CN107184585B - Hc-067047在制备抗胶质瘤的药物中的应用 - Google Patents
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Abstract
本发明公开了瞬时感受器电位辣椒素4型(TRPV4)的阻滞剂HC‑067047在制备抗胶质瘤的药物中的应用，扩大了HC‑067047的应用范围，提高了其应用价值，给胶质瘤的治疗或保健带来了新的希望，并且，以HC‑067047作为先导化合物，通过结构修饰或改造提高活性或降低副作用，还有助于进一步开发新的抗胶质瘤的药物。
Description
技术领域
本发明属于含有机有效成分的医药配制品技术领域，涉及HC-067047在药物制备领域的新用途。
背景技术
胶质瘤是颅内最多的肿瘤类型。据最新的统计显示：胶质瘤约占原发颅内肿瘤的26.9％，而占颅内原发恶性肿瘤的71％左右。近年来，胶质瘤的发病率呈逐年增加的趋势。由于胶质瘤的侵袭性生长方式，与周围的正常脑组织界限模糊，很难通过手术彻底清除。目前针对术后残留的胶质瘤细胞，主要采用放疗、化疗等措施来加以杀灭，然而这些术后残留病变对放疗、化疗等措施表现出显著的抵抗性，导致复发率明显高过其他肿瘤。过去的几十年来，尽管多种新兴的治疗手段不断取得进展，但胶质瘤的预后仍不尽如人意，胶质瘤患者的生存期依然未有明显改善。因此，针对胶质瘤的新且有效治疗策略和治疗药物是亟待解决的难题。
瞬时感受器电位辣椒素4型(TRPV4:transient receptor potential,vanilloidtype 4)属于TRP超家族的一员，为非选择型阳离子通道受体，主要调节钙离子的细胞内外转运。HC-067047是新发现的高效特异的TRPV4阻滞剂，目前作为一种科学研究试剂，主要用于TRPV4的阻断后效应的研究。迄今为止，尚无文献报道TRPV4及其阻滞剂HC-067047在胶质瘤中的作用。
发明内容
有鉴于此，本发明的目的在于考察TRPV4及其阻滞剂HC-067047对胶质瘤的作用，以期开发出新的抗胶质瘤的药物，从而为胶质瘤的临床治疗或日常保健提供更多有效的备选药物。
本发明研究发现，TRPV4在胶质瘤中高表达并促进细胞侵袭，而TRPV4的阻滞剂HC-067047具有抵抗胶质瘤的功能，主要通过阻断small Rho-GTPase家族信号通路，导致胶质瘤细胞的骨架重塑，减少细胞伪足，进而抑制胶质瘤细胞的迁移和侵袭，在进一步的胶质瘤动物模型实验中也证实了上述结论。
因此，本发明提供如下技术方案：
HC-067047在制备抗胶质瘤的药物中的应用，所述HC-067047的结构如下所示：
进一步，所述抗胶质瘤是抑制胶质瘤细胞的迁移和侵袭。
进一步，所述抗胶质瘤是通过阻断small Rho-GTPase家族信号通路引起胶质瘤细胞骨架重塑进而抑制胶质瘤细胞的迁移和侵袭。
本发明的有益效果在于，本发明公开了TRPV4的阻滞剂HC-067047用于制备抗胶质瘤的药物的新用途，不仅扩大了HC-067047的应用范围，提高了其应用价值，给胶质瘤的治疗或保健带来了新的希望，并且，以HC-067047作为先导化合物，通过结构修饰或改造提高活性或降低副作用，还有助于进一步开发新的抗胶质瘤的药物。
附图说明
为了使本发明的目的、技术方案和有益效果更加清楚，本发明提供如下附图进行说明：
图1显示了HC-067047能够有效抑制胶质瘤细胞的迁移和侵袭，而对胶质瘤细胞的增殖无影响，其中A为划痕实验结果(与DMSO组比较，p<0.01)，B为Transwell小室侵袭实验结果(与DMSO组比较，p<0.01)，C为流式细胞术检测细胞周期实验结果，D为CCK-8生长曲线测定结果。
图2显示了HC-067047在胶质瘤细胞中能阻断small Rho-GTPase家族信号通路引起细胞骨架重塑进而抑制细胞的迁移和侵袭，其中A为100nM的HC-067047处理胶质瘤细胞后的免疫荧光染色实验结果，B为不同浓度(50nM、100nM)的HC-067047处理胶质瘤细胞后的免疫蛋白印记实验结果(*p<0.05；#p<0.01)。
图3显示了HC-067047在皮下接种和原位接种的鼠模型上具有抑制胶质瘤迁移和侵袭的功能，其中A为小鼠皮下成瘤的在体图片及肿瘤体积示意图，B为小鼠颅内成瘤磁共振检测结果，C为颅内原位成瘤的小鼠的生活状态以及小鼠生存曲线，D为颅内原位成瘤的死亡小鼠大脑的HE染色结果。
具体实施方式
