基于3D微流控芯片分析鸡血藤总鞣质对宫颈癌细胞HeLa的药理作用
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摘要
目的:研究鸡血藤总鞣质的化学成分,并分析该有效部位对宫颈癌细胞HeLa的药效与作用机制,为鸡血藤抗宫颈癌提供实验依据。方法:运用UPLC-Q-TOF/MS对鸡血藤鞣质类化学成分进行定性分析。将不同质量浓度的鸡血藤总鞣质作用于宫颈癌细胞HeLa,通过3维(3D)微流控芯片筛选合适的给药浓度及时间。运用流式细胞仪测定鸡血藤总鞣质对宫颈癌细胞HeLa周期与凋亡的影响,分别应用Flow Jo v10. 0. 7及ModFit LT 3. 2软件进行分析。采用酶联免疫吸附测定(ELISA)测定经鸡血藤总鞣质作用后,宫颈癌细胞HeLa上清液中血管内皮生长因子-A(VEGF-A)与半胱天冬酶-3(Caspase-3)因子的含量变化。结果:鉴定并推断了鸡血藤总鞣质中15种成分。最终确定鸡血藤总鞣质低、中、高给药质量浓度分别为0. 5,1. 0,2. 0 g·L-1,36 h为最佳给药时间。经鸡血藤总鞣质作用后宫颈癌细胞HeLa早凋与晚凋比例明显增加,宫颈癌细胞HeLa DNA合成前期(G0/G1期)明显增加,DNA合成期(S期)与DNA合成后期(G2/M期)减小。经鸡血藤总鞣质作用后,宫颈癌细胞HeLa上清液中VEGF-A因子显著降低,Caspase-3因子显著增加。结论:鸡血藤中含有丰富的鞣质类成分,此类成分对宫颈癌细胞HeLa有明显的抑制其增殖并促进其凋亡的作用,为该有效部位的后续研究与开发提供了实验依据。
Objective: To study on the chemical constituents of total tannins from Spatholobi Caulis,and to analyze the pharmacodynamics and mechanism of total tannins from Spatholobi Caulis on cervical cancer HeLa cells. Method: UPLC-Q-TOF/MS was used to qualitatively analyze the composition of total tannins from Spatholobi Caulis. The appropriate concentration and time of administration were screened by 3 dimensional( 3 D)microfluidic chip. Flow cytometry was used to determine the effect of total tannins from Spatholobi Caulis on the cell cycle and apoptosis of cervical cancer HeLa cells and analyzed by FlowJo v10. 0. 7 and ModFit LT 3. 2 software. Enzyme-linked immunosorbent assay( ELISA) was used to determine the changes of vascular endothelial growth factor( VEGF)-A and Caspase-3 factors in cervical cancer HeLa cells supernatant treated with total tannins from Spatholobi Caulis. Result: Total of 15 components in total tannins from Spatholobi Caulis were identified or inferred. The low,medium and high dosages of total tannins from Spatholobi Caulis were 0. 5,1. 0,2. 0 g·L-1 and the best time of administration was 36 h. The proportions of early and late apoptosis of cervical cancer HeLa cells increased significantly in the apoptosis analysis after being treated by total tannins from Spatholobi Caulis. The DNA synthesis early phase( G_0/G_1 phase) of cervical cancer HeLa cells significantly increased,and the DNA synthesis phase( S phase) and the DNA synthesis late phase( G_2/M phase) reduced.After being treated with total tannins from Spatholobi Caulis,the expression of VEGF-A in cervical cancer HeLa cells supernatant was significantly decreased and the expression of Caspase-3 was significantly increased.Conclusion: Spatholobi Caulis is rich in tannins, which can significantly inhibit the proliferation of cervical cancer HeLa cells and promote its apoptosis. This paper can provide the basis for further research of total tannins from Spatholobi Caulis.
Objective: To study on the chemical constituents of total tannins from Spatholobi Caulis,and to analyze the pharmacodynamics and mechanism of total tannins from Spatholobi Caulis on cervical cancer HeLa cells. Method: UPLC-Q-TOF/MS was used to qualitatively analyze the composition of total tannins from Spatholobi Caulis. The appropriate concentration and time of administration were screened by 3 dimensional( 3 D)microfluidic chip. Flow cytometry was used to determine the effect of total tannins from Spatholobi Caulis on the cell cycle and apoptosis of cervical cancer HeLa cells and analyzed by FlowJo v10. 0. 7 and ModFit LT 3. 2 software. Enzyme-linked immunosorbent assay( ELISA) was used to determine the changes of vascular endothelial growth factor( VEGF)-A and Caspase-3 factors in cervical cancer HeLa cells supernatant treated with total tannins from Spatholobi Caulis. Result: Total of 15 components in total tannins from Spatholobi Caulis were identified or inferred. The low,medium and high dosages of total tannins from Spatholobi Caulis were 0. 5,1. 0,2. 0 g·L-1 and the best time of administration was 36 h. The proportions of early and late apoptosis of cervical cancer HeLa cells increased significantly in the apoptosis analysis after being treated by total tannins from Spatholobi Caulis. The DNA synthesis early phase( G_0/G_1 phase) of cervical cancer HeLa cells significantly increased,and the DNA synthesis phase( S phase) and the DNA synthesis late phase( G_2/M phase) reduced.After being treated with total tannins from Spatholobi Caulis,the expression of VEGF-A in cervical cancer HeLa cells supernatant was significantly decreased and the expression of Caspase-3 was significantly increased.Conclusion: Spatholobi Caulis is rich in tannins, which can significantly inhibit the proliferation of cervical cancer HeLa cells and promote its apoptosis. This paper can provide the basis for further research of total tannins from Spatholobi Caulis.
