荞麦中活性成分对Hep G2细胞作用的初步研究
摘要
荞麦不仅营养价值高,而且保健功能强,是最受欢迎的绿色食品。研究表明荞麦中的主要活性成分如胰蛋白酶抑制剂和黄酮类物质具有降低血液胆固醇,抑制脂肪蓄积,抑制大肠癌和胆结石,改善便秘及抗衰老等作用。为进一步揭示其抑制细胞增殖、诱导肿瘤细胞凋亡机理及荞麦药用价值的开发利用,本研究以荞麦胰蛋白酶抑制剂、槲皮素和芦丁为实验材料,对它们的抗肿瘤活性进行了深入研究。
荞麦BTI基因转染对人肝癌Hep G2细胞的作用研究实验中,利用先前已构建的pEGFP-N1-BTI真核表达质粒,脂质体介导转染Hep G2细胞,分析BTI诱导细胞凋亡作用及对细胞周期调控因子及细胞侵袭迁移的影响。MTT比色法检测BTI对HepG2细胞的毒理效应,结果显示BTI能够明显降低细胞的存活率,并具有时间依赖性,当作用48h后,抑制率达到35%以上。DAPI染色及流式细胞术检测细胞凋亡,实验结果表明经BTI处理的Hep G2细胞出现凋亡信号并产生凋亡小体。RT-PCR和Westem Blot法检测表明BTI在转染细胞中成功转录和表达。细胞周期调控因子检测结果显示转染BTI的细胞中CyclinDl、CyelinE1、CDK2及CDK7转录减少,而P21和P53的转录增加。Westem B10t研究发现BTI可能通过下调CyclinD1表达和上调P21表达,来抑制细胞增殖,诱导细胞凋亡。另外研究还发现经BTI转染后Hep G2细胞的黏附和侵袭迁移能力均有所降低。
荞麦槲皮素及芦丁对Hep G2细胞作用的比较研究实验中,MTT法检测结果表明槲皮素和芦丁对Hep G2细胞增长均具有抑制作用,槲皮素的抑制效果较芦丁大,并具有时间和浓度依赖性。DAPI染色发现细胞核凝集并有凋亡小体产生。划痕实验表明槲皮素和芦丁在一定浓度范围内能够抑制细胞迁移,并且槲皮素对细胞迁移的抑制作用更明显。流式细胞仪检测细胞内活性氧(ROS)水平,结果发现经槲皮素和芦丁处理后细胞内的ROS均有明显提高,并且经槲皮素作用细胞后ROS变化更明显,推断引发ROS水平升高并诱导细胞凋亡可能是黄酮类物质发挥抗肿瘤作用的一个重要机制。总抗氧化能力(T-AOC)检测结果发现芦丁溶液的T-AOC较槲皮素溶液的T-AOC大,而当二者作用Hep G2细胞后,槲皮素作用后细胞内及培养基中的T-AOC要比芦丁作用后的T-AOC大,这表明两种黄酮化合物都具有很好的抗氧化性,在不同体系中它们的抗氧化活性大小不同。
真核细胞凋亡的信号转导途径是非常复杂和极其精细的调控网络,需要许多信号分子的参与,胰蛋白酶抑制剂和黄酮类物质诱导细胞凋亡的分子机理仍需要进一步深入地研究,而本文为这个复杂机制的研究及荞麦药用价值的开发利用奠定一定的理论和实验基础,该研究具有一定创新性,研究意义深远。
Buckwheat is of high nutritional values and powerful healthy functions. It is known as the most popular green food. In recent years, some researchs had shown that the main active ingredients of buckwheat, Such as trypsin inhibitor and flavonoids, can cut down blood cholesterol, suppress lipopexia, restrain cancer of colon and gallstones, improve constipation, anti-aging, et al. In order to further reveal the molecular mechanism of anticancer drugs which inhibit cell proliferation and induce cells apoptosis, we used trypsin inhibitor BTI, quercetin and rutin from buckwheat seeds as experimental materials and deeply researched their antitumor effect.
