黄芪甲甙协同吉非替尼对非小细胞肺癌细胞敏感性的作用研究

发布时间：2018-06-02 17:27

本文选题：非小细胞肺癌 + 黄芪甲甙　；参考：《成都中医药大学》2016年博士论文

【摘要】：研究背景与目的：肺癌(Lung cancer)是我国最常见的恶性肿瘤,2015年国家癌症中心发布的数据显示,肺癌是发病率最高的肿瘤,也是癌症死因之首。其中以非小细胞肺癌(non-small cell lung cancer. NSCLC)最为多见,约占肺癌总数的80%-87%。随着对NSCLC研究的深入,表皮生长因子(Epidermal Growth Factor Receptor. EGFR,又称ErbB1或HER1)在NSCLC的发病机制、启动基因、肿瘤细胞生长、细胞异质性和侵袭力增加过程中的作用被逐渐阐明,使EGFR成为靶向治疗的重要靶点之一。尽管靶向药物能够使肿瘤缓解率增加、无症状生存期延长,但随着TKIS使用时间的延长,不可避免的出现耐药。因此,研发延缓或者逆转靶向治疗耐药的方法是靶向治疗的热门领域。黄芪是常用的中药材,含有许多化学成分,大量基础及临床研究报道,黄芪在体内和体外均有抗肿瘤作用,黄芪甲甙(Astragaloside Ⅳ,AS-Ⅳ)是黄芪的主要成分之一,研究表明,AS-IV可以通过调节性T细胞和细胞毒性T淋巴细胞以及通过相关的信号传导途径抑制肺癌细胞的增殖、迁移和侵袭。SIRT6是NAD+依赖性第Ⅲ类组蛋白去乙酞化酶家族(Sirtuin家族)中重要的一份子。表观遗传调控是目前肿瘤研究的热点,通过DNA、组蛋白等甲基化、乙酞化、磷酸化以及染色体重塑等一系列生物过程来调节基因表达。现在研究发现,SIRT6在肿瘤组织中的表达情况与肿瘤发生、肿瘤转移、术后生存率、术后复发率以及化疗敏感性密切相关,目前认为SIRT6是一个新的肿瘤相关基因、减轻或者抑制,甚至逆转化疗耐药的新靶点。研究目的是(1)探究SIRT6在NSCLC组织中的表达丰度及表达定位,以及SIRT6在人肺癌NCI-H1299、PC9/GR、A549细胞中的表达；(2)不同浓度黄芪甲甙作用于人肺癌NCI-H1299、PC9/GR、A549细胞,观察其对不同突变状态的肺癌细胞增殖抑制作用；(3)不同浓度黄芪甲甙联合吉非替尼作用于人肺癌NCI-H1299、PC9/GR、A549细胞,观察其对不同突变状态的肺癌细胞增殖抑制作用；(4)AS-Ⅳ单独干预细胞以及AS-Ⅳ联合吉非替尼联合干预的细胞后SIRT6的表达水平变化;(5)慢病毒转染PC9/GR细胞,检测AS-Ⅳ联合吉非替尼对转染后PC9/GR细胞增殖水平的影响；(6)探讨在耐药的肺癌细胞中SIRT6和NF-κB信号通路之间相互作用。研究方法：1、利用免疫组化法检测NSCLC肿瘤组织、癌旁组织及良性病变组织中SIRT6的表达量,并收集患者临床资料,分析SIRT6与临床参数的关系；2、采用qRT-PCR和Western blot实验方法检测人正常肺组织腺上皮细胞BEAS-2B、NCI-H1299、PC9/GR、A549细胞中SIRT6的表达；3、不同浓度吉非替尼作用于人肺癌NCI-H1299、PC9/GR、A549细胞,以CCK-8法检测细胞增殖抑制率,使用酶标仪读取OD值,按公式计算细胞生长抑制率,描绘细胞生长曲线；4、不同浓度AS-Ⅳ作用于人肺癌NCI-H1299、PC9/GR、A549细胞,以CCK-8法检测细胞增殖抑制率,使用酶标仪读取OD值,按公式计算细胞生长抑制率,描绘细胞生长曲线；5、不同浓度AS-Ⅳ联合吉非替尼作用于人肺癌NCI-H1299、PC9/GR、A549细胞,以CCK-8法检测细胞增殖抑制率,计算细胞生长抑制率,描绘细胞生长曲线；6、不同浓度AS-Ⅳ联合吉非替尼,吉非替尼不同浓度单独作用于人肺癌PC9/GR细胞,使用qRT-PCR检测SERT6 mRNA表达变化；使用Western-blot技术检测SIRT6的蛋白表达变化；7、选择慢病毒进行介导转染,使SIRT6基因真核表达载体转染PC9/GR细胞,倒置荧光显微镜观察荧光表达,测定病毒滴度。Western Blot检测,SIRT6蛋白的表达。检测AS-Ⅳ联合吉非替尼对转染后PC9/GR细胞增殖水平的影响；8、Western-blot检测IκB及NF-κBp65,探讨在耐药的肺癌细胞中SIRT6和NF-κB信号通路之间相互作用。结果：1、结果SIRT6在NSCLC组织中低于癌旁组织及良性病变组织；分化程度高者平均IOD高于分化程度中、低的患者。发生淋巴结转移和未发生淋巴结转移的患者SIRT6的平均IOD之间存在显著性差异；2、通过CCK-8实验,A549、PC9/GR和H1299经不同浓度吉非替尼干预48h后,细胞增殖抑制率,随着浓度的增高,体现剂量依赖性抑制作用。与对照相比,吉非替尼的细胞抑制作用显著差异。吉非替尼半抑制浓度分别为21.29μM、 45.98±1.62μM、26.29±2.25μM;3、采用CCK-8法,选取不同浓度的AS-Ⅳ作用于A549和PC9/GR细胞株,研究发现,当干预时间为48h时,3ng/ml和6ng/ml浓度的AS-Ⅳ对A549和PC9/GR细胞均不能产生明显的抑制作用,而大于12ng/ml的AS-Ⅳ可明显抑制细胞是的生长。选取不同浓度的AS-Ⅳ作用于H1299细胞株,当干预时间为48h时,大于6ng/ml的AS-Ⅳ可明显抑制细胞是的生长；4、用含4个浓度(3ng/ml、6ng/ml、12ng/ml、24ng/ml)的AS-Ⅳ和10μM的吉非替尼培养液联合作用于A549、H11299、PC9/GR细胞,48小时后使用CCK-8试剂酶标仪检测,同一grfitinib浓度,不同浓度的AS-Ⅳ作用于同一细胞株,细胞增殖抑制率显著不同,细胞增殖抑制率随AS-Ⅳ浓度增加而增加。3种细胞均可见gefitinib联合AS-Ⅳ浓度为24ng/ml时细胞增值抑制率最高,但同一grfitinib浓度下,随药物浓度增加,不同细胞的增殖能力不同,PC9/GR细胞增殖能力最差。在12ng/ml的AS-Ⅳ联合10 μ M的吉非替尼作用下Q值最大；5、通过qRT-PCR和Western blotting技术,肺癌PC9/GR、A549、H1299细胞与正常肺组织腺上皮BEAS-2B细胞相比,SIRT6 mRNA和蛋白的表达水平显著下降；6. qRT-PCR分析结果显示,与对照组相比,AS-Ⅳ 12ng/ml联合吉非替尼10μM干预PC9/GR, SITR6mRNA的表达增加,P0.01。单独AS-Ⅳ 12ng/ml单独干预PC9/GR,与对照组比较,而吉非替尼单独干预PC9/GR,与对照组比较,无统计学意义；Western blotting检测结果,与对照组相比,AS-Ⅳ 12ng/ml联合吉非替尼10μ M干预PC9/GR, SITR6蛋白的表达增加。