CN112451662A - 一种gli蛋白抑制剂及其应用 - Google Patents
一种gli蛋白抑制剂及其应用 Download PDFInfo
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Abstract
本发明公开了一种GLI蛋白抑制剂及其应用，该GLI蛋白抑制剂是由GLI1/3抗体蛋白制成的抑制剂。该GLI蛋白抑制剂及其应用，明确GLI1/3在肝细胞癌中对CD90+细胞的促进作用和肿瘤微环境中癌症的启动作用，提出并阐明IL‑6以全新的作用方式调节肝细胞癌微环境中JAK/STAT3通路的观点，以及评价肿瘤微环境中CD90细胞作为肿瘤进程标志及治疗靶点的理论基础，从而明确GLI1/3对肿瘤中CD90干细胞样细胞诱导，且CD90在肿瘤微环境中的作用及能通过与IL‑6的互作实现。
Description
技术领域
本发明涉及生物工程技术领域，具体为一种GLI蛋白抑制剂及其应用。
背景技术
肝癌是异质性强、死亡率高的难治性恶性肿瘤。原发性肝癌包括肝细胞癌(HCC)、胆管细胞肝癌和混合型肝癌，HCC占比85～90％。肝癌成因主要有病毒感染和代谢性疾病对肝脏的长期损伤，乙肝病毒(HBV)和丙肝病毒(HCV)导致慢性肝脏炎症的持续进展和恶性转化，可诱发肝癌；近年来，包括非酒精性代谢性肝病在内的代谢类肝病导致的慢性肝炎逐年增加，成为导致慢性肝炎的另一个重要原因，HBV慢性持续感染是我国肝细胞癌最重要的危险因素。我国肝炎患者数量庞大，且感染多发生在早期，由于免疫机制的不健全，容易发展为慢性肝炎，导致肝炎病毒持续复制，发展为肝癌，幼儿感染HBV后发展为慢性肝炎的比例是20～30％。我国肝癌的病死率位居肿瘤第二位，仅次于肺癌。
目前肝癌主要以甲胎蛋白(AFP)，GPC-3/MXR7，DCP，GP-73以及胰岛素样生长因子(IGF)作为诊断指标，并结合影像学检测以确定，但由于肝癌早期发病分子机理的不明确，早期症状的不明显，确诊时多处在肝癌晚期，这对于肝癌的治疗造成很大困难，所以，了解肝癌的发病机制特别是早期过程的分子机制显得格外重要。
对于肝癌发展分子机理的研究，基因突变和分子通路的失调成为关注的重点。一些癌症相关基因如N-ras、c-fms、p53、c-myc、IGF-2、IGF-2R、p16、p21、DCC、TGFα等的突变，被认为是肝癌发生的分子基础。多基因的突变以及相关分子通路诸如IL-6/STAT3，COX-2/PGE2/VEGF(EGFR)，Wnt/β-catenin信号通路紊乱造成肿瘤细胞不受控制的生长、分化、迁移及侵入正常组织，是肝癌发展的重要分子基础。
IL-6/STAT3信号通路在肝细胞癌中能够刺激DNA合成和肿瘤细胞复制增殖，促进肿瘤环境中的血管发生，是非常重要的肿瘤信号通路，也是长期关注的肿瘤治疗潜在靶点通路。
现有的GLI蛋白抑制剂中，不明确GLI1/3对肿瘤中CD90干细胞样细胞是否诱导，且CD90在肿瘤微环境中的作用及是否能通过与IL-6的互作实现，以及GLI1/3对IL-6和CD90的表达促进是否提供肿瘤细胞微环境的适应条件和肿瘤增殖、转移的基础。
本发明明确GLI1/3在肝细胞癌中对CD90+细胞的促进作用和肿瘤微环境中癌症的启动作用，提出并阐明IL-6以全新的作用方式调节肝细胞癌微环境中JAK/STAT3通路的观点，以及评价肿瘤微环境中CD90细胞作为肿瘤进程标志及治疗靶点的理论基础。
发明内容
(一)解决的技术问题
针对现有技术的不足，本发明提供了一种GLI蛋白抑制剂及其应用，解决了现有的GLI蛋白抑制剂中，不明确GLI1/3对肿瘤中CD90干细胞样细胞是否诱导，且CD90在肿瘤微环境中的作用及是否能通过与IL-6的互作实现，以及GLI1/3对IL-6和CD90的表达促进是否提供肿瘤细胞微环境的适应条件和肿瘤增殖、转移基础的问题。
