Inhibitory Effect of Cantharidin on Proliferation of A549 Cells

Objective:To study the inhibition of Cantharidin against the proliferation of human lung cancer A549 cells and its mechanism.Methods:MTT assay was employed to determine the inhibition of Cantharidin against proliferation of A549 cells and flow Cytometry was applied to analyze A549 cell cycle and the effect of Cantharidin on cell cycle.Results:Cantharidin showed inhibition against the proliferation of A549 cells,and the inhibition was mediated by blocking A549 cell cycle at G2/M phase significantly.Conclusion:Cantharidin exhibits inhibition against the proliferation of human lung cancer A549 cells.     

转染外源CC10对肺腺癌细胞系A549的CyclinD1蛋白及mRNA的影响

Objective： To investigate the effects of exogenous CC10 gene transfection on cell cycle and the expression of cyclinD1 protein and mRNA in A549 cells. Methods： A549 cells in all test groups （group A to E） and control group （group F） were transfected with exogenous CC10 gene by liposome for 8, 16, 24, 36, 48 and 0 h respectively. CC10 protein expression was detected in A549 cells by Western blot. The growth inhibitory rate was detected by MTT method. Flow cyometry analysis （FCS） and AnnexinV-PI staining were used to determine the changes of cell cycle progression and apoptosis rate in all groups. CyclinD1 protein and mRNA expression in A549 cells was detected by the methods of immunocytochemistry and RT-PCR. Results： Exogenous CC10 gene could inhibit the growth of A549 cells, and the growth inhibitory rates in all test groups （from group A to E） were 24.7%, 33.1%, 44.3%, 61.7% and 74.2% respectively, and that in group F was 6.24%. CC10 blocked the cell cycle progression at G0/G1 and induced apoptosis gradually. In A549 cells of test groups, the expression of cyclinD1 protein and mRNA was significantly decreased. Conclusion： The inhibitory effects of the transfection of exogenous CC10 gene on G0/G1 cycle of lung cancer cells might be related with the down-regulation of cyclinD1 gene.     

Transfection of the Human Sodium/Iodide Symporter （NIS） Gene with Liposomes and the Expression of the NIS Protein in Human Lung A549 Cancer Cells

OBJECTIVE To examine the possibility of human sodium iodide symporter （hNIS） protein expression in lung cancer cells. METHODS Human lung A549 cancer cells were thawed and cultured in vitro. The cells were divided into an experimental group transfected with a recombinant pcDNA3-hNIS plasmid and a control group transfected only with a pcDNA3 plasmid. The recombinant plasmid vector encoding the hNIS gene （pcDNA3-hNIS） was amplified, purified and identified. The hNIS gene was followed by DNA sequencing. A Western blot and an immunohistochemical assay were applied to detect the hNIS protein expression in the transfected human lung A549 cancer cells. RESULTS Restriction enzyme digestion and DNA sequencing results showed the size and direction of the inserted gene in the recombinant pcD- NA3-hNIS plasmid was correct. The Western blot method and immunohistochemical analysis showed a positive NIS protein expression in the experimental group. The NIS protein was detected mainly in the cell membranes showing a positive rate up to 70.6% with no expression of the NIS protein in the control group. There was a significant difference between two groups （P=0.000）. CONCLUSION The hNIS gene was transfected effectively into human lung A549 cancer cells mediated by Lipofectamine 2000, and was expressed with its protein in vitro.     

In vitro study of radiosensitization by β-Elemene in A549 cell line from adenocarcinoma of lung

Objective: To explore the effect and possible mechanism in vitro of radiosensitization by β-Elemene in A549 cell line from adenocarcinoma of lung. Methods: The A549 cell line from adenocarcinoma of lung was chosen for the study to determine the inhibition ratio by using MTT assay. Morphologic change, growth curve, cloning efficiency, divisional index were observed. Change of cell cycle and apoptosis rate were analyzed by FCM and the expressions of gene P53 and Bcl-2 were detected. Results: Reproductive activity of the group which was under irradiation and β-Elemene was significantly sup-pressed and its cloning efficiency and divisional index also declined. The apoptosis rate of the group which was under irradia-tion and β-Elemene was significantly higher at 48 h and 72 h, which was analyzed by FCM. The expression of P53 without Bcl-2 was observed in the group under irradiation and β-Elemene and the group under β-Elemene only at the 48th hour point, while the expression of Bcl-2 without p53 was observed in the group under irradiation only and the control group. Conclusion: β-Elemene is good at radiosensitization and its mechanism may be relevant to the up-regulation of P53, down-regulation of Bcl-2 and inducing apoptosis.     

