CN114053314A - 一种抗肿瘤灵芝孢子油番茄红素复合物制剂的实验研究方法 - Google Patents
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Abstract
本发明公开了一种抗肿瘤灵芝孢子油番茄红素复合物制剂的实验研究方法，包括LZFQ对4种肿瘤细胞的抗增殖作用的实验研究步骤、LZFQ对A549荷瘤小鼠肿瘤质量的影响实验研究步骤以及不同浓度LZFQ对A549细胞凋亡的影响实验研究步骤；本发明的实验研究方式，简便且直观，能够有效且直观的将抗肿瘤灵芝孢子油番茄红素复合物制剂的优势与康肿瘤疗效直观的展示出来，从而得出灵芝孢子油和番茄红素具有优越的抗肿瘤疗效。
Description
技术领域
本发明涉及中药保健品技术领域，具体为一种抗肿瘤灵芝孢子油番茄红素复合物制剂的实验研究方法。
背景技术
灵芝孢子油是通过二氧化碳超临界萃取技术从破壁的灵芝孢子中提取的脂质活性物，富含有三萜类、核苷类及部分多糖类化合物，是灵芝孢子有效成分的集合体。现代药理研究表明，灵芝孢子油具有抗肿瘤、免疫调节、神经调节、降血脂、保肝护肝等活性。在抗肿瘤方面，灵芝孢子油能显著诱导多种肿瘤细胞凋亡，这与其上调Bax同时下调Bcl-2和VEGF的mRNA 表达水平，降低线粒体膜电位和细胞色素c的释放等关键因素有关。
番茄红素是一种天然类胡萝卜素，主要存在于成熟的番茄、西瓜、番石榴、玫瑰果、木瓜和葡萄柚等果实中。人体自身不能合成番茄红素，其摄入量的85％来源于番茄和以番茄为基础的产品。药理研究表明，番茄红素在抗氧化、降脂、抗炎、抗肿瘤等方面具有较好的作用。在抗肿瘤方面，番茄红素与降低前列腺癌、乳腺癌、胃癌、肺癌、结肠癌等癌症的风险有关，其可通过上调PPARγ、Caspase-3蛋白表达，下调NF-κB、COX-2蛋白表达来抑制癌症细胞增殖。
灵芝孢子油番茄红素复合物(LZFQ)由灵芝孢子油和番茄红素按重量6：1搅拌混合而成，该复合物有望通过灵芝孢子油和番茄红素的协同作用发挥更加有效的抗肿瘤活性。本专利旨在研究LZFQ对4种人源肿瘤细胞的活力进行检测，通过裸鼠移植瘤模型研究LZFQ在体内对肝肿瘤的抑制作用，并对LZFQ的抗肿瘤活性机制进行初步探讨，以期为灵芝孢子油和番茄红素类保健食品的研发提供实验依据。
发明内容
本发明的目的在于提供一种抗肿瘤灵芝孢子油番茄红素复合物制剂的实验研究方法，以解决上述背景技术中提出的问题。
为实现上述目的，本发明提供如下技术方案：一种抗肿瘤灵芝孢子油番茄红素复合物制剂的实验研究方法，LZFQ对4种肿瘤细胞的抗增殖作用的实验研究包括以下步骤：
A.在细胞状态处于指数生长期时准备接种，先倾倒培养液，PBS洗涤1次，加入1mL含0.25％胰蛋白酶消化液，消化时长为2min，加入1mL含10％胎牛血清的完全培养基，再通过离心机进行1000r/min的离心处理，处理时长为5min，然后倾倒上清液，将细胞计数稀释至5×103个/mL，并将细胞悬液以每孔200μL接种于96孔板上，置恒温CO2培养箱中培养24h；
B.根据步骤A得出的产物，向其加入一定浓度LZFQ对照药物灵芝孢子油LZBZ，每组设3个复孔，培养24h；
C.再将MTT试剂加入96孔板中，加入量为20μL/孔，培养箱孵育4h；
D.用酶标仪在570nm波长处测吸光值为OD值，并计算细胞抑制率和受试化合物的IC50值。
优选的，LZFQ对A549荷瘤小鼠肿瘤质量的影响实验研究包括以下步骤：
A.分别将6×106个A549细胞皮下接种于Balb/c裸鼠的背部近腋窝皮下；
B.饲养7天后，取小鼠称重，随机分为6组，每组6只；
C.第1组为模型对照组，第2组为LZFQ低剂量组，所用剂量为0.5g/kg，第 3组为LZFQ中剂量组，所用剂量为1.0g/kg，第4组为LZFQ高剂量组，所用剂量为2.0g/kg，第5组为灵芝孢子油组，所用剂量为2.0g/kg，第6组为环磷酰胺组，所用剂量为0.075g/kg；
