CN117298107A - 洛美他派在制备治疗神经胶质瘤药物中的应用 - Google Patents
洛美他派在制备治疗神经胶质瘤药物中的应用 Download PDFInfo
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Abstract
本发明涉及医药领域,具体涉及洛美他派在制备治疗神经胶质瘤药物中的应用。而本发明首次发现洛美他派可以透过血脑屏障,且通过抑制MMP3激活炎性细胞因子信号通路,促进神经胶质瘤细胞发生焦亡,而具有抑制恶性神经胶质瘤生长的作用,增加了洛美他派新用途。
Description
技术领域
本发明涉及医药领域,具体涉及洛美他派在制备治疗神经胶质瘤药物中的应用。
背景技术
原发性中枢神经系统癌严重地影响着儿童和成人的健康,该疾病发生于中枢神经系统的各个区域中,其中绝大多数发生在大脑中,其余发生在脑膜、脊髓和脑神经等部位。中枢神经系统肿瘤在成年人中比较罕见,但它是引起儿童和青少年癌症发病和死亡的重要原因,分别占全球癌儿童和青少年症死亡的约30%和20%。恶性神经胶质瘤是常见的原发性中枢神经系统癌症,包括高级别胶质瘤或胶质母细胞瘤和低级别胶质瘤。其余中枢神经系统癌症包括其它胶质来源的肿瘤、髓母细胞瘤、中枢神经系统淋巴瘤和脑膜瘤。神经胶质瘤作为患病率、死亡率最高的原发性脑癌,即便进行最大限度的手术和药物治疗,该疾病导致的2年内致死率仍居高不下。
目前针对神经胶质瘤的治疗手段主要包括手术治疗、放射治疗、化学治疗和免疫治疗等。由于神经胶质瘤细胞与正常的大脑神经细胞缠绕并粘附在一起,这使得它们很难被手术清除。用于治疗神经胶质瘤的化疗药物包括TMZ、PCZ、CCNU、VCR等,而这些化疗药物均会引起不同程度的副作用。因此寻找新的抗神经胶质瘤成为本领域的技术难题。
美他派是一种微粒体甘油三脂转移蛋白酶抑制剂,对HoFH治疗疗效较好,不良反应相对较少。作用机制为在内质网的腔内,该药直接与甘油三脂转移蛋白酶结合并抑制其活性,阻止小肠上皮细胞和肝细胞内载脂蛋白B的组装和分泌,抑制乳糜微粒和极低密脂蛋白的合成,从而使血浆低密脂蛋白胆固醇水平降低,由此减少体内的总胆固醇值和低密度脂蛋白胆固醇值,降低心血管疾病风险。2012年12月,洛美他派以甲磺酸盐形式被美国FDA批准用于治疗由于常染色体基因缺陷而导致的罕见病纯合子型家族性高胆固醇血症,但未见在抗恶性神经胶质瘤的应用。
发明内容
本发明目的之一,提供洛美他派的新用途,即提供了洛美他派在抗恶性神经胶质瘤中的应用。
本发明的目的可以通过以下技术方案实现:
洛美他派在制备治疗神经胶质瘤药物中的应用,所述洛美他派的结构式如式Ⅰ所示:
本发明技术方案中,所述洛美他派用于抑制神经胶质瘤中的应用。
本发明技术方案中,所述洛美他派的给药剂量为20mg/kg。
本发明技术方案中,取对数生长期的U87MG、U251MG细胞,用适量预冷PBS重悬细胞,使细胞密度保持在2×107个/mL左右。每只裸鼠接种体积为0.1mL,将细胞通过皮下注射于裸鼠的腋窝中后部,构建异位胶质瘤模型。通过灌胃洛美他派,评估其对恶性神经胶质瘤的治疗效果。经研究发现,洛美他派可以抑制恶性神经胶质瘤的生长,上调焦亡基因的表达。
本发明的有益效果:
