【作者】 张敏；

【导师】 张秀琴；

【作者基本信息】 苏州大学 ， 内科学（呼吸系病学）， 2020， 硕士

【摘要】 目的:研究姜黄素对非小细胞肺癌(non small cell lung cancer,NSCLC)细胞活性的影响,和对 NSCLC 上皮间质转化(epithelial mesenchymal transition,EMT)的作用及其可能的作用机制。方法:1.通过MTT试验,检测三种非小细胞肺癌细胞HCI-H1299、A549和YTMLC-90在经过不同浓度的姜黄素处理后,测量其各自的OD值,与空白对照组(未经姜黄素处理的)相比较,观察姜黄素对NSCLC细胞活性的影响。2.通过倒置显微镜观察经过CoCl2诱导的三种NSCLC细胞与未经CoCl2诱导的三种NSCLC细胞其各自的细胞形态差异,初步构建EMT模型。将CoCl2诱导后的三种NSCLC细胞分成两组,观察组经过姜黄素处理,对照组未经过姜黄素处理,通过Transwell侵袭实验、划痕实验,比较两组的细胞侵袭抑制率及转移侵袭能力。3.把经过CoCl2诱导发生EMT的NSCLC细胞经过姜黄素处理72h,作为观察组。把未经CoCl2诱导的NSCLC细胞作为对照组1,把经过CoC12诱导发生EMT的但未经姜黄素处理的NSCLC细胞作为对照组2,通过western blot、RT-PCR试验检测姜黄素对CoCl2诱导的NSCLC细胞EMT的作用。结果:1.姜黄素以剂量依赖方式及时间依赖方式显著抑制三种NSCLC的细胞活性。自1μmol/L 至 100μmol/L 的六种浓度(1μmol/L,5μmol/L,10μmol/L,20μmol/L,50μmol/L,100μmol/L)的姜黄素对三种NSCLC细胞作用24h、48h、72h的细胞活性抑制率均与对照组比较均有极显著的差异(p<0.05)。6种浓度的姜黄素对NSCLC细胞的抑瘤率之间的差异也具有统计学意义(p<0.05)。其中100μmol/L姜黄素对NCI-H1299细胞、A549腺癌细胞、YTMLC-90鳞癌细胞作用72h的抑制率分别为82.44%(P=0.000)、86.47%(P=0.000)、85.70%(P=0.000)。2.200μmol/L的氯化钴处理后的NSCLC细胞,细胞轮廓模糊不清,细胞显著发生梭型变化,细胞间距离较远,部分细胞开始脱壁。说明氯化钴处理后的NSCLC细胞发生EMT转变,形成了具有间质特征的细胞。3.姜黄素能显著抑制三种发生EMT转化的NSCLC细胞的侵袭、迁徙能力。100μmol/L的姜黄素对NCI-H1299细胞、A549腺癌细胞、YTMLC-90鳞癌细胞处理24h后,与不加姜黄素的对照组比较,侵袭抑制率分别为60.77%,59.02%,58.41%。4.姜黄素能显著上调三组EMT化NSCLC细胞的E-钙粘蛋白表达,显著下调其N-钙粘蛋白、Vimentin蛋白、MMP-2、MMP-9、MMP-14、Snail 蛋白、Twist 蛋白的表达,差异均具有统计学意义(P均<0.05)。说明姜黄素能逆转NSCLC细胞的EMT化。结论:1.姜黄素对NSCLC细胞HCI-H1299、A549和YTMLC-90的细胞活性有较强的抑制作用,作用呈剂量依赖及时间依赖性。2.姜黄素能抑制EMT化的HCI-H1299、A549和YTMLC-90细胞的侵袭、迁徙能力。3.利用CoCl2诱导NSCLC细胞发生EMT的模型构建成功。4.姜黄素能抑制NSCLC细胞的EMT,其机制可能是通过调节EMT相关基因的表达量(上调 E-cadherin 表达,下调 N-cadherin、Vimentin、MMP-2、MMP-9、MMP-14、Snail及Twist表达)来实现的。更多还原

【Abstract】 ObjectiveTo study the inhibitory effect of curcumin on NSCLC cells and EMT of NSCLC and its possible mechanism.Methods1.Three types of non-small cell lung cancer cells HCI-H1299、A549 and YTMLC-90were treated with different concentrations of curcumin,and measured the OD value and matched with the blank group(not treated with curcumin).To observe the effect of curcumin on proliferation activity of NSCLC cells.2.Three kinds of NSCLC cells were induced into EMT by CoCl2.The morphological changes of NSCLC cells were observed under microscope and Western Blot to verify the EMT model construction.The next three NSCLC cells were devided into two groups.The observation group was treated with curcumin,while the control group was not treated with curcumin.Transwell invasion test and scratch test were conducted to compare the inhibition rate and metastasis of cellinvasion between the two groups.3.NSCLC cells induced by CoCl2 to produce EMT were treated with curcumin for 72h as the observation group.Uninduced NSCLC cells were treated as control groupl,and the EMT NSCLC cells induced by CoCl2 without curcumin treatment were used as control group2.The effects of curcumin on EMT in CoCl2-induced NSCLC cells were measured by RT-PCR and western blot.Results1.Curcumin significantly inhibited the activity of three NSCLC cells in a dose-dependent and time-dependent manner.The inhibition rates of curcumin at the concentration of 1 to 100μmol(1、5、10、20、50、100)for 24h,48h and 72h were significantly different from those of the control group.The inhibition rates of curcumin on NCI-H1299 cells,A549 cells and YTMLC-90 cells were 82.44%(P=0.000),86.47%(P=0.000)and 85.70%(P=0.000),respectively.2.NSCLC cells treated with 200mol/L of CoCl2 were indistinct in cell contour,with significantly fusiform changes in the cells,the distance between the cells was relatively far,and some cells began to dewall.This indicated that EMT transformation occurred in NSCLC cells treated with CoCl2,and cells with interstitial characteristics were formed.3.Curcumin can significantly inhibit invasion and migration of three types of NSCLC cells undergoing EMT transformation capacity.The inhibition rates of invasion capacity by curcumin on NCI-H1299 cells,A549 cells and YTMLC-90 cells were 60.77%,59.02%,58.41%respectively after 24 hours of treatment with curcumin,compared with the control group without curcumin.4.Curcumin can significantly up-regulate E-cadherin expression and down-regulate the expressions of N-cadherin,vimentin,MMP-2,MMP-9,MMP-14,Snail and Twist in three EMT-induced NSCLC cells(all P<0.05),thus reversing EMT in NSCLC cells.Conclusions1.Curcumin could significantly inhibit the activity of HCI-H1299,A549 and YTMLC-90 in NSCLC cells in a dose-and time-dependent manner.2.Curcumin can inhibit the invasion and migration of emt-induced HCI-H1299,A549 and YTMLC-90 cells.3.CoCl2 was successfully used to induce EMT in NSCLC cells.4.Curcumin inhibits EMT of NSCLC cells,the mechanism may be by regulating the expression of emt-related genes(up-regulating the expression levels of E-cadherin,down-regulating the expression levels of N-cadherin,Vimentin,MMP-2,MMP-9,MMP-14,Snail and Twist).更多还原