(二)技术方案
为实现以上目的，本发明通过以下技术方案予以实现：一种GLI蛋白抑制剂，该GLI蛋白抑制剂是由GLI1/3抗体蛋白制成的抑制剂。
本发明还公开了一种GLI蛋白抑制剂在治疗肝癌中的应用，通过使用所述GLI蛋白抑制剂，使GLI1/3下调抑制CD90的转录，CD90+细胞分泌IL-6低，在IL-6R或sIL-6R的相对缺乏、快速生长的肿瘤细胞中，CD90与IL-6的相互作用被抑制，不能激活JAK/STAT3下游通路，抑制肿瘤细胞的生长。
优选的，所述肝细胞癌中GLI1/3对CD90+细胞亚群的数量及肿瘤微环境中IL-6的表达的应用，具体包括以下步骤：
S1、将在前期肿瘤样本的基础上，扩大现有样本组织数量，并严格纳入标准，检测GLI1/3和CD90的mRNA表达，同时检测IL-6和IL-6R的mRNA表达；
S2、根据步骤S1中临床验证的结果，选择两个肝细胞癌细胞系干预GLI1/3的表达，检测CD90 mRNA的表达(已在一个细胞系中做验证)，检测CD90阳性细胞的数量变化和IL-6和IL6R的表达差异；
S3、拟比较CD90+和CD90-肿瘤细胞中IL-6的表达差异，并联合GLI1/3的配体SHH对细胞进行干预，分析IL-6与IL-6R的表达及IL-6的信号转导方式；
S4、拟通过干预GLI1/3的表达，并结合SHH配体处理细胞，在mRNA及蛋白表达等多个水平检测CD90、IL-6及IL-6R下游通路分子的表达，分析GLI1/3对CD90、IL-6的转录激活及对IL-6/STAT3信号通路影响的结果。
优选的，所述步骤S1中，使用统计学方法分析GLI/3对CD90和IL-6的表达的影响，以及这三个分子在肿瘤组织和癌旁组织的表达差异，提供相对充实的临床病例验证数据。
优选的，所述步骤S2中，对IL-6R的定位进行验证和讨论，分析IL-6激活下游分子的分子通路。
优选的，所述GLI1/3调节的CD90+细胞亚群通过IL-6促进肿瘤进程的具体步骤如下：
T1、在对肝癌组织样本分子表达检测的基础上，检测CD90+肿瘤细胞系IL-6的表达，讨论CD90与IL-6在肿瘤细胞中表达的相关性；
T2、干预肝癌细胞GLI1/3的表达，考查CD90+细胞数量，及与细胞增殖、迁移、侵袭和细胞周期等的关系，考查IL-6的分泌及下游通路的激活情况；
T3、构建慢病毒过表达GLI1/3肝细胞癌细胞，结合裸鼠体内置瘤的实验手段，并同时通过干预CD90或IL-6及JAK2/STAT3抑制剂的使用证明CD90+细胞中GLI1/3-IL6-STAT3通路的完整性，并考查肿瘤在体内的生长状况，讨论该通路与肿瘤进展的相关性。
优选的，所述步骤T2中，结合内源性抗体消耗IL-6表达的方式，考查对细胞功能和IL-6下游信号的影响。
优选的，所述CD90和IL-6的相互作用的应用，具体步骤如下：
E1、利用生物信息学完善CD90和IL-6相互作用的预测数据，并结合组织原位免疫显色(IHC)的检验方法定位CD90和IL-6的表达；
E2、拟使用免疫共沉淀(CoIP)分别调取CD90和IL-6，检验两分子在物理层面的结合；
E3、干预GLI1/3表达与重组融合蛋白sgp130Fc处理相结合，并拟使用免疫共沉淀(CoIP)分别调取CD90和IL-6，检验两分子在物理层面的结合，并判断CD90+细胞中IL-6的主要结合蛋白，同时监测JAK/STAT3通路激活情况；
E4、敲除癌细胞IL-6R的表达，并结合GLI1/3干预等手段，验证CD90和IL-6的互作及下游通路JAK/STAT3的激活情况。
优选的，所述步骤E3中，在干预GLI1/3表达与重组融合蛋白sgp130Fc处理相结合，能够选择性抑制IL-6/sIL-6R的互作方式。
