CN108998415A - 蟛蜞菊内酯在制备治疗肺癌产品中应用的研究方法 - Google Patents
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Abstract
本发明属于生物科技领域，具体涉及一种蟛蜞菊内酯在制备治疗肺癌产品中应用的研究方法。本发明研究了蟛蜞菊内酯对非小细胞肺癌A549细胞活性、周期和凋亡的影响，以及对细胞p‑Caspase3、p53、p‑STAT1、STAT1基因表达的干预作用，探讨蟛蜞菊内酯对A549细胞的抗肿瘤作用及其机制，提供一种蟛蜞菊内酯在制备治疗肺癌产品中应用的研究方法，为今后中药治疗非小细胞肺癌的机制研究提供理论基础。本发明包括细胞培养的步骤、用CCK‑8法检测细胞增殖抑制率的步骤、用碘化丙啶及Annexin‑PI染色检测细胞周期及凋亡的步骤以及用RT‑PCR及Western blot检测细胞基因转录及翻译的步骤。
Description
技术领域
本发明属于生物科技领域，具体涉及一种蟛蜞菊内酯在制备治疗肺癌产品中应用的研究方法。
背景技术
非小细胞肺癌是最常见的肺癌类型，目前已居我国恶性肿瘤死亡率之首。因此，采取积极有效的措施防治非小细胞肺癌迫在眉睫。近年来，临床上主要以西药治疗非小细胞肺癌，而大多数西药长期服用易产生较强依赖性和严重毒副作用，因此研究中药治疗非小细胞肺癌的重要性日益凸显。
自1956年首次从金盏蟛蜞菊中分离纯化得到蟛蜞菊内酯后，人们一直将其视为潜在药物单体进行研究，其抗癌功效也已渐渐被发现。研究表明，蟛蜞菊内酯能有效抑制前列腺癌、乳腺癌、垂体腺瘤等多种癌细胞活性，杀伤肿瘤细胞并诱导其凋亡。然而蟛蜞菊内酯对肺癌细胞的抑制活性和诱导凋亡机制尚不明确。
发明内容
本发明研究了蟛蜞菊内酯对非小细胞肺癌A549细胞活性、周期和凋亡的影响，以及对细胞p-Caspase3、p53、p-STAT1、STAT1基因表达的干预作用，提供一种蟛蜞菊内酯在制备治疗肺癌产品中应用的研究方法，研究蟛蜞菊内酯对A549细胞的抗肿瘤作用及其机制，为今后中药治疗非小细胞肺癌的机制研究提供理论基础。
本发明提供的蟛蜞菊内酯在制备治疗肺癌产品中应用的研究方法，包括以下步骤：
细胞培养的步骤：复苏A549细胞后，每2～4天继代培养一次；
CCK-8法检测细胞增殖抑制率的步骤：在孔板上接种生长期细胞，之后分别用10、20、30μmol/L蟛蜞菊内酯作用细胞12、24、36h，每孔加CCK-8孵育，之后采集各孔光密度值；
碘化丙啶及Annexin-PI染色检测细胞周期及凋亡的步骤：在孔板上接种生长期细胞后分为对照组和蟛蜞菊内酯组，对照组每孔添加新鲜培养液，蟛蜞菊内酯组每孔加含蟛蜞菊内酯的新鲜培养液，之后收集细胞，一部分样本乙醇固定过夜，RNase A水浴，PI孵育，流式检测周期；剩余样本结合缓冲液重悬，取悬液于流式管中，Annexin V-FITC和PI孵育后，流式检测凋亡；
RT-PCR及Western blot检测细胞基因转录及翻译的步骤：细胞接种、培养、分对照组和蟛蜞菊内酯组，对照组每孔添加新鲜培养液，蟛蜞菊内酯组每孔加含蟛蜞菊内酯的新鲜培养液，之后收集细胞，一部分样本提总RNA并反转录，cDNA扩增，95℃预变性3min，95℃变性7s，56℃退火延伸30s，45个循环，凝胶成像；剩余样本RIPA提取总蛋白，SDS-PAGE后，以p-Caspase3、p53、p-STAT1、STAT1、GAPDH为一抗，孵育过夜，室温孵育二抗，化学发光仪曝光条带。作为本发明的进一步优化，细胞培养的步骤中，A549细胞复苏后在含10％FBS的RPMI-1640培养基中培养。
作为本发明的进一步优化，CCK-8法检测细胞增殖抑制率的步骤中，各孔光密度值在550nm波长处采集。
作为本发明的进一步优化，碘化丙啶及Annexin-PI染色检测细胞周期及凋亡的步骤中以及RT-PCR及Western blot检测细胞基因转录及翻译的步骤中，蟛蜞菊内酯的终浓度为30μmol/L。
本发明提供的蟛蜞菊内酯在制备治疗肺癌产品中应用的研究方法，为今后中药治疗非小细胞肺癌的机制研究提供理论基础。
附图说明
图1是蟛蜞菊内酯对A549细胞增殖的影响图；
图2是蟛蜞菊内酯对A549细胞周期的影响图；
图3是蟛蜞菊内酯对A549细胞凋亡的影响图；
图4是蟛蜞菊内酯对A549细胞凋亡相关蛋白表达的影响图；
