CN113244214A - 黄酮类衍生物cpu-008在制备抗淋巴瘤药物中的应用 - Google Patents
黄酮类衍生物cpu-008在制备抗淋巴瘤药物中的应用 Download PDFInfo
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Abstract
本申请涉及黄酮类衍生物CPU‑008(7‑{4‑[双‑(2‑羟基‑乙基)‑氨基]‑丁氧基}‑5‑羟基‑8‑甲氧基‑2‑苯基‑苯并吡喃‑4‑酮)在制备抗淋巴瘤药物中的应用。CPU‑008可以抑制淋巴瘤细胞的生长活力，可以通过降低淋巴瘤细胞的线粒体膜电位，引起淋巴瘤细胞的凋亡。
Description
技术领域
本发明属于抗肿瘤药物领域，具体涉及一种黄酮类衍生物CPU-008在制备抗淋巴瘤药物中的应用。
背景技术
淋巴瘤是起源于淋巴造血系统的恶性肿瘤，主要表现为无痛性淋巴结肿大，肝脾肿大，全身各组织器官均可受累，伴发热、盗汗、消瘦、瘙痒等全身症状。恶性淋巴瘤是具有相当异质性的一大类肿瘤，虽然好发于淋巴结，但是由于淋巴系统的分布特点，使得淋巴瘤属于全身性疾病，几乎可以侵犯到全身任何组织和器官。
淋巴瘤具有高度异质性，治疗效果差别很大，不同病理类型和分期的淋巴瘤无论从治疗强度和预后上都存在很大差别，但具体患者还应根据患者实际情况具体分析。因为淋巴分布在动物体的身体各处，参与各种各样的生理反应，因此淋巴癌细胞更易转移且难以治疗。目前放疗化疗是主要治疗手段，但化疗引起的毒副作用给患者造成了极大的痛苦，甚至影响治疗依从性和效果。因而能寻找到有效、毒副作用较低的天然抗肿瘤药物意义重大，也是临床治疗的迫切需求。
发明内容
为改善上述技术问题，本发明提供了一种黄酮类衍生物CPU-008在制备抗淋巴瘤药物中的应用，
其中，CPU-008的化学名称为7-{4-[双-(2-羟基-乙基)-氨基]-丁氧基}-5-羟基-8-甲氧基-2-苯基-苯并吡喃-4-酮。
根据本发明的实施方案，所述淋巴瘤可以为非霍奇金淋巴瘤或霍奇金淋巴瘤。
根据本发明的实施方案，所述CPU-008可以抑制淋巴瘤细胞的生长活力。
根据本发明的实施方案，所述CPU-008可以诱导淋巴瘤细胞发生凋亡。
根据本发明的实施方案，所述CPU-008可以通过降低淋巴瘤细胞的线粒体膜电位，引起淋巴瘤细胞的凋亡。
有益效果
本发明研究意外发现黄酮类衍生物CPU-008能显著抑制人淋巴瘤Hut-102细胞的生长活力，进一步通过细胞凋亡实验发现，CPU-008能显著促进Hut-102细胞发生早期及晚期凋亡，表明CPU-008可用于淋巴瘤的治疗，具有开发抗淋巴瘤药物的应用前景。
附图说明
图1示出了不同浓度CPU-008对Hut-102的活力抑制率折线图。
图2示出了CPU-008对Hut-102的凋亡诱导作用随时间的变化情况。
图3示出了不同浓度CPU-008对Hut-102的线粒体膜电位的影响。
具体实施方式
下文将结合具体实施例对本发明的技术方案做更进一步的详细说明。应当理解，下列实施例仅为示例性地说明和解释本发明，而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。
除非另有说明，以下实施例中使用的原料和试剂均为市售商品，或者可以通过已知方法制备。
实验材料
1.1药物
CPU-008(7-{4-[双-(2-羟基-乙基)-氨基]-丁氧基}-5-羟基-8-甲氧基-2-苯基-苯并吡喃-4-酮，C24H29NO7，分子量：443.49)由中国药科大学提供，外观为淡黄色粉末，纯度>99％。通过二甲亚砜(DMSO)将药物粉末配制为0.02M的储备液，置于-20℃保存。临用前用含10％胎牛血清的RPMI-1640培养液配成所需浓度。