优选的，所述IL-6/CD90相互作用对肝细胞癌进程的应用，是通过体内置瘤，细胞内IL-6R敲除及慢病毒过表达GLI1/3的实验手段相结合，分别分析GLI/IL-6/CD90信号通路微环境对肝细胞癌生长、转移的影响，对罹患肝细胞癌预后的评价，以及对CD90作为肝细胞癌治疗靶点的可行性做评估。
(三)有益效果
本发明提供了一种GLI蛋白抑制剂及其应用。与现有技术相比具备以下有益效果：该GLI蛋白抑制剂及其应用，明确GLI1/3在肝细胞癌中对CD90+细胞的促进作用和肿瘤微环境中癌症的启动作用，提出并阐明IL-6以全新的作用方式调节肝细胞癌微环境中JAK/STAT3通路的观点，以及评价肿瘤微环境中CD90细胞作为肿瘤进程标志及治疗靶点的理论基础，GLI蛋白抑制剂使GLI1/3下调抑制CD90的转录，CD90+细胞分泌IL-6低，在IL-6R或sIL-6R的相对缺乏、快速生长的肿瘤细胞中，CD90与IL-6的相互作用被抑制，不能激活JAK/STAT3下游通路，抑制肿瘤细胞的生长，从而明确GLI1/3对肿瘤中CD90干细胞样细胞诱导，且CD90在肿瘤微环境中的作用及能通过与IL-6的互作实现，以及GLI1/3对IL-6和CD90的表达促进提供肿瘤细胞微环境的适应条件和肿瘤增殖、转移的基础。
附图说明
图1为本发明的流程图；
图2为本发明GLI1和GLI3 mRNA在肝细胞癌组织中的表达示意图；
图3为本发明IL-6及CD90 mRNA在肝细胞癌组织中的表达及相关性示意图；
图4为本发明不同肝癌细胞系中GLI1/3、CD90和IL-6mRNA的表达示意图；
图5为本发明细胞分选后CD90+/CD90-肿瘤细胞GLI1/3mRNA的表达示意图；
图6为本发明细胞分选后CD90+/CD90-肿瘤细胞IL-6mRNA的表达示意图；
图7为本发明干涉GLI1/3后IL-6和CD90等干细胞因子mRNA的表达示意图；
图8为本发明干涉GLI1/3后肿瘤细胞增殖、迁移能力的变化示意图；
图9为本发明SHH处理激活Huh7和97L细胞GLI1/3后干细胞因子及IL-6/IL-6RmRNA的表达示意图。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中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，本领域普通技术人员在没有做出创造性劳动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。
请参阅图1-9，本发明实施例提供一种技术方案：一种GLI蛋白抑制剂，该GLI蛋白抑制剂是由GLI1/3抗体蛋白制成的抑制剂。
本发明实施例还公开了一种GLI蛋白抑制剂在治疗肝癌中的应用，通过使用GLI蛋白抑制剂，使GLI1/3下调抑制CD90的转录，CD90+细胞分泌IL-6低，在IL-6R或sIL-6R的相对缺乏、快速生长的肿瘤细胞中，CD90与IL-6的相互作用被抑制，不能激活JAK/STAT3下游通路，抑制肿瘤细胞的生长。
本发明实施例中，肝细胞癌中GLI1/3对CD90+细胞亚群的数量及肿瘤微环境中IL-6的表达的应用，具体包括以下步骤：
S1、将在前期肿瘤样本的基础上，扩大现有样本组织数量，并严格纳入标准，检测GLI1/3和CD90的mRNA表达，同时检测IL-6和IL-6R的mRNA表达，使用统计学方法分析GLI/3对CD90和IL-6的表达的影响，以及这三个分子在肿瘤组织和癌旁组织的表达差异，提供相对充实的临床病例验证数据；
S2、根据步骤S1中临床验证的结果，选择两个肝细胞癌细胞系干预GLI1/3的表达，检测CD90 mRNA的表达(已在一个细胞系中做验证)，检测CD90阳性细胞的数量变化和IL-6和IL6R的表达差异，对IL-6R的定位进行验证和讨论，分析IL-6激活下游分子的分子通路；
S3、拟比较CD90+和CD90-肿瘤细胞中IL-6的表达差异，并联合GLI1/3的配体SHH对细胞进行干预，分析IL-6与IL-6R的表达及IL-6的信号转导方式；