The experiment maked use of the gene of Buckwheat trypsin inhibitor (BTI) to transfect the human hepatocellular carcinoma cells Hep G2, then studied the influence of BTI to cells growth and migration preliminarily. MTT assay results indicated that BTI could inhibit the growth of Hep G2in a time dependent manner. The inhibition rate reached35%when Hep G2cells were treated for48h. Through DAPI nuclear staining and flow cytometry analysis, we observed that Hep G2cells showed death signals, and produced apoptotic bodies after they were induced by BTI. The cell cycle regulation factor test showed that the mRNA of CyclinD1, CyclinE1, CDK2, CDK7was decreased, but the mRNA of P21, P53was increased in transfected pEGFP-N1-BT1cells. Western Blot analysis showed that BTI may inhibit cells proliferation and induce cells apoptosis through decreasing the expression of CyclinD1and increasing the expression of P21. Then it can achieve the purpose of cancer therapy. Besides, the adhesion and migration capacities of Hep G2cells were decreased when cells were transfected with BTI.
In another experiment, we compared and studied the antitumor effects of quercetin and rutin from tartary buckwheat seeds. MTT assay showed that quercetin and rutin could inhibit the growth of Hep G2cells in time and concentration dependent manners. DAPI nuclear staining showed that nuclear condensed and apoptotic bodies produced. Cell injury healing experiment indicated that quercetin and rutin could suppress cells migration in certain concentration range. And the inhibiting migration capacity of quercetin was bigger than that of rutin. Through flow cytometry analysis, we can test the intracellular reactive oxygen species (ROS) level. We found that quercetin and rutin could induce the increase of ROS levels significantly, and the ROS level of quercetin was higher than that of rutin. We used the total antioxidant competency (T-AOC) detection Kit to detect the T-AOC of quercetin and rutin, and found that the T-AOC of rutin solution was higher than that of quercetin. Two kinds of flavonoids treate Hep G2cells, the T-AOC of the cells and culture medium of quercetin was higher than that of rutin. The results indicate that quercetin and rutin both have good antioxidant competency, and the antioxidant competency is different in different systems.
The signal transduction pathways of eukaryotic cells apoptosis are very complex and extremely subtle, it needs many signal molecules to participate. The molecular mechanism of trypsin inhibitors and flavonoids inducing cells apoptosis still need to be further studied, and the thesis laid a experimental basis for this complex mechanism and the exploitation of the value of buckwheat as drugs, the study is originality and meaningful.
荞麦BTI基因转染对人肝癌Hep G2细胞的作用研究实验中,利用先前已构建的pEGFP-N1-BTI真核表达质粒,脂质体介导转染Hep G2细胞,分析BTI诱导细胞凋亡作用及对细胞周期调控因子及细胞侵袭迁移的影响。MTT比色法检测BTI对HepG2细胞的毒理效应,结果显示BTI能够明显降低细胞的存活率,并具有时间依赖性,当作用48h后,抑制率达到35%以上。DAPI染色及流式细胞术检测细胞凋亡,实验结果表明经BTI处理的Hep G2细胞出现凋亡信号并产生凋亡小体。RT-PCR和Westem Blot法检测表明BTI在转染细胞中成功转录和表达。细胞周期调控因子检测结果显示转染BTI的细胞中CyclinDl、CyelinE1、CDK2及CDK7转录减少,而P21和P53的转录增加。Westem B10t研究发现BTI可能通过下调CyclinD1表达和上调P21表达,来抑制细胞增殖,诱导细胞凋亡。另外研究还发现经BTI转染后Hep G2细胞的黏附和侵袭迁移能力均有所降低。
荞麦槲皮素及芦丁对Hep G2细胞作用的比较研究实验中,MTT法检测结果表明槲皮素和芦丁对Hep G2细胞增长均具有抑制作用,槲皮素的抑制效果较芦丁大,并具有时间和浓度依赖性。DAPI染色发现细胞核凝集并有凋亡小体产生。划痕实验表明槲皮素和芦丁在一定浓度范围内能够抑制细胞迁移,并且槲皮素对细胞迁移的抑制作用更明显。流式细胞仪检测细胞内活性氧(ROS)水平,结果发现经槲皮素和芦丁处理后细胞内的ROS均有明显提高,并且经槲皮素作用细胞后ROS变化更明显,推断引发ROS水平升高并诱导细胞凋亡可能是黄酮类物质发挥抗肿瘤作用的一个重要机制。总抗氧化能力(T-AOC)检测结果发现芦丁溶液的T-AOC较槲皮素溶液的T-AOC大,而当二者作用Hep G2细胞后,槲皮素作用后细胞内及培养基中的T-AOC要比芦丁作用后的T-AOC大,这表明两种黄酮化合物都具有很好的抗氧化性,在不同体系中它们的抗氧化活性大小不同。
