CN116622848A - 长链非编码rnatug1的应用及产品 - Google Patents
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Abstract
本发明公开了一种长链非编码RNA TUG1的应用及产品,属于生物医药技术领域。其应用为用于制备或筛选诊断消化系统肿瘤的产品,或者为用于制备诊断消化系统肿瘤的检测标志物,或者为用于制备消化系统肿瘤预后的产品,或者为用于制备治疗消化系统肿瘤的药物,或者用于筛选治疗消化系统肿瘤的药物。其产品包括肿瘤诊断试剂盒、肿瘤预后评估试剂盒和消化系统肿瘤的免疫治疗药剂。本发明提出的TUG1在制备诊断、预后产品或药物产品方面的相关应用,为肿瘤免疫治疗提供新的免疫治疗靶点和策略,提高肿瘤免疫治疗效果。
Description
技术领域
本发明涉及长链非编码RNA TUG1的应用及产品,属于生物医药技术领域。
背景技术
消化系统肿瘤是我国主要的恶性肿瘤,发病率和死亡率大多居于各类肿瘤前列,严重危害着人民的生命健康。由于消化道肿瘤早期诊断困难,大多患者直到晚期才被确诊,常规手术和化疗效果较差且存在抗药性,5年生存率较低。因此,为了进一步提高消化系统肿瘤患者的临床治疗效果,迫切需要开发新的、更有效的治疗策略,探讨消化系统肿瘤发生发展机制与治疗靶点尤为重要。
近年来,免疫疗法发展迅速,以T细胞为核心的靶向免疫检查点的单克隆抗体疗法已经被批准应用于临床治疗,对消化系统肿瘤的治疗有明显改善,但仅能使部分患者收益。因此,进一步开发新的靶点和治疗策略以提高免疫细胞免疫效应,仍是目前基础研究和临床研究的焦点。近期研究表明,抗肿瘤髓系细胞的激活可提高肿瘤对免疫检查点阻断治疗的敏感性,故探索靶向巨噬细胞和T细胞增强抗肿瘤功能的新靶点对解决现有技术中免疫治疗受抑制的问题至关重要。
发明内容
本发明的目的在于克服现有技术的不足,提供一种长链非编码RNA TUG1的应用及产品,通过抑制肿瘤细胞中TUG1表达,提高CD8+ T细胞杀伤效应以及巨噬细胞吞噬功能,促进T细胞和巨噬细胞的免疫效应和抗肿瘤作用,以提高肿瘤免疫治疗效果,为肿瘤免疫治疗提供新的免疫治疗靶点和策略。
本发明的第一方面提供长链非编码RNA TUG1作为消化系统肿瘤免疫治疗靶点的应用,所述应用为用于制备或筛选诊断消化系统肿瘤的产品,或者为用于制备诊断消化系统肿瘤的检测标志物,或者为用于制备消化系统肿瘤预后的产品,或者为用于制备治疗消化系统肿瘤的药物,或者用于筛选治疗消化系统肿瘤的药物。
在本发明的一种实施方式中,所述药物为免疫治疗药物。
本发明的第二方面提供一种肿瘤诊断试剂盒,包括用于定量检测权利要求1所述的TUG1的试剂。
在本发明的一种实施方式中,肿瘤诊断试剂盒包括实时荧光定量检测TUG1的引物序列。
本发明的第三方面提供一种肿瘤预后评估试剂盒,包括用于定量检测权利要求1所述的TUG1的试剂。
在本发明的一种实施方式中,肿瘤预后评估试剂盒包括实时荧光定量检测TUG1的引物序列。
本发明的第四方面提供一种消化系统肿瘤的免疫治疗药剂,其特征在于:包括抑制权利要求1所述的TUG1表达的试剂。
本发明的第五方面提供一种长链非编码RNA TUG1的检测方法,其特征在于:采用权利要求3或4所述的肿瘤诊断试剂盒,或者采用权利要求5或6所述的肿瘤预后评估试剂盒。
本发明的有益效果是:
提供通过免疫检查点对免疫细胞效应、抗肿瘤活性具有调控作用的长链非编码RNA TUG1的应用及产品,为肿瘤免疫治疗提供新的策略和靶点,填补其在肿瘤免疫逃逸方面研究的空白,克服了现有技术存在的技术偏见。本发明发现了TUG1与患者生存预后的负相关性,肿瘤细胞中TUG1下调可抑制肿瘤生长,抑制肿瘤细胞的TUG1可促进肿瘤微环境中M1型巨噬细胞及CD8+ T细胞浸润增加,并促进CD8+ T细胞活化及分泌杀伤性细胞因子,TUG1与PD-L1和CD47的表达均呈正相关,依赖于PD-L1而直接影响CD8+ T细胞的杀伤功能,同时依赖于CD47而直接影响巨噬细胞的吞噬功能,通过抑制肿瘤组织的TUG1,改善其在肿瘤组织中的高表达状态,能够增强免疫细胞效应功能、抑制肿瘤生长。因此,TUG1能够作为肿瘤细胞靶点被调节以改善CD8+ T细胞的杀伤力以及巨噬细胞的吞噬功能,从而促进抗肿瘤作用。当其应用于制备肿瘤免疫治疗药物时,有助于显著提高CD8+ T细胞抗肿瘤活性,提高肿瘤免疫治疗效果,延长病人生存期。此外,对TUG1的抑制还能联合免疫检查点阻断剂抗PD-L1抗体,进一步提高免疫治疗效果。本发明为肿瘤免疫治疗提供新的治疗方案及靶点,有利于改善诊断、预后,提高肿瘤治疗效果,本发明揭示的TUG1对免疫检查点PD-L1以及CD47的调控作用,也为开发新的免疫治疗策略提供研究基础,有助于促进肿瘤免疫治疗领域的进步。