D.每2天测量一次体重和肿瘤体积，在连续14天的灌胃喂食后，将所有小鼠处死并称重，并采用公式计算肿瘤体积：肿瘤体积＝长×1/2宽2。
F.肿瘤抑制率的计算方式为：肿瘤抑制率＝(1-给药组平均肿瘤重量/对照组平均肿瘤重量)×100％。
优选的，不同浓度LZFQ对A549细胞凋亡的影响实验研究包括以下步骤：
A.将A549细胞接种于6孔板中培养24h，每个孔板中所接种的A549细胞为 4×105/孔，然后分别用DMSO和LZFQ再培养48h，并分别收集培养后的细胞，再用PBS洗涤两次,每次洗涤机的运转功率为2000rpm,洗涤时长为5min； B.然后将细胞重悬于500μL缓冲液中，依次加入Annexin V-FITC，所用剂量为5μL，以及碘化丙啶PI，所用剂量为5μL，室温避光条件下孵育15min 后用流式细胞仪检测细胞凋亡。
与现有技术相比，本发明的有益效果是：本发明的实验研究方式，简便且直观，能够有效且直观的将抗肿瘤灵芝孢子油番茄红素复合物制剂的优势与康肿瘤疗效直观的展示出来；因此本发明的复合物制剂中灵芝孢子油能与番茄红素协同作用，具有传统中医药复方复合性治疗的优势，充分发挥了灵芝孢子油和番茄红素的抗肿瘤疗效，可以协助肿瘤患者提高免疫力，抑制肿瘤手术后肿瘤复发的目的。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
本发明提供一种技术方案：一种抗肿瘤灵芝孢子油番茄红素复合物制剂的实验研究方法：
实施例1：
A.在细胞状态处于指数生长期时准备接种，先倾倒培养液，PBS洗涤1次，加入1mL含0.25％胰蛋白酶消化液，消化时长为2min，加入1mL含10％胎牛血清的完全培养基，再通过离心机进行1000r/min的离心处理，处理时长为5min，然后倾倒上清液，将细胞计数稀释至5×103个/mL，并将细胞悬液以每孔200μL接种于96孔板上，置恒温CO2培养箱中培养24h；
B.根据步骤A得出的产物，向其加入一定浓度LZFQ对照药物灵芝孢子油LZBZ，每组设3个复孔，培养24h；
C.再将MTT试剂加入96孔板中，加入量为20μL/孔，培养箱孵育4h；
D.用酶标仪在570nm波长处测吸光值为OD值，并计算细胞抑制率和受试化合物的IC50值；
表1为LZFQ对4种肿瘤细胞的抗增殖作用(x±s,n＝3)
根据表1可以看出，LZFQ对四种肿瘤细胞均表现出较好的抑制作用，其中对 A549细胞的IC50值在0.49mg/mL，优于灵芝孢子油(IC50值为0.54mg/mL)；因此，我们选取A549细胞对LZFQ进行抗肿瘤机制研究。
实施例2：
A.分别将6×106个A549细胞皮下接种于Balb/c裸鼠的背部近腋窝皮下；
B.饲养7天后，取小鼠称重，随机分为6组，每组6只；
C.第1组为模型对照组，第2组为LZFQ低剂量组，所用剂量为0.5g/kg，第 3组为LZFQ中剂量组，所用剂量为1.0g/kg，第4组为LZFQ高剂量组，所用剂量为2.0g/kg，第5组为灵芝孢子油组，所用剂量为2.0g/kg，第6组为环磷酰胺组，所用剂量为0.075g/kg；
D.每2天测量一次体重和肿瘤体积，在连续14天的灌胃喂食后，将所有小鼠处死并称重，并采用公式计算肿瘤体积：肿瘤体积＝长×1/2宽2；
F.肿瘤抑制率的计算方式为：肿瘤抑制率＝(1-给药组平均肿瘤重量/对照组平均肿瘤重量)×100％；
表2为LZFQ对A549荷瘤小鼠肿瘤质量的影响(x±s,n＝6)
注：与模型对照组相比*p<0.05,**p<0.01；
如表2所示，LZFQ低、中、高剂量组对A549荷瘤小鼠抑瘤率分别为18.67％、37.24％、58.12％，中、高剂量组明显减小了肿瘤体积。阳性对照组肿瘤显著减小，与模型对照组相比差异有统计学意义(p<0.01)；
另外，LZFQ低、中、高剂量给药荷瘤小鼠与模型组实验前后各组体重基本一致，而环磷酰胺组体重有所降低，表明LZFQ对体重没有显著影响。
