CN112891376B - 羊油在制备治疗肺癌药品中的应用 - Google Patents
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Abstract
本发明公开了羊油在制备治疗肺癌药品中的应用,以及将羊油与吉非替尼联用,用于促进吉非替尼对非小细胞肺癌细胞的抑制作用的应用。羊油属于食用油脂的一种,本发明首次探明了羊油在肺癌治疗中发挥的积极作用,尤其是针对非小细胞肺癌,为今后肺癌的治疗提供了成本更低廉、效果更优化的药品,具有重要意义;同时,本发明验证了羊油与吉非替尼联用能够进一步促进吉非替尼的癌症抑制效果。
Description
技术领域
本发明涉及食用油脂技术领域,具体涉及羊油在制备治疗肺癌药品中的应用。
背景技术
肺癌是目前世界范围内发病率和致死率最高的癌症种类,其中非小细胞肺癌(non-small-cell lung cancer,NSCLC)约占总数的80%。针对表皮生长因子受体(epidermal growth factor receptor,EGFR)的抑制剂,如gefitinib等对NSCLC具有显著的疗效,然而肺癌细胞的高突变特性使得患者对这些抑制剂往往表现出获得性耐受,导致肺癌患者的5年生存率往往较低。近年来,食疗对疾病的预防和辅助治疗效果受到了广泛关注,而油脂是饮食的重要部分。目前较多研究报道了植物油具有良好的抗癌活性,如核桃油对食管腺癌细胞表现出良好的抗癌活性(Antitumor and antimetastatic effects ofwalnut oil in esophageal adenocarcinoma cells,Clinical Nutrition,2018,37:2166-2171)。橘皮精油能抑制HCT116和HepG2两种癌细胞的增殖和迁移(Extraction of 'Gannanzao' Orange Peel Essential Oil by Response Surface Methodology and itsEffect on Cancer Cell,Molecules,2019,24:499)。
根据文献调研发现羊油具有重要的药用价值,可以作为炮制辅料用于中药材炮制,在哈萨克医学中还用于治疗多种疾病。羊油中含有C16:0、C17:0和C18:0,同时羊油中还含有部分的支链脂肪酸和共轭亚油酸。而含有这些脂肪酸的羊油是否具有抗癌作用未见报道,因此研究羊油在癌症中的作用具有重要的意义。
发明内容
本发明的目的在于提供了一种羊油在制备治疗肺癌药品中的应用,以充分利用羊油的药用价值以及提供更多的药品。
本发明通过以下技术方案来实现上述目的:
本发明要求保护羊油在制备治疗肺癌药品中的应用,羊油属于食用油脂的一种,直接探索羊油应用于肺癌的治疗具有重要的意义。
进一步改进在于,所述应用包括降低肺癌细胞活力、抑制肺癌细胞迁移、加速肺癌细胞凋亡、促进肺癌细胞中活性氧生成、下调肺癌细胞生长代谢相关信号通路活性水平以及移植瘤的生长速度。
进一步改进在于,所述羊油均含有脂肪酸C17:0,可以为草原领头羊、蒙鑫伊族羊、阿勒泰羊、哈萨克羊、苏尼特羊或乌珠穆沁羊等品种,其中草原领头羊和蒙鑫伊族羊的羊油中C17:0含量较高。
进一步改进在于,所述羊油是通过加热熬制、除菌、过滤的工序处理后获得,且羊油的理化特性符合食品安全国家标准。
进一步改进在于,所述肺癌为非小细胞肺癌细胞。
进一步改进在于,所述肺癌为非小细胞肺癌A549、PC-9细胞系。
进一步改进在于,将羊油与吉非替尼联用,用于促进吉非替尼对非小细胞肺癌细胞的抑制作用。
本发明的有益效果在于:羊油属于食用油脂的一种,本发明首次探明了羊油在肺癌治疗中发挥的积极作用,尤其是针对非小细胞肺癌,为今后肺癌的治疗提供了成本更低廉、效果更优化的特医/药品,具有重要意义;同时,本发明验证了羊油与吉非替尼联用能够进一步促进吉非替尼的癌症抑制效果。