神经胶质瘤具有局部异质性和高度侵袭性,生长速度快,使得手术彻底切除困难,极易复发,且极易产生耐药性。并且由于血脑屏障的存在,使得绝大多数药物不能直接到达肿瘤部位,导致恶性胶质瘤治疗困难,预后差。血-脑屏障是血液与脑组织之间的屏障,可限制物质在血液和脑组织之间的自由交换,可防止有害物质进入脑组织,对脑、脊髓起到保护作用。一般来说对于脂溶性高、分子量比较小、化学结构比较简单的药物、游离的药物其血脑屏障通透率一般较高。在药物治疗学上面,血脑屏障的存在会使几乎所有大分子药物和98%小分子药物都不能进入大脑及中枢神经系统,根据文献(Pardridge WM,Drugtransport across the blood-brain barrier.J Cereb Blood Flow Metab.2012Nov;32(11):1959-72.doi:10.1038/jcbfm.2012.126.Epub 2012 Aug 29.)记载脂溶性小分子化合物透过血脑屏障需要具备高脂溶性,分子量小于400Da和形成小于8个氢键。此外小分子化合物进入血脑屏障与CMT系统有关。满足上述要求的药物少之又少,6000个化合物中只有6%可以作用于脑部。因此如抗肿瘤药物如紫杉醇,顺铂,长春新碱等其对如肺癌,肝癌等是有效的,但由于无法穿过血脑屏障,对脑部如神经胶质瘤治疗效果差或无效,故寻找既有抗肿瘤活性又可以穿过血脑屏障成为本领域的技术难题。
尽管有文献报道洛美他派有抗肿瘤作用,但基于前述多数抗肿瘤药物实际对神经胶质瘤是无效的,而本发明首次发现洛美他派可以透过血脑屏障,且通过抑制MMP3激活炎性细胞因子信号通路,促进神经胶质瘤细胞发生焦亡,而具有抑制恶性神经胶质瘤生长的作用,增加了洛美他派新用途。
具体来说,首先通过CCK-8法从FDA批准的1430种小分子化合物中筛选对U118MG细胞有抑制作用的药物,并首次发现了家族性高胆固醇血症治疗药物洛美他派(Lomitapide,LM)可剂量依赖性地抑制U87MG、U118MG与U251MG神经胶质瘤细胞的增长。另一方面,通过裸鼠异位瘤实验也发现,LM在体内也能有效的抑制U87MG、U251MG神经胶质瘤的生长,而不会影响小鼠的体重,具有良好的安全性。研究发现,LM使U87MG、U1187MG和U251MG细胞的早期凋亡率显著增加。药物刺激后,细胞中坏死性凋亡标志蛋白(MLKL)、凋亡标志蛋白(Caspase3)和焦亡蛋白(GSDMD)表达含量显著上升。细胞形态SEM观察发现,加药刺激后细胞膜表面出现“煎蛋”形状和孔洞,呈现明显的细胞焦亡形态。因此,我们认为LM可以同时触发GSDMD的剪切,以促进神经胶质瘤细胞发生焦亡,从而有效地杀伤神经胶质瘤细胞。对LM抑制神经胶质瘤的分子机制的研究发现,LM可通过抑制MMP3杀伤神经胶质瘤细胞,MMP3是LM发挥药效的潜在作用靶点。通过裸鼠原位瘤实验发现,在第18天时CTL组裸鼠已全部死亡,而给药组裸鼠还有一半的生存率,说明与对照组相比游离LM可以通过血脑屏障到达胶质瘤部位并抑制肿瘤的生长。
附图说明
图1神经胶质瘤药物的筛选。A.药物筛选流程图;B.不同药物对U118MG细胞抑制率排名。
图2 LM剂量依赖性地抑制神经胶质瘤细胞增长;A.CCK-8法测定不同浓度LM对人脑神经胶质瘤细胞活力影响;B.加药后细胞形态学观测;C.A375、A549、H460、HepG2、小鼠原代肝细胞(PHs)及AML-12细胞加药后细胞活力;*P<0.05,**P<0.01vs.CTL。
图3 LM抑制裸鼠神经胶质异位瘤的生长。A.将接种同种肿瘤细胞的裸鼠随机分成2组,每48h口服给药(玉米油、20mg/kg LM)1次,持续给药3周(每组n=7),裸鼠异位瘤拍照;B.裸鼠神经胶质瘤重量统计;C.给药期间裸鼠异位瘤体积统计;D.肿瘤组织的H&E染色;*P<0.05,**P<0.01vs.CTL.
图4 LM在体内具有良好的安全性。A.B.裸鼠给药期间体重检测;裸鼠移植U87MG细胞异位瘤模型建造成功后,收集小鼠血清测定AST(C)、ALT(D)含量;U251MG细胞异位瘤血清中AST(E)、ALT(F)含量测定;*P<0.05,**P<0.01vs.CTL.