附图说明
图1为本发明实施例一中的分析结果,其中,图1A为TUG1在肝癌中表达上调的分析结果;图1B为TUG1在胰腺癌中表达上调的分析结果;图1C为TUG1与肝癌患者预后相关性分析结果;图1D为TUG1与胰腺癌患者预后相关性分析结果;图1E为肝癌组织中TUG1的表达与PD-L1相关性分析结果;图1F为肝癌组织中TUG1的表达与CD47相关性分析结果;图1G为胰腺癌组织中TUG1的表达与PD-L1相关性分析结果;图1H为胰腺癌组织中TUG1的表达与CD47相关性分析结果。
图2为本发明实施例二和实施例三的检测对比结果,其中,图2A为实施例二Vector/Hepa1-6(sh-NC)和sh-TUG1/Hepa1-6的TUG1的相对表达值对比;图2B为实施例二Vector/Panc02(sh-NC)和sh-TUG1/Panc02的TUG1的相对表达值对比;图2C为实施例三对照肝癌细胞系(sh-NC)及 TUG1 降表达 Hep1-6 细胞系(sh-TUG1)的肿瘤生长差异;图2D为对照肝癌细胞系(sh-NC)及 TUG1 降表达 Hep1-6 细胞系(sh-TUG1)的肿瘤体积数据对比;图2E为实施例三对照肝癌细胞系(sh-NC)及 TUG1 降表达 Hep1-6 细胞系(sh-TUG1)的肿瘤重量数据对比;图2F为实施例三对照胰腺癌细胞系(sh-NC)及 TUG1 降表达 Panc02细胞系(sh-TUG1)的肿瘤生长差异;图2G为对照胰腺癌细胞系(sh-NC)及 TUG1 降表达Panc02细胞系(sh-TUG1)的肿瘤体积数据对比;图2H为对照胰腺癌细胞系(sh-NC)及 TUG1 降表达Panc02细胞系(sh-TUG1)的肿瘤重量数据对比。
图3为本发明实施例四的实验结果,其中,图3A为敲降TUG1前后肝原位荷瘤小鼠脾脏中M1型巨噬细胞比例变化;图3B为敲降TUG1前后肝原位荷瘤小鼠肿瘤免疫微环境中M1型巨噬细胞比例变化;图3C为敲降TUG1前后肝原位荷瘤小鼠脾脏中CD8+T细胞比例变化;图3D为敲降TUG1前后肝原位荷瘤小鼠肿瘤免疫微环境中CD8+T细胞比例变化;图3E为敲降TUG1前后肝原位荷瘤小鼠脾脏中CD8+T细胞分泌IFN-γ能力变化;图3F为敲降TUG1前后肝原位荷瘤小鼠脾脏中CD8+T细胞分泌IL-2能力变化;图3G为敲降TUG1前后肝原位荷瘤小鼠脾脏中CD8+T细胞分泌TNF-α能力变化;图3H为敲降TUG1前后肝原位荷瘤小鼠肿瘤免疫微环境中CD8+T细胞分泌IFN-γ能力变化;图3I为敲降TUG1前后肝原位荷瘤小鼠肿瘤免疫微环境中CD8+T细胞分泌IL-2能力变化;图3J为敲降TUG1前后肝原位荷瘤小鼠肿瘤免疫微环境中CD8+T细胞分泌TNF-α能力变化;图3K为敲降TUG1前后胰腺原位荷瘤小鼠肿瘤免疫微环境中M1型巨噬细胞比例变化;图3L为敲降TUG1前后胰腺原位荷瘤小鼠肿瘤免疫微环境中CD8+T细胞比例变化;图3M为敲降TUG1前后胰腺原位荷瘤小鼠肿瘤免疫微环境中CD44比例变化;图3N为敲降TUG1前后胰腺原位荷瘤小鼠肿瘤免疫微环境中CD8+T细胞分泌TNF-α能力变化。
图4为本发明实施例五的检测结果,其中,图4A为肝癌细胞敲降TUG1前后CD8+ T细胞分泌 GzmB能力变化;图4B为肝癌细胞敲降TUG1前后CD8+ T 细胞分泌 GzmB数据变化;图4C为肝癌细胞敲降TUG1前后CD8+ T 细胞分泌 IFN-γ能力变化;图4D为肝癌细胞敲降TUG1前后CD8+ T 细胞分泌 IFN-γ数据变化;图4E为肝癌细胞敲降TUG1前后CD8+ T 细胞分泌 TNF-α能力变化;图4F为肝癌细胞敲降TUG1前后CD8+ T 细胞分泌 TNF-α数据变化。