引文
[1]符影,程悦,陈建萍,等.鸡血藤化学成分及药理作用研究进展[J].中草药,2011,42(6):1229-1234.
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[8]潘国凤,杨庆,刘安,等.瑞香狼毒药效组分诱导人肝癌细胞凋亡及对细胞周期依赖性蛋白激酶CDK2的影响[J].中药药理与临床,2011,27(5):41-45.
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[10]张彩霞,张亚杰,江滨,等.白术内酯Ⅱ促进大肠癌Lovo细胞凋亡及对PARP1和Caspase-3表达的影响[J].中国实验方剂学杂志,2017,23(5):157-161.
[11]刘梦娇,王云,李玲云,等.三黄片化学成分的UPLCQ-TOF-MS/MS快速鉴定分析[J].中国中药杂志,2017,42(9):1685-1692.
[12]周本宏,易慧兰,郭咸希,等. HPLC-ESI-MS对石榴皮中鞣质类化学成分的初步分析[J].中国药师,2015,18(2):201-204.
[13]富臻,李波,王源,等.塔拉豆荚中鞣质化合物及其抗氧化活性[J].中国实验方剂学杂志,2016,22(10):56-59.
[14]黄西,李国春,尹莲,等.凉血通瘀方凉血活血有效部位及其化学成分的HPLC-MS和GC-MS分析[J].药学学报,2015,50(1):86-93.
[15]潘海峰,杨明宇,徐宝欣,等.基于HPLC-MS/MS比较不同产地山楂叶中8个成分含量[J].药物分析杂志,2015,37(12):2173-2179.
[16]董红娇,陈晓虎,曾锐,等. UPLC-Q-Exactive四级杆-静电场轨道阱高分辨质谱联用快速分析民族药小大黄的化学成分[J].中草药,2016,47(14):2428-2435.
[17] Navarro-González J F,Mora-Fernández C,Muros de Fuentes M,et al. Inflammatory molecules and pathways in the pathogenesis of diabetic nephropathy[J]. Nat Rev Nephrol,2011,7(6):327-340.
[18]吴志敏,袁克厚. Caspase蛋白酶的结构及其在CNS损伤中的作用[J].中国临床神经外科杂志,2003,8(2):155-156.
[2]国家药典委员会.中华人民共和国药典.一部[M].北京:中国医药科技出版社,2015:194.
[3]刘伟桥,于澎.中药黄酮类化合物的抗肿瘤机制[J].长春中医药大学学报,2015,31(2):254-255.
[4]程悦,符影,王志宇,等.鸡血藤提取物中缩合鞣质的含量测定及其抗肿瘤活性初步研究[J].中山大学学报:自然科学版,2011,50(2):75-80.
[5]徐为峰,王帅,孟宪生,等.用于肿瘤细胞三维培养的微流控芯片构建及培养条件研究[J].中国药理学通报,2016,32(4):581-584.
[6]樊佳新,包永睿,孟宪生,等.基于细胞凋亡和细胞周期研究荆芥总黄酮对Caco-2细胞的影响[J].中药材,2016,39(8):1876-1878.
[7]包永睿,王帅,孟宪生,等.薏苡仁脂肪酸类成分对人肝癌细胞株SMMC-7721细胞周期及细胞凋亡的影响[J].中成药,2014,36(2):235-239.
[8]潘国凤,杨庆,刘安,等.瑞香狼毒药效组分诱导人肝癌细胞凋亡及对细胞周期依赖性蛋白激酶CDK2的影响[J].中药药理与临床,2011,27(5):41-45.
[9]曾志涛,傅缨,熊耀斌,等.发酵虫草菌粉对人肝癌细胞增殖、凋亡及VEGF,MMP-2表达的影响[J].中国实验方剂学杂志,2017,21(9):141-144.
[10]张彩霞,张亚杰,江滨,等.白术内酯Ⅱ促进大肠癌Lovo细胞凋亡及对PARP1和Caspase-3表达的影响[J].中国实验方剂学杂志,2017,23(5):157-161.
[11]刘梦娇,王云,李玲云,等.三黄片化学成分的UPLCQ-TOF-MS/MS快速鉴定分析[J].中国中药杂志,2017,42(9):1685-1692.
[12]周本宏,易慧兰,郭咸希,等. HPLC-ESI-MS对石榴皮中鞣质类化学成分的初步分析[J].中国药师,2015,18(2):201-204.
[13]富臻,李波,王源,等.塔拉豆荚中鞣质化合物及其抗氧化活性[J].中国实验方剂学杂志,2016,22(10):56-59.
[14]黄西,李国春,尹莲,等.凉血通瘀方凉血活血有效部位及其化学成分的HPLC-MS和GC-MS分析[J].药学学报,2015,50(1):86-93.
[15]潘海峰,杨明宇,徐宝欣,等.基于HPLC-MS/MS比较不同产地山楂叶中8个成分含量[J].药物分析杂志,2015,37(12):2173-2179.
[16]董红娇,陈晓虎,曾锐,等. UPLC-Q-Exactive四级杆-静电场轨道阱高分辨质谱联用快速分析民族药小大黄的化学成分[J].中草药,2016,47(14):2428-2435.
[17] Navarro-González J F,Mora-Fernández C,Muros de Fuentes M,et al. Inflammatory molecules and pathways in the pathogenesis of diabetic nephropathy[J]. Nat Rev Nephrol,2011,7(6):327-340.
[18]吴志敏,袁克厚. Caspase蛋白酶的结构及其在CNS损伤中的作用[J].中国临床神经外科杂志,2003,8(2):155-156.