图5 LM促进神经胶质瘤细胞发生程序性死亡;Annexin V-FITC/PI双染法测定检测IC50浓度的LM对神经胶质瘤细胞影响。
图6 LM促进神经胶质瘤细胞发生程序性死亡;A.LM对人脑神经胶质瘤细胞中各种蛋白表达的影响;B.MLKL、GSDMD、Caspase3蛋白量统计;*P<0.05,**P<0.01vs.CTL.
图7 LM起神经胶质瘤细胞发生细胞焦亡。A.U87MG、U118MG、U251MG细胞分别转染mNeon-GSDMD质粒后,加入IC50剂量的LM处理固定后免疫荧光拍照结果;B.三种神经胶质瘤细胞分别加入IC50剂量LM处理,TUNEL染色拍照结果;C.三种神经胶质瘤细胞加入LM孵育,扫描电镜成像结果。
图8 LM激活神经胶质瘤细胞的炎性细胞因子相关通路;A.U118MG细胞加入6μM LM处理24h后转录组学测序结果;B.差异基因分析后,行GO富集分析得到的相关通路;C.关键差异基因基因GO富集分析;D.关键差异基因KEGG富集分析;E.GDF15、IL-1β、IL-6mRNA表达水平变化。*P<0.05,**P<0.01vs.CTL.
图9 LM杀伤神经胶质瘤细胞的作用靶点;A.U118MG细胞转染siMTTP质粒36h后,加入6μM LM孵育24h测定细胞活力结果;B.MAPK8和MMP3蛋白与LM分子对接结果。*P<0.05,**P<0.01vs.NC。
图10 MAPK8和MMP3是LM杀伤神经胶质瘤细胞的作用靶点。A.U87MG、U118MG、U251MG细胞中MAPK8、MMP3的相对表达量;B.U87MG、U118MG、U251MG细胞分别加入SP600125、GM6001与药物刺激24h后细胞活力检测;*P<0.05,**P<0.01vs.LM-IC50.
图11原位瘤实验结果。
具体实施方式
下面结合实施例对本发明做进一步说明,但本发明的保护范围不限于此:
洛美他派(Lomitapide,LM)
实施例1药物筛选确定LM在抑制神经胶质瘤生长中的应用
1.脑星形胶质母细胞瘤细胞系及小鼠原代肝细胞的培养
人脑星形胶质母细胞瘤细胞系U87MG、U118MG、U251MG均购自美国菌种保藏中心。BALB/c-Nude小鼠及C57品系小鼠购自杭州子源实验动物科技有限公司。细胞培养箱保持37℃、5%CO2、恒温、恒湿条件下培养U87MG、U118MG及U251MG细胞。分离小鼠原代肝细胞接种至96孔板,12h后加药处理。
2.药物筛选实验(CCK-8检测法)
CCK-8试剂盒购自南京建成生物工程研究所。将U118MG细胞接种于96孔板中,密度约为1×104/孔,培养箱过夜。使用无血清DMEM换液处理2h,使细胞同步化。替换培养基为包含10μM药物(1430种小分子化合物库,美国selleck公司,货号:L1300)的无血清DMEM培养基或包含0.1%DMSO(对照组)的无血清培养基,继续在37℃培养箱中孵育24h。每孔逐一加入10μLCCK-8(2-(2-甲氧基-4-硝苯基)-3-(4-硝苯基)-5-(2,4-二磺基苯)-2H-四唑单钠盐)试剂,轻轻混匀后将培养板至于培养箱中继续孵育1~4h。使用微孔板酶标仪检测细胞在450nm处的吸光值,统计并汇总。
3.细胞形态学观察
使用不同浓度(0、2.5μM、5μM、7.5μM、10μM)的LM处理人脑星形胶质母细胞瘤细胞系U87MG、U118MG、U251MG,孵育24h。使用4%多聚甲醛固定细胞30min,PBS清洗2次,每次5min。倒置显微镜观察细胞形态并拍照。
4.实验动物饲养及处理