图5是蟛蜞菊内酯对A549细胞基因的转录翻译效果图。
具体实施方式
以下结合具体实施例对本发明作详细说明。
本实施例包括以下步骤：
材料准备步骤，材料包括
人非小细胞肺癌A549细胞，购自中国科学院细胞库；
蟛蜞菊内酯，购自中国标准物质网；
RPMI-1640、FBS，购自Life公司；
p-Caspase3抗体、p53抗体、p-STAT1抗体、STAT1抗体，购自Abcam公司；
CCK-8试剂盒、AnnexinV/PI试剂盒、反转录试剂盒、RIPA裂解液，购自碧云天。
细胞培养的步骤，包括复苏A549细胞，含10％FBS的RPMI-1640培养，条件37℃、5％CO2，每2～4天，优选3天继代一次；
CCK-8法检测细胞增殖抑制率的步骤，如下：
(1)在96孔板上每孔接种100μL 2×104个对数生长期细胞；
(2)24h后分别用10、20、30μmol/L蟛蜞菊内酯作用细胞12、24、36h；
(3)每孔加10μL CCK-8孵育2h；
(4)最后在550nm波长处采集各孔光密度值。
如图1所示，采用该步骤能测得不同浓度的蟛蜞菊内酯作用于A549细胞不同时间对细胞增殖的影响，发现蟛蜞菊内酯在各浓度与时间观察点均对细胞具有抑制增殖效果，随着作用时间加长，蟛蜞菊内酯对细胞的抑制作用逐渐上升，各时间观察点间差异显著，并呈剂量、时间依赖性，初步结果表明蟛蜞菊内酯可能是A549细胞增殖的一种有效抑制剂，对非小细胞肺癌的治疗具有积极作用。由于蟛蜞菊内酯在30μmol/L、36h时对A549细胞增殖抑制达到最高峰为49.65％，因此选取该点作为蟛蜞菊内酯抑制A549细胞增殖的最佳作用时间与浓度。
用碘化丙啶及Annexin-PI染色检测细胞周期及凋亡的步骤，如下：
(1)在6孔板上每孔接种2mL 2×105个对数生长期细胞；
(2)24h后分为对照组和蟛蜞菊内酯组，对照组每孔直接添加2mL新鲜培养液，蟛蜞菊内酯组每孔加2mL含蟛蜞菊内酯的新鲜培养液，优选的蟛蜞菊内酯终浓度为30μmol/L，36h后收集细胞；
(3)一部分样本65～75％乙醇固定过夜，优选70％，RNase A37℃水浴30min，PI 4℃孵育30min，流式检测周期；
(4)剩余样本结合缓冲液重悬，并调整悬液密度至2.5×105个/mL，取200μL悬液于流式管中，Annexin V-FITC和PI 25℃孵育20min，流式检测凋亡。
进行后续碘化丙啶染色、AnnexinV-PI双染色实验，能测得蟛蜞菊内酯对A549细胞周期与凋亡的影响，如图2、图3所示，以进一步研究蟛蜞菊内酯抑制A549细胞生长的机制，发现蟛蜞菊内酯组较对照组A549细胞被阻滞在G1期，且B2、B4区早晚凋细胞明显增加，整体凋亡率达30.71％，蟛蜞菊内酯组的细胞凋亡率显著上升至30.71％，蟛蜞菊内酯对A549细胞表现出良好的阻滞周期与促凋亡作用，再次验证其对非小细胞肺癌的积极治疗效果。
用RT-PCR及Western blot检测细胞基因转录及翻译的步骤，如下：
(1)细胞接种、培养、分对照组和蟛蜞菊内酯组，对照组每孔直接添加2mL新鲜培养液，蟛蜞菊内酯组每孔加2mL含蟛蜞菊内酯的新鲜培养液，优选的蟛蜞菊内酯终浓度为30μmol/L，36h后收集细胞；
(2)一部分样本提总RNA并反转录，cDNA扩增，条件为95℃预变性3min，95℃变性7s，56℃退火延伸30s，45个循环，凝胶成像系统观察结果，相应引物序列见下表：
(3)剩余样本RIPA提取总蛋白，SDS-PAGE后，以p-Caspase3(1:800)、p53(1:800)、p-STAT1(1:800)、STAT1(1:800)、GAPDH(1:1000)为一抗，4℃孵育过夜；室温孵育二抗1h，化学发光仪曝光条带。
采用上述方法，能测得蟛蜞菊内酯对A549细胞基因转录及翻译的影响，相较于对照组，蟛蜞菊内酯组各mRNA指标表达量分别达到对照组表达量的(15.38±1.03)、(7.81±0.53)、(1.96±0.13)和(0.89±0.06)倍；各蛋白指标表达量分别达到对照组表达量的(3.93±0.12)、(4.30±0.15)、(1.95±0.05)和(0.99±0.03)倍，具体如图4及下表所示：
*表示与同指标正常对照组相比P＜0.01