单独AS-IV 12ng/ml单独干预PC9/GR,与对照组比较；7、吉非替尼浓度梯度0、5、10、20、40μM单独干预PC9/GR,与对照组比较,细胞的SIRT6mRNA及蛋白水平均有所提高,但没有明显统计意义；8、构建了稳定转染SIRT6过表达载体的PC/GR,倒置荧光显微镜下观察,细胞转染效率估计大约在80%-85%,通过Western Blot检测,与对照比较,SIRT6蛋白的表达量有大幅度的提高；9、予以10rtM的吉非替尼和浓度梯度的AS-IV干预pLVTHM-SIRT6和pLVTHM-scramble细胞,SIRT6、吉非替尼和AS-IV联合作用时细胞的生长抑制现象,与其他三组比较明显增强；pLVTHM-SIRT6组与pLVTHM-scramble+吉非替尼组比较无明显统计意义,但与pLVTHM-scramble组比较有统计意义；予以12ng/ml的AS-IV和浓度梯度吉非替尼干预pLVTHM-SIRT6和pLVTHM-scramble细胞,SIRT6、AS-IV和吉非替尼联合作用时细胞的生长抑制现象明显,pLVTHM-SIRT6组与pLVTHM-scramble+AS-Ⅳ比较无明显统计意义,但与pLVTHM-scramble组比较有差异；10、Western blot检测pLVTHM-scramble细胞和Plvthm-SIRT6细胞,IκB在pLVTHM-scramble细胞中的表达水平低于Plvthm-SIRT6细胞,予以AS-IV24ng/ml干预pLVTHM-scramble细胞和Plvthm-SIRT6细胞,IκB在pLVTHM-scramble细胞表达水平低于Plvthm-SIRT6细胞,但与未干预细胞相比,两种细胞IκB水平均有上调：予以AS-IV24ng/m干预pLVTHM-scramble细胞和Plvthm-SIRT6细胞48h后,提取各组细胞核蛋白,Western blot检测NF-κBP65亚单位的表达,NF-κBp65亚单位在Plvthm-SIRT6+AS-IV干预细胞中表达最低,与pLVTHM-scramble细胞相比,SIRT6过表达降低了NF-κBp65亚单位在Plvthm-SIRT6细胞核中的积累。结论：研究表明,1、SIRT6在NSCLC组织及肺癌细胞中的表达下降；2、AS-IV对肺癌细胞有抑制作用,呈现浓度趋势；3、AS-IV联合吉非替尼可增加肿瘤细胞抑制率；4、过表达SIRT6可以抑制NF-κB的活化；5、AS-IV可通过上调SITR6,抑制NF-κB活化,促进了吉非替尼的敏感性。
[Abstract]:Background and objective: lung cancer (Lung cancer) is the most common malignant tumor in China. The data released by the National Cancer Center in 2015 showed that lung cancer is the highest incidence of cancer and the leading cause of cancer death. Non small cell lung cancer (non-small cell lung cancer. NSCLC) is the most common, and the 80%-87%. of the total number of lung cancer is associated with NSC In LC research, the role of epidermal growth factor (Epidermal Growth Factor Receptor. EGFR, also known as ErbB1 or HER1) is gradually clarified in the pathogenesis of NSCLC, the promoter gene, tumor cell growth, cell heterogeneity and increase of invasiveness, which makes EGFR become one of the important targets in target therapy. Although targeted drugs can make tumors The remission rate increases and the asymptomatic life prolongs, but with the prolonged use of TKIS, drug resistance inevitably occurs. Therefore, developing a method to postpone or reverse the drug resistance is a hot field in target therapy. Astragalus is a common medicinal herb with many chemical components, a large number of basic and clinical research reports, Astragalus membranaceus in the body and in the body. There are anti-tumor effects in vitro. Astragalin (Astragaloside IV, AS- IV) is one of the main components of Astragalus membranaceus. The study shows that AS-IV can inhibit the proliferation of lung cancer cells through regulatory T cells and cytotoxic T lymphocytes and the related signal transduction pathway. Migration and invasion of.SIRT6 are NAD+ dependent group III histone group B An important part of the phthalidase family (Sirtuin family). Epigenetic regulation is a hot spot in cancer research. Through a series of biological processes such as DNA, histone methylation, phthalide, phosphorylation and chromosome remodeling, the expression of SIRT6 in tumor tissue and tumor, tumor, tumor, and tumor