取对数生长期的U87MG、U251MG细胞,细胞生长融合度达到80~90%后收集细胞。消化并收集细胞后,使用PBS清洗2次,以去除溶液中的胰酶与血清。用适量预冷PBS重悬细胞,使细胞密度保持在2×107个/mL左右。每只裸鼠接种体积为0.1mL,将细胞通过皮下注射于裸鼠的腋窝中后部。接种过程中,将细胞悬液置于冰上以降低细胞代谢,提高细胞存活率,整个接种操作尽量在半小时内完成。待接种一周后,可观察到裸鼠明显的肿瘤。将接种同种细胞的裸鼠随机分成2组,每48h口服给药(玉米油、20mg/kgLM)1次,持续给药3。每次给药前用游标卡尺测量小鼠肿瘤大小。待给药实验结束后,脱脊安乐死小鼠,剥离肿瘤拍照并称重。肿瘤体积计算公式:肿瘤体积=长径×短径2×0.5(mm)。
5.丙氨酸氨基转移酶、天冬氨酸氨基转移酶测定
小鼠血清收集与预处理:使用1.5mL离心管收集小鼠血液,置于4℃静置4h以上,可观察到血清析出。将离心管4000rpm,4℃,10min离心,吸取血清上清液至新管中,分离后的血清储存于-80℃。按照南京建成公司的谷丙转氨酶(Alanine Transaminase,ALT)和天冬氨酸转氨酶(Aspartate Transaminase,AST)检测试剂盒中的说明操作,准备相应试剂与耗材。吸取20μL预热至37℃的ALT/AST基质液至96孔板中,加入5μL待测血清样品,用枪头反复吹打混匀,避免出现气泡。轻轻震荡孔板,置于37℃气浴30min。向每个孔中逐一加入苯肼显色液20μL,再次用枪头反复吹打混匀,避免出现气泡。轻轻震荡孔板,置于37℃气浴20min。向每个孔中逐一加入0.4M氢氧化钠溶液200μL,终止反应并显色。轻轻震荡孔板,室温静置15min后,使用多功能酶标仪测定OD510 nm,计算出血清中AST/ALT含量。
6.苏木精-伊红(Hematoxylin–Eosin,H&E)染色分析
取出4%多聚甲醛固定的组织,用石蜡进行包埋,然后使用切片机进行切片,切片厚度为4μm。烘烤切片至切片中的石蜡融化,约30min。将切片转移至二甲苯中脱蜡5min,然后用滤纸吸干多余液体。再将切片转移至二甲苯中继续脱蜡10min至切片透明,然后用吸水纸吸干多余液体。将切片转移至乙醇中处理5min,吸干多余液体,重复2次,每次更换新鲜的乙醇。将切片置于95%的乙醇中浸泡3min后,用流水洗5min,滤纸吸干多余水分。将苏木精染液滴加至切片样本上,完全覆盖,室温条件下染色5min,稍微水洗。使用1%的HCl溶液分化8s至切片颜色由蓝色转变为红色,然后自来水冲洗30min左右,使得切片返蓝。向切片样本中滴加0.5%的伊红染色1min,然后分别使用95%和100%的乙醇各清洗切片2次,每次5min;将切片浸泡于二甲苯中脱色5min。取出切片,吸干多余液体并用中性树脂封片。镜检,细胞核呈现蓝色,其它组织成分深浅不一的红色。通过显微镜对H&E染色后的切片进行观察,拍照。
7.免疫组化分析
按照H&E染色的操作,将石蜡切片进行脱蜡和水化处理后,然后使用3%医用过氧化氢浸泡切片30min,除去内源性过氧化氢酶。将水化好的切片拎出,浸泡于预热后1×枸橼酸抗原修复液中,在100℃的水浴锅中加热30min后,从水浴锅取出后,让其自然冷却至室温。将切片拎至PBS中,洗涤3次,每次5min,然后取出切片,用吸水纸小心擦去载玻片周围的水,用组化笔圈定组织的位置,再在组织上滴上3%医用过氧化氢,室温孵育30min,清除内源性的过氧化氢酶活性。将切片拎至PBS中,洗涤3次,每次5min,然后取出切片,用吸水纸擦去组化圈外的水,在圈内滴入山羊血清进行封闭,室温下孵育60min。封闭结束后,使用PBS洗3次,每次5min。然后,滴加一抗工作液(MLKL一抗工作液由5%BSA配制,稀释比例1:500),并在室温条件下孵育120min。孵育结束后,使用PBS洗3次,每次5min。然后,滴加二抗工作液,继续在室温条件下孵育1h。孵育结束后,使用PBS洗3次,每次5min。向处理好的切片上滴加新鲜配制的DAB溶液显色,1~2min。使用双蒸水缓慢清洗,滴加苏木精复染,然后水洗返蓝。再次使用梯度乙醇和二甲苯进行进脱水,干燥处理,中性树脂封片。通过显微镜对切片免疫组化结果进行观察,拍照。
8.实验结果
