CN115969976B - Fbxo6蛋白或其编码基因作为靶点的应用 - Google Patents
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Abstract
本发明公开了FBXO6蛋白或其编码基因作为靶点的应用，属于医药领域。本发明提供的靶向FBXO6在制备用于治疗急性肺损伤的药物中的应用，FBXO6敲减抑制肺泡巨噬细胞的凋亡，抑制炎细胞向肺部浸润，抑制肺内的病毒负荷。靶向FBXO6对病毒诱导的急性肺损伤的保护作用与抑制肺泡巨噬的凋亡有关。因此，本发明为临床病毒性肺炎的治疗提供了新的方向和具体措施。
Description
技术领域
本发明属于医药领域，具体涉及FBXO6蛋白或其编码基因作为靶点的应用。
背景技术
蛋白质降解对于信号转导的快速反应和氨基酸的再循环都是必不可少的，在各种生理病理过程中发挥着重要作用。E3连接酶能够促进泛素部分与底物蛋白的连接，决定了蛋白降解的特异性，在蛋白酶体介导的蛋白质降解中起着关键作用。
FBXO6蛋白，属于F-box蛋白家族的新成员之一，属于连接酶-复合物SCF(SKP1/CDC53-Cullin1/F-box)的关键组成蛋白。SCF是一种进化相对保守的泛素蛋白复合物，F-box蛋白可以通过与不同的泛素化受体蛋白组相互结合来发挥其功能，介导SCF E3连接酶复合物泛素化。已有研究表明，FBXO6蛋白的表达被抑制后可以通过诱导泛素化的多个靶分子蛋白降解，进一步引起肿瘤细胞的化疗敏感性降低。研究发现FBXO6蛋白可以与Chk1直接结合，促进Chk1的泛素化并降解Chk1。最近的研究表明Chk1不仅在药物处理的细胞中，而且在未作干扰的细胞中也发挥重要的生物学功能，这可能与FBXO6蛋白可以参与调解DNA损伤应答反应、细胞纺锤体复制检查点、细胞周期过程的停滞、DNA修复和细胞的死亡有关。有文献报道FBXO6蛋白促进了胃癌细胞的生长和增殖，但同时减弱了胃癌细胞的凋亡水平以及细胞侵袭能力(Chan C H，Li C F，Yang W L，et al.The Skp2-SCF E3 ligase regulatesAkt ubiquitination，glycolysis，herceptin sensitivity，and tumorigenesis.Cell，2012，149(5)：1098-1111.)。然而目前尚没有FBXO6蛋白的靶向药物在治疗流感病毒诱导的急性肺损伤中的应用。
病毒引起的呼吸道感染是一个重要的公共卫生问题，根据世界卫生组织的报告，季节性流感病毒，包括甲型流感病毒以及乙型流感病毒等，每年会导致全球约三百至五百万例严重感染和29-65万例死亡。甲型流感病毒(Influenza A virus)属于正粘病毒科，流感病毒属；基因组由大小不同的8个独立的单股负链RNA片段组成，是引起人类流感的重要病原体之一。另外，部分动物来源的病毒能够跨越物种屏障，通常是通过禽流感病毒和人类流感病毒之间的交互发生抗原转变，不定期地引起大流行病，造成人员死亡和重大的社会损失。到目前为止，甲型流感病毒已经多次引起世界范围内的流感大爆发，如1918年的甲型HINI流感的大流行，据保守估计造成了约4000万人的死亡。因此，除了加紧研发新的抗病毒药物外，如何通过调节机体天然免疫系统来抵御病毒感染，是感染免疫学领域的重要科学问题。
发明内容
发明的目的是针对现有技术中的不足，提供FBXO6蛋白或其编码基因作为靶点在制备用于治疗急性肺损伤的药物中的应用。
本发明提供了FBXO6蛋白或其编码基因作为靶点在制备用于抗流感病毒感染或者治疗由流感病毒感染引起的急性肺损伤的药物中的应用。
人FBXO6蛋白编码基因序列的Gene ID：26270，人FBXO6蛋白编码基因的核苷酸序列的GenBank号：NM_018438.6。
所述应用为增加肺组织中的I型干扰素水平。
I型干扰素是参与抗病毒免疫的重要效应分子。它的产生主要由先天性免疫细胞(主要是巨噬细胞)表面或内部受体(Toll like receptor，NOD like receptor，RIG-Ireceptor，cGAS等)接触到病毒特异性的抗原物质(DNA，RNA)，然后通过胞内的信号分子传递(MAVS，STING，TBK，IKK等)，最终激活转录因子IRF3/7从而启动I型干扰素基因的表达。