are found. Metastasis, postoperative survival rate, postoperative recurrence rate and chemosensitivity are closely related. At present, SIRT6 is considered as a new tumor related gene, alleviating or inhibiting, and even reversing the new target of chemotherapeutic resistance. The purpose of this study is (1) to explore the expression and expression of SIRT6 in NSCLC tissues, and SIRT6 in human lung cancer NCI-H1299, PC9/GR, A54 The expression of 9 cells; (2) different concentrations of astragalin acted on human lung cancer NCI-H1299, PC9/GR, A549 cells, and observed its inhibitory effect on the proliferation of lung cancer cells with different mutant states. (3) different concentrations of astragalin combined with gefitinib on human lung cancer NCI-H1299, PC9/ GR, A549 cells, and observed their different mutant states of lung cancer cells. Proliferation inhibition; (4) AS- IV alone intervention cells and AS- IV combined with gefitinib combined with the intervention of the expression level of SIRT6; (5) lentivirus transfected PC9/GR cells, AS- IV combined with gefitinib on the proliferation of PC9/GR cells after transfection; (6) to explore the resistance of lung cancer cells in the SIRT6 and NF- kappa B signal Interaction between roads. 1. 1, using immunohistochemical method to detect the expression of SIRT6 in NSCLC tumor tissue, paracancerous tissue and benign pathological tissue, and collect the clinical data of patients, analyze the relationship between SIRT6 and clinical parameters. 2, using qRT-PCR and Western blot test method to detect human normal lung gland epithelial cells BEAS-2B, NCI-H 1299, PC9/GR, A549 cells in the expression of SIRT6; 3, different concentrations of gefitinib in human lung cancer NCI-H1299, PC9/GR, A549 cells, CCK-8 method to detect cell proliferation inhibition rate, use an enzyme labeled instrument to read the OD value, calculate cell growth inhibition rate and describe cell growth curve according to formula; 4, different concentration AS- IV action on human lung cancer NCI-H1299, PC9/G R, A549 cells, the cell proliferation inhibition rate was detected by CCK-8 method, the OD values were read by the enzyme labeling instrument, the cell growth inhibition rate and the cell growth curve were calculated according to the formula. 5, different concentrations AS- IV combined with gefitinib to the human lung cancer NCI-H1299, PC9/GR, A549 cells, and the cell proliferation inhibition rate was detected by CCK-8 method and the cell growth inhibition rate was calculated. Draw the cell growth curve; 6, different concentrations AS- IV combined gefitinib, gefitinib acts on human lung cancer PC9/GR cells separately, using qRT-PCR to detect the expression of SERT6 mRNA, and Western-blot technique to detect the protein expression changes of SIRT6; 7, select the lentivirus to mediate transfection and make the SIRT6 gene eukaryotic expression vector. PC9/GR cells were stained, fluorescence microscopy was inverted to observe fluorescence expression,.Western Blot detection of virus titer and expression of SIRT6 protein. The effect of AS- IV combined with gefitinib on the proliferation of PC9/GR cells after transfection was detected. 