如图1所示,通过CCK-8法从FDA批准的1430种小分子化合物中筛选对U118MG细胞有抑制作用的药物,并首次发现了家族性高胆固醇血症治疗药物LM可剂量依赖性地抑制U87MG、U118MG与U251MG神经胶质瘤细胞的增长。如图2所示,LM也能剂量依赖性地杀伤人黑色素瘤细胞A375及人非小细胞肺癌细胞A549,而该药物并不会杀伤正常细胞,如小鼠原代肝细胞和肝细胞系AML-12。这说明LM具有较好的安全性。如图3所示,我们通过裸鼠异位瘤实验也发现,LM在体内也能有效的抑制U87MG、U251MG神经胶质瘤的生长。如图4所示,LM也不会影响小鼠的体重,具有良好的安全性。
实施例2 LM抑制神经胶质瘤的药效研究
1.Annexin V-FITC/PI双染法测定细胞凋亡
以6孔板为例,对U87MG、U118MG和U251MG细胞分别加入药物半抑制浓度的LM刺激24h。将细胞培养上清培养基液吸入离心管中,使用PBS缓冲液清洗细胞一次,加入300μL不含EDTA的胰蛋白酶消化细胞。细胞消化下来后,加入上一步收集的培养基终止胰酶消化功能,重悬细胞后转移至离心管中,1000g,离心5min,收集细胞。用PBS轻轻重悬,并进行细胞计数。取约1~5×105个重悬细胞,1000g,离心5min,沉淀细胞,加入500μL染色结合液重悬细胞。在离心管中分别加入5μLAnnexinV-FITC和5μL碘化丙啶,轻轻混匀。室温静置避光孵育10min。使用流式细胞仪对细胞进行检测,AnnexinV-FITC为绿色荧光,碘化丙啶为红色荧光。使用CytExpert软件对流式细胞术结果进行分析。
2.Western Blot
药物处理U87MG、U118MG和U251MG细胞后,6孔板细胞每孔加100μL裂解液,充分裂解细胞后,于4℃,13000rpm离心10min,收集上清。使用BCA测定试剂盒测定蛋白浓度。调整蛋白浓度后,与6×Sample buffer混匀,置于100℃条件下加热7min,使蛋白质彻底变性,最后将提取好的蛋白样品放在-80℃冰箱中保存备用。使用10%或8%SDS-PAGE凝胶电泳,将目的条带转至PVDF膜上,在5%(W/V)的脱脂牛奶中室温封闭1h。4℃孵育一抗过夜。孵育结束后,PBST清洗3次,每次10min。二抗在室温条件下孵育1h后,PBST清洗3次,每次10min。将PVDF膜浸泡在显色液中,通过Tanon5200化学发光成像系统观察目的蛋白条带。
3.扫描电子显微镜
将U87MG、U118MG和U251MG细胞分别铺至细胞爬片,培养过夜。在细胞中加入药物半抑制浓度的LM刺激24h。前固定:使用PBS缓冲液轻轻清洗细胞一次,加入2.5%戊二醛电镜固定液,4℃固定2h以上。使用0.1M pH=7.4磷酸盐缓冲液漂洗细胞,每次大约10min,共洗3次。后固定:加入1%锇酸固定液,4℃固定1~2h。使用0.1M pH=7.4磷酸盐缓冲液漂洗细胞,每次大约10min,共洗3次。脱水:玻片——30%乙醇,处理5min——50%乙醇,处理5min——70%乙醇,处理10min——80%乙醇,处理10min——95%乙醇,处理15min——无水乙醇(无水硫酸钠处理),处理15min两次——叔丁醇,处理15min两次;冷冻干燥过夜;喷金;扫描电镜观察,拍摄照片。样本的扫描电镜拍摄工作主要委托杭州科学指南针有限公司完成。
4.Neon-GSDMD质粒转染(以35mm玻底/共聚焦培养皿为例)
4%多聚甲醛溶液:称取4g多聚甲醛溶到100mL的PBS缓冲液中,用NaOH调至pH=7.4后,60℃水浴并磁力搅拌,直至完全溶解。分别配制转染混合液。a:50μL无血清无抗生素的基础培养基、适量Lipofectamine3000(转染试剂)及P3000(转染辅助试剂)(对于siRNA转染,无需加入P3000辅助试剂):b:50μL无血清培养基及适量质粒DNA,室温静置5min,然后将B管中的溶液逐滴加入转移至A管中。轻弹管壁使液体混匀,再次室温静置10min。将混合液逐滴加入,使液体均匀铺满皿底。将培养皿放入培养箱中,3min后取出,加入培养基使终体积达到100μL。6~8h后更换为含有10%FBS的培养基。转染48h后,加入4%多聚甲醛溶液固定细胞,进行荧光拍照等其它相关实验。