实时荧光PCR法检测肺癌细胞系中吉西他滨耐药相关基因的表达

Objective: To explore the molecular mechanism of gemcitabine-resistance, the relative mRNA expression of five genes related to gemcitabine-resistance was detected in six lung cancer cell lines. Methods: The total RNA was extracted from six lung cancer cell lines GLC-82, NCI-H460, A549, 95-C, 95-D and QG56. Then the cDNA was amplified by real-time quantitative PCR method to quantify the gene expression of RRM1, PTEN, ERCC1, dCK and CDA. The cytotoxicity of gem- citabine to cell lines was tested by MTT method. Results: Among the detected six lung cancer cell lines, the mRNA level of RRM1, PTEN and ERCC1 in lung squamous cell line QG56 was highest, and the IC50 of gemcitabine to QG56 cell line was also highest. Conclusion: The mRNA expression of RRM1, PTEN and ERCC1 was correlated, and the high expression of RRM1 was related to gemcitabine resistance of lung cancer.     

DF3调控下的白喉毒素A片段对人乳腺癌细胞的特异性杀伤作用

Objective： To study the effects of recombinant expression vector containing human breast cancer DF3 promotor and diphtheria toxin A fragment on human breast cancer cells. Methods： Constructing recombinant expression vector PGL3- DF3-DTA and transfecting it into human breast cancer cells of DF3 positive and negative. By means of RT-PCR to measure the expression of DTA in human breast cancer cells. MTT color-imetry was used to examine the effect of PGL3-DF3-DTA on growth of human breast cancer cells. By experiment on nude mice to observe the killing effect of PGL3-DF3-DTA on human breast cancer cells. Results： Recombinant expression vector PGL3-DF3-DTA was highly expressed in human breast cancer cell line of DF3 positive, and it could kill the human breast cancer cells not only in vitro but also in vivo. Conclusion： Recombinant expression vector PGL3-DF3-DTA could produce specific killing effect on human breast cancer cell line of DF3 positive.     

Effect of 125I seed brachytherapy and radiotherapy on the growth of transplanted human lung cancer cell line A549 in nude mice and the impact of HIF-1α expression after therapy

Objective To study the inhibitory effects of radiotherapy and 125I seed brachytherapy on the growth of transplanted human lung cancer cell line A549 in nude mice and the impact of HIF-1αexpression after therapy.Methods Forty nude mice bearing human lung cancer cell line A549 were randomly divided into control group,radiotherapy group,125I seed brachytherapy group and radiotherapy + 125I seed group when tumor volume achieved (300±50) mm3.The tumor growth was observed and the alteration of tumor size was calculated at different time.On 15th day,the expression of HIF-1α was detected by immunohistochemistry and western blot.Results When eighth day after treatment,compared with the control group,the tumor volume of the combined treatment group was significantly smaller (t = 46.4,P ＜0.05).After fifteenth day after treatment,compared with control group,the group of radiotherapy,125I seed brachytherapy and radiotherapy + 125I seed gained the tumor control rate of 45.9 %,44.4 %,69.4 % respectively.Compared with other groups,the change of expression of HIF-1α in the combined treatment group was not significant (P ＞0.05).Conclusion Radiotherapy combined with 125I seed brachytherapy can inhibit the growth of transplanted human lung cancer cell line A549 in nude mice,and the tumor regression can be observed in early stage.But in our study,the expression of HIF-1α in tumors cannot be inhibited by 125I seed.     