图5为本发明实施例六检测结果,其中,图5A为与腹腔来源巨噬细胞共培养的TUG1降表达前后肝癌细胞组F4/80+GFP+细胞占 F4/80+巨噬细胞的比例变化;图5B为与骨髓来源巨噬细胞共培养的TUG1降表达前后肝癌细胞组F4/80+GFP+细胞占 F4/80+巨噬细胞的比例变化;图5C为与腹腔来源巨噬细胞共培养的TUG1降表达前后肝癌细胞组F4/80+GFP+细胞占 F4/80+巨噬细胞的数据变化;图5D为与骨髓来源巨噬细胞共培养的TUG1降表达前后肝癌细胞组F4/80+GFP+细胞占 F4/80+巨噬细胞的数据变化;图5E显示与腹腔来源巨噬细胞共培养的TUG1降表达前后肝癌细胞组巨噬细胞的吞噬能力;图5F显示与骨髓来源巨噬细胞共培养的TUG1降表达前后肝癌细胞组巨噬细胞的吞噬能力;图5G为与腹腔来源巨噬细胞共培养的TUG1降表达前后肝癌细胞组巨噬细胞的吞噬比例;图5H为与骨髓来源巨噬细胞共培养的TUG1降表达前后肝癌细胞组巨噬细胞的吞噬比例;图5I为与腹腔来源巨噬细胞共培养的TUG1降表达前后胰腺癌细胞组F4/80+GFP+细胞占 F4/80+巨噬细胞的比例变化;图5J为与腹腔来源巨噬细胞共培养的TUG1降表达前后胰腺癌细胞组F4/80+GFP+细胞占 F4/80+巨噬细胞的数据变化;图5K为与骨髓来源巨噬细胞共培养的TUG1降表达前后胰腺癌细胞组F4/80+GFP+细胞占 F4/80+巨噬细胞的比例变化;图5L为与骨髓来源巨噬细胞共培养的TUG1降表达前后胰腺癌细胞组F4/80+GFP+细胞占 F4/80+巨噬细胞的数据变化。
图6为本发明实施例七的检测结果,其中,图6A为肝癌细胞中TUG1降表达前后的比例变化;图6B为肝癌细胞中TUG1降表达前后PD-L1的表达情况;图6C为肝癌细胞中TUG1降表达前后CD47的表达情况;图6D为肝癌细胞中TUG1降表达前后的对PD-L1和CD47的表达影响对比;图6E为胰腺癌细胞中TUG1降表达前后的比例变化;图6F为胰腺癌细胞中TUG1降表达前后PD-L1的表达情况;图6G为胰腺癌细胞中TUG1降表达前后CD47的表达情况;图6H为胰腺癌细胞中TUG1降表达前后的对PD-L1和CD47的表达影响对比。
图7为本发明实施例八的联合治疗方案及结果,其中,图7A为联合治疗示意图;图7B为不同治疗方案下小鼠的肿瘤生长情况;图7C为不同治疗方案下小鼠的肿瘤体积数据对比;图7D为不同治疗方案下小鼠的肿瘤重量数据对比。
具体实施方式
下面将结合实施例,对本发明的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域技术人员在没有付出创造性劳动的前提下所获得的所有其他实施例,都属于本发明保护的范围。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
实施例一 TUG1在消化系统肿瘤中表达上调并与免疫检查点PD-L1及CD47表达呈正相关
1.TUG1在肝癌、胰腺癌中表达上调
通过GSE107170, GSE87630, GSE14323, GSE190967 和GSE159088共五个数据集分析TUG1在112例HBV相关肝癌组织、 91例HCV相关肝癌组织、112例非病毒性肝癌组织、76例非酒精性脂肪肝组织以及91例健康人正常肝组织中的表达情况。分析结果如图1A所示,TUG1在HBV相关肝癌组织(HBV-HCC)、HCV相关肝癌组织(HCV-HCC)以及非病毒性肝癌组织(Non-viral-HCC)中的均显著高于健康人正常肝组织(Normal)和非酒精性脂肪肝组织(NAFLD)。其中,TUG1在HBV相关肝癌组织表达最高。
通过GEPIA数据库分析TUG1在179例胰腺癌组织以及171例健康人正常胰腺组织中的表达情况。分析结果如图1B所示,TUG1在胰腺癌组织(左边)中的表达显著高于健康人正常胰腺组织(右边)。
2.TUG1与肝癌、胰腺癌患者预后的临床相关性
通过GEPIA数据库分析TUG1与肝癌患者临床预后的相关性,分析结果如图1C所示,结果显示肝癌患者组织中TUG1表达越高,患者预后越差(图中在下的线为high,在上的线为low)。
通过The Cancer Genome Altas Project (TCGA)中分析TUG1与胰腺癌患者临床预后的相关性,分析结果如图1D所示,结果也显示,胰腺癌中TUG1的高表达预示了病人较差的预后(图中在下的线为High groups,在上的线为Low groups)。
