CN108342446A - 一种促进肝癌细胞凋亡和抑制肝癌细胞增殖的方法 - Google Patents

一种促进肝癌细胞凋亡和抑制肝癌细胞增殖的方法 Download PDF

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CN108342446A
CN108342446A CN201810440035.5A CN201810440035A CN108342446A CN 108342446 A CN108342446 A CN 108342446A CN 201810440035 A CN201810440035 A CN 201810440035A CN 108342446 A CN108342446 A CN 108342446A
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张秀娟
李文娟
查纯婷
南德
贺高红
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Abstract

本发明属于细胞治疗技术领域肝癌细胞技术领域，具体为一种促进肝癌细胞凋亡和抑制肝癌细胞增殖的方法。将肝癌细胞培养至80％融合，再进行传代培养，选用生长良好的传代细胞，用含有载虾青素海藻酸钙微球的细胞培养液培养细胞，最后进行实验组和对照组的细胞增殖、细胞凋亡及Real‑time PCR实验对比检测。在虾青素的干预下，肝癌细胞有明显的凋亡现象，且肝癌细胞增殖明显受到抑制，并且使肝癌细胞代谢过程中的糖酵解过程及三羧酸循环过程发生改变，总体表现出更强的抑制肝癌细胞增殖的功能。

Description

一种促进肝癌细胞凋亡和抑制肝癌细胞增殖的方法
技术领域
本发明属于细胞治疗技术领域肝癌细胞技术领域，具体为一种促进肝癌细胞凋亡和抑制肝癌细胞增殖的方法。
背景技术
HepG2细胞系是常用于体外研究肝癌的理想肿瘤模型，故选择HepG2细胞系作为本研究的一种肝癌细胞的细胞模型。虾青素分子结构中碳-碳共轭双键长链及其连接的不饱和α-羟基酮的存在使虾青素有较强的电子效应，提供给自由基电子并与之发生反应，从而清除自由基并发挥抗氧化的作用，由于其自身的优点而在癌症治疗方面有很好的应用前景。然而，由于虾青素自身的生物局限性阻碍了人们对其抗癌效果及抗癌作用机制的探究，为有效探究虾青素在癌症治疗方面的治疗潜力及治疗方法，亟需探究虾青素抗癌效果及抗癌机理，使其在癌症治疗方面取得有效应用。
目前癌细胞代谢过程以有氧糖酵解假说认可度最高，有氧糖酵解是肿瘤细胞的主要代谢方式，糖酵解提供核酸、蛋白质、脂肪酸等合成所需的主要碳前体，癌细胞通过快速的糖酵解过程，完成癌细胞快速增殖过程中的能量供给，糖酵解过程产生的中间体作为新增殖细胞的碳前体被利用，从而阻碍了糖酵解后续步骤三羧酸循环(正常细胞主要供能过程)的正常进行。因此，本发明探究了虾青素的干预对癌细胞供能方式的异常情况有无缓解。
发明内容
本发明为了抑制肝癌细胞增殖，促进肝癌细胞在体内凋亡率的提升，提出了一种促进肝癌细胞凋亡和抑制肝癌细胞增殖的方法，通过一种唯一能够穿过人类血脑屏障的类胡萝卜素-虾青素的干预，通过对癌细胞供能异常的缓解来促进肝癌细胞凋亡和抑制肝癌细胞增殖。
本发明的技术方案：
一种促进肝癌细胞凋亡和抑制肝癌细胞增殖的方法，将肝癌细胞培养至80％融合，再进行传代培养，选用生长良好的传代细胞，用含有载虾青素海藻酸钙微球的细胞培养液培养细胞，最后进行实验组和对照组的细胞增殖、细胞凋亡及Real-time PCR实验对比检测；步骤如下：
(1)肝癌细胞的培养
将原代肝癌细胞培养至80％融合，用含0.25g/100mL EDTA的胰蛋白酶液进行消化，重悬肝癌细胞，然后接种到培养皿中进行传代培养，选取传代细胞中状态优良的细胞进行实验；
(2)虾青素对肝癌细胞活性的干预
首先利用双乳液法制备载虾青素海藻酸钙微球(CN 104856963A)，以改善虾青素水溶性，同时使虾青素的稳定性得到提升，保护抗癌活性；将载虾青素微球按照0-40μM超声溶解入肝癌细胞培养基中，用肝癌细胞培养基培养细胞1-16天，用虾青素对肝癌细胞活性进行干预；
(3)肝癌细胞增殖动力学测试及凋亡测试
虾青素干预完成后，以未干预的肝癌细胞作同期对照，用计数法记录虾青素干预后肝癌细胞的死亡率；在凋亡试剂盒的辅助下，用流式细胞仪测试肝癌细胞凋亡率；
(4)肝癌细胞代谢相关酶的表达
利用Real-time PCR方法分别对经过虾青素干预后的肝癌细胞以及相应对照组进行对比实验，对二者糖酵解过程中的PKM2(丙酮酸激酶M2)、LDHA(乳酸脱脱氢酶A)以及三羧酸循环过程中的PDHA1(丙酮酸脱氢酶复合体)、SDHA(琥珀酸脱氢酶)的mRNA表达水平进行检测。
所述的虾青素作为抗氧化清除自由基药物采用药用级别。
所述的肝癌细胞选用HepG2细胞系。
本发明的有益效果：
(1)虾青素的干预使肝癌细胞的死亡率明显增加，成功抑制了肝癌细胞的快速增殖。
(2)虾青素的干预使肝癌细胞凋亡现象明显增加，成功促进了肝癌细胞的自然凋亡，抑制了肝癌细胞无限增殖。
(3)虾青素的干预使糖酵解过程关键酶PKM2、LDHA表达明显水平下降，三羧酸循环过程关键酶PDHA1、SDHA表达水平上调，诱导肝癌细胞从异常供能方式有氧糖酵解向正常供能方式氧化磷酸化转化，使细胞逐渐将主要供能过程转变为正常的线粒体中的三羧酸循环。
附图说明
图1是虾青素不同浓度干预下肝癌细胞活性图。
图2是虾青素干预后肝癌细胞凋亡图，其中，Q1为坏死；Q2为晚期凋亡；Q3为正常细胞；Q4为早期凋亡。
图3是虾青素干预下代谢相关酶基因表达图。其中，AST为虾青素，Normal为对照组。
具体实施方式
以下结合附图和技术方案，进一步说明本发明的具体实施方式。
实施例一：
选择一批质量较好的载虾青素海藻酸钙微球，根据其载药量和包载率将培养基配置成含30μM虾青素的培养基，在肝癌细胞培养至80％融合后，用含虾青素的培养基培养细胞48h后收集细胞并测试其细胞活性、凋亡率以及RT-PCR测其相关基因表达。
根据图1，可以看出虾青素的加入明显抑制了HepG2增殖，在干预96h后，HepG2增殖率抑制了约40％，且其明显呈现时间及浓度依赖性。
根据图2，可以得出，虾青素干预96h后，HepG2细胞中，早期凋亡细胞占14.48％，晚期凋亡细胞占17.15％。
根据图3，虾青素的干预96h使糖酵解过程关键酶PKM2、LDHA表达明显水平下降，三羧酸循环过程关键酶PDHA1、SDHA表达水平上调。这充分表明虾青素的干预能使HepG2细胞的糖代谢过程逐渐恢复正常。