Construction and Biological Effects in vitro of Double－Directed Tissue－Specific HSV－tk／GCV Anti－Tumor System

Objective: To construct a specifically targeted gene delivery and expression system, and to investigate the special killing effect of the HSV-tk/GCV system on human liver cancer cells in vitro.Methods: The anti-transferrin receptor (TfR) ScFv-GAL4 fusion protein expression vector ScFv-GAL4-pET28a and the eukaryotic expression plasmid pEBAF/tk-GAL4rec were constructed by recombinant DNA technology. After the induction by IPTG, we obtained the anti-TfR ScFv-GAL4 fusion protein as delivery vector to transfect pEBAF/tk-GAL4rec into the human liver cancer cell lines HepG2 and SMMC7721 and the human lung cancer cell line A549 that overexpress TfR via receptor-mediated endocytosis. The positive cell clones were selected by hygromycin and named HepG2/tk, SMMC7721/tk and A549/tk,respectively. Cell killing after GCV application was determined by MTT. Results: The correct structure of the ScFv-Gal4 fusion protein and the plasmid pEBAF/tk-GAL4rec was confirmed by double enzyme digestion, SDS-PAGE and sequencing. HepG2/tk cells that express alphafetoprotein (AFP) to high levels(845 ng/ml) were very sensitive to GCV, while SMMC7721/tk cells that express AFP at low levels (2ng/ml) and AFP-negative A549/tk cells were only slightly or not sensitive to GCV. Conclusion: The double-directed and tissue-specific HSV-tk/GCV anti-tumor system shows good targeting to tumor cells.     

nm23-H1小干扰RNA增强紫杉醇脂质体对肺腺癌细胞化疗的敏感性

目的 探讨抑制nm23-H1基因的表达后,紫杉醇脂质体对人肺腺癌A549细胞化疗敏感性的影响.方法 以人肺腺癌A549细胞为研究对象,将其分为nm23-H1-小干扰RNA(siRNA)转染组和未转染组.采用Western blot法检测两组A549细胞中nm23-H1蛋白的表达情况,采用四甲基偶氮唑蓝(MTT)法检测紫杉醇脂质体对两组A549细胞的生长抑制率,以流式细胞仪检测A549细胞的凋亡和细胞周期的变化.结果 转染nm23-H1-siRNA后,A549细胞中nm23-H1蛋白的表达水平明显降低.紫杉醇脂质体作用48 h后,A549细胞的生长抑制率随药物浓度的升高而升高,且转染组细胞的抑制率更高.当紫杉醇脂质体浓度≥5 μg/ml时,转染组的抑制效率明显高于未转染组(均P＜0.05).转染nm23-H1-siRNA后,A549细胞的凋亡率[(65.62±4.36)%]较未转染组明显增加[(43.78±5.56)%,P＜0.05],S期和G2/M期细胞所占的比例则有所下降[S期:分别为(15.73±3.21)%和(25.56±4.01)%,P＜0.05;G2/M期:分别为(31.91±3.12)%和(39.41±4.21)%,P＜0.05].结论 nm23-H1基因与紫杉醇脂质体的化疗抵抗有关,抑制nm23-H1基因的表达可以增强紫杉醇脂质体的化疗敏感性.

Abstract：

Objective To study the chemosensitivity of lung adenocarcinoma cell line A549 cells to liposome-encapsulated paclitaxel after treatment by nm23-H1-small interference RNA (nm23-H1-siRNA) in vitro. Methods The A549 cells were divided into two groups: non-transfected group and nm23-H1-siRNA-transfected group. Western blot analysis was used to detect the expression of nm23-H1. MTT and flow cytometry were used to determine the cell mortality rate, apoptosis rate and cell cycle after liposome-encapsulated paclitaxel treatment in both groups. Results The expression of nm23-H1 in A549 cells was significantly decreased after transfection with nm23-H1-siRNA. After treatment for 48 hours with liposome-encapsulated paclitaxel, the cell mortality rate was increased with the increasing concentration of liposome-encapsulated paclitaxel in both groups, but increased higher in the nm23-H1-siRNA-transfected group. When the concentration of liposome-encapsulated paclitaxel was above 5 μg/ml, the cell mortality rate was significantly higher than that in the non-transfected group (P<0.05). The proportion of apoptotic cells also increased in the nm23-H1-siRNA-transfected group, compared with that of the non-transfected group (t=3.812,P<0.05), while the proportion of cells at S and G2/M phase decreased after transfection with nm23-H1-siRNA (S phase:t=8.356,P<0.05;G2/M phase:t=7.256,P<0.05). Conclusions Nm23-H1 is related with the chemoresistance to liposome-encapsulated paclitaxel in lung adenocarcinoma cell line A549 cells. Inhibition of the expression of nm23-H1 by nm23-H1-siRNA can improve the in vitro chemosensitivity of A549 cells to liposome-encapsulated paclitaxel.     

