阿帕替尼联合紫杉醇对A-549肺癌小鼠移植瘤生长及生物钟基因表达的影响

目的分析阿帕替尼联合紫杉醇对A-549肺癌小鼠移植瘤生长及生物钟基因表达的影响。方法无特定病原体(SPF)级BALB/c裸鼠40只,雌雄各半,6~8周龄,体重18~20 g。于裸鼠右侧腋窝皮下注射0.2 mL A-549肿瘤细胞悬液建立裸鼠肺癌移植瘤模型。将建模成功的40只裸鼠按随机数字表法分为模型组、阿帕替尼组、紫杉醇组和联合给药组,每组各10只。模型组每日给予0.2 mL 0.9%氯化钠溶液灌胃,并经静脉注射0.2 mL 0.9%氯化钠溶液;阿帕替尼组每日给予0.2 mL 50 mg/kg阿帕替尼灌胃;紫杉醇组每日经静脉注射0.2 mL 20 mg/kg紫杉醇;联合给药组每日给予0.2 mL 50 mg/kg阿帕替尼灌胃,并经静脉注射0.2 mL 20 mg/kg紫杉醇。各组大鼠均连续给药21 d。给药期间,于规定时间对裸鼠的一般情况、肿瘤体积、肿瘤重量、肿瘤抑制率、肿瘤细胞凋亡率、移植瘤组中半胱氨酸蛋白酶-3(Caspase-3)和Ki-67的表达水平以及移植瘤组织中生物钟基因,如周期基因(Per)家族、时钟基因(CLOCK)、隐花色素基因(Cry)家族、脑和肌肉ARNT样蛋白1(BMAL1)、酪蛋白激酶1ε(CKIε)mRNA表达水平进行检测。结果给药后21 d,与模型组相比,阿帕替尼组、紫杉醇组和联合给药组小鼠精神状态、毛发、活动、进食状态均有所改善;小鼠体重、肿瘤抑制率、肿瘤细胞凋亡率、肿瘤组织中Per2、Per3、Cry1、Cry2、CKIεmRNA表达水平以及Caspase-3、Ki-67蛋白表达水平均显著升高,裸鼠移植瘤体积、肿瘤重量均显著减小,CLOCK的mRNA表达水平显著降低,差异均有统计学意义(P<0.05),且联合给药组较阿帕替尼组、紫杉醇组变化更显著。结论阿帕替尼与紫杉醇联合治疗对A-549肺癌移植瘤生长有较好的抑制作用,其机制可能与上调肿瘤细胞生物钟基因Per2、Per3、Cry1、Cry2和CKIεmRNA表达,下调基因CLOCK的mRNA表达及肿瘤细胞凋亡有关。

肺癌抗原组中强免疫原性抗原的筛选

目的筛选肺癌抗原组中强免疫原性抗原,以期为肺癌的早期诊断、疫苗研制提供参考。方法选取5份异体肺癌患者血清以重组cDNA表达文库血清学分析(SEREX)技术筛选肺癌cDNA表达文库,并进行生物信息学分析。此外,选取2017年2月至2018年2月收治的肺癌患者60例为肺癌组,另取同期进行体检的正常人员60例为对照组。分别采集两组人员血清标本,对比相关肺癌抗原的异体血清反应结果。并对差异有统计学意义的肺癌抗原进行受试者工作特征(ROC)曲线分析,明确其在肺癌早期诊断中的价值。结果本实验共完成3轮血清学筛选,最终得到70个阳性克隆。采用NCBI网上序列分析软件BLAST对各cDNA序列的同源性进行比对,结果显示上述70个阳性克隆分别表示63个不同基因,包括35个已知基因、26个功能未知基因以及2个新基因。其中35个已知基因中仅有DNA拓扑异构酶Ⅱα(Top2A)基因与肺癌相关,2个新基因分别为热休克蛋白90α(HSP90α)、真核翻译延长因子1γ(EF-1γ),功能未知基因中可能与肺癌相关的包括二羟基苯甲酸内置结合蛋白1配偶体(POB1)、中枢神经系肽酶Ⅰ(TPP1),共5种肺癌相关抗原。肺癌组Top2A、HSP90α、POB1抗原克隆的IgG血清抗体阳性率高于对照组(P<0.01)。Top2A、HSP90α、POB1抗原克隆联合检测诊断肺癌的曲线下面积、敏感性、特异性高于上述三项指标单独检测(P<0.05)。结论SEREX技术可用于肺癌抗原组中强免疫原性抗原的筛选、鉴定,临床工作中可将Top2A、HSP90α、POB1基因作为肺癌早期诊断以及免疫治疗的靶向基因。

Detection of lung adenocarcinoma using magnetic beads based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry serum protein profiling

