CN114736917B - Usp11在抑制细胞因子il6降解中的应用 - Google Patents
Usp11在抑制细胞因子il6降解中的应用 Download PDFInfo
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Abstract
IL6是一种重要的细胞因子，在抗感染，肿瘤免疫，免疫细胞发育等过程中发挥着重要的作用，因此维持一定水平的IL6水平，使其免于过早被降解在上述过程中显得非常重要。USP11是参与机体泛素化过程的基因，研究发现其在多种肿瘤中表达异常，未见其参与IL6降解的研究。本发明公开了USP11新的功能‑保护IL6避免降解的作用，为抗感染和肿瘤的治疗提供新的靶点与思路。
Description
技术领域
本发明属于生物技术，具体公开了USP11在抑制细胞因子IL6 降解中的应用。
背景技术
IL6是一种重要的细胞因子，在抗感染，肿瘤免疫，免疫细胞发育等过程中发挥着重要的作用，因此维持一定的IL6水平，使其免于过早被降解在上述过程中显得非常重要。USP11是参与机体泛素化过程的基因，研究发现其在多种肿瘤中表达异常，未见其参与IL6降解的研究。
发明内容
现有技术针对IL6降解的研究较少，本发明公开了USP11新的功能-保护IL6避免降解的作用，为抗感染和肿瘤的治疗提供新的靶点与思路。
本发明采用如下技术方案：
USP11在抑制细胞因子IL6 降解中的应用。
USP11在制备抑制细胞因子IL6 降解药物中的应用。
过表达USP11的试剂在抑制细胞因子IL6 降解中的应用，或者在制备抑制细胞因子IL6 降解药物中的应用。
本发明中，USP11以病毒载体或者质粒的形式作为药物，比如慢病毒、腺病毒等常规病毒载体。
本发明分别取USP11基因敲除C57小鼠为实验组，对照组为USP11野生型C57小鼠。皮下注射5mg/kg LPS，特定时间点取小鼠血清，ELISA检测IL-6含量，证实USP11能避免IL6过早降解，为抗感染和肿瘤治疗提供更有效的治疗策略及思路。
附图说明
图1为LPS（5mg/kg）处理后小鼠血清内IL-6表达水平。
图2为人重组腺病毒感染293细胞。
图3为Ad-USP11和Ad-NC组细胞IL-6 mRNA表达水平差异。
图4为Ad-USP11和Ad-NC细胞上清液内IL-6表达差异。
图5为慢病毒感染293细胞。
图6为sh-USP11和sh-NC组细胞IL-6 mRNA表达水平差异。
具体实施方式
C57BL/6小鼠(H-2b)为雌性小鼠，周龄为6-8周，体重为17-22 克，购买上海斯莱克实验动物公司。C57BL/6 遗传背景的 USP11-/-小鼠、人293细胞源自苏州大学医学部放射医学与防护学院。小鼠的饮用水、饲料和垫料均经过灭菌处理。所有小鼠均饲养于SPF级别的隔离笼内。所有动物实验均按照苏州大学实验与动物伦理委员要求操作。
超净台消毒灭菌，快速复苏293细胞。用10%血清DMEM培养基（青霉素+链霉素）于无菌培养皿内培养。定期观察。在超净台内用无菌PBS将LPS粉末溶解，配制为1mg/kg的试剂。分装置于-80℃冰箱内待用。
ELSIA检测细胞因子。
血清获得方法。摘眼球取小鼠外周血至于于无热源和内毒素的试管内，室温下静置2h，离心，1000g，30min。取上清液于-80℃待用。
细胞上清液获得方法。观察细胞状态，取细胞融合度约为80%时进行实验。加入胰蛋白酶消化细胞。用预冷的PBS洗涤细胞。加入细胞裂解液（临用前一秒加入蛋白酶抑制剂）进行细胞重悬。离心，4℃ ，10000×g ，10min。取细胞上清，-80℃保存，避免反复冻融。
ELSIA测试。预先进行标号，A1-A6为标准品孔，其他为样品孔，每个样品加3个复孔。按照说明书进行操作，分别加入标准品和样品5ul,样品稀释液45ul。加入酶标抗体100ul,37℃ 30min。洗涤，每次1分钟，洗涤5次。加入显色液A液和B液于37℃孵育15分钟，加入终止液。在酶标仪上于450nm检测各组OD值。