Aurora A反义寡核苷酸对人肺癌细胞系A549细胞生长与细胞周期的影响

目的：探讨Aurora A基因表达的下调对肺癌细胞生长和细胞周期分布的影响。方法：用脂质体瞬时转染法介导Aurora A反义硫代磷酸寡核苷酸（antisense oligodeoxynucleotides,ASODN）处理肺腺癌A549细胞,RT-PCR及Western-blot方法检测Aurora A mRNA和蛋白表达,流式细胞仪检测细胞周期分布的变化,四氮唑盐法（MTT）检测转染前后细胞生长抑制率变化。结果：Aurora A反义寡核苷酸作用于A549细胞后,细胞的生长抑制率在一定范围内呈剂量和时间依赖性,其中48h的IC50值约为300nmol/L,在此浓度和时间下,Aurora A反义寡核苷酸可明显降低Aurora A mRNA和蛋白的表达,且细胞周期阻滞于G2/M期和S期。结论：下调Aurora A表达可抑制肺腺癌细胞系A549的增殖,同时导致细胞阻滞于G2/M期及S期。     

Testin在非小细胞肺癌组织和细胞株中的表达及其对癌细胞恶性生物学行为的影响

目的:探讨Testin基因(TES)在非小细胞肺癌(NSCLC)组织和细胞株中的表达及其对人肺癌A549细胞增殖、迁移、侵袭和凋亡的影响.方法:收集2015年1月至2015年12月在华中科技大学同济医学院附属同济医院手术切除的27例NSCLC患者的癌组织及癌旁组织标本,用Western blotting法检测癌组织和癌旁组织,以及正常人胚肺成纤维细胞株MRC5和肺癌细胞株A427、A549、H1299、LK2、PC9和SW900中TES蛋白的表达水平.应用短发卡RNA(shRNA)瞬时转染肺癌细胞株A549干扰TES基因的表达,并进一步检测TES低表达对A549细胞增殖、迁移、侵袭以及凋亡的影响,同时检测凋亡相关蛋白Bax、Bcl-2和Cas-pase-3的表达.结果:在NSCLC组织和细胞株中TES蛋白的表达明显下降(均P＜0.05).shTES干扰A549细胞后,TES mRNA和蛋白表达水平均显著下降(均P＜0.05).抑制TES表达显著增强A549细胞的增殖[(2.75±0.04) vs (1.79±0.06),P＜0.05]、迁移[(52.3±2.6)％s(19.7±1.4)%,P＜0.05]和侵袭能力[(31.2±3.9)％vs(14.5±4.1)％,P＜0.05],同时降低了细胞凋亡率[(8.2±1.1)％s(23.1±1.7)％,P＜0.05].TES低表达使A549细胞Bax和Caspase-3蛋白表达明显下降(P＜0.05)、Bcl-2蛋白表达明显升高(P＜0.05).结论:TES在NSCLC组织中呈低表达,TES表达下调具有促进肺癌细胞的增殖、迁移、侵袭并抑制凋亡等生物学效应,其有可能成为肺癌治疗一个新靶点.     

