肺癌H460细胞与单个核细胞相互作用对MMP-1、-2、-9表达的影响

目的：研究单个核细胞和肺癌H460细胞三维立体混合培养对MMP-1、MMP-2和MMP-9表达的影响,并观察MMPs对胶原的降解作用。方法：活性胶原立体培养分为空白对照组（CG）、H460细胞组（HG）、单个核细胞组（MG）、混合组（HMG）共4组,观察癌细胞的生长状况。用明胶酶谱法测定不同时间点各组MMP-2和MMP-9表达,用Western blotting法测定MMP-1。结果：各组细胞在立体胶原内生长良好。MG未明显分泌MMP-1、MMP-2和MMP-9,HG和HMG均分泌MMP-1、MMP-2和MMP-9,但HMG分泌量明显多于HG。结论：炎症细胞和癌细胞混合培养后,细胞间相互作用促进MMPs分泌增多。基质金属蛋白酶可降解细胞外基质,进而促进肺癌的侵袭和转移。     

MiR-148a对肺癌细胞放射敏感性的影响

目的探讨miR-148a对肺癌A549、H460以及H1299细胞放射敏感性的影响。方法根据不同的处理方法,分别按以下方式将肺癌A549、H460和H1299细胞进行分组。（1）将肺癌A549和H460细胞分为3组:空白对照组、4 Gy γ射线照射组和8 Gy γ射线照射组。采用实时定量PCR检测miR-148a的表达水平;（2）将肺癌A549细胞分为2组:空白对照组、miR-148a转染组;同时,将肺癌H460细胞分为2组:空白对照组、anti-miR-148a转染组。分别对转染2组细胞进行不同剂量的γ射线照射,并采用克隆形成实验方法来检测细胞增殖;（3）将肺癌A549和H1299细胞分为3组:空白对照组、miR-148a单独处理组、miR-148a和雌激素受体（ER）共转染组。分别对3组细胞进行不同剂量的γ射线照射,并采用克隆形成实验方法检测细胞生长情况。采用Student t-test对数据进行统计学分析,P < 0.05表示差异有统计学意义。结果实时定量PCR实验结果显示,γ射线照射能够显著下调肺癌A549和H460细胞中miR-148a的表达水平。克隆形成实验结果显示,与空白对照组相比,miR-148a转染能够显著增强肺癌A549细胞的放射敏感性,差异有统计学意义（t=12.16,P < 0.01）,而anti-miR-148a转染能够显著降低肺癌H460细胞的放射敏感性,差异有统计学意义（t=11.93,P < 0.01）。同时,miR-148a过表达可以明显下调肺癌A549细胞中ER的mRNA及蛋白表达水平;而anti-miR-148a能够显著上调肺癌H460细胞中ER的mRNA及蛋白表达水平。另外,与miR-148a单独处理组相比,miR-148a和ER共转染组中miR-148a对肺癌A549和H1299细胞增殖的影响显著降低,差异有统计学意义（t=11.34、12.68,均P < 0.01）。结论照射可以诱导miR-148a的表达水平降低,人为过表达miR-148a能够抑制ER的蛋白表达水平,进而降低肺癌A549和H1299细胞的增殖能力,同时增强其放射敏感性。     

CRIM1对非小细胞肺癌辐射敏感性和转移的影响

目的:探讨富含半胱氨酸型运动神经元蛋白1（CRIM1）对非小细胞肺癌（NSCLC）辐射敏感性和转移的影响及可能的机制。方法:采用短发夹RNA（shRNA）敲降NSCLC H460、H358细胞中CRIM1的表达,并将H460、H358细胞分别分为3组：H460细胞、H460-shCRIM1细胞、H460-shNC细胞和...     

端粒酶催化亚基基因表达激活人胚肺成纤维细胞端粒酶活性

目的 :通过端粒酶催化亚基基因转入人胚肺成纤维细胞 ,激活人胚肺成纤维细胞端粒酶活性 ,探讨其在延缓细胞衰老中的作用。方法 :用PCI_hTRT质粒转染人胚肺成纤维细胞 ,G4 18筛选获得稳定表达端粒酶活性的人胚肺成纤维细胞 ,采用RT_PCR和TRAP法分别测定端粒酶催化亚基mRNA和端粒酶活性表达情况。结果 :获得了表达端粒酶活性的人胚肺成纤维细胞 ,其寿限长于正常人胚肺成纤维细胞。结论 :端粒酶催化亚基异位表达可以激活人胚肺成纤维细胞的端粒酶活性 ,并延长人胚肺成纤维细胞的寿命     