5.TUNEL染色(以35mm玻底/共聚焦培养皿为例)
用TUNEL法(TdT-mediated dUTP Nick End Labeling,TUNEL)检测细胞凋亡,具体步骤详见南京诺唯赞生物科技股份有限公司TUNEL BrightGreen Apoptosis DetectionKit说明书。
6.实验结果
6.1 LM诱导神经胶质瘤细胞发生程序性死亡
如图5所示,在U87MG、U118MG与U251MG三种细胞中,半抑制浓度的LM引起U87MG、U118MG与U251MG细胞早期凋亡比率分别从1.18%、1.63%和0.53%上升到3.36%、3.97%和2.78%,给药组和CTL间存在极显著性差异。同时,LM也极大增加了细胞发生晚期凋亡的比率,分别从5.55%、3.67%和2.35%上升到30.2%、20.8%和18.2%。t检验分析给药组和CTL的细胞晚期凋亡率也出现极显著性差。因此我们认为,LM可通过促进细胞程序性死亡抑制神经胶质瘤细胞的增殖。
6.2 LM促进神经胶质瘤细胞发生焦亡
如图6所示,通过Western Blot分析三种神经胶质瘤细胞加药前后细胞坏死性凋亡标志蛋白MLKL,细胞凋亡标志蛋白Caspase3,细胞焦亡标志蛋白GSDMD的表达变化。在三种神经胶质瘤细胞中加入半抑制浓度的LM后,GSDMD蛋白的表达量均显著升高。而体内实验也观察到相同的现象,裸鼠异位瘤切片IHC结果均显示,LM给药组中MLKL的表达量显著上升。分析上述结果我们认为,LM可以显著的促进神经胶质瘤细胞发生焦亡。此外,U118MG和U251MG细胞加药刺激后,细胞焦亡标志蛋GSDMD(53kD)的表达量也显著的上升。
6.3 LM引起神经胶质瘤细胞发生细胞焦亡
如图7A所示,在三种胶质瘤细胞中,LM处理后,细胞中绿色荧光的GSDMD蛋白聚集于细胞膜上,证实半抑制浓度剂量的LM药物处理并会引起神经胶质瘤细胞发生细胞焦亡,这一结果也与上文中GSDMD蛋白变化结果相符。TUNEL染色结果显示(图7B),U87MG细胞和U118MG细胞在LM处理后,出现明显的红色荧光阳性细胞,说明LM刺激细胞发生早期凋亡。而U251MG细胞中发生凋亡的细胞数量较少,该结果也与三种细胞加药后细胞的Caspase3蛋白表达水平相符。最后,我们也对三种细胞的细胞形态通过扫描电镜进行观察。如图7C所示,LM药物刺激后,细胞膜表面可观察到泡状突起的形成,细胞出现“煎蛋形”与穿孔,细胞膜下陷。我们认为该现象是由于细胞发生了焦亡所引起。综合以上结果推测,LM通过激活细胞焦亡杀伤神经胶质瘤细胞。
实施例3 LM抑制神经胶质瘤的分子机制研究
1.MTTP siRNA转染
所用转染方法同实施例2.4所述。(MTTP siRNA委托上海吉玛制药技术有限公司设计并合成)。
2.总RNA提取(以6孔板为例)
细胞经加药处理后,用PBS缓冲液清洗细胞3次,直接向孔板中加入1mLTrizol,用移液器剧烈反复吹打,收集裂解液。加入200μL三氯甲烷,vortex10s,使样品充分混匀,13000rpm,4℃,10min离心。吸取上清液400μL,转移至新的RNase-free离心管中,而后加入等体积异丙醇,vortex10 s使其充分混匀,-20℃静置10min。13000rpm,4℃,10min离心,管底出现白色絮状沉淀,弃去上清,并用倒扣的吸水纸尽量吸去上清,注意防止白色沉淀滑落。加入1mL75%乙醇DEPC水溶液,vortex使白色沉淀悬浮起来,13000rpm,4℃,10min离心,尽可能弃去上清,并倒扣,用吸水纸尽量吸去上清,注意防止白色沉淀滑落,在通风橱中室温放置约10min晾干。乙醇挥发完全后,RNA白色沉淀会变成透明,根据前一步RNA白色团块大小加入25~100μLDEPC水,vortex混匀,放入65℃中金属浴7min,观察RNA是否全部溶解,vortex10 s,瞬时离心,-80℃储存。以琼脂糖胶法检测提取的RNA是否合格。