附图说明
图1为羊油脂肪酸组成峰图;
图2为羊油对肺癌细胞增殖的影响图;
图3为羊油对肺癌细胞迁移能力的影响图;
图4为羊油对肺癌细胞凋亡的影响图;
图5为羊油对肺癌细胞产生ROS的影响图;
图6为羊油对肺癌细胞中相应信号通路的影响图。
图7为羊油对肺癌细胞移植瘤的抑制影响图。
具体实施方式
下面结合附图对本申请作进一步详细描述,有必要在此指出的是,以下具体实施方式只用于对本申请进行进一步的说明,不能理解为对本申请保护范围的限制,该领域的技术人员可以根据上述申请内容对本申请作出一些非本质的改进和调整。
下述实施例中的实验方法,如无特殊说明均为常规生化方法;下述实施例中所用的试验材料如无特殊说明均为常规生化试剂公司购买所得。其中,实验所用A549(人肺腺癌细胞)、PC-9(非小细胞肺癌细胞)和MRC-5(人肺成纤维细胞)均可在市面购买得到,PC-9/GR(人肺腺癌细胞吉非替尼耐药株)这个细胞市面上没有,由本实验室保存,其他试验材料见表1。以下实施例中的实验均设置三次重复实验,结果取平均值,*表示P<0.05,**表示P<0.01,***表示P<0.001。
羊油可以来源于草原领头羊、蒙鑫伊族羊、阿勒泰羊、哈萨克羊、苏尼特羊或乌珠穆沁羊等品种,加热熬制,并用纱布过滤,4 ℃分装保存,这些油的理化性质,如酸值、碘值等均符合动物油脂食品安全国家标准并与文献报道基本一致。4 ℃分装保存6个月后,12个月后再次检测性状稳定。
表1:实验材料的来源
一、羊油脂肪酸组分检测
取0.06 g油脂于15 mL离心管中,加入8 mL的正己烷,待其充分溶解后,再加入2mL KOH-CH3OH饱和溶液,最后混合均匀,40 ℃静置30 min,待分层后,取上层液于样品瓶中,上机检测。
GC-MS检测条件:SP-2560强极性毛细管气相色谱柱(100 m×0.25 mm,0.2 µm),载气为:氮气;汽化室温度:280 ℃;分流进样,分流比20:1;进样口和检测器温度均为250 ℃,柱前压278.6 kPa;柱温100 ℃保留5 min,以4 ℃/min的速率升温至220 ℃并保留10 min,4 ℃/min升温至240 ℃并保留20 min;进样量1 µL。电离方式:EI源,单四极杆检测器;电子能量:70 eV;离子源温度和接口温度均为250 ℃。
检测结果表明羊油中的脂肪酸主要由C16:0、C18:0、C18:1组成,还有反刍动物中特有的脂肪酸C17:0,同时含有一些支链脂肪酸。不同品种羊来源的羊油检测结果大体相同(如图1所示)。并将羊油用DMSO进行溶解,用于后续实验分析。
二、羊油对肺癌细胞活力的影响
取对数生长期细胞,制备细胞悬液,按照每孔100 μL的细胞悬液,细胞密度为4000-6000个/孔,待细胞贴壁,血清饥饿处理12 h后,进行加药处理,加入用完全培养基配制的不同浓度的羊油,羊油的浓度设置为0、0.5、1.0、1.5、2.0和2.5 mg/mL,置于培养箱培养48 h。MTT实验结果表明这些浓度下的溶剂DMSO对处理细胞均无显著影响,而羊油对非小细胞肺癌细胞A549、PC-9和PC-9/GR细胞均有抑制作用,但对正常人肺成纤维细胞MRC-5的生长无影响(如图2所示)。为了进一步探究羊油对这三种细胞的影响,后续实验选用了1和2mg/mL的羊油浓度。
三、羊油对肺癌细胞迁移速度的影响