采用RT-PCR与Western blot检测A549细胞p-Caspase-3、P53、p-STAT1、STAT1的mRNA和蛋白表达情况，进一步在凋亡相关基因转录和翻译水平对蟛蜞菊内酯作用机制进行研究。Caspase-3是Caspase家族中直接参与细胞凋亡执行的核心激酶，活化后切割多种结构和功能蛋白致细胞解体，其表达强度与细胞凋亡水平相关。p53转录因子是与肿瘤相关性最高的抑癌基因，活化p53阻滞细胞在G1或G2期，增加p53稳定性可不可逆抑制细胞生长、诱发凋亡，p53基因缺失对肿瘤形成起重要作用。STAT1是STAT家族中第1个被发现的成员，其是T细胞膜上干扰素受体与选择效应器之间信号转导的主要蛋白，STAT1被激活后形成二聚体，可抑制肿瘤细胞增殖、促进凋亡，STAT1基因缺失易发生肿瘤。研究证实STAT1可以诱导Caspase-3的表达，STAT1参与到TNF-α激活Caspase-3的过程中。STAT1的功能不仅仅是激活Caspase-3，STAT1还通过与p53相互作用，参与到不同信号通路细胞死亡或周期停滞等。STAT1可通过抑制其抑制子MDM2的表达增加p53表达量，STAT1调控p53丝氨酸15位和20位的磷酸化，同时STAT1的激活也依赖于p53。
如图5所示，30μmol/L蟛蜞菊内酯作用A549细胞36h后可显著提升p-Caspase3、p53、p-STAT1基因的转录翻译，而对STAT1的mRNA及蛋白表达无明显影响，这表示经蟛蜞菊内酯处理可活化A549细胞中p-Caspase3、p53、p-STAT1等基因表达，促使细胞凋亡。
蟛蜞菊内酯呈一定剂量、时间依赖性降低A549细胞存活率，并阻滞细胞于G1期，增加细胞凋亡数量。蜞菊内酯可上调A549细胞p-Caspase3、p53、p-STAT1等基因转录翻译水平，而对STAT1基因表达无明显影响，说明其对非小细胞肺癌有一定积极的作用，其作用机制与阻滞周期和促凋亡作用相关。
最后应当说明的是，以上实施例仅用以说明本发明的技术方案，而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细地说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。
Claims (4)
1.蟛蜞菊内酯在制备治疗肺癌产品中应用的研究方法，其特征在于，包括以下步骤：
细胞培养的步骤：复苏A549细胞后，每2～4天继代培养一次；
CCK-8法检测细胞增殖抑制率的步骤：在孔板上接种生长期细胞，之后分别用10、20、30μmol/L蟛蜞菊内酯作用细胞12、24、36h，每孔加CCK-8孵育，之后采集各孔光密度值；
碘化丙啶及Annexin-PI染色检测细胞周期及凋亡的步骤：在孔板上接种生长期细胞后分为对照组和蟛蜞菊内酯组，对照组每孔添加新鲜培养液，蟛蜞菊内酯组每孔加含蟛蜞菊内酯的新鲜培养液，之后收集细胞，一部分样本乙醇固定过夜，RNase A水浴，PI孵育，流式检测周期；剩余样本结合缓冲液重悬，取悬液于流式管中，Annexin V-FITC和PI孵育后，流式检测凋亡；
RT-PCR及Western blot检测细胞基因转录及翻译的步骤：细胞接种、培养、分对照组和蟛蜞菊内酯组，对照组每孔添加新鲜培养液，蟛蜞菊内酯组每孔加含蟛蜞菊内酯的新鲜培养液，之后收集细胞，一部分样本提总RNA并反转录，cDNA扩增，95℃预变性3min，95℃变性7s，56℃退火延伸30s，45个循环，凝胶成像；剩余样本RIPA提取总蛋白，SDS-PAGE后，以p-Caspase3、p53、p-STAT1、STAT1、GAPDH为一抗，孵育过夜，室温孵育二抗，化学发光仪曝光条带。
2.根据权利要求1所述蟛蜞菊内酯在制备治疗肺癌产品中应用的研究方法，其特征在于，细胞培养的步骤中，A549细胞复苏后在含10％FBS的RPMI-1640培养基中培养。
3.根据权利要求1所述蟛蜞菊内酯在制备治疗肺癌产品中应用的研究方法，其特征在于，CCK-8法检测细胞增殖抑制率的步骤中以及，各孔光密度值在550nm波长处采集。
4.根据权利要求1所述蟛蜞菊内酯在制备治疗肺癌产品中应用的研究方法，其特征在于，碘化丙啶及Annexin-PI染色检测细胞周期及凋亡的步骤中以及RT-PCR及Western blot检测细胞基因转录及翻译的步骤中，蟛蜞菊内酯的终浓度为30μmol/L。
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