RT-PCR检测mRNA水平。总RNA的获取，小鼠外周血静置2h后进行离心，上层为血清，下层为单核细胞。将上层血清吸取掉后向剩余组织内加入1ml Trizol溶液室温下颠倒混匀15min。加入200ul氯仿，震动1min后静置5min，离心,12000rpm,15min,4℃。将上层吸取至新EP管内，加入500ul异丙醇，冰上30min。离心。弃上清，加1ml 75%乙醇，离心。弃上清液，于通风橱中冰上30min，加入25ul的DEPC水溶解置于-20℃冰箱。RNA浓度的测定，将RNA于-20℃冰箱取出，于震动涡旋器上震荡，吸取2ul的DEPC水做空白对照，其余样品吸取2ul于板上，使用全自动酶标仪进行检测。
本发明具体操作方法以及测试方法为常规技术，GraphPad Prism 8.0作图，SPSS20统计分析。P<0.05定义为有显著性差异。
实施例一
小鼠分组及腹腔注射LPS诱导脓毒血症模型。实验小鼠分组：WT小鼠（USP11野生型）和KO小鼠（USP11基因敲除）各10只，分别接受LPS腹腔注射剂量为5mg/kg。
1.分别取USP11基因敲除C57小鼠为实验组，对照组为USP11野生型C57小鼠，皮下注射5mg/kg LPS。
2. 特定时间点取小鼠血清，行ELISA检测IL-6含量。
3. USP11基因敲除后，小鼠血液中IL-6含量，在LPS刺激4小时后，明显低于未敲除小鼠。说明，USP11有效降低IL-6的降解速度，参见图1。
实施例二
重组腺病毒Ad-USP11转染293细胞。显微镜下观察293细胞细胞融合至70%后，消化细胞，离心。分为实验组（Ad-USP11）和对照组（Nc-USP11）弃掉上清液，向每个孔内一次加入病毒稀释液和细胞培养基，混匀，培养24h后更换正常培养基，观察细胞状态。48h后，胰蛋白酶消化细胞，离心，重悬细胞观察状态，用于后续实验。取上述培养好的细胞，加入LPS（1ug/ml）刺激293细胞产生IL-6。
实施例三
慢病毒sh-USP11转染293细胞。293细胞融合度为70%时，使用胰酶将细胞下滑下来，计数，调整细胞浓度。使用培养基重悬细胞，滴加慢病毒试剂。实验组为（sh-USP11），对照组为（sh-NC）。慢病毒用量：病毒体积=（MOI×细胞数）/病毒滴度。12h后更换为正常培养基，继续培养，观察细胞状态。感染72小时后，qRT-PCR检测感染效率用于后续实验。取上述培养好的细胞，加入LPS（1ug/ml）刺激293细胞产生IL-6。
观测LPS（1ug/ml）处理实施例二及实施例三细胞后IL-6表达水平的变化，在细胞水平验证USP11和IL-6的相关性。使用LPS刺激过表达USP11和对照组的293细胞，测IL-6的表达量。从图2可以看到USP11过表达成功，图3看到高表达USP11组IL-6的mRNA水平较正常对照组增高（P<0.05），同时图4看到ELSIA检测细胞上清液测高表达USP11组的IL-6水平明显高于对照组（P<0.05）。同样的，取293细胞进行慢病毒包装，设置USP11低表达组（sh-USP11）和对照组（sh-NC），从图5可以得出慢病毒下调USP11表达量模型建立成功，图6可以看到下调USP11后IL-6的mRNA表达水平随之下降（P<0.05）。
IL-6 可由多种细胞分泌，其负责协调T细胞扩增和分化，USP11作为一种重要的去泛素化酶，在免疫细胞的活化起着重要作用；脂多糖(LPS)诱导的细胞分泌IL-6等，本发明通过实验公开了USP11新的功能-保护IL6，避免过早降解。
Claims (3)
1.USP11在制备抑制细胞因子IL6 降解药物中的应用，其特征在于，所述药物使USP11过表达。
2.根据权利要求1所述的应用，其特征在于，药物为病毒载体药物或者质粒药物。
3.过表达USP11的试剂在制备抑制细胞因子IL6 降解药物中的应用，其特征在于，过表达USP11的试剂为病毒载体试剂或者质粒试剂。
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