肝癌和胰腺癌中TUG1与免疫检查点PD-L1及CD47呈显著正相关
通过Tumor IMmune Estimation Resource(TIMER)在线数据库分析肝癌以及胰腺癌中TUG1的表达与免疫检查点PD-L1以及CD47的临床相关性。如图1E和图1F所示,结果显示肝癌组织中,TUG1的表达与PD-L1以及CD47呈显著正相关。如图1G和图1H所示,结果显示胰腺癌组织中,TUG1的表达与PD-L1以及CD47呈显著正相关。
由上述分析结果可见,消化系统肿瘤组织中TUG1的表达量不仅可以作为辅助诊断的指标,还可以作为独立的预后指标,肿瘤组织TUG1的高表达表明病人较差的预后。TUG1与免疫检查点PD-L1以及CD47的密切相关,表明TUG1可能通过调节PD-L1以及CD47调控肿瘤免疫逃逸,在肿瘤免疫治疗中发挥关键作用,为肿瘤免疫治疗提供新思路。
实施例二 构建TUG1稳定降表达细胞株
1.构建TUG1的降表达质粒
针对TUG1靶序列(5’-CCATCTCACAAGGCTTCAA-3’)合成片段,并将合成的包含TUG1靶序列的片段构建到LV3(H1/GFP&Puro)表达载体中,获得TUG1干扰质粒sh-TUG1。
2.转染肝癌Hepal-6细胞系以及胰腺癌Panc02细胞系
利用PEI将上述干扰质粒sh-TUG1和空质粒LV3(H1/GFP&Puro)Vector分别转入HEK293T细胞中,并于6小时后更换含有10%的胎牛血清的DMEM培养基(Gibco)。48小时及72小时后收集慢病毒上清,并感染Hepal-6细胞及Panc02细胞。用嘌呤毒素(8μg/ml; Sigma-aldrich)筛选10天得到TUG1稳定降表达细胞sh-TUG1/Hepa1-6以及sh-TUG1/Panc02,对照细胞分别为Vector/Hepa1-6以及Vector/Panc02。
3.qRT-PCR检测TUG1稳转株降表达情况
应用qRT-PCR检测Vector/Hepa1-6细胞、sh-TUG1/Hepa1-6细胞、Vector/Panc02细胞以及sh-TUG1/Panc02细胞中TUG1的表达情况。如图2A所示,Vector/Hepa1-6(sh-NC)的TUG1的表达值为1,sh-TUG1/Hepa1-6的TUG1的相对表达值显著低于Vector/Hepa1-6的表达值,证明构建了TUG1稳定低表达的Hepa1-6细胞。如图2B所示,以Vector/Panc02(sh-NC)的TUG1的表达值为1,sh-TUG1/Panc02的TUG1的相对表达值显著低于Vector/Panc02的表达值,证明构建了TUG1稳定低表达的Panc02细胞。
实施例三 TUG1降表达肿瘤细胞肝原位以及胰腺原位荷瘤小鼠肿瘤生长受到明显抑制
1.肝癌原位模型建立
通过慢病毒包装体系构建对照肝癌细胞系(sh-NC)及 TUG1 降表达 Hep1-6 细胞系(sh-TUG1),分别将 100μL 2×107/mL 的上述细胞注射至 6-8 周龄野生型 C57BL/6 小鼠背部皮下,出瘤 10 天后取下肿瘤,切成同样大小的瘤块,移植入6-8 周龄野生型小鼠肝内,构建肝癌原位成瘤模型,4 周后处死小鼠,取肿瘤,比较肝原位肿瘤生长差异。结果如图2C和图2D所示,敲降TUG1的Hep1-6细胞肝原位荷瘤小鼠肿瘤生长受到明显抑制,如图2E所示,sh-TUG1的肿瘤重量明显减轻,表明TUG1在肝癌的生长及免疫逃逸中具有关键作用。
2.胰腺癌原位模型建立
通过慢病毒体系构建小鼠胰腺癌sh-NC 和 sh-TUG1 Panc02 细胞系,通过胰腺原位注射 50μL 2×106个细胞至 C57BL/6小鼠体内,构建胰腺癌原位成瘤模型,3 周后处死小鼠,比较肿瘤生长差异。比较结果如图2F和图2G所示,敲降TUG1的Panc02 细胞胰腺原位荷瘤小鼠肿瘤生长受到明显抑制,如图2H所示,sh-TUG1的肿瘤重量明显减轻,表明TUG1在胰腺癌的生长及免疫逃逸中具有关键作用。
实施例四 TUG1在肝癌和胰腺癌免疫逃逸中的功能
1.流式细胞术检测TUG1对肝癌免疫微环境中关键免疫细胞比例及功能影响