转录因子Oct-4在肺癌中的表达及对其生长、增殖的影响

目的观察Oct-4蛋白在肺癌组织及肺癌细胞系中的表达,探讨Oct-4对肺癌细胞生长、增殖的影响。方法比较正常组织、肺癌组织,正常肺细胞系和肺癌细胞系中Oct-4的表达水平;选取高表达Oct-4的肺癌细胞系,运用siRNA技术降低Oct-4表达,通过MTT实验、BrdU掺入实验等技术检测肺癌细胞的生长、增殖,并检测PI3K分子的表达变化。结果 Oct-4在肺癌组织及肺癌细胞中表达上调,分别与正常组织及HBE比较差异有统计学意义(P<0.05);降低Oct-4表达后,细胞BrdU掺入率明显降低(P<0.05),生长速度减慢(P<0.05),PI3K水平明显降低。结论 Oct-4在肺癌组织及肺癌细胞中表达升高,并通过PI3K促进肺癌细胞生长及增殖。     

一氧化氮拮抗肺成纤维细胞增殖的量效关系

目的：应用硝普钠（SNP）作为一氧化氮（NO）供体观察NO对^60Coγ射线照射的人胚肺成纤维细胞（HELF）增殖活力及其分泌功能的影响。以探讨NO的抗纤维化机理。方法：应用MTT比色法，Griess一氧化氮浓度测定法，免疫组化和原位杂交等方法检测成纤维细胞增殖活性及内皮素（ET）、血管紧张素Ⅱ（AⅡ）和转化生长因子β（TGFβ）的表达情况。结果：低剂量的^60Coγ射线照射对人胚肺成纤维细胞有促增殖作用，以5Gy剂量的促增殖作用最强，30h以内，硝普钠基本以线性方式释放NO，平均增幅为4．25μmol/L，初步认为4－5μmol/L的NO抑制成纤维细胞增生的最佳浓度，未照射成纤维细胞不表或仅微弱表达ET、AⅡ和TGFβ；照后细胞ET和TGFβmRNA及其蛋白表达显著增强，AⅡ合成增加，结论：NO供体硝普钠可明显抑制成纤维细胞的过度增殖和分泌活动，随浓度的加大，抑制作用增强，呈明显的剂量依赖关系。     

两种纳米SiO_2刺激巨噬细胞上清液对成纤维细胞的作用

目的探讨20-nm、60-nm Si O_2诱导巨噬细胞生成细胞因子在中国仓鼠肺成纤维细胞(Chinese hamster lung fibroblast,CHL细胞)增殖、胶原合成和基质金属蛋白酶-2(matrix metalloproteinase-2,MMP-2)表达中的作用。方法采用酶联免疫法(enzyme-linked immunosorbent assay,ELISA)测定2种纳米Si O_2染毒RAW264.7细胞生成转化生长因子-β1(transforming growth factor-β1,TGF-β1)、白细胞介素-1β(interleukin-1β,IL-1β)的量。CHL细胞与两种纳米Si O_2染毒RAW264.7细胞培养上清液共同孵育后,分别用MTT、消化和免疫组化法测定CHL细胞增殖率、羟脯氨酸(hydroxyproline,Hyp)生成量及MMP-2表达。结果与阴性对照组比较,RAW264.7细胞生成TGF-β1,IL-1β增加;CHL细胞增殖率、Hyp生成量和MMP-2表达率增加。结论两种粒径纳米Si O_2与微米Si O_2一样,可通过刺激巨噬细胞释放细胞因子促进成纤维细胞增殖,胶原合成和MMP-2增加,启动肺纤维化的发生。     

内皮抑素对裸鼠肺癌生长和转移的抑制作用

目的:观察内皮抑素(Endostatin,ES)对裸鼠肺癌生长和转移的抑制作用.方法:将胸腔内接种了H460大细胞肺癌的裸鼠随机分成实验组和对照组.分别给予ES和等体积PBS皮下注射,共20 d.测量瘤重,计算动物生存率,测定动物血液中基质金属蛋白酶2(Matrix metalloproteinase-2,MMP-2)的表达情况.结果:对照组瘤重均高于实验组(P＜0.05);对照组裸鼠死亡时间均较实验组早;实验组动物血液中MMP-2的表达明显低于对照组.结论:ES可抑制肺肿瘤生长和转移,其作用机制之一与抑制MMP-2的表达活性有关.     