下面将结合附图对本发明进行详细的描述。实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件进行。
一、HC-067047能够有效抑制胶质瘤细胞的迁移和侵袭，而对胶质瘤细胞的增殖无影响
通过划痕实验与Transwell小室培养细胞穿透实验检测HC-067047对胶质瘤细胞的迁移和侵袭能力的影响。通过流式细胞周期检测和CCK-8实验检测HC-067047对胶质瘤细胞增殖的影响。
划痕实验：用0.25％的胰酶消化原代胶质瘤细胞(U87细胞)，计数适量的细胞数用完全培养基重悬后，加入6孔板中培养，至次日细胞完全贴壁后，PBS洗去漂浮细胞，分为两组(HC-067047组和DMSO组)，每组3个孔，每孔作3条划痕(用10μl移液器套枪头以直尺倚靠作划痕)，HC-067047组每孔加入100nM的HC-067047(以DMSO为溶剂)，DMSO组每孔加入等体积的溶剂DMSO，两组每孔再加入无血清F12/DMEM培养基继续培养，分别于划痕后0小时和24小时于显微镜下拍照，每孔随机选取5个视野，利用Image J分析细胞划痕的愈合面积。结果见图1A，原代胶质瘤细胞经100nM的HC-067047处理24小时后，划痕面积缩小了12％；而DMSO组划痕愈合情况良好，面积缩小了32％。
Transwell小室培养细胞穿透实验：用0.25％的胰酶消化原代胶质瘤细胞(U87细胞)，计数适量的细胞数用无血清培养基重悬，使细胞密度达到1×106个/ml，分为两组(HC-067047组和DMSO组)，每组3个小室(8.0um)，每个预包被基质胶的小室的上室内加入上述细胞悬液200μl，HC-067047组的下室内加入10％血清培养基600μl和100nM的HC-067047(以DMSO为溶剂)，DMSO组的下室内加入10％血清培养基600μl和等体积的溶剂DMSO，24小时后取出小室，用棉签将小室内的基质胶和未穿过的细胞全部擦除，将小室放入4％多聚甲醛内固定15分钟，PBS洗涤2次，甲醇通透10分钟，PBS洗涤2次，结晶紫染色5分钟，PBS洗涤2次，将小室倒扣晾干后，小心取下小室下端的膜，用中性树脂封片，于显微镜下观察，随机采取5个视野的图像，Image-pro-plus 6.0计数细胞。结果见图1B，原代胶质瘤细胞经100nM的HC-067047处理24小时后，穿过Transwell微孔的细胞数量较DMSO组减少了89％。
流式细胞周期检测：将原代胶质瘤细胞(U87细胞)按1×106个细胞/孔培养在6孔板内，次日于细胞贴壁后，将六孔分为两组(HC-067047组和DMSO组)，HC-067047组每孔加入100nM的HC-067047(以DMSO为溶剂)，DMSO组每孔加入等体积的溶剂DMSO，24小时后弃去培养基，PBS洗涤2次，胰酶消化收集细胞，PBS洗涤2次，用4℃预冷的70％乙醇重悬并固定细胞，4℃冷藏过夜，次日取出细胞悬液，离心去固定液，细胞沉淀用PBS洗涤2次，用细胞周期测定试剂盒中的碘化丙啶染色液于室温下避光染色30分钟后，在流式细胞仪上检测细胞周期，分析细胞周期变化。结果见图1C，原代胶质瘤细胞经100nM的HC-067047处理24小时后，相对于DMSO组，细胞周期没有受到明显影响。
CCK-8实验：将原代胶质瘤细胞(U87细胞)分两组(HC-067047组和DMSO组)接种于96孔细胞培养板，每组5个复孔，细胞密度为2×103个细胞/孔，每孔加入含10％胎牛血清的DMEM/F12培养基100μl，于37℃二氧化碳培养箱培养过夜，次日将培养基更换为无血清DMEM/F12培养基，继续培养12小时后，向HC-067047组加入100nM的HC-067047(以DMSO为溶剂)，DMSO组加入等体积的溶剂DMSO，然后分别在0、1、3、5和7天的同一时间点向两组的5个复孔中加入CCK-8(Cell Counting Kit-8)，孵育2小时后测定细胞在波长450nm处的吸光值，利用GRAPHPAD5.0绘制细胞生长曲线。结果见图1D，HC-067047对原代胶质瘤细胞的增殖无明显的抑制作用。
二、HC-067047通过阻断small Rho-GTPase家族信号通路引起胶质瘤细胞骨架重塑进而抑制胶质瘤细胞的迁移和侵袭
通过免疫荧光染色实验检测HC-067047处理胶质瘤细胞后细胞的骨架结构改变。通过western-blot免疫蛋白印记实验检测HC-067047处理胶质瘤细胞后细胞内small Rho-GTPase家族信号通路的变化。