3.反转录PCR
用Nanodrop检测所提取总RNA浓度,按照TaKaRa反转录试剂盒说明书操作。PCR反转录程序设置如下:
表3-1PCR反转录程序
程序结束后,加入90μL RNase-free水,可立即进行RT-qPCR检测或者长期储存于-20℃中。
4.实时荧光定量PCR(RT-qPCR)
使用PrimerBank网站(https://pga.mgh.harvard.edu/primerbank/)或者Primerpremier 5软件设计引物,委托北京擎科新业生物技术有限公司合成,引物序列参见表3-3。PCR反应产物进行电泳以及溶解曲线分析验证引物的特异性。从Ct值来确定cDNA的表达丰度,以β-actin作为内参,通过2-△△Ct法,计算得出基因相对表达量。
使用2×ChamQ Universal SYBR qPCR Master Mix试剂盒(购自南京诺唯赞生物科技有限公司),按照说明书配制反应体系,如下表所示:
表3-2荧光定量PCR体系配方
混匀后瞬时离心,放入RT-qPCR扩增管中进行反应,程序设置如下:
表3-3荧光定量PCR程序
5.高通量RNA测序
为了在研究LM杀伤神经胶质瘤细胞的具体分子机制,在U118MG细胞中加入6μM LM孵育24h后。如上所述,收集细胞样本,提取总RNA,构建RNA-seq文库。使用Agilent 2100生物分析仪对RNA文库的质量进行评估。文库准备在Illumina Hiseq平台上测序。计算每百万映射读数(FPKM)每千碱基的片段进行额外的统计数据。所有读数都被映射到人类基因组(GRCh38)。差异基因的可视化采用R软件的ggplot2包进行,使用R语言对差异基因的GO和KEGG进行分析和可视化。P<0.05为GO富集分析的阈值。样本的高通量RNA测序工作主要委托北京诺禾致源科技股份有限公司完成。
6.分子对接分析(Molecular docking analysis)
PDB(http://www.rcsb.org/)数据库中获取MMP3(PDBID:2JT6)及MAPK8(PDB ID:4G1W)的蛋白解析结构,在PyMOL 2.4软件中删除受体中的水分子以及小分子配体,并进行加氢等预处理。在PubChem(https://pubchem.ncbi.nlm.nih.gov/)数据库下载LM的分子结构。以蛋白为受体,LM为配体用MOE(version 2015.10)进行分子对接,分析结合能数值,数值为负表示配体和蛋白受体能够结合,且数值越小表明结合可能性越大,并对结果进行分子对接图的绘制。
7.实验结果
7.1 LM激活神经胶质瘤细胞的炎性细胞因子相关通路
如图8所示,通过转录组学研究证实,LM激活了神经胶质瘤细胞内炎性细胞因子相关通路,从而诱发细胞发生细胞焦亡,最终抑制其生长。随后我们对LM的药物作用靶点进行了研究,首先,我们研究了该药物的经典作用靶点MTTP。通过siRNA干扰MTTP后加药物刺激并测定细胞活力发现,干扰掉原有靶点MTTP并不会对LM杀伤神经胶质瘤细胞产生影响,排除了MTTP作为药物靶点的可能性。
7.2 MTTP不是LM杀伤神经胶质瘤细胞的作用靶点
如图9所示,我们通过生物信息学手段预测药物作用靶点,并将转录组学结果与预测药物靶点间进行hub gene分析,筛选到潜在的药物作用靶点MAPK8和MMP3。运用MOE软件进行分子对接结果显示,MAPK8、MMP3蛋白与LM的结合能分别为-9.2077kcal/mol和-9.0625kcal/mol,说明其具有较强的结合能。
7.3 LM杀伤神经胶质瘤细胞的作用靶点研究