S4、拟通过干预GLI1/3的表达，并结合SHH配体处理细胞，在mRNA及蛋白表达等多个水平检测CD90、IL-6及IL-6R下游通路分子的表达，分析GLI1/3对CD90、IL-6的转录激活及对IL-6/STAT3信号通路影响的结果。
本发明实施例中，GLI1/3调节的CD90+细胞亚群通过IL-6促进肿瘤进程的具体步骤如下：
T1、在对肝癌组织样本分子表达检测的基础上，检测CD90+肿瘤细胞系IL-6的表达，讨论CD90与IL-6在肿瘤细胞中表达的相关性；
T2、干预肝癌细胞GLI1/3的表达，考查CD90+细胞数量，及与细胞增殖、迁移、侵袭和细胞周期等的关系，考查IL-6的分泌及下游通路的激活情况，结合内源性抗体消耗IL-6表达的方式，考查对细胞功能和IL-6下游信号的影响；
T3、构建慢病毒过表达GLI1/3肝细胞癌细胞，结合裸鼠体内置瘤的实验手段，并同时通过干预CD90或IL-6及JAK2/STAT3抑制剂的使用证明CD90+细胞中GLI1/3-IL6-STAT3通路的完整性，并考查肿瘤在体内的生长状况，讨论该通路与肿瘤进展的相关性。
本发明实施例中，CD90和IL-6的相互作用的应用，具体步骤如下：
E1、利用生物信息学完善CD90和IL-6相互作用的预测数据，并结合组织原位免疫显色(IHC)的检验方法定位CD90和IL-6的表达；
E2、拟使用免疫共沉淀(CoIP)分别调取CD90和IL-6，检验两分子在物理层面的结合；
E3、干预GLI1/3表达与重组融合蛋白sgp130Fc处理相结合，并拟使用免疫共沉淀(CoIP)分别调取CD90和IL-6，检验两分子在物理层面的结合，并判断CD90+细胞中IL-6的主要结合蛋白，同时监测JAK/STAT3通路激活情况，在干预GLI1/3表达与重组融合蛋白sgp130Fc处理相结合，能够选择性抑制IL-6/sIL-6R的互作方式；
E4、敲除癌细胞IL-6R的表达，并结合GLI1/3干预等手段，验证CD90和IL-6的互作及下游通路JAK/STAT3的激活情况。
本发明实施例中，IL-6/CD90相互作用对肝细胞癌进程的应用，是通过体内置瘤，细胞内IL-6R敲除及慢病毒过表达GLI1/3的实验手段相结合，分别分析GLI/IL-6/CD90信号通路微环境对肝细胞癌生长、转移的影响，对罹患肝细胞癌预后的评价，以及对CD90作为肝细胞癌治疗靶点的可行性做评估。
实验方案
1、GLI1/3对CD90+细胞亚群的数量及肿瘤微环境中IL-6的表达的影响
1)严格肝细胞癌临床样本的纳入和收集标准，从肿瘤分期、分化程度、转移能力等方面选择划分，并及时处理组织标本，(1)使用核酸提取试剂TRIzol提取肿瘤组织及癌旁组织的总RNA，反转录试剂盒反转为cDNA，荧光染料法QPCR检测IL-6，GLI1/3，IL-6R，CD90mRNA水平的表达；(2)统计学方法分析CD90与IL-6的表达关联，同时统计CD90、IL-6的表达与癌症预后(肿瘤大小、恶性程度和转移灶)的相关性。
2)肝癌细胞系LM3、HepG2、Huh7、SK-hep-1和MHCC97L培养于37℃，5％CO2的恒温培养箱中，收获细胞后，(1)用QPCR的方法检测IL-6，GLI1/3，IL-6R，CD90 mRNA水平的表达；(2)选择其中两个细胞系，构建GLI1/3慢病毒过表达体系，通过QPCR、Western Blot和ELISA检测CD90和IL-6(细胞上清)的表达变化，并结合流式细胞术(FCM)检测CD90+细胞的比重变化，结合荧光型免疫细胞化学(IF)检测CD90、IL-6和IL-6R的表达定位情况。
