导致的ARDS进行性发展,是急性肺损伤风险最高的诱因(40%),尤其是败血症相关导致了最高的死亡率。
作用于免疫系统调控的关键位置,耐受性树突状细胞治疗,具有安全性高,疗效明显等特点,近年来,成为研发的热点。然而,由于体内环境的复杂,体外衍生的目标免疫细胞,容易被体内的病毒或细菌,而成为其临床开发的重要障碍。
树突状细胞(dendritic cell,DCs)数量虽少,但能通过吞噬抗原并对T细胞进行递呈;既能发展获得性免疫反应,又能引发快速的天然免疫反应;处于启动、调控、并维持免疫应答的中心环节。由此DCs成为了各类免疫性疾病的细胞治疗热点细胞。事实上,DCs的极化状态对免疫方向的进程起着指导性的作用,当DCs上调成熟标记和共刺激分子时引导效应性免疫反应,DCs维持低表达成熟标记和共刺激分子时引导耐受性免疫反应,后者也被称作耐受性树突状细胞(tolerogenic dendritic cell,tDCs)。
本申请提供一种mTOR抑制剂或mTOR抑制剂体外诱导树突状细胞在制备抗输血相关急性肺损伤预防/治疗药物中的用途。哺乳动物雷帕霉素靶蛋白(mammalian target ofrapamycin,mTOR)是细胞生长和增殖的重要调节因子。大量研究显示mTOR信号途径调控异常与细胞增殖密切相关。
如本文所用,术语“预防/治疗”(及其语法变体)指试图改变治疗个体中疾病的自然进程,并且可以是为了预防或在临床病理学的过程期间实施的临床干预。治疗的期望效果包括但不限于预防疾病的发生或复发、缓解症状、降低疾病的任何直接或间接病理学后果、预防转移、减缓疾病进展的速率、改善或减轻疾病状态、及免除或改善预后。在一些实施方案中,本申请的mTOR抑制剂或mTOR抑制剂体外诱导树突状细胞用于延迟疾病的形成或延缓病症的进展。
在一些实施例中,所述抗输血相关急性肺损伤预防/治疗药物可以阻止肺组织病变。在一些实施例中,所述抗输血相关急性肺损伤预防/治疗药物可以降低胸腔积液。在一些实施例中,所述抗输血相关急性肺损伤预防/治疗药物可以降低胸腔积液。在一些实施例中,所述抗输血相关急性肺损伤预防/治疗药物可以提高生存率。在一些实施例中,所述抗输血相关急性肺损伤预防/治疗药物可以降低炎症区粒细胞的侵润,并控制肺泡的蛋白粘液析出和肺泡壁增厚。
在一些实施例中,所述mTOR抑制剂可以包括Rapamycin、Temisirolimus或Everolimus。
在一些实施例中,所述mTOR抑制剂体外诱导树突状细胞可以为经mTOR抑制剂体外处理的树突状细胞。在一些实施例中,mTOR抑制剂体外处理的树突状细胞为未成熟的树突状细胞。在一些实施例中,mTOR抑制剂体外处理的树突状细胞为成熟相关蛋白表达量低的树突状细胞。在一些实施例中,mTOR抑制剂体外处理的树突状细胞与T细胞混合后,不能引起T细胞的大量增殖。
本申请还提供一种体外筛选抗输血相关急性肺损伤预防/治疗药物的方法,包括以下步骤:以mTOR作为药物作用对象,挑选出mTOR抑制剂作为抗输血相关急性肺损伤治疗候选初筛药物。
在一些实施例中,体外筛选抗输血相关急性肺损伤预防/治疗药物的方法,还包括,将所述候选药物处理树突状细胞后,与T细胞混合培养后分析mTOR信号通路磷酸化水平,mTOR信号通路磷酸化水平越低,抑制效果越好。
在一些实施例中,所述mTOR抑制剂可以抑制所述mTOR的活性,或抑制mTOR的基因转录或表达。在一些实施例中,所述mTOR抑制剂可以抑制mTOR通路中上游蛋白或下游蛋白的表达,例如PI3K、Akt、PTEN、TSC1/2和LKB1。mTOR作为一种非典型丝氨酸/苏氨酸蛋白激酶,在哺乳动物发育中发挥核心作用。它是细胞生长和增殖以及转录、mRNA转换、翻译、核糖体生物发生、囊泡运输、自噬、细胞骨架组织等生理事件的关键调节剂。
