CN116531364B - 扁柏双黄酮在制备用以抑制C-Myc表达的药物中的应用 - Google Patents
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Abstract
本发明涉及医学技术领域，具体地说，涉及扁柏双黄酮在制备用以抑制c‑Myc表达的药物中的应用，其可以抑制细胞内c‑Myc的转录水平，抑制其蛋白质表达，进而发挥抗白血病作用；实验证明，扁柏双黄酮可以抑制白血病细胞增殖，诱导其凋亡，并抑制细胞内c‑Myc的表达。因此，本发明为c‑Myc过表达的血液系统恶性肿瘤患者提供了一种新的治疗选择。
Description
技术领域
本发明涉及医学技术领域，具体地说，涉及扁柏双黄酮在制备用以抑制c-Myc表达的药物中的应用。
背景技术
白血病是一类人类血液系统恶性肿瘤，目前白血病的标准治疗措施包括化疗和干细胞移植。现有的化疗手段与造血干细胞移植在一定程度改善了患者的预后，然而化疗耐药以及造血干细胞供体来源有限仍是制约该病治疗效果的难题。因此，急需新的治疗手段的出现。
c-Myc作为一种转录因子，可与多个基因位点结合并调节这些基因的表达，参与调节细胞生长、分化、凋亡、代谢、DNA损伤、蛋白翻译、免疫应答与血管再生等多种生理过程，并驱动肿瘤发生[Kress TR, Sabo A, Amati B. MYC: connecting selectivetranscriptional control to global RNA production [J]. Nat Rev Cancer, 2015,15(10): 593－607.]。正常生理条件下，c-Myc的表达受到严格调控，当受到生长因子等细胞外刺激时表达升高。当染色体易位或信号通路基因突变等情况发生时，c-Myc会发生不依赖于生长因子刺激的扩增，导致不受控制的细胞增殖和肿瘤产生，尤其是在一些恶性、低分化的肿瘤中[Duffy MJ, O'Grady S, Tang M, Crown J. MYC as a target for cancertreatment. Cancer Treat Rev. 2021 Mar;94:102154.]。大约70%的人类肿瘤中存在c-Myc不受控制和过度表达现象，广泛见于白血病、结直肠癌、乳腺癌、宫颈癌、前列腺癌、睾丸癌和卵巢癌等恶性肿瘤中[Wang C, Zhang J, Yin J, Gan Y, Xu S, Gu Y, Huang W.Alternative approaches to target Myc for cancer treatment. Signal TransductTarget Ther. 2021 Mar 10;6(1):117; Dang CV. MYC on the path to cancer. Cell.2012 Mar 30;149(1):22-35; Meyer N, Penn LZ. Reflecting on 25 years with MYC.Nat Rev Cancer. 2008 Dec;8(12):976-90. ]。因此，C-Myc是肿瘤治疗的潜在靶点：通过下调c-Myc的过表达，可以有效抑制肿瘤细胞的增殖。
扁柏双黄酮是一种双黄酮类药物，可通过靶向TP53、NF-κB、PI3K/Akt、JAK2/STAT3、TNF-α等多种通路诱导细胞分化与凋亡，进而抑制肿瘤细胞生长（Goossens JF,Goossens L, Bailly C. Hinokiflavone and Related C-O-C-Type Biflavonoids asAnti-cancer Compounds: Properties and Mechanism of Action. Nat ProdBioprospect. 2021 Aug;11(4):365-377.）。
然而，目前尚未见扁柏双黄酮调节c-Myc表达的报道，更没有关于扁柏双黄酮应用于抑制白血病细胞c-Myc的报道。
发明内容
为了解决上述问题，本发明提供了扁柏双黄酮在制备用以抑制C-Myc表达的药物中的应用，其不仅可以用于抑制c-Myc的表达，也可以用于治疗伴有c-Myc异常表达的肿瘤的治疗。
为解决以上技术问题，本发明采用的技术方案为：
扁柏双黄酮在制备用以抑制c-Myc表达的药物中的应用；
所述扁柏双黄酮的化学式为：
扁柏双黄酮是一种双黄酮类化合物，存在于多种植物中。
进一步的，扁柏双黄酮在制备用以抑制细胞内c-Myc表达的有效药物中的用途。