Polo-like激酶1反义RNA对肺癌细胞A549细胞周期的影响

背景与目的Polo-Like激酶1(Polo-likekinase 1,Plk1)是参与有丝分裂调控的重要分子,已在肺腺癌细胞株A549中检测到Plk1的高表达,并认为Plk1高表达与肺癌患者的放化疗耐受和预后相关。本研究利用反义RNA技术,探讨Plk1基因表达下调对肺癌细胞细胞周期的影响。方法培养肺腺癌细胞株A549,构建表达Plk1反义RNA的质粒pcDNA3.0-Plk1(pc3.0P),通过脂质体介导转入A549细胞,Westernblot、RT-PCR检测Plk1的表达,BrdU脉冲标记和流式细胞术分析细胞周期变化;免疫荧光染色检测α微管蛋白的表达。结果A549细胞转染pc3.0P后,Plk1mRNA的表达较对照组显著下降(P<0.05),转染24h后Plk1mRNA的表达下降46.75%,转染48h后下降61.84%;蛋白表达亦有下降;S期细胞百分数(BrdU标记指数)较对照组明显下降(P<0.05),转染后48h仅有25.59%;转染后72h出现G2/M期阻滞(P<0.05),并出现细胞凋亡;微管染色显示细胞周边缺乏微管的聚集,单极纺锤体形成。结论Plk1影响纺锤体微管的形成,使A549细胞增殖速度减慢,细胞周期阻滞并发生凋亡。     

慢病毒沉默PRDX4抑制肺癌细胞A549迁移和侵袭及其机制的探讨北大核心

目的:研究慢病毒沉默过氧化物还原酶4(peroxiredoxin 4,PRDX4)对肺癌细胞迁移和侵袭能力的影响,并探讨其分子机制。方法:采用shRNA干扰技术敲低肺癌细胞系A549中PRDX4的表达,Western blot和qRT-PCR验证转染效果;划痕愈合实验检测细胞迁移能力;Transwell细胞侵袭实验检测细胞侵袭能力;Western blot分析p-ERK1/2、N-cadherin、Vimentin蛋白表达。结果:成功构建沉默PRDX4的肺癌细胞系;慢病毒感染后A549细胞PRDX4蛋白和mRNA表达水平显著低于对照组(P<0.05);与BC组和NC组相比,Sh组细胞迁移、侵袭能力显著降低(P<0.05);与BC组和NC组相比,Sh组细胞p-ERK1/2、N-cadherin、Vimentin蛋白表达水平显著降低。结论:沉默PRDX4可能通过ERK信号通路抑制肺癌细胞的迁移和侵袭。     

Rb94对人肺腺癌细胞系A549放射增敏作用的实验研究

目的 构建人视网膜母细胞瘤基因(Rb94)真核表达载体,观察其对人肺腺癌细胞系A549的放射增敏作用并探讨其机制.方法 构建重组表达质粒pIPES-Rb94,经脂质体转染A549细胞、G418筛选获得稳定转染的细胞系.细胞计数法绘制生长曲线、计算细胞倍增时间;实时RT-PCR检测hTERT、Bcl-2mRNA的表达;成克隆实验检测pIRES-Rb94对A549细胞放射敏感性影响,计算放射增敏比(SER);流式细胞仪分析细胞周期.结果 成功构建pIRES-Rb94质粒,转染A549细胞后获得稳定转染细胞系pIRES-Rb94-A549.与A549细胞相比,pIRES-Rb94-A549细胞倍增时间延长(31.5h:39.5 h;t=5.15,P<0.01);hTERT、Bcl-2 mRNA表达下降(0.02:1.00;t=18.99,P<0.01;0.01:1.00;t=13.73,P<0.01);G2+M期细胞增加(35.91%:4.53%;t=36.78,P=0.00),G0+G1期和S期细胞减少(47.02%:74.07%;t=11.71,P=0.00和17.07%:22.32%;t=2.30,P<0.05);由D0值计算的SER为1.30.结论 Rb94对A549细胞具有明显放射增敏作用,其机制可能是G2+M期阻滞作用以及下调hTERT、Bcl-2 mRNA表达,并可能通过调节细胞周期来影响细胞增殖.     