在实施例三中的肝癌原位成瘤模型构建 4 周后处死小鼠,取脾脏,将脾脏研磨离心后得到脾细胞悬液,裂解红细胞;同时取肿瘤,并使用胶原酶消化肿瘤组织,研磨离心后使用淋巴细胞分离液分离肿瘤浸润单个核细胞,通过流式分析肝原位荷瘤小鼠外周免疫器官脾脏及免疫抑制性肿瘤微环境中M1(CD45+CD11b+F4/80+MHC-II+)以及CD8+T细胞(CD45+CD3+CD8+)等免疫细胞的比例变化,研究降表达TUG1对各种免疫细胞及其亚群含量变化的影响。分离肿瘤组织中的单个核细胞,检测 TUG1 对 CD8+ T 细胞分泌 IFN-γ、TNF-α、IL-2等细胞因子能力的影响。
由图3A至图3D可见,敲降TUG1的Hep1-6细胞肝原位荷瘤小鼠脾脏以及肿瘤免疫微环境中M1型巨噬细胞、CD8+T细胞的浸润明显增加。如图3E至图3J所示,荷瘤小鼠脾脏以及肿瘤中CD8+T细胞分泌肿瘤杀伤相关细胞因子IFN-γ、TNF-α、IL-2的能力增强。表明敲降TUG1的肝癌细胞原位荷瘤小鼠抗肿瘤免疫作用显著增强。
2.流式细胞术检测TUG1对胰腺癌免疫微环境中关键免疫细胞比例及功能影响
通过慢病毒体系构建小鼠胰腺癌sh-NC和sh-TUG1 Panc02,通过胰腺原位注射50μL 2×106个细胞至 C57BL/6小鼠体内,构建胰腺癌原位成瘤模型,3周后处死小鼠,比较肿瘤生长差异。使用胶原酶消化肿瘤组织,分离肿瘤浸润单个核细胞,通过流式分析肿瘤微环境中M1(CD45+CD11b+F4/80+MHC-II+)以及CD8+T细胞(CD45+CD3+CD8+)等免疫细胞的比例变化。分离肿瘤组织中的单个核细胞,检测TUG1对CD8+T细胞分泌细胞因子能力的影响。
如图3K和图3L所示,敲降TUG1的Panc02细胞胰腺原位荷瘤小鼠肿瘤免疫微环境中M1型巨噬细胞以及CD8+T细胞的浸润明显增加。如图3M和图3N所示,CD8+T细胞的活化水平以及分泌肿瘤杀伤相关细胞因子TNF-α的能力增强。表明敲降TUG1的胰腺癌细胞原位荷瘤小鼠抗肿瘤免疫作用显著增强。
实施例五 干扰肿瘤细胞中TUG1可促进CD8+T细胞的杀伤功能
1.应用磁珠分选CD8+T细胞
用外科方法无菌摘取小鼠脾脏及腹股沟、肠系膜淋巴结,剪去结缔组织及脂肪,置于盛有预冷的1×PBS的15ml离心管中。将脾脏及淋巴结置于滤网中,快速研磨,收集脾细胞悬液,裂红,离心结束后,将上清完全弃掉,根据细胞数量加入一定体积的缓冲液,加入CD8a磁珠(美天旎),4℃冰箱中孵育15分钟。用缓冲液调整细胞浓度,随后进行磁柱分离。分离结束后将收集的细胞离心,细胞计数。取少量细胞做CD8抗体标记,应用流式细胞术检测分选细胞的纯度。
2.干扰肿瘤细胞中TUG1对共培养体系中CD8+T细胞功能的影响
使用磁珠从C57BL/6小鼠脾脏中分选得到CD8+ T 细胞,将 2×105个 CD8+ T 细胞分别与 2×104 个 sh-NC 或sh-TUG1 肝癌细胞系共培养(10:1),使用 anti-CD3(5μg/mL)和 anti-CD28(2μg/mL)激活 T 细胞,48h 后收集细胞进行染色,使用流式细胞仪检测干扰肝癌细胞中TUG1对CD8+ T 细胞分泌 GzmB、TNF-α、IFN-γ等细胞因子能力的影响。
如图4A至4F所示,敲降肝癌细胞中的TUG1 可显著促进 CD8+T 细胞分泌肿瘤杀伤相关细胞因子GzmB(图 4A和图4B)、IFN-γ(图4C和图4D)以及 TNF-α(图4E和图4F),从而增强 CD8+ T 细胞的抗肿瘤活性。
实施例6 干扰肿瘤细胞中TUG1可促进巨噬细胞的吞噬功能
1.腹腔来源巨噬细胞诱导方案
通过向小鼠腹腔注射 2 mL 的 3 % 硫乙醇酸盐(TG)溶液诱导小鼠腹腔产生巨噬细胞,3 天后脱颈处死麻醉后的C57BL/6 小鼠,用 75%酒精对小鼠腹部进行消毒,用消毒过的剪刀剪开皮肤,不剪开腹膜,用注射器吸取 5 mL RPMI-1640 无血清培养基,经腹膜注入腹腔中,轻揉小鼠腹部,使培养基充分灌入腹腔各部位,通过收集腹腔冲洗液并离心得到巨噬细胞。
2.骨髓来源巨噬细胞诱导方案
脱颈处死麻醉后的 C57BL/6小鼠,用消毒过的剪刀剪开皮肤,剥离肌肉组织,暴露股骨及胫骨,用剪刀沿关节剪开骨两端,将针尖插入骨髓腔中使用无血清1640培养基冲洗得到全骨髓细胞,使用终浓度为 10 ng/mL 的 M-CSF 将其诱导分化为骨髓来源巨噬细胞(BMDMs)。
3.腹腔或骨髓来源巨噬细胞与肿瘤细胞共培养,检测干扰肿瘤细胞中TUG1对巨噬细胞吞噬功能的影响
