CN116509877B - 一种化合物t-mas在制备治疗乳腺癌药物中的应用 - Google Patents
一种化合物t-mas在制备治疗乳腺癌药物中的应用 Download PDFInfo
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Abstract
本发明首次揭示了化合物T‑MAS具有治疗乳腺癌的作用，可以用在制备治疗乳腺癌药物中的应用；该药物单独使用有效果，也可以联合其他抗肿瘤药物，例如紫杉醇、卡铂共同用在制备治疗乳腺癌药物中，能够抑制肿瘤细胞增殖、促进肿瘤细胞凋亡，具有广泛的临床意义。
Description
技术领域
本发明涉及医药领域，具体涉及一种化合物T-MAS在制备治疗乳腺癌药物中的应用。
背景技术
哺乳动物卵母细胞从开始形成到生长成熟，和减数分裂过程是密不可分的。在体内，卵原细胞会停止有丝分裂、启动并进入减数分裂而转变为卵母细胞，进而被周围的体细胞包围形成卵泡。卵母细胞减数分裂随之停滞于第一次减数分裂的前期，在卵泡中通过漫长的生长发育过程后,在一定的刺激条件下才恢复减数分裂而成熟。因此减数分裂恢复的调控研究对于了解卵母细胞的形成、生长发育和成熟都是至关重妥的。促减数分裂甾醇(meiosis-activatingsteorl,MAS)能诱导体外培养的卵母细胞恢复减数分裂成熟，因此自从MAS被发现以来就引起了研究者的注意，但是对于其具体的作用和机制还在争议之中。
MAS有两种，即FF-MAS(follicle fluid-meiosisactivatingsterol)和-TMAS(testis-meiosisacitviatingsetorl)，都是胆固醇生物合成途径中的中间产物，FF-MAS由羊毛甾醇14α-脱甲基酶(CY5P1)合成,然后由△14-还原酶进步转化为T-MAS，在卵巢中土要积累FF-MAS。抑制CYP51酶的抑制剂，如azalanstat(RS-21607)和RS-21745等,均能抑制MAS的合成，降低MAS的积累；而抑制△14-还原酶的抑制剂，如AY9944-A-7等，能抑制FF-MAS向-TMAS的转化而造成FF-MAS的积累；还有一些物质,如制霉菌素，能阻止MAS向下游的代谢而造成其积累。
乳腺癌是女性最常见的恶性肿瘤，是女性癌症死亡的主要原因。2020年全球最新癌症负担数据显示，乳腺癌已成为全球发病率最高的癌症。乳腺癌可根据雌激素受体(estrogen receptor，ER)、孕激素受体(progesterone receptor，PR)、人表皮生长因子受体2(human epidermal growth factor receptor 2，HER2)的表达情况及Ki-67增殖指数进行分子分型，其中HER2阳性型乳腺癌恶性程度高，易转移，易复发，预后差，是乳腺癌治疗的重点和难点。
由此可见，现有技术中，并未发现采用甾体激素类药物治疗乳腺癌较为成功的范例。
发明内容
为克服现有技术缺陷，本发明提供了以下技术方案：
本发明提供了化合物T-MAS在制备治疗乳腺癌药物中的应用；
进一步的，所述化合物T-MAS如式Ⅰ所示：
进一步的，所述化合物可以联合抗肿瘤药物在制备治疗乳腺癌药物中的应用；
进一步的，所述抗肿瘤药物是紫杉醇；
进一步的，所述抗肿瘤药物是卡铂。
附图说明
图1.MDA-MB-231细胞系不同浓度T-MAS处理下UPR通路Western Blot实验结果
图2.MDA-MB-231细胞系不同浓度T-MAS处理下Thioflavin T染色实验结果
图3.MDA-MB-231细胞系在T-MAS以及4-PBA处理下的流式凋亡实验结果
图4.MDA-MB-231细胞系中紫杉醇、卡铂和T-MAS处理下的流式凋亡实验结果
图5.T-MAS对化疗药物卡铂的增敏实验。
有益效果
本发明首次揭示了化合物T-MAS具有治疗乳腺癌的作用，可以用在制备治疗乳腺癌药物中的应用；该药物单独使用有效果，也可以联合其他抗肿瘤药物，例如紫杉醇、卡铂共同用在制备治疗乳腺癌药物中，能够抑制肿瘤细胞增殖、促进肿瘤细胞凋亡，具有广泛的临床意义。
具体实施方式
实施例1Western Blot实验
如图1所示，根据实验需要，预先处理细胞，对于T-MAS，选取0，1μg/ml,2ug/ml,4μg/ml处理细胞2d后收集蛋白；配置SDS-PAGE凝胶；拔出上样梳，根据蛋白浓度调平蛋白量后利用移液枪上样；向电泳槽内倒入1X电泳缓冲液，接通电源，75V恒压电泳，待蛋白Marker分层后，调整电压为120V，待溴酚蓝跑到适当位置后停止电泳；激活PVDF膜，250mA横流转膜，根据蛋白分子量大小设置合适的转膜时间；转膜结束后，用5％脱脂奶粉封闭，室温封闭1小时；封闭结束后将条带放入对应的一抗内，4℃孵育过夜；次日利用TBST洗膜，随后加入对应的二抗，室温孵育1h；TBST洗膜，配置曝光液，使用成像仪显影。图1中，分别利用不含T-MAS培液，以及含1μg/ml,2μg/ml和4μg/mlT-MAS的培液处理细胞，Western Blot结果提示T-MAS可诱导内质网应激，并激活PERK/p-eIF2α/ATF-4/CHOP通路。
实施例2Thioflavin T染色实验