番茄红素对肺癌A549细胞增殖和凋亡的影响及其机制研究

目的:研究番茄红素(lycopene,LP)对人非小细胞肺癌A549细胞增殖和凋亡的影响并探讨其机制.方法:体外培养并取对数生长期人非小细胞肺癌A549细胞,分别给予不同浓度的LP(2.5、5、10、20 μg/ml)和顺铂(40 μg/ml)进行干预,48 h后采用四甲基偶氮唑盐(MTT)比色法测定吸光度值并计算细胞增殖抑制率,流式细胞术(FCM)检测细胞周期和细胞凋亡状况,逆转录PCR法(RT-PCR)检测细胞中凋亡相关基因(BaxmRNA、bcl-2 mRNA)表达,免疫印迹法(Western blot)检测凋亡相关蛋白(caspase-3)表达.结果:与空白对照组比较,LP能够剂量依赖性地提高人非小细胞肺癌A549细胞增殖抑制率,延长细胞周期中G0/G1期并缩短G2/M期,提高A549细胞凋亡率(AI),上调Bax mRNA表达、下调bcl-2 mRNA表达,提高Bax/bcl-2比值,上调caspase-3蛋白表达.结论:LP具有抑制人非小细胞肺癌A549细胞增殖并促进其凋亡的作用,其机制可能与LP能够阻滞细胞周期并调节凋亡相关基因蛋白表达有关.     

单唾液酸神经节苷脂3对人肺腺癌细胞增殖凋亡及血管内皮生长因子表达的影响

目的 研究外源性单唾液酸神经节苷脂3(GM3)对人肺腺癌细胞增殖、凋亡和血管内皮生长因子(VEGF)表达的影响,探讨GM3的抗肿瘤作用.方法 以不同浓度外源性GM3干预人肺腺癌A549细胞48 h,采用四甲基偶氮唑蓝(MTT)法检测细胞增殖变化,Annexin V-FITC/PI双染流式细胞学技术检测细胞凋亡变化,逆转录聚合酶链反应(RT-PCR)检测细胞VEGF mRNA的表达水平,激光共聚焦显微镜观察细胞VEGF蛋白的表达变化.结果 2.5、10、40、160和640 μmol/L GM3干预A549细胞48 h,增殖抑制率分别为4.1%、8.9%、29.9%、34.2%和52.6%,GM3作用于A549细胞48 h的半数抑制浓度(IC50)为412 μmol/L.与对照组相比,当GM3＞10 μmol/L时,对细胞增殖抑制作用明显(P＜0.05),且具有浓度依赖性.10、40和160μmol/L GM3干预A549细胞48 h,A549细胞的凋亡率分别为(1.3±0.6)%、(4.8±0.4)%、(14.2±1.0)%.与对照组相比,当GM3＞40 μmol/L时,其促进细胞凋亡作用明显(P＜0.05).与对照组相比,经浓度＞40 μmol/L的GM3干预后,A549细胞的VEGF mRNA表达水平显著下降(P＜0.05).随着GM3浓度增高,A549细胞的VEGF荧光强度明显减弱.结论 GM3能呈浓度依赖性抑制人肺腺癌细胞株A549增殖,促进A549细胞凋亡,下调A549细胞VEGF mRNA和蛋白水平的表达,提示GM3可能通过调节肿瘤细胞凋亡和肿瘤血管生成而发挥双重抗肿瘤作用.

Abstract：

Objective To determine the effect of exogenous GM3 on proliferation, apoptosis and VEGF expression in human lung adenocarcinoma cell line A549 cells.Methods A549 cells were treated with GM3 at different concentrations for 48 hours.MTT assay was used to detect the cell proliferation and flow cytometry was applied to analyze cell apoptosis.RT-PCR was used to detect the expression level of VEGF mRNA and confocal laser scanning microscopy was applied to observe the localization and fluorescence intensity of VEGF.Results Comparing with the control, being treated with higher than 10μmol/L GM3 significantly inhibited A549 cell proliferation ( P ＜ 0.05 ), and the suppressive effect could be enhanced following increasing doses.The IC50 was 412 μ mol/L.Comparing with the control, being treated with higher than 40 μmol/L GM3 significantly promoted the apoptotic rate of A549 cells ( P ＜ 0.05 ).Comparing with the control, being treated with higher than 40 μ mol/L GM3 significantly decreased the VEGF mRNA level of A549 cells (P＜0.05), and the fluorescence intensity of VEGF distinctly weakened.Conclusions Exogenous ganglioside GM3 can inhibit the proliferation, promote apoptosis, and down-regulate the VEGF expression level in A549 cells.This may be considered as two mechanisms of GM3 for its anti-tumor effect by modulating cell apoptosis and angiogenesis.     