本文所用的术语“抑制”或这些术语的任何变体,是指mTOR抑制剂的能力,其通过直接或间接与靶点相互作用,降低目标靶点的信号传导活性,且是指目标靶点活性的任何可以测量的减少或完全抑制。例如,与正常情况相比,可以是活性降低量约、至多约或至少约5%、10%、15%、20%、25%、30%、35%、40%、45%、50%、55%、60%、65%、70%、75%、80%、85%、90%、95%、99%或更多、或其中可衍生的任何范围。
本申请还提供mTOR信号通路作为靶标在筛选抗输血相关急性肺损伤预防/治疗药物中的用途。哺乳动物(mTOR)靶点是一种大型(~289kDa)非典型激酶,与磷脂酰肌醇激酶相关激酶(PIKK)家族的其他成员一样,它包含一个羧基末端丝氨酸/苏氨酸蛋白激酶结构域。同样与其他PIKK一致的是,mTOR包含一个FRAP-ATM-TTRAP(FAT)域和一个羧基末端FAT域(FATC),它们可能在mTOR结构和稳定性中起作用。
本申请还提供mTOR抑制剂在制备抗输血相关急性肺损伤预防/治疗药物中的用途。
本申请还提供一种抗输血相关急性肺损伤预防/治疗药物,包括有效量的mTOR抑制剂、mTOR抑制剂体外诱导树突状细胞和药学上可接受的载体或辅料。
本文所用的术语“有效量”是指通常足以对需要治疗的输血相关急性肺损伤患者产生有益治疗效果的量或剂量。本领域技术人员可以通过常规方法、结合常规影响因素来确定本发明中活性成分的有效量或剂量。
“药学上可接受的”是指当分子本体和组合物适当地给予动物或人时,它们不会产生不利的、过敏的或其它不良反应。“药学上可接受的载体或辅料”应当与mTOR抑制剂相容,即能与其共混而不会在通常情况下大幅度降低药物组合物的效果。可作为药学上可接受的载体或辅料的一些物质的具体例子是糖类,如乳糖、葡萄糖和蔗糖;淀粉,如玉米淀粉和土豆淀粉;纤维素及其衍生物,如甲基纤维素钠、乙基纤维素和甲基纤维素;西黄蓍胶粉末;麦芽;明胶;滑石;固体润滑剂,如硬脂酸和硬脂酸镁;硫酸钙;植物油,如花生油、棉籽油、芝麻油、橄榄油、玉米油和可可油;多元醇,如丙二醉、甘油、山梨糖醇、甘露糖醇和聚乙二醇;海藻酸;乳化剂,如Tween;润湿剂,如月桂基硫酸钠;着色剂;调味剂;压片剂、稳定剂;抗氧化剂;防腐剂;无热原水;等渗盐溶液;和磷酸盐缓冲液等。这些物质根据需要用于帮助配方的稳定性或有助于提高活性或它的生物有效性或在口服的情况下产生可接受的口感或气味。
下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的试验材料,如无特殊说明,均为自常规生化试剂公司购买得到的。以下实施例中的定量试验,均设置三次重复实验,结果取平均值。
实施例
材料:
1.纯化的人未成熟树突状细胞(imDC)(纯化的人单核细胞用细胞因子诱导而得,或者外周血PBMC诱导后再纯化,以及其他)。
2.具有mTOR信号抑制能力的小分子化合物(Rapamycin,Temisirolimus,Everolimus)。
3.完全细胞培养基。
实施例1
mTOR-tDC细胞制备:
取Balb/c小鼠骨髓,Ficoll分离取得干净的单个核细胞,PBS洗涤3次后,加入含rmGM-CSF,rmIL-4的完全培养基,置于37℃5%CO2培养箱中,每3天换液一次,培养第8天收获细胞,PBS洗涤一次。CD11c阳性分选纯化小鼠DC。用mTOR抑制剂处理后(同之前浓度和时间技术指标),收集并低速洗涤两次后备用。