进一步的，扁柏双黄酮在制备用以治疗白血病的有效药物中的用途。
本发明采取以上技术方案，与现有技术相比，具有以下优点：
1． 扁柏双黄酮能从mRNA与蛋白水平下调急性髓系白血病细胞系THP-1、U-937以及急性淋巴细胞白血病细胞系Jurkat内c-Myc的表达。
2． 扁柏双黄酮可以诱导THP-1、U-937和Jurkat细胞凋亡，24小时IC50分别为9.68±0.25μmol/L、20.7±1.64μmol/L、17.84±0.68μmol/L。
3． 扁柏双黄酮能以剂量依赖的方式诱导细胞凋亡：伴随着该药浓度增加，凋亡细胞比例也不断增加。同时，它还能诱导THP-1、U-937和Jurkat细胞周期停滞：用该药处理后，THP-1和U-937细胞停滞在S期，Jurkat细胞停滞在G0/G1期。
因此，扁柏双黄酮不仅可抑制c-Myc的表达，也可以用于治疗伴有c-Myc异常表达的肿瘤。
附图说明
图1为本发明实施例中AML细胞系THP-1、U-937和Jurkat经不同浓度扁柏双黄酮处理24小时后的细胞活率结果图；
图2为本发明实施例中扁柏双黄酮诱导THP-1细胞凋亡结果及统计结果图；
图3为本发明实施例中扁柏双黄酮诱导U-937细胞凋亡结果及统计结果图；
图4为本发明实施例中扁柏双黄酮诱导Jurkat细胞凋亡结果及统计结果图；
图5为本发明实施例中扁柏双黄酮处理THP-1、U-937和Jurkat细胞24h后的细胞周期结果及统计结果图；
图6为本发明实施例中扁柏双黄酮处理24h后，THP-1、U-937和Jurkat细胞内c-MycmRNA表达结果图；
图7为本发明实施例中扁柏双黄酮处理24h后，THP-1、U-937和Jurkat细胞内c-Myc蛋白表达结果图；
图8为本发明实施例中扁柏双黄酮处理THP-1、U-937和Jurkat细胞0小时、3小时、6小时、12小时、24小时后c-Myc蛋白表达结果图。
具体实施方式
下对本发明的优选实施例进行说明，应当理解，此处所描述的优选实施例仅用于说明和解释本发明，并不用于限定本发明。
实施例，扁柏双黄酮在制备用以抑制c-Myc表达的药物中的应用；
所述扁柏双黄酮的化学式为：
扁柏双黄酮是一种双黄酮类化合物，存在于多种植物中。
扁柏双黄酮在制备用以抑制细胞内c-Myc的有效药物中的用途。
扁柏双黄酮在制备用以治疗伴有c-Myc异常表达的肿瘤有效药物中的用途。
实验证实，扁柏双黄酮能抑制急性髓系白血病细胞THP-1、U-937和急性淋巴细胞白血病Jurkat细胞活性，诱导这些细胞凋亡与周期阻滞，进而发挥抗白血病作用。
白血病细胞系的培养
从液氮中取出白血病细胞，迅速置于水浴锅内，使其融化。将解冻后的白血病细胞悬液转移到15ml离心管中，补充RPMI-1640培养基至10ml，混匀，离心除去上清液。用含10%胎牛血清的RPMI 1640培养基重悬细胞，将细胞接种至细胞培养瓶中，置于37℃，含5%的CO2培养箱中培养。待其进入对数生长期时收集细胞并计数后，再进行后续实验，包括细胞增殖试验、细胞凋亡试验、细胞周期试验。
CCK8法检测细胞增殖
将THP-1、U-937和Jurkat细胞接种在96孔板中，用不同浓度的扁柏双黄酮处理细胞24小时，每个剂量做3个复孔，1×104个细胞/孔，终体积为100μL/孔，于37℃、5% CO2中分别培养24h，而后每孔内加入10μL CCK-8，于培养结束前4h内向每孔内加入10μL CCK-8，将培养板置于培养箱内孵育2~4小时，用酶标仪测定OD450nm，计算IC50，选择IC50所对应的处理浓度作为后续实验的处理浓度。
流式细胞术检测细胞凋亡
将处于对数生长期的THP-1、U-937和Jurkat细胞接种到6孔板中，加入不同浓度的扁柏双黄酮，同时设立溶剂对照及复孔，于37℃、5% CO2中培养24h后，收集细胞，用结合缓冲液洗涤，分别加入5μL Annexin-V-FITC和PI（Propidium Iodide），避光孵育30min，上流式细胞仪检测，从而确定细胞凋亡情况。
（4）流式细胞术检测细胞周期
将处于对数生长期的THP-1、U-937和Jurkat细胞接种到6孔板中，加入不同浓度的扁柏双黄酮，同时设立溶剂对照及复孔，于37℃、5% CO2中培养24h后，收集细胞，用70%冷乙醇溶液固定过夜，然后加入含RNase的PI溶液，室温避光孵育15min，上流式细胞仪检测，从而确定细胞周期分布情况。
（5）荧光实时定量PCR（RT-qPCR）
