海兔素对人肝癌细胞及小鼠肝癌移植瘤抑制作用的研究
发布时间：2018-09-18 16:39
【摘要】：目的:本实验通过观察海兔素对人肝癌Hep G2细胞株增殖以及对小鼠H22肝癌移植瘤的抑制效应,以此探讨海兔素对肝癌的抑制作用及可能的分子机制。方法:1.人肝癌Hep G2细胞株常规培养于含10%胎牛血清的DMEM/HIGH GLUCOSE完全培养基,置于37℃、体积分数为0.05 CO2的细胞恒温培养箱中培养。取对数生长期细胞,分别加入终末浓度为20、40、60、80、100、120mg/L的海兔素溶解液对其进行干预,Cell Counting Kit-8(CCK-8)法检测海兔素作用24h、48h后对肝癌Hep G2细胞株增殖的影响;荧光显微镜Annexin V-FITC/PI双染实验检测海兔素对Hep G2肝癌细胞株凋亡的诱导作用。2.昆明种雄性小鼠40只,将其按体重随机分为模型组和海兔素低、中、高剂量组,每组10只。将以生理盐水稀释的H22瘤细胞悬液接种于各组小鼠左前肢腋下皮下组织,建立小鼠H22皮下移植瘤模型。次日,海兔素低、中、高剂量组分别给予海兔素25、50、100mg·kg-1·d-1 0.5m L灌胃;模型组动物则予以等体积大豆色拉油灌胃,实验进行2周。小鼠末次灌胃24h后,称取体质量,摘眼球法取全血,颈椎脱臼法处死动物。完整剥离新鲜移植瘤组织,称重,计算抑瘤率;免疫组化法检测肿瘤组织中基质金属蛋白酶-9(MMP-9)、血管内皮生长因子(VEGF)及增殖细胞核抗原(PCNA)的表达情况;酶联免疫吸附试验(ELISA法)测定小鼠血清中白介素6(IL-6)和肿瘤坏死因子α(TNF-α)的含量。结果:1.海兔素对人肝癌Hep G2细胞株抑制作用的体外试验效果评价CCK-8实验结果表明,Hep G2肝癌细胞经海兔素干预后,生长增殖能力显著受到抑制,且随药物剂量的增加和干预时间的延长,抑制作用增强(P0.05),其24h IC25值和IC50值分别为65.36mg/L和103.77mg/L。荧光显微镜Annexin V-FITC/I双染实验检测发现,经65mg/L和105mg/L海兔素干预24h后,Hep G2肝癌细胞凋亡数目较对照组明显增加,差异均具有统计学意义(P0.05)。2.海兔素对H22荷瘤小鼠体内抑瘤试验效果评价H22荷瘤小鼠经低、中、高剂量海兔素处理后,肿瘤的生长水平显著受到抑制,与模型组相比,瘤块质量呈剂量依赖性显著减轻(P0.05),其抑瘤率分别为28.31%、33.84%、42.96%。海兔素各剂量干预组小鼠移植瘤组织中MMP-9的阳性表达率与模型组相比,均有不同程度地降低,尤其是50mg/kg、100mg/kg剂量组降低更为明显,差异具有统计学意义(P0.05);各海兔素组与模型动物组相比,小鼠肿瘤组织中VEGF和PCNA的阳性表达率逐渐降低,并呈一定的剂量依赖关系(P0.05)。经50mg/kg、100mg/kg海兔素处理后,小鼠血清中IL-6及TNF-α表达水平均显著高于模型组,差异具有统计学意义(P0.05)。结论:1.通过体外细胞培养的方法,运用CCK-8法及荧光显微镜Annexin V-FITC/PI双染实验法,初步证实了海兔素能够有效抑制肝癌细胞的增殖作用,同时显著诱导肝癌细胞发生凋亡。2.海兔素能够在体内抑制小鼠H22肝癌移植瘤的生长,其作用机制主要表现为以下几个方面:①抑制肿瘤组织周围细胞外基质降解过程②阻碍新血管形成过程③提高机体免疫功能。3.体内、外研究结果显示,海兔素对肝癌具有明显的抑制作用,有望成为一种新型的天然抗癌药物。然而海兔素能否成为有效逆转和治疗肝癌的药物,尚需要进一步的实验研究进行佐证。
[Abstract]:OBJECTIVE: To investigate the inhibitory effect of pirarin on the proliferation of human hepatocellular carcinoma Hep G2 cell line and H22 transplanted tumor in mice, and to explore the possible molecular mechanism of pirarin on hepatocellular carcinoma. Methods: 1. The human hepatocellular carcinoma Hep G2 cell line was cultured in DMEM / HIGH GLUCOSE complete medium containing 10% fetal bovine serum and placed in 37% medium. Cells in logarithmic growth phase were cultured in a constant temperature incubator with 0.05 CO2 volume fraction and 20,40,60,80,100,120 mg/L Kainin solution respectively. Cell Counting Kit-8 (CCK-8) assay was used to detect the effect of Kainin on proliferation of Hep G2 cell line after 24 hours and 48 hours. V-FITC/PI double staining assay was used to detect the induction of apoptosis of Hep G2 hepatoma cell line. 2. Forty Kunming male mice were randomly divided into two groups according to their body weight: model group and low, medium and high dosage groups, 10 in each group. The next day, the mice were given 25,50,100 mg kg 1 D 10.5 ml of kaempferol by gastric lavage respectively, and the mice in the model group were given soybean salad oil by gastric lavage for 2 weeks. After 24 hours of the last gastric lavage, the mice were weighed, eyeballs were taken out, and the animals were killed by cervical vertebra dislocation. The expression of matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor (VEGF) and proliferating cell nuclear antigen (PCNA) in tumor tissues were detected by immunohistochemistry, and the levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) in serum of mice were determined by enzyme linked immunosorbent assay (ELISA). The results of CCK-8 assay showed that the growth and proliferation of Hep G2 hepatoma cells were significantly inhibited after the intervention of