细胞过继输注:
经尾静脉输注同型小鼠,细胞悬液浓度为5*104/ml-5*106/ml。同时,设置多重对照组。
TRALI疾病小鼠模型验证:
DC输注1h后,腹腔注射细菌脂多糖(LPS)0.1-3mg/kg,次日对小鼠输注抗MHC-I分子抗体或其同型对照抗体,以小鼠死亡或抗体输注2h作为试验的终点,安乐死处死小鼠(麻醉剂注射致死:腹腔注射4%四水合氯醛250ul/10g),麻醉起效后,颈椎脱臼处死小鼠。对照组小鼠与其他组小鼠在同一环境、观察相同时间后处理。
各组小鼠体温测定:
各组小鼠注射抗体后,根据时间需要,使用动物电子体温仪,进行肛门测温并记录。
各组小鼠生存率检测:
分别观察每组小鼠的生存状态,在试验终点记录生存时间,利用RPISM软件绘制生存曲线,如图1所示。
各组小鼠肺脏湿/干比率:
各组小鼠肺脏组织用吸水纸吸干表面液体后,置于35mm培养皿中,称量总重,并减去小皿重量,作为湿重。将肺组织与小皿放入电热恒温干燥箱内,65℃烘干72小时后取出测重,并减去小皿重量,作为干重。肺湿/干比率=湿重/干重。
各组小鼠胸腔积液测定:
小鼠处死后,打开胸腔,取出肺脏后,吸取胸腔积液至小试管中,称重,并减去小试管重量,为胸腔积液重量,实验结果如图1所示。
各组小鼠肺脏组织病理检测:
每组取小鼠右下肺叶置于10%福尔马林固定24h后,脱水,包埋切片,苏木精-伊红(hematoxylin-eosin,HE)染色,用显微摄像系统采集图像,如图2所示。
各组免疫组织化学检测:
每组取小鼠右下肺置于4%多聚甲醛固定24h后,脱水、透明、包埋、切片、封闭、抗原修复、封闭,各个标本中分别加入抗Foxp3兔多克隆抗体(1∶200)、抗CD11c兔多克隆抗体(1∶200)、抗CD11b兔多克隆抗体(1∶200)4℃孵育过夜后,加入山羊抗兔工作液完成水洗、显色、复染、封片程序;采集图像,如图3所示。
实施例2-结果分析
如图1A所示,本试验的观察时间内,TRALI发病组的小鼠生存率为36.4%,本技术制备的mTOR-tDC干预组的小鼠生存率为100%,并优于mTOR药物干预组。
本技术制备的mTOR-tDC干预组小鼠,体温变化与肺干湿比与TRALI发病组小鼠,没有显著性差异(图1B)。但是两组干预组的胸腔积液重量与TRALI发病组有显著性差异(P<0.05)。说明本技术制备的mTOR-tDC通过降低胸腔积液缓解了TRALI小鼠的病情进展。
与对照组相比(图2),TRALI发病组小鼠的肺组织肺泡结构破坏,并伴有大量粘液渗出,大量细胞溶解坏死,小血管中充血,肺泡腔大量红细胞和炎性细胞侵润。
经本技术制备的mTOR-tDC干预后,小鼠的肺组织中的上述病变显著缓解。与未经处理过的DC干预组小鼠相比,本技术制备的mTOR-tDC干预组小鼠的肺泡结构被保护的更为完好。
由于TRALI已报到的主要相关炎性细胞为中性粒细胞,而主要的耐受调节细胞为调节性T细胞(regular T cells,Treg)。因此我们对肺组织进行的主要免疫细胞的抗体标记示踪,CD11b为粒细胞标记,CD11c为DC标记,FOXP3为Treg标记。
经本技术制备的mTOR-tDC干预后,DC能大量出现在肺泡区域,并大幅度降低了炎症区粒细胞的侵润,并控制了肺泡的蛋白粘液析出和肺泡壁增厚。与此同时,FOXP3+的T细胞并未大量出现在炎症灶。
本发明技术,通过适宜浓度mTOR抑制剂对DC进行短效处理,一方面,保证了细胞制品的存活率,一方面,有效介导了DC耐受极化,并能在炎症环境下维持稳定耐受状态。
通过本技术制备的mTOR-tDC输注,在动物模型体内有效的干预和控制了感染后TRALI的疾病进展。希望通过本技术的开发,制备有效稳定的细胞,而将该技术为基础的治疗手段尽快造福于患者。