3)利用磁珠偶联的CD90抗体分选GLI1/3过表达处理的肿瘤细胞：(1)过表达细胞培养72hr后，移除细胞培养基，用无菌PBS清洗一遍，0.25％的胰酶消化细胞，用含血清的HBSS终止胰酶反应；(2)收集细胞，用HBSS清洗两遍，分选缓冲液重悬，200目无菌滤网过滤制备单细胞悬液，并用细胞计数仪计数；(3)将细胞和抗体溶液按比例混合，快速轻盈混匀，4℃孵育20分钟，期间轻混一次；(4)缓冲液清洗细胞，同时安装磁珠分离装置，润湿清洗柱子一遍，过柱分离细胞；(5)用缓冲液清洗柱子三遍，将柱子移开磁场环境，用缓冲液用力冲洗柱子，收集CD90+细胞和CD90-细胞，流式细胞术(FCM)检测分选纯度；(6)在分离的两类细胞中用QPCR、Western Blot和ELISA的实验手段对IL-6和(s)IL-6R及下游分子JAK、STAT3的表达进行检测。
4)培养分离的CD90+细胞，并用细胞因子SHH维持GLI1/3的激活，培养一段时间后，收集细胞上清和细胞，用ELISA及Western Blot检测IL-6(培养基上清)/(s)IL-6R的表达，与CD90-细胞及SHH处理的对照比较差异。
5)构建慢病毒的GLI1/3干扰片段体系，转染两株肿瘤细胞，QPCR、ELISA和WesternBlot的实验方法检测IL-6(培养上清)、CD90、(s)IL-6R及下游分子JAK、STAT3的激活情况。
2、GLI1/3调节的CD90+细胞亚群通过IL-6促进肿瘤的进程
1)培养两株肝细胞癌细胞系，用GLI1/3的慢病毒过表达载体上调GLI1/3的表达，(1)用FCM检测CD90+细胞亚群的数量变化；MTS检测癌细胞在24hr、48hr及72hr的细胞活性情况；(2)用平板克隆形成实验检测细胞的增殖能力；(3)用Trans well实验检验癌细胞24hr和48hr的迁移和侵袭能力；(4)用Western Blot和ELISA的方法检测IL-6和(s)IL-6R的表达及下游通路JAK/STAT3的激活情况。
2)在过表达GLI1/3或SHH诱导GLI1/3激活的肝癌细胞系中，干涉CD90mRNA在癌细胞中的表达或使用内源性消耗抗体anti-IL6处理，继续培养48hr后，收集细胞，(1)用FCM检测CD90+细胞的数量变化；(2)考查细胞的活性、迁移、侵袭等能力及JAK/STAT3信号激活情况。
3)筛选GLI1/3过表达的MHCC97L稳定株：(1)确定抗生素杀伤细胞浓度曲线，以在5天左右出现细胞大批死亡，2周全部死亡的最低药物浓度作为筛选浓度；(2)转染GLI1/3慢病毒载入肝癌细胞，铺板后使用筛选浓度药物反复筛选；(3)将存活的细胞用有限稀释法接种到96孔板中，并用含筛选浓度药物培养基培养；(4)筛选出单克隆细胞后扩大培养，并用Western Blot鉴定GLI1/3的表达。
4)利用筛选的稳定株细胞，构建裸鼠皮下肿瘤模型：(1)裸鼠麻醉后皮下注射1x10^5/0.1ml稳定GLI1/3过表达株细胞，7～15天后处死小鼠，取肿瘤组织称重，检测肿瘤大小；(2)裸鼠麻醉后，肝叶注射1x10^5/0.1ml稳定GLI1/3过表达株细胞，15～20天后，处死小鼠，取肝脏及肺组织，HE着色观察癌细胞的生长和扩散情况，Western及IHC检测下游JAK/STAT3的激活情况；(3)构建肝脏的裸鼠成瘤模型，并检测小鼠的存活率，绘制生存曲线；(4)裸鼠成瘤实验中，同时设置干预CD90或IL-6表达组和JAK/STAT3抑制剂组，并进行以上肿瘤相关指标的检测。
3、CD90和IL-6的相互作用
1)基于生物信息学预测的结果，分别使用IL-6和CD90作为IP，进行免疫共沉淀(CoIP)实验：(1)培养磁珠分选或扩大培养的GLI1/3过表达细胞系，收集细胞，使用RIPA-非变性型蛋白裂解液裂解细胞，并收集总蛋白，BCA法测定蛋白浓度；(2)将蛋白稀释到适当浓度，取出部分作为Input对照，其余加IP抗体，混匀，4℃孵育过夜；(3)用磷酸盐缓冲液(PBS)清洗CoIP Protein A/G磁珠，然后用PBS配制成50％浓度；(4)磁珠加入过夜反应的蛋白中，4℃摇晃10分钟；(5)将磁珠和蛋白混合物安置在磁场中，去除上清，保留磁珠，并用预冷的PBS清洗三遍；(6)用RIPA蛋白裂解液重悬磁珠，并加入loading Buffer，5分钟煮沸，使蛋白变性，用Western Blot检测CD90(及IL-6R)或IL-6蛋白表达。
