摘要
目的利用植物化学物(phytochemicals)中的越橘提取物,制备大肠可利用越橘提取物(Colon-available cranberry extract,CACE),并以此观察CACE对H2O2诱导的DNA损伤的保护作用;并研究CACE对人大肠癌细胞的增殖和侵袭能力的影响及其分子机制。方法经模拟上消化道环境消化吸收后,将越橘提取物制备成CACE。首先以MTT法检测CACE对大肠癌Lovo细胞的生长抑制作用。然后利用单细胞凝胶电泳实验检测CACE对于过氧化氢诱导的大肠癌细胞核损伤的保护作用;进而用Matrigel基底膜侵袭实验检测CACE对Lovo细胞侵袭能力的影响,并通过流式细胞技术观察CACE对于Lovo细胞周期的影响。最后利用Western-blot方法检测CACE对Lovo细胞P53及VEGF蛋白表达的影响,同时使用实时荧光定量聚合酶链反应方法检测CACE对于Lovo细胞VEGF表达的影响。结果 MTT实验结果示:CACE浓度从20μg/ml增至120μg/ml,Lovo细胞增殖率从90.64±3.11%降至38.54±3.13%。单细胞凝胶电泳实验结果发现:以相同浓度的H2O2损伤Lovo细胞,CACE干预组较对照组的细胞DNA损伤明显减轻,而且随CACE浓度增加损伤进一步减轻。流式细胞仪检测细胞周期结果发现:CACE增加了Lovo细胞在G1期的比例,但没有明显的统计学意义(P>0.05)。穿膜侵袭实验显示:穿膜细胞数量随CACE浓度增加而减少;Western-blot结果:CACE能显著降低大肠癌Lovo细胞P53和VEGF蛋白的表达,且随浓度增加该作用进一步增强;Real-time PCR实验结果显示,CACE抑制了VEGF的mRNA的表达。结论 CACE在体外模型中能够显著抑制人大肠癌细胞系Lovo的增殖和侵袭,且对DNA损伤具有保护作用,其作用机制可能是通过抑制P53和VEGF的表达来实现。
Objective In recent years,phytochemicals as cancer chemopreventive treatment of colorectal cancer have been widespread concerned. In this study,we used cranberry extract,which is one of phytochemicals,to prepare Colon-available cranberry extract( CACE),and to observe protection effect of CACE on the DNA damage induced by H2O2. The effects of CACE on colorectal cancer cell proliferation and invasive ability were observed. Moreover,the molecular mechanisms of CACE in colorectal cancer cell proliferation and invasion were detected. Methods By imitating the environment of upper gastrointestinal tract digestion and absorption,cranberry extract was prepared into CACE. First of all,the effect of CACE on growth inhibition was detected in colorectal cancer Lovo cell line determined by the MTT methood. Single cell gel electrophoresis experiment was used to test the protective effects of nucleus by CACE in colorectal cancer cells induced by hydrogen peroxide damage. Next,the Matrigel basement membrane invasion experiment was carried to exam CACE influence on Lovo cell invasive ability. Flow cytometry technique was also used to observe the influence of CACE on Lovo cell cycle. Western blot was further used to detect CACE impact on Lovo cell P53 and VEGF protein expression. Meanwhile,real-time fluorescent quantitative PCR method was used to detect CACE VEGF expression in Lovo cells. Results The MTT experiment results showed that,with CACE concentr ation increasing from 20 ug / ml to 120 ug / ml,proliferation rate of Lovo cells falls from 90. 64± 3. 11% to 38. 54 ± 3. 13%. Single cell gel electrophoresis experiment showed that DNA damage of Lovo cells significantly reduces in the CACE intervention group compared to the control group after the cells were damaged with the same concentration of H2O2. Moreover,with increase in the CACE concentration the damage further reduced. The cell cycle was identified by Flow cytometry,and the results showed that CACE increased the proportion of Lovo cells in G1 phase. However,there was no discernible statistical significance( P > 0. 05). The Matrigel basement membrane invasion experiment showed that membrane cells reduced with the increase of concentration of CACE. CACE can significantly reduce the expression of colorectal cancer Lovo cells P53 and VEGF protein by Western blot results. With the increasing of concentration,the effect was further strengthened. The Real-time PCR experimental results showed that CACE inhibits VEGF mRNA expression. Conclusion The CACE can significantly inhibit the proliferation and invasion of colorectal cancer in Lovo cell line,and CACE plays a protective role in DNA damage. The anti-proliferation mechanisms of CACE may be through inhibiting the expression of P53 and VEGF.
出处
《中华结直肠疾病电子杂志》
2015年第2期27-31,共5页
Chinese Journal of Colorectal Diseases(Electronic Edition)
基金
国家自然科学基金(81372612)
国家自然科学青年基金(81302059)
黑龙江省留学归国科学基金(LC2013C35)
黑龙江省教育厅面上项目(12541300)
哈尔滨医科大学附属第四医院杰出青年基金
关键词
越橘
中草药
结直肠肿瘤
抑制
分子机制
Vaccinium vitis-idaea
Drugs,chinese herbal
Colorectal neoplasms
Inhibition
Molecular mechanism
