摘要
目的探讨土荆皮乙酸(PAB)对TGF-β1介导的人肺癌细胞A549上皮间质转化(EMT)的影响以及与其迁移侵袭的关系,以期找到该单体抗肺癌的作用机制,或为临床肺癌治疗新方案提供实验依据。方法应用CCK8法检测不同浓度(0、10、20、40、60、80μmol/L)PAB对人肺癌A549细胞增殖的影响,分别干预(0、12、24、36、48 h)确定最佳作用浓度及最佳作用时间。将A549细胞分组:对照组(添加培养基)、诱导组(添加5μg/L TGF-β1)、实验组(最佳浓度PAB+5μg/L TGF-β1)。Transwell小室实验检测PAB对肺癌A549细胞迁移、侵袭力的影响。采用免疫印迹法、细胞免疫荧光法以及实时定量PCR法检测EMT相关分子标记物E-钙黏蛋白(E-cadherin)和N-钙黏蛋白(N-cadherin)表达和mRNA水平。采用SPSS 16.0对实验数据进行统计学分析,GraphPad PHsm 6.0作图软件制图,应用单因素方差分析法进行多组数据比较。结果CCK-8结果显示,不同浓度PAB对人肺癌细胞A549均具有增殖抑制作用,且抑制细胞增殖具有时间和浓度依赖性。当PAB浓度40μmol/L时对肺癌A549细胞具有明显增殖抑制作用,达到肿瘤半数抑制率,且细胞毒性较低,因而选用该浓度作用48 h作为后续实验的最佳作用浓度及时间。蛋白质印迹法检测结果显示,PAB能下调N-Ca的表达,差异有统计学意义。对照组肺癌A549细胞N-Ca蛋白量为1.080±0.015,诱导组为2.777±0.018,实验组为1.807±0.032,其中诱导组与对照组比较,差异有统计学意义,t=-13.863,P=0.001;实验组与对照组比较,差异有统计学意义,t=-6.853,P=0.002;实验组与诱导组比较,差异有统计学意义,t=-6.183,P=0.009。RT-PCR显示结果,PAB能增加E-Ca的表达而下调N-Ca的表达,差异有统计学意义。对照组肺癌A549细胞E-Ca蛋白量为1.000±0.001,诱导组为0.477±0.007,实验组为0.847±0.012,其中诱导组与对照组比较,差异有统计学意义,t=21.772,P=0.002;实验组与对照组比较,差异有统计学意义,t=2.484,P=0.048;实验组与诱导组比较,差异有统计学意义,t=-10.566,P=0.002。对照组肺癌A549细胞N-Ca蛋白量为1.000±0.001,诱导组为3.273±0.022,实验组为1.613±0.003,其中诱导组与对照组比较,差异有统计学意义,t=-24.690,P=0.002;实验组与对照组比较,差异有统计学意义,t=-6.635,P=0.003;实验组与诱导组比较,差异有统计学意义,t=9.878,P=0.002。上述结果说明与对照组相比,诱导组的肺癌A549细胞上皮标记蛋白E-ca表达下降而间质标记蛋白N-ca表达增强,同时迁移和侵袭能力也增强。与诱导组对比,实验组的肺癌A549细胞经过PAB干预后相关上皮性标记蛋白E-ca表达增加,间质标记蛋白N-ca表达下降,且迁移和侵袭能力也明显减弱。结论40μmol/L PAB作用48 h可以抑制TGF-β1介导的肺癌A549细胞的迁移和侵袭能力,这可能与抑制TGF-β1介导的肺癌A549细胞EMT的过程有关。
Objective The effect of pseudolaric acid B was studied TGF-β1 mediated epithelial mesenchymal transformation of human lung cancer cell line A549 and its relationship with migration and invasion,in order to find the anti-lung cancer mechanism of the monomer,or provide experimental basis for new clinical treatment of lung cancer.Methods CCK8 method was used to detect different concentrations(0,10,20,40,60,80)μmol/L pseudolaric acid B on the proliferation of lung cancer A549 cells.The optimal concentration and time were determined by intervention(0,12,24,36,48 h).Then A549 cells were divided into control group(added medium)and induction group(added 5μg/L TGF-β1),experimental group(the best concentration of pseudolaric acid B+5μg/L TGF-β1).Transwell chamber experiment was used to detect the effect of pseudolaric acid B on the migration and invasion of lung cancer A549cells.The protein expression and mRNA level of E-cadherin and N-cadherin were detected by Western Blot,cellular immunofluorescence and realtime quantitative PCR.SPSS16.0was used to analyze the experimental data;GraphPad PHsm6.0,a mapping software was used for drafting,one-way analysis of variance was used to compare multiple groups of data.Results CCK-8showed that different concentrations of pseudolaric acid B inhibited the proliferation of lung cancer A549cells in a time and concentration dependent manner.The pseudolaric acid B at a concentration of 40μmol/L has a significant inhibitory effect on