首先使用记号笔在六孔板底部画出三条平行且笔直的直线。制备细胞悬液,细胞密度约为1×106个/mL,均匀铺到6孔板中,放入培养箱培养,待细胞密度达到90%时,血清饥饿处理12 h,用20 μL的枪头在细胞表面划直线,保持与6孔板背面的直线垂直。用PBS清洗漂浮细胞,加入提前用含2% FBS的完全培养基配制的羊油浓度为0、1和2 mg/mL的培养液。用荧光倒置显微镜拍摄0 h和48 h同一位置的图片。用Image J软件分析细胞愈合程度,细胞愈合率=(0 h划痕面积-48 h划痕面积)/0 h划痕面积。实验结果表明,羊油显著降低了A549、PC-9和PC-9/GR细胞的迁移能力,并且具有浓度梯度效应(如图3所示)。
四、羊油对肺癌细胞凋亡的影响
取对数生长期细胞,制备成1×105个/mL细胞悬液,接种到6孔板中,呈十字型摇晃,使细胞均匀分布,置于培养箱中培养。待细胞贴壁后,进行12 h的血清饥饿处理,再进行加药处理,将培养液替换成提前配制好的含羊油的完全培养基,羊油的浓度为0、1和2 mg/mL,置于培养箱培养48 h。收集细胞:扔弃6孔板中的培养液,PBS洗涤一遍,胰酶消化,将细胞吹打下来,移至1.5 mL的离心管,3000 rpm,4 ℃离心5 min,弃上清,用预冷的PBS洗涤2次,收集1-5×105个细胞。100 μL的1×Binding Buffer重悬细胞沉淀,实验组分别加5 μL的Annexin V-FITC和PI,用移液枪轻轻混匀。同时设置不加任何染料的阴性对照以及单染Annexin V-FITC和单染PI的阳性对照。室温避光孵育10 min,再加入400 μL的1×BindingBuffer,用移液枪轻轻混匀,置于冰上,1 h内完成检测,同时注意避光。流式细胞术检测结果发现羊油显著促进了A549、PC-9和PC-9/GR细胞的凋亡发生(如图4所示)。
五、羊油对肺癌细胞活性氧水平的影响
取对数生长期细胞,制备约1×106个/mL的细胞悬液,接种到6孔板中,呈十字型摇晃,使细胞均匀分布,置于培养箱培养。待细胞贴壁后,血清饥饿处理12 h。再进行加药处理,将培养液替换成提前配制好的含羊油的完全培养基,羊油的浓度为0、1和2 mg/mL,置于培养箱培养48 h。收集细胞:扔弃6孔板中的培养液,PBS洗涤一遍,胰酶消化,将细胞吹打下来,移至1.5 mL的离心管,3000 rpm,4 ℃离心5 min,弃上清,用预冷的PBS洗涤3次。探针标记:提前将DCFH-DA用以1:1000的比例,用无FBS的培养基进行稀释,注意用锡箔纸包裹进行避光。每个1.5 mL的离心管中加入1 mL稀释好的DCFH-DA重悬收集的细胞,细胞浓度在1×106个/mL以上,2×107个/mL以下,37℃培养箱避光孵育20 min,每孵育3-5 min,上下颠倒混匀,保证探针与细胞充分接触,3000 rpm,4 ℃离心5 min,弃上清,用无血清的培养液洗涤细胞3次,洗去残留的未进入细胞的DCFH-DA。用无FBS的培养基将细胞沉淀重悬,移至流式的管中,上机检测。结果表明羊油促进肺癌细胞中ROS的生成(如图5所示)。
六、羊油对肺癌细胞相关信号通路的影响
取对数生长期细胞,制备约1×106个/mL的细胞悬液,接种到6孔板中,呈十字型摇晃,使细胞均匀分布,置于培养箱培养。待细胞贴壁后,血清饥饿处理12 h。再进行加药处理,将培养液替换成提前配制好的含羊油的完全培养基,羊油的浓度为0、1和2 mg/mL,置于培养箱培养48 h。收集细胞:扔弃6孔板中的培养液,PBS洗涤一遍,胰酶消化,将细胞吹打下来,移至1.5 mL的离心管,3000 rpm,4 ℃离心5 min,弃上清,用预冷的PBS洗涤3次。在收集的细胞沉淀中加适量的3体积的PBS,再加入1体积的4×蛋白上样缓冲液,用振荡涡旋仪使其均匀后,沸水煮沸10 min,待样品冷却后,进行电泳、转膜、孵育一抗和相应的二抗,最后显影。结果表明羊油下调PI3K/Akt信号通路的关键因子Akt和S6K的磷酸化水平,同时下调NF-κB信号通路的主要蛋白因子p65的磷酸化水平,并且影响了葡萄糖转运蛋白GLUT1的表达(如图6所示)。