1.2细胞株
人TCL细胞株Hut-102购自上海中国科学院细胞所。细胞用含100U/ml青霉素、100mg/ml链霉素和10％胎牛血清的RPMI1640培养液培养。
1.3试剂
1.3.1细胞培养及实验试剂
(1)培养液：RPMI-1640培养基，购自美国GIBCO公司。取RPMI-1640粉末10.39g溶于1000mL灭菌三蒸水中，并加入2.0g NaHCO3，用1M盐酸调pH值至7.0，圆筒式过滤器过滤除菌、分装，4℃冰箱保存。使用前加入100U/ml的青霉素和100U/ml的链霉素。
(2)胎牛血清：美国GIBCO公司产品。经56℃水浴灭活30min，分装并保存于-20℃低温冰箱中。
(3)磷酸盐(PBS)缓冲液：称取NaCl 8.0g、KCl 0.20g、Na2HPO4·H2O 1.56g、KH2PO42.0g，溶于1000mL三蒸水中，高压灭菌，4℃冰箱保存。
(4)试剂盒：Cell Counting Kit-8(CCK8)试剂盒，购买于上海翊圣生物公司；Annexin V/PI双染细胞凋亡检测试剂盒，购买自南京诺唯赞生物科技有限公司；JC-1线粒体膜电位试剂盒，购买于南京凯基生物科技发展有限公司。
1.4实验仪器
(1)YJ-875型医用净化工作台：苏州净化设备厂生产。
(2)3111型水套式CO2培养箱：美国Thermo electron公司产品。
(3)电子天平：北京赛多利斯仪器系统有限公司产品。
(4)QIUJING血细胞计数版：中国上海。
(5)LD4-2普通离心机：北京医用离心机厂产品。
(6)YG-2000型圆筒式过滤器：绍兴市卫星医疗设备制造公司产品。
(7)KY-111型微型空气压缩机：绍兴市卫星医疗设备制造公司产品。
(8)5417R型台式冷冻高速离心机：德国Eppendorf公司产品。
(9)Research型单道可调移液器：德国Eppendorf公司产品。
(10)Varioskan全波长酶标仪：美国Thermo公司。
(11)流式细胞仪：美国Becton Dickinson公司。
实施例1 CPU-008对TCL细胞株Hut-102细胞活力的抑制作用
细胞生长活力检测
CCK-8是一种基于水溶性四唑盐(WST-8)的高灵敏度试剂盒，常用于检测细胞的增殖。其作用原理是WST-8在电子耦合试剂存在下可以被线粒体内的脱氢酶还原生成橙黄色的甲臜染料。使用酶标仪在450nm波长处测定其OD值，通过比色可以动态地量化活细胞的数量，从而间接反映活细胞的数量。
将处于对数生长期的人TCL细胞株Hut-102按照一定的细胞密度培养在96孔酶标板内，每孔内的细胞体积为100μL，并加入100μL不同浓度的CPU-008，继续置于孵箱培养12和24h后，每孔加入20μL CCK-8溶液。继续孵育1h后使用酶标仪在450nM波长检测其吸光度。药物对细胞的抑制率计算如下：
抑制率％＝(1－给药组细胞平均吸光值/对照组细胞平均吸光值)×100％
利用Graphpad Prism 7.0软件计算可得CPU-008对Hut-102的半数抑制浓度(IC50)。
实验结果
通过CCK8实验测定12和24h内天然产物衍生物CPU-008 Hut-102细胞活力的抑制作用。结果表明，CPU-008能显著抑制Hut-102的细胞活力，且呈时间和浓度依赖。CPU-008对Hut-102细胞12、24h的半数抑制浓度分别为4.034±0.429μM，2.567±0.483μM。该结果说明CPU-008具有对Hut-102细胞的活力抑制作用(图1)。
实施例2 CPU-008诱导AML和TCL细胞凋亡
细胞凋亡检测