种植合适数目细胞于细胞爬片，24h后于显微镜下观察，待细胞贴壁后根据实验需要对细胞进行处理，对于T-MAS，选择2μg/ml，4μg/ml处理细胞2d；待细胞预处理结束后，使用多聚甲醛常温固定细胞1h；PBS慢摇清洗爬片；配置10μM浓度Thioflavin T，避光，室温孵育10min；PBS慢摇清洗爬片；DAPI复染，封片；于荧光显微镜下观察，拍照。Thiflavin T是一种细胞膜通透性苯并噻唑染料，与堆叠的蛋白质β折叠结合可导致最大激发波长位移和发射增强。其最大激发波长为： (游离)；(结合)；最大发射波长为：(游离)；(结合)。Thiflavin T染色后，共聚焦显微镜检测荧光阳性细胞比例以及荧光强度可反应细胞内未折叠蛋白水平。图2中，分别利用不含T-MAS培液，以及含2μg/ml和4μg/mlT-MAS的培液处理细胞，图2中，小圆圈内均表示Thioflavin T染色的荧光斑点。Thioflavin T染色实验显示T-MAS处理后细胞内未折叠蛋白含量上升，结合图1结果提示T-MAS可促进未折叠蛋白堆积，进而诱导未折叠蛋白反应。
实施例3：流式凋亡实验
如图3所示，提前利用不同浓度药物处理细胞，对于T-MAS，选择4μg/ml处理细胞2d；对于4-PBA，选择1mM 4-PBA预处理细胞3h后更换正常培液。
利用Annexin V-FITC/PI Apoptosis Detection Kit(Vazyme)检测细胞凋亡：利用不含EDTA的胰酶消化细胞，1000rpm，4℃离心5min，弃上清；用预冷的PBS洗涤细胞两次，每次1000rpm，4℃离心5min；加入100μl1X Binding Buffer，轻轻吹匀至单细胞悬液；加入5μl Annexin V-FITC和5ul PI Staining Solution，轻轻吹匀；避光，室温孵育10min，加入400μl1X Binding Buffer，轻轻混匀。染色后样品在1h内用流式细胞仪检测。流式细胞仪分析：流式细胞仪激发波长为488nm；FITC的绿色荧光在FL1通道检测；PI的红色荧光在FL2或FL3通道检测，建议使用FL3，每个样本采集10,000events。用FlowJo等软件进行数据分析，FL1为横坐标，FL3为纵坐标，根据FITC和PI荧光值确定两荧光参数阴阳界限，划定十字门。典型的实验中细胞可分为三个亚群：活细胞为双阴性(Annexin V-FITC-/PI-)；早期凋亡细胞为Annexin V-FITC单阳性(Annexin V-FITC+/PI-)；晚期凋亡细胞为Annexin V-FITC和PI双阳性(Annexin V-FITC+/PI+)。
通过流式技术发现T-MAS可诱导细胞凋亡，且内质网应激抑制剂4-PBA可抑制由T-MAS诱导的细胞凋亡，提示T-MAS通过内质网应激途径诱导细胞凋亡。
实验例4：增敏化疗药物的流式凋亡实验
如图4所示，提前利用不同浓度药物处理细胞，对于T-MAS，选择4μg/ml处理细胞2d；紫杉醇(PAX)，选择150nM处理细胞2d；卡铂(CBP)，选择100μM处理细胞2d；联合应用T-MAS与化疗药物紫杉醇或卡铂，通过与实验例3同样的方法检测细胞凋亡，发现T-MAS可显著提高由紫杉醇及卡铂诱导的细胞凋亡比例，提示T-MAS具有化疗增敏效果。
实施例5：类器官死亡/存活分析
如图5所示，乳腺癌类器官分别提前于不含药物培养基、含4μg/ml T-MAS培养基、含300μM卡铂(CBP)培养基以及含4μg/ml T-MAS和300μMCBP的培养基中培养1周。小心吸去培养基，加入40μl(对于96孔板)NexcelomViaStainAO/PI染液(Nexcelom，CS2-0106)对类器官进行染色(室温，避光孵育20min)。吖啶橙(AO)是一种荧光色素，与细胞内DNA结合后产生绿色荧光。碘化丙啶(PI)不能通过活细胞膜，但可穿过破损细胞膜使死亡细胞显红色荧光。利用共聚焦显微镜拍摄图像，并使用Z-Stack功能拍摄多层面荧光，拼接多个不同视野以获得完整图像。类器官存活和死亡细胞的定量是通过计算每种荧光染料的面积与总细胞面积之比实现的。
图5a中，于低倍视野下拍摄类器官荧光，通过ImageJ分析各荧光染料所占面积，利用荧光染料面积之比反应类器官死亡/存活情况。图5b为高倍镜视野下，具有代表性的类器官荧光染色结果。总之，类器官死亡/存活实验结果提示，乳腺癌类器官中单独使用4μg/mlT-MAS尚不足以导致乳腺癌类器官明显死亡，然而联合应用T-MAS和化疗药物卡铂可显著诱导类器官死亡，证明T-MAS在乳腺癌类器官中具有化疗增敏效果。
Claims (5)
1.化合物T-MAS在制备治疗乳腺癌药物中的应用。
2.根据权利要求1所述的应用，其特征在于，所述化合物T-MAS如式Ⅰ所示：
3.根据权利要求1或2所述的应用，其特征在于，所述化合物联合抗肿瘤药物用在制备治疗乳腺癌的药物中。
4.根据权利要求3所述的应用，其特征在于，所述抗肿瘤药物是紫杉醇。
5.根据权利要求3所述的应用，其特征在于，所述抗肿瘤药物是卡铂。
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