将处于对数生长期的THP-1、U-937和Jurkat细胞与不同浓度的扁柏双黄酮一起孵育，24小时后收集细胞，加入TRIzol，提取总RNA，并逆转录成cDNA。基于SYBR Green方法，使用Applied Biosystems 7500 Fast Real-Time PCR系统进行RT-qPCR，使用2-△△Ct方法对细胞内c-Myc mRNA水平进行定量检测。
（6）Western blot
将处于对数生长期的THP-1、U-937和Jurkat细胞与一定浓度的扁柏双黄酮孵育，在合适的时间收集细胞，用PBS洗涤两次，加入RIPA缓冲液，冰上孵育30min，13000rpm离心10min，收集上清，加入2×Loading buffer，于沸水中煮5min，上样，行SDS-PAGE电泳，转膜，封闭，再加入兔抗人C-myc抗体，孵育过夜后洗膜，然后加入HRP标记的羊抗兔IgG二抗，孵育30min，加入化学发光液曝光，采集结果。
实施例一：细胞增殖实验
材料与试剂：
扁柏双黄酮溶液（用DMSO溶解成终浓度为100mM，购自成都植标化纯有限公司）、RPMI 1640培养基（Gibco）、胎牛血清（Hyclone）、CCK-8（索莱宝）、青链霉素、15ml无菌离心管、1.5ml离心管、10μL、200μL和1mL无菌吸头、96孔细胞培养板。
操作步骤：
用含10%胎牛血清的RPMI 1640培养基培养THP-1、U-937和Jurkat细胞；待其进入对数生长期时收集细胞并计数，接种于96孔板中，1×104/孔，100μL/孔；加入不同浓度扁柏双黄酮（0，5，10，20，40，60，80，100μM），以加DMSO组为对照，于37℃、5% CO2细胞培养箱中培养24小时，于结束培养前4h加入CCK-8，10μL/孔，2~4h后于450nm测定吸光度。实验重复3次，每次实验做3个复孔。
实验结果：
图1是白血病细胞系THP-1、U-937和Jurkat经不同浓度扁柏双黄酮处理后的细胞增殖结果图（CCK-8法）。从图中可以看出，伴随着扁柏双黄酮剂量的增加，THP-1、U-937与Jurkat这3种细胞活率均呈下降趋势。统计学结果显示该药在这3种细胞中的IC50分别为THP-1：（9.68±0.25）μM，U-937：（20.7±1.64）μM，Jurkat：（17.84±0.68）μM。
实施例二：细胞凋亡与细胞周期实验
材料与试剂：
扁柏双黄酮溶液（用DMSO溶解成终浓度为100mM，购自成都植标化纯有限公司）、RPMI 1640培养基（Gibco）、胎牛血清（Hyclone）、青链霉素、Annexin-V-FITC/PI细胞凋亡试剂盒（购自BD Bioscience）、PI（含RNase，购自BD Bioscience）、15ml无菌离心管、1．5ml离心管、10μL、200μL和1mL无菌吸头、6孔细胞培养板。
操作步骤：
用含10%胎牛血清的RPMI 1640培养基培养THP-1、U-937和Jurkat细胞；待其进入对数生长期时收集细胞并计数，接种于6孔板中，1×106/孔；加入不同浓度的扁柏双黄酮，以加DMSO组为对照，于37℃、5% CO2的细胞培养箱中培养24h；然后收集细胞，将其分为2份；其中，第一份细胞于1000rpm离心5min，弃上清，在细胞沉淀中加入1ml 1×BindingBuffer, 重悬细胞沉淀，分别加入5μL Annexin-V-FITC和PI，于冰上避光染色15min，用流式细胞仪进行检测；第二份细胞于1000rpm离心5min，弃上清，用PBS洗涤1次，加入70%的冷乙醇溶液，充分重悬细胞，于4℃孵育过夜，1000rpm离心5min，弃上清，再用PBS洗涤2次，然后加入500 μL含RNase的PI溶液，于冰上避光染色15min，用流式细胞仪进行检测。
实验结果：
由图2的结果中可以看出，使用6μM和7μM扁柏双黄酮处理THP-1，24小时凋亡率分别为8.56%±0.87%，11.30%±0.78%，均高于未加药组4.31%±0.23%，48小时THP-1的凋亡率分别为28.47%±5.08%，44.04%±1.60%，均高于48小时未加药对照组；由图3中结果可以看出，使用6μM和10μM扁柏双黄酮处理U-937，24小时凋亡率分别为13.63%±0.72%，19.47%±0.52%，均高于未加药组10．08%±0．27%，48小时凋亡率分别为13.58%±1.26，27.59%±1.86%，均高于未加药组；由图4中结果可以看出，使用15μM和25μM扁柏双黄酮处理Jurkat，24小时凋亡率分别为28.28%±2.81%，41.53%±6.82%，均高于未加药组11.67%±0.97%；48小时凋亡率分别为43.93%±0.68，55.15%±2.08%，也高于未加药组。从以上数据可以看出，扁柏双黄酮以剂量与时间依赖性的方式诱导白血病细胞凋亡。