8, Western-blot was used to detect I kappa B and NF- kappa Bp65, and to explore the phase between SIRT6 and nuclear kappa signaling pathway in drug-resistant lung cancer cells. Results: results: results: 1, SIRT6 was lower in NSCLC tissues than para cancerous tissue and benign lesion tissue; the average IOD of high differentiated patients was higher than that of differentiation. The average IOD of SIRT6 in patients with lymph node metastasis and non lymph node metastasis was significantly different; 2, A549, PC9/GR, and H1299 via CCK-8 experiment. After different concentrations of gefitinib intervened for 48h, the inhibitory rate of cell proliferation was dose-dependent. Compared with the control, the inhibitory effects of gefitinib were significantly different. The hemi inhibitory concentration of gefitinib was 21.29 mu M, 45.98 + 1.62 micron M, 26.29 + 2.25 micron M; 3, the CCK-8 method was used to select AS- IV of different concentrations. As the A549 and PC9/GR cell lines, it was found that when the intervention time was 48h, the AS- IV concentration of 3ng/ml and 6ng/ml had no obvious inhibitory effect on A549 and PC9/GR cells, while AS- IV greater than 12ng/ml could inhibit the growth of the cells. AS- IV in 6ng/ml could obviously inhibit the growth of cells; 4, a gefitinib culture containing 4 concentrations (3ng/ml, 6ng/ml, 12ng/ml, 24ng/ml) combined with a gefitinib culture of 10 mu M to act on A549, H11299, PC9/GR cells. 48 hours later, a CCK-8 reagent enzyme scale was used to detect the same concentration, and the same cell strain was acted on the same cell strain. The inhibitory rate of cell proliferation was significantly different, the proliferation inhibition rate of cell proliferation was increased with the concentration of AS- IV, and the proliferation inhibition rate of.3 cells was the highest when the concentration of gefitinib combined with AS- IV was 24ng/ml, but with the same grfitinib concentration, the proliferation ability of different cells was different and the proliferation ability of PC9/GR cells was the worst with the same grfitinib concentration. A in 12ng/ml was in A. The Q value of S- IV combined with gefitinib was the largest. 5, the expression level of SIRT6 mRNA and protein decreased significantly compared with BEAS-2B cells of normal lung tissue by qRT-PCR and Western Blotting Technology. The results showed that compared with the control group, the expression of Q, PC9/GR, A549, H1299 cells and BEAS-2B cells in normal lung tissue showed a significant decrease. The expression of PC9/GR, SITR6mRNA increased and P0.01. alone AS- IV 12ng/ml intervened PC9/GR alone, compared with the control group, while