所述应用为抑制肺泡巨噬细胞发生凋亡，减少炎症细胞向肺部浸润，降低肺组织中的病毒负荷。
优选的，所述应用为抑制FBXO6蛋白活性或降低Fbxo6基因表达。
本发明还提供了用于抑制FBXO6蛋白活性或降低Fbxo6基因表达的化合物在制备用于抗流感病毒感染或者治疗由流感病毒感染引起的急性肺损伤的药物中的应用。
优选的，所述化合物是阻碍FBXO6蛋白表达或转录的DNA或RNA。
优选的，所述化合物是针对FBXO6蛋白的抑制剂或其药学上可接受的盐、溶剂合物或水合物的药物制剂。
优选的，所述化合物是针对FBXO6蛋白的抗体。
本发明还提供了一种用于抗流感病毒感染或者治疗由流感病毒感染引起的急性肺损伤的药物，所述药物是以下一种：
(1)针对FBXO6蛋白的抗体；
(2)针对FBXO6蛋白的抑制剂或其药学上可接受的盐、溶剂合物或水合物的药物制剂；
(3)阻碍FBXO6蛋白表达或转录的DNA或RNA。
本发明提供的一种用于抗流感病毒感染或者治疗由流感病毒感染引起的急性肺损伤的药物，所述药物是用于敲除Fbxo6基因的siRNA或用于表达该siRNA的载体，所述siRNA的序列为正义链5’-CCCACACCUUCUCUGAUUATT-3’和/或反义链5’-UAAUCAGAGAAGGUGUGGGTT-3’。
本发明的有益效果：
(1)本发明研究发现抑制FBXO6蛋白的表达可以显著降低病毒诱导的肺泡巨噬细胞凋亡，增加肺组织中的I型干扰素的表达，抑制病毒的早期增殖，从而抑制炎细胞向肺部浸润。
(2)抑制FBXO6蛋白的表达对病毒诱导肺炎的保护作用与抑制肺泡巨噬细胞凋亡有关。因此，本发明为临床病毒诱导的急性肺损伤治疗提供了新的可能的方向和具体措施。
附图说明
图1为感染病毒后FBXO6蛋白敲减和野生肺泡巨噬细胞的I型干扰素及下游基因的表达差异图；“**”代表p＜0.01。
图2为感染病毒后FBXO6蛋白敲减和野生肺泡巨噬的凋亡情况图；“**”代表p＜0.01。
图3为感染病毒后FBXO6蛋白敲减和野生肺泡巨噬的凋亡蛋白caspase3的激活情况图；“*”代表p＜0.05，“**”代表p＜0.01。
图4为Fbxo6基因敲除对病毒诱导急性肺损伤的保护作用图；其中，A为感染病毒患者的Fbxo6基因的表达图；B为Fbxo6基因敲除对小鼠的发病率及生存率的影响图；“*”代表p＜0.05，“**”代表p＜0.01。
图5为Fbxo6基因敲除对病毒性肺炎小鼠肺部病理变化的影响图。
图6为Fbxo6基因敲除对病毒性肺炎小鼠肺部炎症细胞的浸润情况和病毒负荷的影响图；“*”代表p＜0.05。
图7为Fbxo6基因敲除对肺内肺泡巨噬细胞的数量影响图。
图8为Fbxo6基因敲除对肺内肺泡巨噬细胞的增殖的影响图。
图9为Fbxo6基因敲除对肺内肺泡巨噬细胞的凋亡的影响图；“*”代表p＜0.05。
图10为Fbxo6基因敲除对肺内肺泡巨噬细胞的凋亡的荧光染色图。
具体实施方式
实施例1
FBXO6蛋白敲减对病毒感染的肺泡巨噬细胞系MH-S的保护实验。
使用小干扰RNA敲减MH～S中的FBXO6蛋白的表达：将永生化HPMEC细胞(人肺微血管内皮细胞)均匀接种于6孔细胞培养板或者transwell小室中，其中6孔板接种(5-10)×105个/孔，培养过夜待细胞完全贴壁后，更换成无抗生素完全培养基，进行后续转染操作。将正义链和反义链浓度各为10μM的siRNA(正义链序列：5’-CCCACACCUUCUCUGAUUATT-3’；反义链序列：5’-UAAUCAGAGAAGGUGUGGGTT-3’)，按照使用量6孔板4μL、transwell小室0.5μL，加入Opti-MEM培养液稀释，混匀静置5min；将转染试剂LipofectamineTM RNA iMAX按照6孔板6μL，加入到Opti-MEM培养基稀释，混匀静置5min；然后将上述siRNA稀释液和RNA iMAX稀释液1∶1混匀后室温孵育5min形成转染复合物，加入细胞培养液中后继续培养，转染24-48h后可提取细胞RNA或蛋白以验证转染效率，验证转染成功后进行相关实验。
(1)PCR检测病毒感染后FBXO6蛋白敲减细胞和野生型细胞的基因表达情况。
(2)美国BD公司的凋亡检测试剂盒检测病毒感染后FBXO6蛋白敲减细胞和野生型细胞的凋亡情况。
(3)用碧云天公司的Caspase 3活性检测试剂盒检测病毒感染后FBXO6蛋白敲减细胞和野生型细胞的Caspase 3激活情况。