从图5的结果中可以看出，伴随着扁柏双黄酮浓度的增加，3种细胞的细胞周期也发生了明显变化：处于S期的THP-1和U-937细胞明显增加，处于G2/M期的U-937和Jurkat细胞明显减少，这说明扁柏双黄酮能通过影响细胞周期而抑制细胞增殖。
实施例三：RT-qPCR
材料与试剂：
扁柏双黄酮溶液（用DMSO溶解成终浓度为100mM，购自成都植标化纯有限公司）、RPMI 1640培养基（Gibco）、胎牛血清（Hyclone）、青链霉素、TRIzol、逆转录酶，SYBR染料，引物：c-Myc (forward): 5′-GGGAGGCTATTCTGCCCATTT-3′，c-Myc (reverse): 5′-CGTAGTCGAGGTCATAGTTCCTG-3′，15ml无菌离心管、1．5ml离心管、10μL、200μL和1mL无菌吸头、6孔细胞培养板。
操作步骤：
RNA的提取：将THP-1、U-937和Jurkat细胞与不同浓度的扁柏双黄酮一起孵育，24小时后收集细胞，并从Trizol样品中提取总RNA。
RT-qPCR反应：从1μg总RNA反向转录cDNA。基于SYBR Green方法，使用ABI 7500快速实时荧光定量PCR系统进行RT-qPCR进行定量分析。
实验结果：
从图6的结果中可以看出，伴随着扁柏双黄酮浓度的增加，THP-1、U-937和Jurkat细胞内c-Myc mRNA表达水平显著下降，并明显低于DMSO组（P<0.05）。
实施例四：WB
材料与试剂：
扁柏双黄酮溶液（用DMSO溶解成终浓度为100mM，购自成都植标化纯有限公司）、RPMI 1640培养基（Gibco）、胎牛血清（Hyclone）、青链霉素、PVDF膜，15ml无菌离心管、1．5ml离心管、10μL、200μL和1mL无菌吸头、6孔细胞培养板，兔抗人c-Myc抗体（购自Proteintech公司）、鼠抗人GAPDH抗体（购自Proteintech公司）、HRP标记的山羊抗兔IgG (H+L)二抗（购自ABclonal）、HRP标记的山羊抗鼠IgG(H+L)二抗（购自ABclonal）、3%胎牛血清的PBS、ECL化学发光底物（购自ABclonal）
操作步骤：
用含10%胎牛血清的RPMI 1640培养基培养THP-1、U-937和Jurkat细胞，待其进入对数生长期时，收集细胞并铺板，THP-1、U-937和Jurkat细胞与不同浓度扁柏双黄酮一起孵育，24小时后收集细胞并进行细胞计数，于1000rpm离心5min，弃上清，用PBS洗涤两次，加入RIPA缓冲液，冰上孵育30min，13000rpm离心10min，收集上清，加入2×Loading buffer，于沸水中煮5min，上样，行SDS-PAGE电泳，转移到0.45μM PVDF膜上，用含5%脱脂奶粉的TBST缓冲液封闭，再加入兔抗人c-myc抗体或鼠抗人GAPDH抗体，孵育过夜后洗膜，然后加入HRP标记的羊抗兔IgG二抗或羊抗鼠IgG二抗，孵育30 min，加入化学发光液曝光，应用AmershamImager 600（GE医疗生物科学公司，美国宾夕法尼亚州匹兹堡）采集结果。
实验结果：
从图7中可以看出，与对照组相比，扁柏双黄酮处理后的THP-1、U-937和Jurkat细胞内C-Myc蛋白表达量表达明显下降，并且与用药浓度负相关，这表明扁柏双黄酮能够下调白血病细胞内C-Myc蛋白表达。
从图8中可以看出，在用药3小时后，THP-1、U-937和Jurkat细胞内c-Myc蛋白表达量已发生改变，12小时后c-Myc蛋白表达量已显著低于未用药处理组。
本领域技术人员应该认识到，上述的具体实施方式只是示例性的，是为了使本领域技术人员能够更好的理解本发明内容，不应理解为是对本发明保护范围的限制，只要是根据本发明技术方案所作的改进，均落入本发明的保护范围。
Claims (2)
1.扁柏双黄酮在制备治疗急性髓系白血病和/或急性淋巴细胞白血病的药物中的应用。
2.如权利要求1所述的应用，其特征在于：扁柏双黄酮用以抑制c-Myc表达。
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