Claims (3)

1.一种促进肝癌细胞凋亡和抑制肝癌细胞增殖的方法，其特征在于，该方法将肝癌细胞培养至80％融合，再进行传代培养，选用生长良好的传代细胞，用含有载虾青素海藻酸钙微球的细胞培养液培养细胞，最后进行实验组和对照组的细胞增殖、细胞凋亡及Real-timePCR实验对比检测；步骤如下：
(1)肝癌细胞的培养
将原代肝癌细胞培养至80％融合，用含0.25g/100mL EDTA的胰蛋白酶液进行消化，重悬肝癌细胞，然后接种到培养皿中进行传代培养，选取传代细胞中状态优良的细胞进行实验；
(2)虾青素对肝癌细胞活性的干预
首先利用双乳液法制备载虾青素海藻酸钙微球，以改善虾青素水溶性，同时使虾青素的稳定性得到提升，保护抗癌活性；将载虾青素微球按照不大于40μM超声溶解入肝癌细胞培养基中，用肝癌细胞培养基培养细胞1-16天，用虾青素对肝癌细胞活性进行干预；
(3)肝癌细胞增殖动力学测试及凋亡测试
虾青素干预完成后，以未干预的肝癌细胞作同期对照，用计数法记录虾青素干预后肝癌细胞的死亡率；在凋亡试剂盒的辅助下，用流式细胞仪测试肝癌细胞凋亡率；
(4)肝癌细胞代谢相关酶的表达
利用Real-time PCR方法分别对经过虾青素干预后的肝癌细胞以及相应对照组进行对比实验，对二者糖酵解过程中的PKM2、LDHA以及三羧酸循环过程中的PDHA1、SDHA的mRNA表达水平进行检测。
2.根据权利要求1所述的一种促进肝癌细胞凋亡和抑制肝癌细胞增殖的方法，其特征在于，所述的虾青素作为抗氧化清除自由基药物采用药用级别。
3.根据权利要求1或2所述的一种促进肝癌细胞凋亡和抑制肝癌细胞增殖的方法，其特征在于，所述的肝癌细胞选用HepG2细胞系。
CN201810440035.5A 2018-05-02 2018-05-02 一种促进肝癌细胞凋亡和抑制肝癌细胞增殖的方法 Withdrawn CN108342446A (zh)

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US20130172407A1 (en) * 2011-12-30 2013-07-04 China Medical University Inhibition of Cancer Cell Proliferation Using Oleoylethanolamide
CN104208117A (zh) * 2014-09-15 2014-12-17 天津蒙特立医疗科技有限责任公司 一种具有维护肝脏功能的植物提取物复方产品

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Application publication date: 20180731