2)过表达GLI1/3在肝癌细胞中的表达，并使用重组融合蛋白Sgp130Fc(Sgp130Fc可选择性抑制IL-6/sIL-6R的互作方式)，(1)用免疫共沉淀(CoIP)以IL-6作为IP，然后检测CD90和IL-6的表达；(2)Western Blot监测JAK/STAT3通路激活情况。
3)利用Crisper/Cas9技术敲除两株肝癌细胞中IL-6R的表达，并过表达GLI1/3，继续培养48hr后(1)CoIP检测CD90和IL-6的相互作用；(2)并检测IL-6/JAK/STAT3通路的激活情况。
4)Crisper/Cas9敲除IL-6R实验方法：(1)利用NCBI的核酸数据库查询IL-6R的基因组DNA，并找到单个外显子所在的CDS区域；(2)通过在线gRNA设计网站，在上一步骤查询的区域内寻找5～10条评分较高的gRNA并委托生物公司合成；(3)运用慢病毒载体将合成的gRNA重组到质粒上，转化至大肠杆菌筛选后扩增培养，提取纯化重组质粒；(4)用重组质粒转染肝癌细胞系MHCC97L，压力筛选后挑选出单克隆细胞，并扩大培养，建立稳定株系；(5)在mRNA和蛋白水平对IL-6R的敲除做鉴定，繁殖细胞以备实验使用。
4、IL-6/CD90相互作用对肝细胞癌的影响
构建GLI1/3过表达(目的：上调CD90和IL-6的表达)、GLI1/3过表达+IL-6R敲除以及GLI1/3过表达+IL-6R敲除后干涉CD90或IL-6的MHCC97L细胞株，分别对裸鼠肝叶置瘤，成瘤后观察：(1)肿瘤组织大小、质量；(2)肿瘤扩散的程度(是否有肺部的转移)；(3)肿瘤组织western Blot及IHC检测下游JAK/STAT3激活情况；(4)绘制生存曲线。
同时本说明书中未作详细描述的内容均属于本领域技术人员公知的现有技术。
需要说明的是，在本文中，诸如第一和第二等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来，而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者设备所固有的要素。
尽管已经示出和描述了本发明的实施例，对于本领域的普通技术人员而言，可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由所附权利要求及其等同物限定。
Claims (10)
1.一种GLI蛋白抑制剂，其特征在于：该GLI蛋白抑制剂是由GLI1/3抗体蛋白制成的抑制剂。
2.一种如权利要求1所述的GLI蛋白抑制剂在治疗肝癌中的应用，其特征在于：通过使用所述GLI蛋白抑制剂，使GLI1/3下调抑制CD90的转录，CD90+细胞分泌IL-6低，在IL-6R或sIL-6R的相对缺乏、快速生长的肿瘤细胞中，CD90与IL-6的相互作用被抑制，不能激活JAK/STAT3下游通路，抑制肿瘤细胞的生长。
3.根据权利要求2所述的一种GLI蛋白抑制剂的应用，其特征在于：所述肝细胞癌中GLI1/3对CD90+细胞亚群的数量及肿瘤微环境中IL-6的表达的应用，具体包括以下步骤：
S1、将在前期肿瘤样本的基础上，扩大现有样本组织数量，并严格纳入标准，检测GLI1/3和CD90的mRNA表达，同时检测IL-6和IL-6R的mRNA表达；
S2、根据步骤S1中临床验证的结果，选择两个肝细胞癌细胞系干预GLI1/3的表达，检测CD90 mRNA的表达，检测CD90阳性细胞的数量变化和IL-6和IL6R的表达差异；