免疫荧光染色：用细胞共聚焦培养皿培养2000个原代胶质瘤细胞(U87细胞)，至正常生长状态后，分别给予100nM的HC-067047(以DMSO为溶剂)和DMSO处理细胞过夜，用4％多聚甲醛固定细胞，0.3％Trixton-100通透细胞，5％BSA封闭半小时，用TRPV4抗体(稀释度为1:100)避光4℃孵育过夜，次日去除抗体孵育液，PBS洗涤3次后，用Cy3标记的二抗(稀释度为1:200)室温下孵育2小时，PBS洗涤3次后，用FITC-phalloidin(稀释度为1:100)室温下避光染色2小时，弃掉染色液，PBS洗涤3次后，用DAPI室温下避光染色5分钟，PBS洗涤3次后，于共聚焦显微镜下观察拍照。结果见图2A，用HC-067047处理原代胶质瘤细胞后，细胞的骨架形态发生明显改变，细胞表面的突起和伪足明显减少，细胞形态由原本的长梭状变成圆而短的形态，这样的结构减少了细胞的迁移和侵袭能力。
Western-blot免疫蛋白印记实验：分别用50nM、100nM的HC-067047(以DMSO为溶剂)和DMSO处理原代胶质瘤细胞(U87细胞)，RIPA细胞裂解液加入磷酸酶抑制剂和蛋白酶抑制剂PMSF混匀后提取细胞总蛋白，测定浓度后电泳、转膜、封闭，分别用RhoA、GTP-RhoA、Cdc42、GTP-Cdc42、Rac1、GTP-Rac1的抗体进行免疫杂交，孵二抗后显示条带，用Image-J软件计算条带灰度值，绘制曲线。结果见图2B，HC-067047处理原代胶质瘤细胞后，细胞内的RhoA、Cdc42和Rac1的活性明显降低，且呈剂量依赖关系。
三、HC-067047在皮下接种和原位接种的鼠模型上具有抑制胶质瘤迁移和侵袭的功能
将4周龄的裸鼠随机分成两组(HC-067047组和生理盐水组)，每组5只，每只裸鼠皮下注射1×105万个原代胶质瘤细胞(U87细胞)，7天后开始给予荷瘤小鼠腹腔注射给药，HC-067047组按0.01mg/kg的剂量给予HC-067047(以生理盐水为溶剂)，生理盐水组给予等体积的溶剂生理盐水，连续给药21天，期间每日观察两组裸鼠的生活状态，每周用游标卡尺测量并记录裸鼠的皮下肿瘤体积大小，将体积大小汇总制作肿瘤体积大小变化曲线。结果见图3A，HC-067047组裸鼠的肿瘤体积大小较生理盐水组明显减小。
将4周龄的裸鼠随机分成两组(HC-067047组和生理盐水组)，每组5只，每只裸鼠颅内原位注射1×105万个原代胶质瘤细胞(U87细胞)，3天后开始给予荷瘤小鼠腹腔注射给药，HC-067047组按0.01mg/kg的剂量给予HC-067047(以生理盐水为溶剂)，生理盐水组给予等体积的溶剂生理盐水，连续给药10天，应用小动物磁共振检测颅内肿瘤大小，每日观察两组裸鼠的生活状态，记录裸鼠死亡时间，至最后1只裸鼠死亡为止，绘制裸鼠生存曲线，然后将每只死亡裸鼠进行解剖，取出整块大脑，肉眼观察外观，再进行HE染色。磁共振检测显示，经HC-067047处理的裸鼠颅内移植瘤的侵袭性生长的体积明显小于生理盐水组(图3B)。颅内移植瘤模型建立后15天，观察裸鼠发现：生理盐水组小鼠出现明显的恶病质状态，体重减轻，活动及进食均减少，而经HC-067047处理的裸鼠生活状态较好，体重正常，活动及进食均正常。生存曲线显示，HC-067047能够明显地延长荷瘤小鼠的生存时间(P<0.01)(图3C)。HE染色结果显示，HC-067047组裸鼠大脑中，胶质瘤向正常脑组织的迁移明显受到抑制(图3D)。
最后说明的是，以上优选实施例仅用以说明本发明的技术方案而非限制，尽管通过上述优选实施例已经对本发明进行了详细的描述，但本领域技术人员应当理解，可以在形式上和细节上对其作出各种各样的改变，而不偏离本发明权利要求书所限定的范围。
Claims (3)
1.HC-067047在制备抗胶质瘤的药物中的应用，其特征在于，所述HC-067047为TRPV4的阻滞剂，所述HC-067047的结构如下所示：
2.根据权利要求1所述的应用，其特征在于，所述抗胶质瘤是抑制胶质瘤细胞的迁移和侵袭。
3.根据权利要求2所述的应用，其特征在于，所述抗胶质瘤是通过阻断small Rho-GTPase家族信号通路引起胶质瘤细胞骨架重塑进而抑制胶质瘤细胞的迁移和侵袭。
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