上文已对基本概念做了描述,显然,对于本领域技术人员来说,上述详细披露仅仅作为示例,而并不构成对本说明书的限定。虽然此处并没有明确说明,本领域技术人员可能会对本说明书进行各种修改、改进和修正。该类修改、改进和修正在本说明书中被建议,所以该类修改、改进、修正仍属于本说明书示范实施例的精神和范围。
同时,本说明书使用了特定词语来描述本说明书的实施例。如“一个实施例”、“一实施例”、和/或“一些实施例”意指与本说明书至少一个实施例相关的某一特征、结构或特点。因此,应强调并注意的是,本说明书中在不同位置两次或多次提及的“一实施例”或“一个实施例”或“一个替代性实施例”并不一定是指同一实施例。此外,本说明书的一个或多个实施例中的某些特征、结构或特点可以进行适当的组合。
一些实施例中使用了描述成分、属性数量的数字,应当理解的是,此类用于实施例描述的数字,在一些示例中使用了修饰词“大约”、“近似”或“大体上”来修饰。除非另外说明,“大约”、“近似”或“大体上”表明所述数字允许有±20%的变化。相应地,在一些实施例中,说明书和权利要求中使用的数值参数均为近似值,该近似值根据个别实施例所需特点可以发生改变。在一些实施例中,数值参数应考虑规定的有效数位并采用一般位数保留的方法。尽管本说明书一些实施例中用于确认其范围广度的数值域和参数为近似值,在具体实施例中,此类数值的设定在可行范围内尽可能精确。
最后,应当理解的是,本说明书中所述实施例仅用以说明本说明书实施例的原则。其他的变形也可能属于本说明书的范围。因此,作为示例而非限制,本说明书实施例的替代配置可视为与本说明书的教导一致。相应地,本说明书的实施例不仅限于本说明书明确介绍和描述的实施例。
Claims (10)
1.一种mTOR抑制剂或mTOR抑制剂体外诱导树突状细胞在制备抗输血相关急性肺损伤预防/治疗药物中的用途。
2.如权利要求1所述的用途,其特征在于,所述抗输血相关急性肺损伤预防/治疗药物可起到以下作用中的一项或多项:
阻止肺组织病变
降低胸腔积液
提高生存率
降低炎症区粒细胞的侵润,并控制肺泡的蛋白粘液析出和肺泡壁增厚。
3.如权利要求1所述的用途,其特征在于,所述mTOR抑制剂包括Rapamycin、Temisirolimus或Everolimus。
4.如权利要求1所述的用途,其特征在于,所述mTOR抑制剂体外诱导树突状细胞为经mTOR抑制剂体外处理的树突状细胞。
5.一种体外筛选抗输血相关急性肺损伤预防/治疗药物的方法,包括以下步骤:以mTOR作为药物作用对象,挑选出mTOR抑制剂作为抗输血相关急性肺损伤预防/治疗候选初筛药物。
6.如权利要求5所述的体外筛选抗输血相关急性肺损伤预防/治疗药物的方法,其特征在于,还包括,将所述候选药物处理树突状细胞后,与T细胞混合培养后分析mTOR信号通路磷酸化水平,mTOR信号通路磷酸化水平越低,抑制效果越好。
7.如权利要求5所述的体外筛选抗输血相关急性肺损伤预防/治疗药物的方法,其特征在于,所述mTOR抑制剂能抑制所述mTOR的活性,或抑制mTOR的基因转录或表达。
8.mTOR信号通路作为靶标在筛选抗输血相关急性肺损伤预防/治疗药物中的用途。
9.mTOR抑制剂在制备抗输血相关急性肺损伤治疗预防/药物中的用途。
10.一种抗输血相关急性肺损伤预防/治疗药物,包括有效量的mTOR抑制剂、mTOR抑制剂体外诱导树突状细胞和药学上可接受的载体或辅料。
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