S3、拟比较CD90+和CD90-肿瘤细胞中IL-6的表达差异，并联合GLI1/3的配体SHH对细胞进行干预，分析IL-6与IL-6R的表达及IL-6的信号转导方式；
S4、拟通过干预GLI1/3的表达，并结合SHH配体处理细胞，在mRNA及蛋白表达等多个水平检测CD90、IL-6及IL-6R下游通路分子的表达，分析GLI1/3对CD90、IL-6的转录激活及对IL-6/STAT3信号通路影响的结果。
4.根据权利要求3所述的一种GLI蛋白抑制剂的应用，其特征在于：所述步骤S1中，使用统计学方法分析GLI/3对CD90和IL-6的表达的影响，以及这三个分子在肿瘤组织和癌旁组织的表达差异，提供相对充实的临床病例验证数据。
5.根据权利要求3所述的一种GLI蛋白抑制剂的应用，其特征在于：所述步骤S2中，对IL-6R的定位进行验证和讨论，分析IL-6激活下游分子的分子通路。
6.根据权利要求2所述的一种GLI蛋白抑制剂及其应用，其特征在于：所述GLI1/3调节的CD90+细胞亚群通过IL-6促进肿瘤进程的具体步骤如下：
T1、在对肝癌组织样本分子表达检测的基础上，检测CD90+肿瘤细胞系IL-6的表达，讨论CD90与IL-6在肿瘤细胞中表达的相关性；
T2、干预肝癌细胞GLI1/3的表达，考查CD90+细胞数量，及与细胞增殖、迁移、侵袭和细胞周期等的关系，考查IL-6的分泌及下游通路的激活情况；
T3、构建慢病毒过表达GLI1/3肝细胞癌细胞，结合裸鼠体内置瘤的实验手段，并同时通过干预CD90或IL-6及JAK2/STAT3抑制剂的使用证明CD90+细胞中GLI1/3-IL6-STAT3通路的完整性，并考查肿瘤在体内的生长状况，讨论该通路与肿瘤进展的相关性。
7.根据权利要求6所述的一种GLI蛋白抑制剂的应用，其特征在于：所述步骤T2中，结合内源性抗体消耗IL-6表达的方式，考查对细胞功能和IL-6下游信号的影响。
8.根据权利要求2所述的一种GLI蛋白抑制剂的应用，其特征在于：所述CD90和IL-6的相互作用的应用，具体步骤如下：
E1、利用生物信息学完善CD90和IL-6相互作用的预测数据，并结合组织原位免疫显色(IHC)的检验方法定位CD90和IL-6的表达；
E2、拟使用免疫共沉淀(CoIP)分别调取CD90和IL-6，检验两分子在物理层面的结合；
E3、干预GLI1/3表达与重组融合蛋白sgp130Fc处理相结合，并拟使用免疫共沉淀(CoIP)分别调取CD90和IL-6，检验两分子在物理层面的结合，并判断CD90+细胞中IL-6的主要结合蛋白，同时监测JAK/STAT3通路激活情况；
E4、敲除癌细胞IL-6R的表达，并结合GLI1/3干预等手段，验证CD90和IL-6的互作及下游通路JAK/STAT3的激活情况。
9.根据权利要求8所述的一种GLI蛋白抑制剂的应用，其特征在于：所述步骤E3中，在干预GLI1/3表达与重组融合蛋白sgp130Fc处理相结合，能够选择性抑制IL-6/sIL-6R的互作方式。
10.根据权利要求2所述的一种GLI蛋白抑制剂的应用，其特征在于：所述IL-6/CD90相互作用对肝细胞癌进程的应用，是通过体内置瘤，细胞内IL-6R敲除及慢病毒过表达GLI1/3的实验手段相结合，分别分析GLI/IL-6/CD90信号通路微环境对肝细胞癌生长、转移的影响，对罹患肝细胞癌预后的评价，以及对CD90作为肝细胞癌治疗靶点的可行性做评估。
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113025614A (zh) * | 2021-03-22 | 2021-06-25 | 四川大学华西医院 | 一种胶质瘤诊断和/或预后评估标志物及其应用 |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107970245A (zh) * | 2018-01-08 | 2018-05-01 | 陈世伟 | 三种大戟烷型三萜用于制备Gli基因抑制剂和防治肝癌药物的生物医药用途 |