the proliferation of lung cancer A549cells,reaching half the tumor inhibition rate,and the cytotoxicity is low.Therefore,48h of this concentration is selected as the best concentration and time for subsequent experiments.Western Blot showed that the amount of n-ca protein in lung cancer A549cells in control group was 1.080±0.015,that in lung cancer A549cells in induction group was 2.777±0.018,and that in experimental group C was 1.807±0.032.Compared with control group,the amount of n-ca protein in induction group was t=-13.863,P=0.001;Compared with control group,t=-6.853,P=0.002;Compared with induction group,t=-6.183,P=0.009.The difference was statistically significant.RT-PCR showed that the pseudolaric acid B could increase the expression of e-ca and down-regulate the expression of n-ca,the difference was statistically significant.The amount of e-ca protein in lung cancer A549cells in control group was 1.000±0.001,the amount of e-ca protein in lung cancer A549 cells in induction group was 0.477±0.007,and the amount of e-ca protein in lung cancer A549 cells in experimental group was 0.847±0.012.Compared with control group,t=21.772,P=0.002;Compared with control group,t=2.484,P=0.048;Compared with induction group,t=-10.566,P=0.002.The amount of n-ca protein in lung cancer A549 cells in control group was 1.000±0.001,the amount of n-ca protein in lung cancer A549 cells in induction group was 3.273±0.022,and the amount of n-ca protein in lung cancer A549 cells in experimental group was 1.613±0.003.Compared with control group,t=-24.690,P=0.002;Compared with control group,t=-6.635,P=0.003;Compared with induction group,t=9.878,P=0.002.The difference of the above experimental results was statistically significant.The above results showed that compared with control group,the expression of epithelial marker protein e-ca decreased and the expression of interstitial marker protein n-ca increased in group B induced lung cancer A549 cells,and the ability of migration and invasion also increased.Compared with group,the expression of epithelial marker protein e-ca increased and interstitial marker protein n-ca decreased in lung cancer A549 cells in group after the intervention of pseudolaric acid B,and the ability of migration and invasion decreased significantly.Conclusion 40μmol/L.pseudolaric acid B could inhibit TGF-β1 after 48hours mediated migration and invasion of lung cancer A549 cells,which may be related to the inhibition of TGF-β1 mediated epithelial mesenchymal transformation of lung cancer A549 cells.
作者
宋佳易
SONG Jia-yi(College of Integrated Traditional Chinese and Western Medicine,Liaoning University of Traditional Chinese Medicine,Shenyang 110847,China)
出处
《社区医学杂志》
CAS
2022年第4期186-192,共7页
Journal Of Community Medicine
关键词
中药抗肿瘤
上皮间质转化
转移
土荆皮乙酸
traditional Chinese medicine anti-tumor
epithelial mesenchymal transition
metastasis
pseudolaric acid B