Annexin V/PI双染用于检测细胞凋亡。正常细胞中，磷脂酰丝氨酸(Phosphatidylserine，PS)分布在细胞膜的磷脂双分子层内侧。细胞凋亡时，PS外翻而暴露出来与Annexin V结合。凋亡早期细胞只会有PS外翻的现象，PI染料无法透过处于凋亡早期以及正常细胞的细胞膜，所以流式检测时凋亡早期的细胞处于右下区域(Annexin V+，PI-)；而晚期凋亡的细胞则由于PI可以透过细胞膜进入核而将DNA着色，处于右上部分(Annexin V+，PI+)。收集不同浓度药物作用后的K562和KU-812细胞，离心去上清，PBS洗一遍，100μL Binding buffer重悬细胞，加入Annexin V以及PI染料各5μL，室温避光孵育10min，PBS洗涤一次，400μL Binding buffer重悬，用流式细胞仪检测。
实验结果
以Hut-102细胞为研究对象，在6μM浓度的CPU-008作用细胞0、3、6、12h时间后，利用Annexin V/PI双染法检测细胞凋亡。结果显示(图2)，与0h组相比，CPU-008能显著诱导Hut-102的凋亡，并呈时间依赖性。该结果说明药物对细胞具有诱导凋亡作用。
实施例3 CPU-008对人TCL细胞株Hut-102细胞线粒体膜电位的影响
线粒体膜电位的测定
JC-1也称CBIC2(3)或5,5′,6,6′-四氯-1,1′,3,3′-四乙基苯并咪唑羰花青碘化物，是一种阳离子脂质荧光染料，有单体和多聚体两种状态，是一种可用于指示线粒体膜电位(mitochondrial membrane potential，△Ψm)的理想荧光探针。在△Ψm较高时，JC-1聚集在线粒体的基质中，形成聚合物，可以产生红色荧光；在△Ψm较低时，JC-1不能聚集在线粒体的基质中，此时JC-1为单体，可以产生绿色荧光。这样就可以非常方便地通过荧光颜色的转变来检测线粒体膜电位的变化。常用红绿荧光的相对比例来衡量线粒体去极化的比例。
将处于对数生长期的Hut-102按照一定的细胞密度培养在6孔细胞培养板中，CPU-008作用于Hut-102孵育一定的时间后收集细胞。按照试剂盒的说明，每组加入1000μL缓冲液和2μL JC-1试剂并混匀，在37℃，5％CO2的培养箱中孵育30min后，以2000rpm离心5min,弃去上清并用PBS清洗。流式细胞仪上机分析。
实验结果
通过JC-1实验测定12h内不同浓度的CPU-008(0，2，4，6μM)对Hut-102线粒体膜电位的变化。实验结果显示(图3)，CPU-008能显著降低Hut-102的线粒体膜电位并呈现浓度依赖性，该结果表明CPU-008影响淋巴肿瘤的线粒体膜电位，导致膜通透性的改变从而引起线粒体内膜外侧的细胞色素C释放进入细胞质并触发Caspase家族介导的细胞凋亡。
以上，对本发明的实施方式进行了说明。但是，本发明不限定于上述实施方式。凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
Claims (5)
1.一种黄酮类衍生物CPU-008在制备抗淋巴瘤药物中的应用，
其中，CPU-008的化学名称为7-{4-[双-(2-羟基-乙基)-氨基]-丁氧基}-5-羟基-8-甲氧基-2-苯基-苯并吡喃-4-酮。
2.根据权利要求1所述的应用，其特征在于，所述淋巴瘤为非霍奇金淋巴瘤或霍奇金淋巴瘤。
3.根据权利要求1或2所述的应用，其特征在于，所述CPU-008可以抑制淋巴瘤细胞的生长活力。
4.根据权利要求1或2所述的应用，其特征在于，所述CPU-008可以诱导淋巴瘤细胞发生凋亡。
5.根据权利要求1或2所述的应用，其特征在于，所述CPU-008可以通过降低淋巴瘤细胞的线粒体膜电位，引起淋巴瘤细胞的凋亡。
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