Background Recently, due to the rapid development of proteomic techniques, great advance has been made in many scientific fields. We aimed to use magnetic beads （liquid chip） based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry （MALDI-TOF-MS） technology to screen distinctive biomarkers for lung adenocarcinoma （adCA）, and to establish the diagnostic protein profiles. Methods Using weak cation exchange magnetic beads （MB-WCX） to isolate and purify low molecular weight proteins from sera of 35 lung adCA, 46 benign lung diseases （BLDs） and 44 healthy individuals. The resulting spectra gained by anchor chip-MALDI-TOF-MS were analyzed by ClinProTools and a pattern recognition genetic algorithm （GA）. Results In the working mass range of 800-10 000 Da, 99 distinctive peaks were resolved in lung adCA versus BLDs, while 101 peaks were resolved in lung adCA versus healthy persons. The profile gained by GA that could distinguish adCA from BLDs was comprised of 4053.88, 4209.57 and 3883.33 Da with sensitivity of 80%, specificity of 93%, while that could separate adCA from healthy control was comprised of 2951.83 Da and 4209.73 Da with sensitivity of 94%, specificity of 95%. The sensitivity provided by carcinoembryonic antigen （CEA） in this experiment was significantly lower than our discriminatory profiles （P 〈0.005）. We further identified a eukaryotic peptide chain release factor GTP-binding subunit （eRF3b） （4209 Da） and a complement C3f （1865 Da） that may serve as candidate biomarkers for lung adCA. Conclusion Magnetic beads based MALDI-TOF-MS technology can rapidly and effectively screen distinctive proteins/polypeptides from sera of lung adCA patients and controls, which has potential value for establishing a new diagnostic method for lung adCA.

miR-10a通过FBXW7-ZEB2轴调控非小细胞肺癌肿瘤化疗耐药的动物实验研究

目的:通过动物实验,具体探讨微小RNA(mi R-10a)通过含F-框WD重复域蛋白7(FBXW7)-E盒锌指结合蛋白2(ZEB2)轴调控非小细胞肺癌的肿瘤化疗耐药作用。方法:非小细胞肺癌模型小鼠(n=42)随机平分为三组-模型组、mi R-10a组与环磷酰胺组,模型组给予生理盐水0.2 m L腹腔注射,环磷酰胺组给予环磷酰胺20 mg/kg腹腔注射,mi R-10a组给hsa-miR-10a mimics15 mg/kg联合环磷酰胺20 mg/kg腹腔注射,1次/d,持续给药14 d。结果:mi R-10a组与环磷酰胺组治疗第7 d与第14 d的肿瘤体积低于模型组,mi R-10a组低于环磷酰胺组(P<0.05)。mi R-10a组与环磷酰胺组治疗第14 d与第28 d的瘤体质量低于模型组,抑瘤率高于模型组,mi R-10a组与环磷酰胺组对比差异也有统计学意义(P<0.05)。mi R-10a组与环磷酰胺组治疗第14 d与第28 d的肿瘤细胞凋亡指数高于模型组,mi R-10a组高于环磷酰胺组(P<0.05)。mi R-10a组与环磷酰胺组治疗第14 d与第28 d的血清FBXW7、ZEB2含量低于模型组,mi R-10a组低于环磷酰胺组(P<0.05)。mi R-10a组与环磷酰胺组治疗第14 d与第28 d的FBXW7、ZEB2 m RNA与蛋白相对表达水平低于模型组,mi R-10a组低于环磷酰胺组(P<0.05)。结论:过表达mi R-10a能抑制非小细胞肺癌小鼠的FBXW7-ZEB2轴的激活,抑制血清FBXW7、ZEB2的表达,从而促进肿瘤细胞凋亡,改善肿瘤化疗耐药性,促进缩小肿瘤体积。

Study of differential proteins in lung adenocarcinoma using laser capture microdissection combined with liquid chip-mass spectrometry technology

Background In recent years the proportion of lung adenocarcinoma （adCA） which occurs in lung cancer patients has increased. Using laser capture microdissection （LCM） combined with liquid chip-mass spectrometry technology, we aimed to screen lung cancer biomarkers by studying the proteins in the tissues of adCA. Methods We used LCM and magnetic bead based weak cation exchange （MB-WCX） to separate and purify the homogeneous adCA cells and normal cells from six cases of fresh adCA and matched normal lung tissues. The proteins were analyzed and identified by matrix assisted laser desorption/ionization time-of-fight mass spectrometry （MALDI-OF-MS）. We screened for the best pattern using a radial basic function neural network algorithm. Results About 2.895x10s and 1.584x10s cells were satisfactorily obtained by LCM from six cases of fresh lung adCA and matched normal lung tissues, respectively. The homogeneities of cell population were estimated to be over 95% as determined by microscopic visualization. Comparing the differentially expressed proteins between the lung adCA and the matched normal lung group, 221 and 239 protein peaks, respectively, were found in the mass-to-charge ration （M/Z） between 800 Da and 10 000 Da. According to ttest, the expression of two protein peaks at 7521.5 M/Zand 5079.3 M/Z had the largest difference between tissues. They were more weakly expressed in the lung adCA compared to the matched normal group. The two protein peaks could accurately separate the lung adCA from the matched normal lung group by the sample distribution chart. A discriminatory pattern which can separate the lung adCA from the matched normal lung tissue consisting of three proteins at 3358.1 M/Z, 5079.3 M/Z and 7521.5 M/Z was established by a radial basic function neural network algorithm with a sensitivity of 100% and a specificity of 100%. Conclusions Differential proteins in lung adCA were screened using LCM combined with liquid chip-mass spectrometry technology, and a biomarker model was established. It is possible that this technology is going to become a powerful tool in screening and early diagnosis of lung adCA.