真核细胞凋亡的信号转导途径是非常复杂和极其精细的调控网络,需要许多信号分子的参与,胰蛋白酶抑制剂和黄酮类物质诱导细胞凋亡的分子机理仍需要进一步深入地研究,而本文为这个复杂机制的研究及荞麦药用价值的开发利用奠定一定的理论和实验基础,该研究具有一定创新性,研究意义深远。
Buckwheat is of high nutritional values and powerful healthy functions. It is known as the most popular green food. In recent years, some researchs had shown that the main active ingredients of buckwheat, Such as trypsin inhibitor and flavonoids, can cut down blood cholesterol, suppress lipopexia, restrain cancer of colon and gallstones, improve constipation, anti-aging, et al. In order to further reveal the molecular mechanism of anticancer drugs which inhibit cell proliferation and induce cells apoptosis, we used trypsin inhibitor BTI, quercetin and rutin from buckwheat seeds as experimental materials and deeply researched their antitumor effect.
The experiment maked use of the gene of Buckwheat trypsin inhibitor (BTI) to transfect the human hepatocellular carcinoma cells Hep G2, then studied the influence of BTI to cells growth and migration preliminarily. MTT assay results indicated that BTI could inhibit the growth of Hep G2in a time dependent manner. The inhibition rate reached35%when Hep G2cells were treated for48h. Through DAPI nuclear staining and flow cytometry analysis, we observed that Hep G2cells showed death signals, and produced apoptotic bodies after they were induced by BTI. The cell cycle regulation factor test showed that the mRNA of CyclinD1, CyclinE1, CDK2, CDK7was decreased, but the mRNA of P21, P53was increased in transfected pEGFP-N1-BT1cells. Western Blot analysis showed that BTI may inhibit cells proliferation and induce cells apoptosis through decreasing the expression of CyclinD1and increasing the expression of P21. Then it can achieve the purpose of cancer therapy. Besides, the adhesion and migration capacities of Hep G2cells were decreased when cells were transfected with BTI.
In another experiment, we compared and studied the antitumor effects of quercetin and rutin from tartary buckwheat seeds. MTT assay showed that quercetin and rutin could inhibit the growth of Hep G2cells in time and concentration dependent manners. DAPI nuclear staining showed that nuclear condensed and apoptotic bodies produced. Cell injury healing experiment indicated that quercetin and rutin could suppress cells migration in certain concentration range. And the inhibiting migration capacity of quercetin was bigger than that of rutin. Through flow cytometry analysis, we can test the intracellular reactive oxygen species (ROS) level. We found that quercetin and rutin could induce the increase of ROS levels significantly, and the ROS level of quercetin was higher than that of rutin. We used the total antioxidant competency (T-AOC) detection Kit to detect the T-AOC of quercetin and rutin, and found that the T-AOC of rutin solution was higher than that of quercetin. Two kinds of flavonoids treate Hep G2cells, the T-AOC of the cells and culture medium of quercetin was higher than that of rutin. The results indicate that quercetin and rutin both have good antioxidant competency, and the antioxidant competency is different in different systems.
The signal transduction pathways of eukaryotic cells apoptosis are very complex and extremely subtle, it needs many signal molecules to participate. The molecular mechanism of trypsin inhibitors and flavonoids inducing cells apoptosis still need to be further studied, and the thesis laid a experimental basis for this complex mechanism and the exploitation of the value of buckwheat as drugs, the study is originality and meaningful.
引文
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