将分化好的腹腔或骨髓来源巨噬细胞分别与sh-NC 或 sh-TUG1 降表达小鼠肝癌或胰腺癌细胞共培养4h,通过流式检测巨噬细胞吞噬能力的变化;由于肿瘤细胞表达绿色荧光蛋白 GFP,因此,F4/80+GFP+细胞占 F4/80+巨噬细胞的比例即为吞噬比例。此外,将 5×104个腹腔或骨髓来源巨噬细胞与 2×104个 sh-NC 或 sh-TUG1 降表达小鼠肿瘤细胞在 37℃孵箱中共培养 4h,将肿瘤细胞与巨噬细胞共培养的玻片用 4%多聚甲醛在室温下固定,经 5% BSA 封闭,孵育 F4/80 抗体,4℃过夜,PBS 清洗后室温避光孵育荧光二抗,DAPI 封片,使用激光共聚焦显微镜观察并分析巨噬细胞吞噬能力的差异,按每 100 个巨噬细胞吞噬 GFP+肿瘤细胞数计算吞噬比例,研究降表达 TUG1 对巨噬细胞吞噬功能的影响。
流式结果显示,如图5A和图5C所示,与腹腔来源巨噬细胞共培养的TUG1降表达肝癌细胞组F4/80+GFP+细胞占 F4/80+巨噬细胞的比例显著增加,如图5B和图5D所示,与骨髓来源巨噬细胞共培养的TUG1降表达肝癌细胞组巨噬细胞吞噬比例也显著增加。同时,免疫荧光结果显示,如图5E和图5G所示,与腹腔来源巨噬细胞共培养的TUG1降表达肝癌细胞组巨噬细胞的吞噬能力显著增强,如图5F和图5H所示,与骨髓来源巨噬细胞共培养的TUG1降表达肝癌细胞组巨噬细胞吞噬比例也显著增加,表明抑制肝癌细胞中的TUG1可显著促进巨噬细胞对肿瘤细胞进行吞噬。此外,流式结果还显示,如图5I和图5J所示,与腹腔来源巨噬细胞共培养的TUG1降表达胰腺癌细胞组F4/80+GFP+细胞占 F4/80+巨噬细胞的比例显著增加。如图5K和图5L所示,与骨髓来源巨噬细胞共培养的TUG1降表达胰腺癌细胞组巨噬细胞吞噬比例也显著增加,表明抑制胰腺癌细胞中的TUG1可显著促进巨噬细胞对肿瘤细胞进行吞噬。
实施例七 敲降肝癌、胰腺癌细胞中TUG1可抑制免疫检查点PD-L1和CD47表达
应用qRT-PCR技术以及蛋白免疫印迹技术检测敲降肝癌、胰腺癌细胞中TUG1对免疫检查点PD-L1和CD47表达的影响。结果显示,如图6A至图6D所示,降表达肝癌细胞中的TUG1可在mRNA和蛋白水平均显著抑制PD-L1和CD47表达。如图6E至图6H所示,降表达胰腺癌细胞中的TUG1可在mRNA和蛋白水平均显著抑制PD-L1和CD47表达。
实施例八 TUG1抑制剂治疗可增强PD-L1阻断抗体的疗效
肝癌原位成瘤联合治疗方案:应用Hepa1-6细胞建立肝癌原位模型,随后将荷瘤小鼠按给药方案随机分为4组:IgG 联合NC-siRNAs组, anti-PD-L1 联合NC-siRNAs组, IgG联合TUG1 siRNAs组, anti-PD-L1 联合TUG1-siRNAs组。从第12天开始,通过尾静脉输送TUG1的siRNAs,10 nmol/只;通过腹腔注射抗PD-L1抗体(克隆号10F.9G2, BioXcell)或IgG同型对照抗体(克隆号;MPC-11, BioXcell),200 μg/d,每4 天一次,联合治疗示意图如图7A所示。所有小鼠在治疗结束后进行安乐死。
实验结果显示,如图7B至7D所示,单独使用抗PD-L1抗体和TUG1-siRNAs治疗均能部分抑制肿瘤生长,将二者联合用于治疗肿瘤显示出较好的协同作用,证实TUG1 siRNAs治疗可提高PD-L1抗体免疫治疗的效果,提示TUG1s tigao PD-L1免疫检查点阻断抗体在抗癌免疫治疗中疗效的潜在治疗靶点。
以上所述仅是本发明的优选实施方式,应当理解本发明并非局限于本文所披露的形式,不应看作是对其他实施例的排除,而可用于各种其他组合、修改和环境,并能够在本文所述构想范围内,通过上述教导或相关领域的技术或知识进行改动。而本领域人员所进行的改动和变化不脱离本发明的精神和范围,则都应在本发明所附权利要求的保护范围内。
Claims (8)
1.长链非编码RNA TUG1作为消化系统肿瘤免疫治疗靶点的应用,其特征在于:所述应用为用于制备或筛选诊断消化系统肿瘤的产品,或者为用于制备诊断消化系统肿瘤的检测标志物,或者为用于制备消化系统肿瘤预后的产品,或者为用于制备治疗消化系统肿瘤的药物,或者用于筛选治疗消化系统肿瘤的药物。
2.根据权利要求1所述的应用,其特征在于:所述药物为免疫治疗药物。