survivin反义寡核苷酸提高人肺腺癌A549耐药细胞系对顺铂敏感性的研究

目的探讨存活素反义寡核苷酸(ASODN)体外转染对耐顺铂人肺腺癌细胞A549/ CDDP凋亡及其对顺铂(CDDP)敏感性的影响。方法常规体外培养A549/CDDP细胞,以脂质体包裹的survivin ASODN转染细胞,采用RT-PCR法和免疫细胞化学法检测survivin mRNA及蛋白表达。通过形态学观察、DNA琼脂糖凝胶电泳、caspase-3酶活性及细胞凋亡率(AI)的测定,评价细胞凋亡程度。采用MTT法测定细胞存活率和生长抑制率,计算半效抑制浓度(IC_(50))及耐药倍数(RI)。结果转染组survivin mRNA及蛋白表达下调明显,分别达41．56%和0．864±0．045,与其他各组比较,差异均有统计学意义(P＜0．05)。细胞形态学显示有细胞凋亡改变,表现为细胞核固缩、核边集和核碎裂等;DNA凝胶电泳可见DNA梯形条带;ASODN转染组细胞凋亡率和caspase-3相对活性分别增高至34．03%和1．1298±0．2502,而ASODN+CDDP组增高更为明显,分别达65．85%和1．6805±0．2758,与其他各组比较,差异均有统计学意义(P＜0．05)。ASODN转染组和ASODN+CDDP组生长抑制率分别增高至59．3%和83．7%(P＜0．05);而ASODN转染组细胞对CDDP的IC_(50)由对照组的(225．03±10．59)μmol/L减低至(158．84±4．26)μmol/L,RI由11．9减至8．4。结论survivin ASODN通过下调survivin的表达,自身诱导了细胞凋亡,并逆转细胞对CDDP诱导的细胞凋亡的耐受,降低凋亡阈值,从而增强了人肺腺癌细胞对CDDP的敏感性。     

沉默CXCR4的表达逆转肺癌细胞顺铂耐药

目的:探讨沉默CXCR4对肺癌细胞顺铂耐药的影响,并研究其分子作用机制。方法:利用RT-qPCR测定肺癌耐药（A549/DDP）及药物敏感细胞株（A549）中CXCR4 mRNA的表达水平;利用LipofectamineTM 2000将siRNA-CXCR4转染至肺癌耐药细胞株中,RT-qPCR及蛋白质印迹法（Western blot）验证siRNA对CXCR4基因的靶向沉默效率;利用CCK-8法检测沉默CXCR4后肺癌耐药细胞的增殖活性;利用流式细胞仪测定沉默CXCR4后肺癌耐药细胞的凋亡率;利用MTT法测定沉默CXCR4后肺癌耐药细胞对顺铂的敏感性;利用Western blot实验测定沉默CXCR4后CYP1B1蛋白的表达水平。结果:RT-qPCR实验结果显示,肺癌耐药细胞株A549/DDP中CXCR4 mRNA的表达量显著高于药物敏感细胞株（P＜0.01）;体外转染siRNA-CXCR4可明显下调A549/DDP细胞株中CXCR4的表达（P＜0.001）;CCK-8实验结果显示,沉默CXCR4的表达抑制了A549/DDP细胞的增殖活性（P＜0.01）;流式细胞仪实验结果显示,沉默CXCR4的表达促进了A549/DDP细胞凋亡（P＜0.01）;MTT实验结果显示,沉默CXCR4的表达增加了A549/DDP细胞对顺铂的敏感性（P＜0.01）;Western blot实验结果显示,沉默CXCR4后CYP1B1蛋白的表达水平显著降低（P＜0.05）。结论:沉默CXCR4的表达抑制肺癌耐药细胞增殖、促进凋亡,逆转肺癌细胞顺铂耐药,CXCR4正向调控CYP1B1的表达,可能是CXCR4逆转肺癌细胞顺铂耐药的作用机制。