| CN110496221A (zh) * | 2019-01-03 | 2019-11-26 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | 抑制dppa3表达的物质在制备预防和治疗癌症的产品中的应用 |
| CN111534859A (zh) * | 2020-05-22 | 2020-08-14 | 中山大学孙逸仙纪念医院 | 一种基于CircRNA群的肝癌细胞分子库建立方法 |
| CN112585160A (zh) * | 2018-08-06 | 2021-03-30 | 麦地金公司 | Il-2受体结合化合物 |
| EP3822284A1 (en) * | 2018-07-11 | 2021-05-19 | Hedgehog, Inc. | Epitope specific to smo protein, antibody recognizing same, and composition comprising same |
-
2020
- 2020-12-22 CN CN202011527459.9A patent/CN112451662A/zh active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107970245A (zh) * | 2018-01-08 | 2018-05-01 | 陈世伟 | 三种大戟烷型三萜用于制备Gli基因抑制剂和防治肝癌药物的生物医药用途 |
| EP3822284A1 (en) * | 2018-07-11 | 2021-05-19 | Hedgehog, Inc. | Epitope specific to smo protein, antibody recognizing same, and composition comprising same |
| CN112585160A (zh) * | 2018-08-06 | 2021-03-30 | 麦地金公司 | Il-2受体结合化合物 |
| CN110496221A (zh) * | 2019-01-03 | 2019-11-26 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | 抑制dppa3表达的物质在制备预防和治疗癌症的产品中的应用 |
| CN111534859A (zh) * | 2020-05-22 | 2020-08-14 | 中山大学孙逸仙纪念医院 | 一种基于CircRNA群的肝癌细胞分子库建立方法 |
Non-Patent Citations (2)
| Title |
|---|
| KETAO ZHANG等: "The SHH/Gli axis regulates CD90‐mediated liver cancer stem cell function by activating the IL6/JAK2 pathway", 《J CELL MOL MED》 * |
| 黄炳御等: "MAOA与肿瘤关系的研究进展", 《生命科学》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113025614A (zh) * | 2021-03-22 | 2021-06-25 | 四川大学华西医院 | 一种胶质瘤诊断和/或预后评估标志物及其应用 |
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