3.一种肿瘤诊断试剂盒,其特征在于:包括用于定量检测权利要求1所述的TUG1的试剂。
4.根据权利要求2所述的肿瘤诊断试剂盒,其特征在于:包括实时荧光定量检测TUG1的引物序列。
5.一种肿瘤预后评估试剂盒,其特征在于:包括用于定量检测权利要求1所述的TUG1的试剂。
6.根据权利要求5所述的肿瘤预后评估试剂盒,其特征在于:包括实时荧光定量检测TUG1的引物序列。
7.一种消化系统肿瘤的免疫治疗药剂,其特征在于:包括抑制权利要求1所述的TUG1表达的试剂。
8.一种长链非编码RNA TUG1的检测方法,其特征在于:采用权利要求3或4所述的肿瘤诊断试剂盒,或者采用权利要求5或6所述的肿瘤预后评估试剂盒。
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Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20160160295A1 (en) * | 2014-12-08 | 2016-06-09 | The Regents Of The University Of Michigan | Non-coding rnas and uses thereof |
CN107320726A (zh) * | 2017-07-11 | 2017-11-07 | 山东大学 | LncRNA‑TUG1在制备调控牙周膜干细胞的干性维持能力的药物中的应用 |
CN107488740A (zh) * | 2017-10-24 | 2017-12-19 | 南阳师范学院 | 检测胃癌预后情况的LncRNA组合及含有该组合的试剂盒 |
CN107619865A (zh) * | 2017-08-02 | 2018-01-23 | 南方医科大学珠江医院 | 一种预测小细胞肺癌患者对化疗药物敏感性的靶点及应用 |
CN108342487A (zh) * | 2018-05-10 | 2018-07-31 | 山东大学第二医院 | 基于血清exosomallncRNAs的食管癌诊断特异性表达图谱及检测分析系统 |
JP2020007260A (ja) * | 2018-07-06 | 2020-01-16 | 北海道公立大学法人 札幌医科大学 | 長鎖非コードrnaを標的とするがん治療剤およびがん診断・予後の予測方法 |
WO2021181221A1 (en) * | 2020-03-11 | 2021-09-16 | Cellido Therapeutics B.V. | Small molecules for regulation of long non-coding rnas |
-
2023
- 2023-05-24 CN CN202310592817.1A patent/CN116622848A/zh active Pending
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20160160295A1 (en) * | 2014-12-08 | 2016-06-09 | The Regents Of The University Of Michigan | Non-coding rnas and uses thereof |
CN107320726A (zh) * | 2017-07-11 | 2017-11-07 | 山东大学 | LncRNA‑TUG1在制备调控牙周膜干细胞的干性维持能力的药物中的应用 |
CN107619865A (zh) * | 2017-08-02 | 2018-01-23 | 南方医科大学珠江医院 | 一种预测小细胞肺癌患者对化疗药物敏感性的靶点及应用 |
CN107488740A (zh) * | 2017-10-24 | 2017-12-19 | 南阳师范学院 | 检测胃癌预后情况的LncRNA组合及含有该组合的试剂盒 |
CN108342487A (zh) * | 2018-05-10 | 2018-07-31 | 山东大学第二医院 | 基于血清exosomallncRNAs的食管癌诊断特异性表达图谱及检测分析系统 |
JP2020007260A (ja) * | 2018-07-06 | 2020-01-16 | 北海道公立大学法人 札幌医科大学 | 長鎖非コードrnaを標的とするがん治療剤およびがん診断・予後の予測方法 |