实施例3：
A.将A549细胞接种于6孔板中培养24h，每个孔板中所接种的A549细胞为 4×105/孔，然后分别用DMSO和LZFQ再培养48h，并分别收集培养后的细胞，再用PBS洗涤两次,每次洗涤机的运转功率为2000rpm,洗涤时长为5min； B.然后将细胞重悬于500μL缓冲液中，依次加入Annexin V-FITC，所用剂量为5μL，以及碘化丙啶PI，所用剂量为5μL，室温避光条件下孵育15min 后用流式细胞仪检测细胞凋亡；
表3为不同浓度LZFQ对A549细胞凋亡的影响(x±s,n＝3)
如表3所示，A549细胞在不同浓度(0.125,0.25,0.5mg/mL)的LZFQ作用48h后，早期(annexin-V+/PI)和晚期(annexin-V+/PI+)凋亡细胞总数分别为11.2％、20.5％和25.3％。结果表明LZFQ可以浓度依赖性的诱导细胞凋亡。
尽管已经示出和描述了本发明的实施例，对于本领域的普通技术人员而言，可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由所附权利要求及其等同物限定。
Claims (3)
1.一种抗肿瘤灵芝孢子油番茄红素复合物制剂的实验研究方法，其特征在于：LZFQ对4种肿瘤细胞的抗增殖作用的实验研究包括以下步骤：
A.在细胞状态处于指数生长期时准备接种，先倾倒培养液，PBS洗涤1次，加入1mL含0.25％胰蛋白酶消化液，消化时长为2min，加入1mL含10％胎牛血清的完全培养基，再通过离心机进行1000r/min的离心处理，处理时长为5min，然后倾倒上清液，将细胞计数稀释至5×103个/mL，并将细胞悬液以每孔200μL接种于96孔板上，置恒温CO2培养箱中培养24h；
B.根据步骤A得出的产物，向其加入一定浓度LZFQ对照药物灵芝孢子油LZBZ，每组设3个复孔，培养24h；
C.再将MTT试剂加入96孔板中，加入量为20μL/孔，培养箱孵育4h；
D.用酶标仪在570nm波长处测吸光值为OD值，并计算细胞抑制率和受试化合物的IC50值。
2.根据权利要求1所述的一种抗肿瘤灵芝孢子油番茄红素复合物制剂的实验研究方法，其特征在于：LZFQ对A549荷瘤小鼠肿瘤质量的影响实验研究包括以下步骤：
A.分别将6×106个A549细胞皮下接种于Balb/c裸鼠的背部近腋窝皮下；
B.饲养7天后，取小鼠称重，随机分为6组，每组6只；
C.第1组为模型对照组，第2组为LZFQ低剂量组，所用剂量为0.5g/kg，第3组为LZFQ中剂量组，所用剂量为1.0g/kg，第4组为LZFQ高剂量组，所用剂量为2.0g/kg，第5组为灵芝孢子油组，所用剂量为2.0g/kg，第6组为环磷酰胺组，所用剂量为0.075g/kg；
D.每2天测量一次体重和肿瘤体积，在连续14天的灌胃喂食后，将所有小鼠处死并称重，并采用公式计算肿瘤体积：肿瘤体积＝长×1/2宽2；
F.肿瘤抑制率的计算方式为：肿瘤抑制率＝(1-给药组平均肿瘤重量/对照组平均肿瘤重量)×100％。
3.根据权利要求1所述的一种抗肿瘤灵芝孢子油番茄红素复合物制剂的实验研究方法，其特征在于：不同浓度LZFQ对A549细胞凋亡的影响实验研究包括以下步骤：
A.将A549细胞接种于6孔板中培养24h，每个孔板中所接种的A549细胞为4×105/孔，然后分别用DMSO和LZFQ再培养48h，并分别收集培养后的细胞，再用PBS洗涤两次,每次洗涤机的运转功率为2000rpm,洗涤时长为5min；
B.然后将细胞重悬于500μL缓冲液中，依次加入Annexin V-FITC，所用剂量为5μL，以及碘化丙啶PI，所用剂量为5μL，室温避光条件下孵育15min后用流式细胞仪检测细胞凋亡。
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