如图10所示,分别使用MAPK8的特异性抑制剂SP600125和MMP的抑制剂GM6001在三种神经胶质瘤细胞上进行靶点验证。通过对细胞活力检测发现,在U87MG和U251MG细胞中抑制MMP3后,LM不再发挥对细胞的抑制作用。且在MMP3低表达的U251MG细胞中,未观察到此现象。这些结果说明了LM可通过抑制MMP3杀伤神经胶质瘤细胞,MMP3是LM发挥药效的潜在作用靶点。
实施例3原位瘤抑制实验(透过血脑屏障实验)
材料:菌种和质粒DH5α感受态大肠埃希菌购买自中国南京诺唯赞生物科技有限公司,pMX载体、pVSVg质粒和psPAX2质粒购自美国Addgene公司,pGL4.17质粒购自Clontech公司。细胞和实验动物293T gag-pol细胞系、BALB/c nude购买自江苏集萃药康生物科技股份有限公司,雌性,6周龄,体重17~19g。试剂和仪器Taq DNA聚合酶、T4 DNA连接酶、HindIII、XbaI、EcoRI、NotI购自NEB公司。
具体方法如下:
(1)pMX-Luc2的构建将pGL4.17[Luc2/neo]质粒经HindIII酶切,从PCI-neo载体中扩增pCMV,再将两者用连接酶连接。转化连接产物,挑选克隆进行测序鉴定,将鉴定正确的pGL4.17[Luc2/pCMV]及pMX进行EcoRI和NotI的双酶切,酶切后的Luc2/pCMV与pMX大片段连接成pMX-Luc2,送测序进行鉴定。
(2)重组质粒共转染293T细胞按照Lipofectamine 3000TM说明书操作,将测序鉴定正确的pMX-Luc2克隆质粒与pVSVg质粒、psPAX2质粒共转染293T gag-pol细胞。转染6h后,加入2mL 37℃新鲜的DMEM完全培养基。
(3)表达Luc2的慢病毒的收获转染48h后,收集含有反转录病毒的培养基上清,加入新鲜DMEM完全培养基继续培养。48h后收集慢病毒上清和细胞,液氮反复冻融3次裂解细胞,释放病毒,将数次收集的慢病毒浓缩后集中放置于-80℃贮存。
(4)Luc2阳性U251细胞系的建立和筛选将100μL表达Luc2的慢病毒上清接种于人脑胶质瘤细胞U251细胞。每12h感染1次,共4次。培养后,每孔加入15mg/ml的D-荧光素(D-Luciferin)1μL,轻轻摇晃细胞板使其均匀分布在培养基中,立即于活体成像系统中进行检测。挑取阳性克隆并进行扩大培养。
(5)活体成像系统检测U251-Luc2细胞荧光素酶的表达
将1×107/mL的U251-Luc2细胞通过尾静脉注射至BALB/c裸鼠体内,其中2只裸鼠注射100μL,另外2只注射300μl。瘤细胞接种后1h及72h经腹腔给予D-Luciferin(150mg/kg),注射后10min在活体成像系统下观察荧光素酶的表达情况。
为了构建携带GBM的裸鼠模型,我们使用胰岛素针注射U251-luc2细胞于裸鼠大脑中,并通过生物荧光信号监测裸鼠脑肿瘤的大小,具体实验操作如下:(1)消化U251-luc2细胞,用PBS重悬,并置于冰上。(2)使用气麻机将裸鼠麻醉,用消毒剂对大脑皮肤进行消毒后,将细胞注射进裸鼠大脑。(3)等待裸鼠清醒后观察裸鼠状态,待其能正常活动进食后即为肿瘤接种成功。(4)肿瘤接种7天后,将0.5mM的LM尾静脉注射150μL到携带GBM的裸鼠体内,每三天给一次药,持续18天,对照组尾静脉注射同样剂量的PBS。在每次给药之前,腹腔注荧光素酶底物(D-Luciferin)150mg/kg,通过生物荧光信号监测肿瘤的大小。
实验结果
如图11所示,通过裸鼠原位瘤实验发现,在第18天时CTL组裸鼠已全部死亡,而给药组裸鼠还有一半的生存率,说明与对照组相比游离LM可以通过血脑屏障到达胶质瘤部位并抑制肿瘤的生长。
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1.洛美他派在制备治疗神经胶质瘤药物中的应用,所述洛美他派的结构式如式Ⅰ所示:
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