WO2021181221A1 (en) * | 2020-03-11 | 2021-09-16 | Cellido Therapeutics B.V. | Small molecules for regulation of long non-coding rnas |
Non-Patent Citations (11)
Title |
---|
HUANG MIND-DE等: "Long non-coding RNA TUG1 is up-regulated in hepatocellular carcinoma and promotes cell growth and apoptosis by epigenetically silencing of KLF2", 《MOLECULAR CANCER》, vol. 14, pages 1 - 12, XP055384143, DOI: 10.1186/s12943-015-0431-0 * |
LI LEI等: "The identification and preliminary study of lncRNA TUG1 and its related genes in hepatocellular carcinoma", 《ARCHIVES OF MEDICAL SCIENCE》, vol. 18, no. 06, pages 1582 - 1595 * |
LIANG ZHAO等: "The Lncrna-TUG1/EZH2 Axis Promotes Pancreatic Cancer Cell Proliferation, Migration and EMT Phenotype Formation Through Sponging Mir-382", 《CELLULAR PHYSIOLOGY AND BIOCHEMISTRY》, vol. 42, no. 06, pages 2145 - 2158 * |
QUN DAI等: "Long non-coding RNA TUG1 promotes cell progression in hepatocellular carcinoma via regulating miR-216b-5p/DLX2 axis", 《CANCER CELL INTERNATIONAL》, vol. 20, pages 1 - 13 * |
YEBIN LU等: "Long Noncoding RNA TUG1/miR-29c Axis Affects Cell Proliferation, Invasion, and Migration in Human Pancreatic Cancer", 《DISEASE MARKERS》, pages 1 - 10 * |
刘维维等: "LncRNA TUG1在肝癌中的研究进展", 《生命科学》, vol. 33, no. 09, pages 1143 - 1152 * |
杨光泽: "LncRNA TUG1调控肝癌生长及肿瘤免疫的作用机制研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技》, no. 2021, pages 15 * |
王伟等: "LncRNA TUG1靶向miR-138-5p调控胰腺癌细胞增殖、迁移和侵袭", 《现代肿瘤医学》, vol. 28, no. 23, pages 4051 - 4056 * |
白永恒等主编: "《肿瘤微环境与免疫耐受》", vol. 2020, 30 June 2020, 天津科学技术出版社, pages: 228 * |
邵旭辉等: "长链非编码RNA牛磺酸上调基因在肝癌组织中表达及对肝癌细胞活性的影响", 《北华大学学报(自然科学版)》, vol. 22, no. 06, pages 748 - 752 * |
陈竺等主编: "《肝癌:基础与临床转化》", vol. 2020, 31 December 2020, 上海交通大学出版社, pages: 100 - 101 * |
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