文章摘要
江昊翼,夏婷婷,王颖.从调控TNKS2/APC蛋白探讨培土生金法对非小细胞肺癌转移干预的体外研究[J].浙江中医药大学学报,2021,(3):214-221.
从调控TNKS2/APC蛋白探讨培土生金法对非小细胞肺癌转移干预的体外研究
In Vitro Study on the Intervention of the Method of Reinforcing Earth to Generate Metal on the Metastasis of Non-small Cell Lung Cancer by Regulating TNKS2/APC Protein
投稿时间:2020-07-13  
DOI:10.16466/j.issn1005-5509.2021.03.002
中文关键词: 培土生金  非小细胞肺癌  转移  机制  黄土汤  含药血清  TNKS2  APC
英文关键词: reinforcing earth to generate metal  non-small cell lung cancer  metastasis  mechanism  Huangtu Decoction  medicated serum  TNKS2  APC
基金项目:国家自然科学基金青年项目(81703961);浙江省中医药科技计划一般项目(2017ZB028)
作者单位E-mail
江昊翼 浙江中医药大学基础医学院 杭州 310053  
夏婷婷 浙江中医药大学基础医学院 杭州 310053  
王颖 浙江中医药大学基础医学院 杭州 310053 wangying0918@163.com 
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中文摘要:
      [目的]基于培土生金理论探讨经方黄土汤含药血清通过调控端锚聚合酶2(tankyrase 2,TNKS2)/腺瘤样息肉病(adenomatous polyposis coli,APC)蛋白抑制非小细胞肺癌(non-small cell lung cancer,NSCLC)转移的作用机制。[方法]采用实时荧光定量聚合酶链式反应(quantitative polymerase chain reaction,qPCR)筛选出人肺癌细胞株A549、PC9、H125、H647细胞中的TNKS2高表达细胞系H647以及低表达细胞系A549,再利用慢病毒构建A549 TNKS2过表达细胞系以及H647 TNKS2干扰细胞系。选取体质量为200g的雄性SD大鼠,以浓度为7.8g·mL-1的黄土汤水煎剂灌胃,1周后分离含药血清。通过细胞划痕实验检测经TNKS2基因表达变化、黄土汤含药血清干预以及黄土汤含药血清联合TNKS2基因表达变化干预后,H647和A549细胞迁移能力变化。将细胞分成黄土汤含药血清+H647siRNA干扰组、H647siRNA干扰组、黄土汤含药血清+A549过表达组及A549过表达组4组,以Western blot法检测TNKS2蛋白和APC蛋白表达变化。[结果]A549、H125、PC9以及H647细胞随着细胞恶性程度增加,qPCR结果显示其TNKS2基因表达量随之增加。H647细胞TNKS2基因表达明显高于A549、H125、PC9细胞系,差异有统计学意义(P<0.001,P<0.001,P<0.01);A549和H125细胞TNKS2基因表达差异无统计学意义(P>0.05)。划痕试验结果显示TNKS2基因高表达细胞系迁移能力较强,黄土汤含药血清能够抑制A549、H647细胞迁移能力(P<0.01,P<0.001),也能抑制经TNKS2基因作用后的A549、H647细胞迁移能力(P<0.05,P<0.001)。Western blot结果显示,黄土汤含药血清能抑制TNKS2基因过表达的A549细胞中TNKS2表达(P<0.05),调高经TNKS2基因干扰的H647细胞中APC表达(P<0.05)。[结论]培土生金法对恶性程度不同的NSCLC细胞转移均有抑制作用,可通过下调TNKS2表达或诱导抑癌蛋白APC表达,从而影响NSCLC的发生、发展、转移。
英文摘要:
      [Objective] To explore the mechanism of Huangtu Decoction medicated serum by regulating tankyrase 2(TNKS2)/adenomatous polyposis coli(APC) protein to inhibit the metastasis of non-small cell lung cancer(NSCLC),based on the theory of reinforcing earth to generate metal.[Methods]High TNKS2 expression cell line H647 and low TNKS2 expression cell line A549 were screened out from A549, PC9, H125 and H647 cell lines by quantitative polymerase chain reaction(qPCR),and the lentivirus was used to construct A549 TNKS2 overexpression cell line and H647 TNKS2 interference cell line.Male SD rats with body weight of 200g were selected and given 7.8g·mL-1 Huangtu Decoction by gavage, and the medicated serum was separated one week later. The scratch test was used to detect changes in the migration ability of H647 and A549 cells after TNKS2 gene expression changes, Huangtu Decoction medicated serum intervention, and Huangtu Decoction medicated serum intervention combined with TNKS2 gene expression changes. Cells were divided into four groups: Huangtu Decoction medicated serum+H647 small interfering RNA(siRNA) group, H647 siRNA group, Huangtu Decoction medicated serum+A549 TNKS2 group, A549 TNKS2 group, Western blot was used to detect the protein expression changes of TNKS2 and APC.[Results] As the malignancy of cell increased in A549,H125,PC9 and H647 cells,the qPCR results showed that the expression of TNKS2 gene increased.The gene expression of TNKS2 in H647 cells was significantly higher than that of other cell lines A549, H125 and PC9, and the difference was statistically significant(P<0.001,P<0.001,P<0.01);there was no significant difference in TNKS2 gene expression in A549 and H125 cells(P>0.05).The results of the scratch test showed that the cell line with high expression of TNKS2 gene had a strong migration ability.Huangtu Decoction medicated serum could inhibit the migration of A549 and H647 cells(P<0.01,P<0.001),and also inhibit the migration ability of A549 and H647 cells after TNKS2 gene action(P<0.05,P<0.001).Western blot results showed that Huangtu Decoction medicated serum could inhibit the expression of TNKS2 in A549 cells after TNKS2 gene overexpression(P<0.05),and increase the expression of APC in H647 cells interfered with TNKS2 gene(P<0.05).[Conclusion] The method of reinforcing earth to generate metal can inhibit the metastasis of NSCLC cells with different malignant degrees by down-regulating the expression of TNKS2 or inducing the expression of the tumor suppressor protein APC, to intervene the development and metastasis of NSCLC.
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