七、羊油与吉非替尼联用对肺癌细胞的影响
取对数生长期PC-9细胞,制备细胞悬液,按照每孔100 μL的细胞悬液,细胞密度为4000-6000个/孔,待细胞贴壁,血清饥饿处理12 h。将细胞悬液均分成11组,分别进行加药处理,加入不同浓度的羊油以及吉非替尼,培养48h,MTT法检测490nm下的OD值,结果经计算后如下表2:
表2:羊油与吉非替尼联用对肺癌细胞的影响结果表
由表2可以看出,在同一有效浓度下,第1组单独采用吉非替尼时抑制率为36%;加入羊油后,从第2组开始抑制率均得到了提升,虽然吉非替尼的浓度下降,但作用效果被加入的羊油补充,并且整体抑制率高于吉非替尼单独处理组,在第4组中可以看到抑制率达到了最大的58%。随着羊油浓度的进一步增加,由于吉非替尼的浓度过小,从第5组开始抑制效果减弱,但抑制作用均高于第1组。综合分析得出,羊油能促进吉非替尼对PC-9细胞的抑制作用,尤其是在羊油浓度为0.75mg/mL、吉非替尼浓度为3.25nM时,促进效果最为明显。
由于吉非替尼等抗癌药物资源匮乏,价格昂贵,并且存在较多副作用,尤其是对人体消化系统。而羊油为天然成分,来源广,成本低,副作用少,所以将羊油与吉非替尼联用,能降低吉非替尼成分的使用量,从而大幅度降低药物或药品的整体成本,同时保证抗癌作用。
八、羊油对非小细胞肺癌移植瘤的抑制影响
正常培养A549细胞于5% CO2、37℃培养箱,培养基为DMEM(含10% FBS)。收集对数生长期细胞,用PBS重悬并计数,调整至1×107 cells/ml。用Matrigel gel等体积混合后进行裸鼠前肢皮下注射,每只裸鼠注射200微升。裸鼠全部为雌性,6-8周龄,体重20±1g,购买自北京维通利华公司。所有实验均经过中国科学院合肥物质科学研究院实验动物伦理委员会批准,依据中华人民共和国科技部所发布的《关于善待实验动物的指导性意见》对实验动物进行处理。细胞成瘤长至200 mm3时进行分组,分为溶剂对照组、羊油组和顺铂组,每组6只。对照组和羊油组每天给药,顺铂组每周给药两次。剂量为羊油组200 mg/kg,顺铂组5mg/kg。给药方式均为腹腔注射。连续给药23天,定期进行肿瘤体积和裸鼠体重测量,结束后进行肿瘤的剥离和数据收集。从图7中我们可以看出,顺铂组和羊油组均能显著抑制A549细胞移植瘤的生长速度,减少最终肿瘤块的重量和肿瘤体积。同时我们还发现顺铂组处理过程中伴随有裸鼠体重的下降,而羊油处理组和对照无显著差异。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对本发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。
Claims (5)
1.羊油在制备治疗肺癌药品中的应用,其特征在于,所述肺癌为非小细胞肺癌细胞,所述羊油是通过加热熬制、除菌、过滤的工序处理后获得,且羊油中含有脂肪酸C14:0、C15:0、C16:0、C17:0、C17:1、C18:0、C18:1 n9t、C18:1 n9c以及C18:2。
2.根据权利要求1所述的应用,其特征在于:所述应用包括降低肺癌细胞活力、抑制肺癌细胞迁移、加速肺癌细胞凋亡、促进肺癌细胞中活性氧生成、下调肺癌细胞生长代谢相关信号通路活性水平以及移植瘤的生长速度。
3.根据权利要求1所述的应用,其特征在于:所述羊油取自草原领头羊、蒙鑫伊族羊、阿勒泰羊、哈萨克羊、苏尼特羊或乌珠穆沁羊品种。
4.根据权利要求1所述的应用,其特征在于:所述肺癌为非小细胞肺癌A549、PC-9细胞系。
5.根据权利要求1所述的应用,其特征在于:将羊油与吉非替尼联用,用于促进吉非替尼对非小细胞肺癌细胞的抑制作用。
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