gefitinib intervened PC9/GR alone, and there was no significant difference between the control group and the control group. The Western blotting test results showed that AS- IV 12ng/ml combined with gefitinib interfered with the control group as compared with the control group. AS-IV 12ng/ml alone intervention PC9/GR, compared with the control group; 7, gefitinib concentration gradient 0,5,10,20,40 M alone intervention PC9/GR, compared with the control group, the level of SIRT6mRNA and protein increased, but no significant statistical significance; 8, the construction of the stable transfection of SIRT6 overexpressed carrier PC/GR, inverted fluorescence display Microscopically, the cell transfection efficiency was estimated at about 80%-85% and detected by Western Blot. Compared with the control, the expression of SIRT6 protein was greatly improved; 9, 10rtM's gefitinib and concentration gradient AS-IV intervention pLVTHM-SIRT6 and pLVTHM-scramble cells, SIRT6, gefitinib and AS-IV were combined to inhibit the growth of cells. The system was significantly enhanced with the other three groups; the pLVTHM-SIRT6 group had no statistical significance compared with the pLVTHM-scramble+ gefitinib group, but had a statistical significance compared with the pLVTHM-scramble group; the AS-IV and concentration gradient gefitinib of 12ng/ml were used to interfere with pLVTHM-SIRT6 and pLVTHM-scramble cells, SIRT6, AS-IV and gefitinib cooperation. The growth inhibition of cell growth was obvious. The pLVTHM-SIRT6 group had no significant statistical significance compared with pLVTHM-scramble+AS- IV, but compared with the pLVTHM-scramble group, 10, Western blot detected pLVTHM-scramble and Plvthm-SIRT6 cells. The expression level of I kappa B in pLVTHM-scramble cells was lower than that of Plvthm-SIRT6 cells. G/ml interfered with pLVTHM-scramble and Plvthm-SIRT6 cells, and the expression level of I kappa B in pLVTHM-scramble cells was lower than that of Plvthm-SIRT6 cells, but the level of I kappa B was up to up in the two cells compared with that of the untreated cells. The expression of NF- kappa BP65 subunit was measured, NF- kappa Bp65 subunit was expressed the lowest in Plvthm-SIRT6+AS-IV intervention cells. Compared with pLVTHM-scramble cells, SIRT6 overexpression reduced the accumulation of NF- kappa Bp65 subunits in Plvthm-SIRT6 nuclei. Conclusion: the study showed that the expression of 1, SIRT6 in NSCLC tissues and lung cancer cells decreased; 2. Cancer cells have inhibitory effect, showing a concentration trend; 3, AS-IV combined with gefitinib can increase tumor cell inhibition rate; 4, overexpression of SIRT6 can inhibit the activation of NF- kappa B; 5, AS-IV can inhibit the activation of NF- kappa B by up regulation of SITR6, and promotes the sensitivity of gefitinib.
【学位授予单位】：成都中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R734.2

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