kaempferol, and the inhibition increased with the increase of drug dosage and the prolongation of intervention time (P 0.05). The 24-hour IC25 and IC50 values of Hep G2 hepatoma cells were 65.36 m, respectively. G/L and 103.77 mg/L. Fluorescence microscope Annexin V-FITC/I double staining assay found that after 65 mg/L and 105 mg/L kaempferol intervention for 24 hours, the number of apoptosis of Hep G2 hepatoma cells was significantly increased compared with the control group, the difference was statistically significant (P 0.05). 2. Compared with the model group, the tumor growth level was significantly inhibited, and the tumor mass was significantly reduced in a dose-dependent manner (P 0.05). The tumor inhibition rates were 28.31%, 33.84% and 42.96% respectively. The positive expression rates of VEGF and PCNA in tumor tissues of mice were gradually decreased and showed a dose-dependent relationship (P 0.05). After treatment with 50 mg/kg and 100 mg/kg Haituansu, the expression of IL-6 and TNF-alpha in serum of mice was significantly decreased (P 0.05). CONCLUSION: 1. The CCK-8 method and fluorescence microscope Annexin V-FITC/PI double staining method were used in vitro cell culture, and the preliminary results showed that pikacin could effectively inhibit the proliferation of hepatocellular carcinoma cells and induce apoptosis of hepatocellular carcinoma cells. It can inhibit the growth of H22 liver cancer xenograft tumor in vivo. The main mechanisms are as follows: 1. Inhibiting the degradation of extracellular matrix around tumor tissue; 2. Inhibiting the formation of new blood vessels; 3. In vivo and in vitro studies show that hippocampin has a significant inhibitory effect on liver cancer. It is expected to become a new natural anticancer drug. However, further experimental studies are needed to confirm whether kaempferol can be an effective drug for reversing and treating liver cancer.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.7
本文编号：2248504
[Abstract]:OBJECTIVE: To investigate the inhibitory effect of pirarin on the proliferation of human hepatocellular carcinoma Hep G2 cell line and H22 transplanted tumor in mice, and to explore the possible molecular mechanism of pirarin on hepatocellular carcinoma. Methods: 1. The human hepatocellular carcinoma Hep G2 cell line was cultured in DMEM / HIGH GLUCOSE complete medium containing 10% fetal bovine serum and placed in 37% medium. Cells in logarithmic growth phase were cultured in a constant temperature incubator with 0.05 CO2 volume fraction and 20,40,60,80,100,120 mg/L Kainin solution respectively. Cell Counting Kit-8 (CCK-8) assay was used to detect the effect of Kainin on proliferation of Hep G2 cell line after 24 hours and 48 hours. V-FITC/PI double staining assay was used to detect the induction of apoptosis of Hep G2 hepatoma cell line. 2. Forty Kunming male mice were randomly divided into two groups according to their body weight: model group and low, medium and high dosage groups, 10 in each group. The next day, the mice were given 25,50,100 mg kg 1 D 10.5 ml of kaempferol by gastric lavage respectively, and the mice in the model group were given soybean salad oil by gastric lavage for 2 weeks. After 24 hours of the last gastric lavage, the mice were weighed, eyeballs were taken out, and the animals were killed by cervical vertebra dislocation. The expression of matrix metalloproteinase-9 (MMP-9), vascular endothelial growth factor (VEGF) and proliferating cell nuclear antigen (PCNA) in tumor tissues were detected by immunohistochemistry, and the levels of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) in serum of mice were determined by enzyme linked immunosorbent assay (ELISA). The results of CCK-8 assay showed that the growth and proliferation of Hep G2 hepatoma cells were significantly inhibited after the intervention of kaempferol, and the inhibition increased with the increase of drug dosage and the prolongation of intervention time (P 0.05). The 24-hour IC25 and IC50 values of Hep G2 hepatoma cells were 65.36 m, respectively. G/L and 103.77 mg/L. Fluorescence microscope Annexin V-FITC/I double staining assay found that after 65 mg/L and 105 mg/L kaempferol intervention for 24 hours, the number of apoptosis of Hep G2 hepatoma cells was significantly increased compared with the control group, the difference was statistically significant (P 0.05). 2. Compared with the model group, the tumor growth level was significantly inhibited, and the tumor mass was significantly reduced in a dose-dependent manner (P 0.05). The tumor inhibition rates were 28.31%, 33.84% and 42.96% respectively. The positive expression rates of VEGF and PCNA in tumor tissues of mice were gradually decreased and showed a dose-dependent relationship (P 0.05). After treatment with 50 mg/kg and 100 mg/kg Haituansu, the expression of IL-6 and TNF-alpha in serum of mice was significantly decreased (P 0.05). CONCLUSION: 1. The CCK-8 method and fluorescence microscope Annexin V-FITC/PI double staining method were used in vitro cell culture, and the preliminary results showed that pikacin could effectively inhibit the proliferation of hepatocellular carcinoma cells and induce apoptosis of hepatocellular carcinoma cells. It can inhibit the growth of H22 liver cancer xenograft tumor in vivo. The main mechanisms are as follows: 1. Inhibiting the degradation of extracellular matrix around tumor tissue; 2. Inhibiting the formation of new blood vessels; 3. In vivo and in vitro studies show that hippocampin has a significant inhibitory effect on liver cancer. It is expected to become a new natural anticancer drug. However, further experimental studies are needed to confirm whether kaempferol can be an effective drug for reversing and treating liver cancer.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.7
【相似文献】
相关期刊论文 前7条
1 贺娟;梁惠;韩磊;马爱国;孙永叶;;海兔素对人乳腺癌细胞增殖及血管内皮生长因子表达的影响[J];营养学报;2009年05期
2 贺娟;梁惠;李元;史大永;马爱国;;海兔素对D-半乳糖诱导衰老小鼠抗氧化作用[J];中国公共卫生;2009年09期
3 梁惠;刘颖;苏爱;韩磊;马爱国;;海兔素对体外人乳腺癌FCM-7细胞的抑制作用[J];营养学报;2013年06期
4 马文龙;梁惠;刘颖;;海兔素对人乳腺癌SK-BR-3细胞的抑制作用及机制研究[J];天然产物研究与开发;2012年09期
5 戈娜;梁惠;刘颖;宫安静;王文成;;海兔素对慢性酒精性肝损伤大鼠肝超微结构及NO和iNOS的影响[J];中国海洋药物;2014年03期
6 丰暖;梁惠;戈娜;刘颖;;海兔素对酒精性肝损伤大鼠Fas/FasL及TNF-α表达的影响[J];中国海洋药物;2014年02期
7 ;[J];;年期
相关会议论文 前4条
1 贺娟;梁惠;史大永;马爱国;;海兔素对D—半乳糖诱导的衰老小鼠抗氧化作用的实验研究[A];中国营养学会第十次全国营养学术会议暨第七届会员代表大会论文摘要汇编[C];2008年
2 贺娟;梁惠;史大永;马爱国;;海兔素对D一半乳糖诱导的衰老小鼠抗氧化作用的实验研究[A];中国营养学会第十次全国营养学术会议公共营养分会论文集[C];2008年
3 梁惠;张秀珍;贺娟;逄丹;马爱国;;海兔素对D-半乳糖诱导的氧化损伤小鼠抗氧化作用的实验研究[A];食物功效成分与健康——达能营养中心第九次学术年会会议论文集[C];2006年
4 梁惠;刘颖;韩磊;贺娟;马爱国;;海兔素阻断EGFR受体通路抑制乳腺癌细胞增殖并诱导凋亡作用研究[A];“细胞活动 生命活力”——中国细胞生物学学会全体会员代表大会暨第十二次学术大会论文摘要集[C];2011年
相关硕士学位论文 前2条
1 王青;海兔素对人肝癌细胞及小鼠肝癌移植瘤抑制作用的研究[D];青岛大学;2015年
2 丰暖;海兔素在肝微粒体中酶动力学及对酒精损伤大鼠Fas/FasL、TNF-α表达影响研究[D];青岛大学;2014年
，本文编号：2248504
本文链接：https://www.wllwen.com/yixuelunwen/zlx/2248504.html