FBXO6蛋白敲减对肺泡巨噬细胞感染病毒后I型干扰素及下游基因、细胞凋亡情况，凋亡蛋白的激活情况的影响如图1-3所示。结果显示，图1中，FBXO6蛋白敲减后肺泡巨噬细胞I型干扰素及下游基因IFN-β、IFIT1、IFIT3、MX1、Cxc110的表达显著上升；图2和图3中，细胞凋亡减少、凋亡蛋白的激活情况减弱。
实施例2
小鼠病毒性肺炎模型的构建。
C57BL/6小鼠和Fbxo6基因(基因序列的Gene ID：50762)敲除小鼠(雌性，18-20g)，经水合氯醛麻醉后，经气道滴注50μL PR8病毒悬液，PR8病毒从中国疾控中心获得，小鼠平躺至苏醒。
(1)每日监测小鼠的体重和生存情况。
(2)5天后收取肺，用4％多聚甲醛固定，HE染色观察肺组织病理变化。
(3)5天后收取肺泡灌洗液(BALF)，取部分BALF进行细胞计数和流式细胞仪检测不同种类免疫细胞的百分比。取部分BALF进行病毒滴度的测定。
病毒感染后Fbxo6基因表达量变化、Fbxo6基因敲除对病毒性肺炎小鼠的生存率、病理变化、肺部炎症细胞浸润及肺组织病毒负荷的影响如图4-6所示。结果显示，图4中，与基线相比，来自甲流病毒感染小鼠的外周血白细胞Fbxo6基因的表达在感染过程的前六天显著增加。随着时间的推移，Fbxo6基因表达逐渐下降，直至第21天恢复到基线值；Fbxo6基因敲除小鼠感染病毒后患病率和死亡率都远低于野生小鼠。图5中，Fbxo6基因敲除小鼠感染病毒后的肺组织损伤低于野生组小鼠。图6中，Fbxo6基因敲除小鼠感染病毒后炎症细胞浸润少于野生小鼠，Fbxo6基因敲除小鼠感染病毒后肺组织病毒负荷低于野生小鼠。
实施例3
小鼠病毒性肺炎模型的构建。
C57BL/6小鼠和Fbxo6基因敲除小鼠(雌性，18-20g)，经水合氯醛麻醉后，经气道滴注50μL PR8病毒悬液，小鼠平躺至苏醒。
(1)3d后流式检测肺内肺泡巨噬细胞的数量。
(2)3d后流式检测肺泡巨噬细胞的增殖情况和凋亡情况。
(3)3d后免疫荧光染色检测肺泡巨噬细胞的凋亡情况；CD11c、F4/80抗体购自CST公司，货号分别为#97585和#30325，Tunel购自recordbio生物科技公司，货号为RC-012。
Fbxo6基因敲除对小鼠肺泡巨噬细胞的数量、增殖和凋亡情况的影响如图7-10所示。结果显示，图7中，Fbxo6基因敲除小鼠感染病毒后肺泡巨噬细胞的数量显著高于野生小鼠组。图8中，Fbxo6基因敲除小鼠和野生小鼠感染病毒后肺泡细胞的增殖没有显著差异。图9和图10中，Fbxo6基因敲除小鼠感染病毒后肺泡细胞的凋亡显著低于野生小鼠。
结论：抑制FBXO6蛋白的表达能抑制病毒诱导的肺泡巨噬细胞凋亡，对病毒诱导的急性肺损伤有显著的保护作用。
Claims (2)
1.用于抑制FBXO6蛋白活性或降低Fbxo6基因表达的化合物在制备用于抗流感病毒感染或者治疗由流感病毒感染引起的急性肺损伤的药物中的应用，所述FBXO6蛋白由Fbxo6基因编码得到；所述Fbxo6基因为人Fbxo6基因或者是小鼠Fbxo6基因；人Fbxo6基因的核苷酸序列的GenBank号：NM_018438.6，或者是小鼠Fbxo6基因序列的Gene ID：50762；
所述化合物是用于敲除Fbxo6基因的siRNA或用于表达该siRNA的载体；
所述siRNA的序列为正义链5’-CCCACACCUUCUCUGAUUATT-3’和/或反义链5’-UAAUCAGAGAAGGUGUGGGTT-3’。
2.一种用于抗流感病毒感染或者治疗由流感病毒感染引起的急性肺损伤的药物，其特征在于，所述药物是阻碍FBXO6蛋白表达或转录的RNA；
所述药物是用于敲除Fbxo6基因的siRNA或用于表达该siRNA的载体；
所述FBXO6蛋白由Fbxo6基因编码得到；所述Fbxo6基因为人Fbxo6基因或者是小鼠Fbxo6基因；
人Fbxo6基因的核苷酸序列的GenBank号：NM_018438.6，或者是小鼠Fbxo6基因序列的Gene ID：50762；
所述siRNA的序列为正义链5’-CCCACACCUUCUCUGAUUATT-3’和/或反义链5’-UAAUCAGAGAAGGUGUGGGTT-3’。
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