染色质结构对辐射所致DNA链断裂频率的影响——用核及拟核单层技术的研究

采用非离子去污剂Triton X-100和NaCl处理人成纤维细胞,造成核及拟核单层,用以研究染色质结构和辐射致DNA损伤的关系.实验方法;1.人VH10成纤维细胞用改良的Eagle培养基培养至单层,1kBq/ml的〔(~14)C〕-胸腺嘧啶标记DNA,37MBq/ml的〔~3H〕混合氨基酸标记蛋白质;2.Triton X-100处理单层细胞获得单层核,Triton X-100及NaCl处理单层细胞得单层拟核;3.所得单层染     

相关生物因子对体外培养关节软骨细胞促增殖作用的比较研究

目的　筛选关节软骨组织工程种子细胞优化获取的高效促增殖剂。　方法　采用培养细胞3H -胸腺嘧啶脱氧核苷 (3H -TdR)掺入实验、四甲基偶氮唑蓝 (MTT)代谢检测及Ⅰ、Ⅱ型胶原mRNA逆转录聚合酶链反应 (RT -PCR)分析方法 ,比较一定浓度维生素C(vitaminC ,VC) +骨形态发生蛋白 2 (bonemorphogeneticprotein 2 ,BMP2 )、类胰岛素生长因子 1(insulin -likegrowthfac tor1,IGF1 ) (体积分数为 2 %的血清培养时 )及碱性成纤维细胞生长因子 (basicfibroblastgrowthfactor,bFGF)、转化生长因子 - β1 (transforminggrowthfactor- β1 ,TGF - β1 ) (体积分数为 10 %的血清培养时 )对适宜密度接种、体外单层传代培养兔关节软骨细胞的促增殖作用。　结果　适宜浓度VC +BMP2 、IGF1 及bFGF、TGF - β1 均可促进体外传代培养关节软骨细胞的分裂增殖。增殖后的培养细胞仍具有其特异性表型表达 ,而IGF1 培养的细胞则有向成骨样细胞分化的趋向。其综合效能依次为 :VC +BMP2 >bFGF >TGF - β1 >IGF1 。　结论　一定浓度VC +BMP2 及bFGF对适宜密度接种、体外单层传代培养关节软骨细胞具有显著的促增殖作用 ;其中VC +BMP2 具有经济、高效的优点。     

Radiation and hypoxia-induced non-targeted effects in normoxic and hypoxic conditions in human lung cancer cells

Purpose: Many cell lines with anaerobic metabolism do not show cytotoxic abscopal effect (AE) following irradiation. Further, there is no existing data on the radiation- and hypoxia (H)-induced AE. The purpose of this study was to investigate and compare the status of radiation-induced abscopal effect (RIAE) in normoxic and hypoxic conditions.

Methods: Lung cancer cells (A549, H460) were exposed either to hypoxia or normoxia and then irradiated (2 or 10?Gy). After 24?h, unirradiated hypoxic (H-CM) or normoxic (N-CM) conditioned media (CM) and irradiated hypoxic (H-RCM) or normoxic (N-RCM) CM was collected. Hypoxia-resistant clones (HR: A549/HR, H460/HR) were generated by continuous exposure of the cells to hypoxia. Unirradiated parental cells or HR were exposed to H-CM, N-CM, H-RCM or N-RCM. In some groups, 24?h after exposure to CM, cells were directly irradiated with 2?Gy. Cell growth was monitored using real-time cell electronic sensing system. Further, levels of hypoxia and HIF1α regulated angiogenesis related growth factors, basic fibroblast growth factor (bFGF), placental growth factor (PlGF), soluble fms-like tyrosine kinase (sFlt-1) and vascular endothelial growth factor (VEGF) were assessed in CM.

Results: In the radio-resistant A549 cells, H-RCM was much more effective in inducing growth delay compared to N-RCM. In the radio-sensitive H460 cells, both N-RCM and H-RCM induced growth delay. Interestingly, effects of N-RCM were completely reversed in HR cells. Exposure of cells to direct irradiation (2?Gy) 24?h after incubation with CM resulted in 50–60% reduction in cell proliferation in A549/HR cells and a very significant induction of death (>95%) in H460/HR cells. Direct irradiation of parental or HR clones of A549 and H460 cells exposed to H-CM 24?h with 2?Gy induced significant reduction in cell proliferation (from 40% to >95%) in all the cells. Further, levels of sFlt-1 correlated with growth delay in all the cells.

Conclusions: These results for the first time demonstrate that irradiation of hypoxic cells and exposing the cells to acute hypoxia lead to significant AE.     

神经纤毛蛋白1对非小细胞肺癌放射敏感性的影响

目的 探讨神经纤毛蛋白1 (neuropilin 1,NRP1)在不同种类非小细胞肺癌(NSCLC)细胞系的表达情况及其与肿瘤放射敏感性的关系.方法 通过Western blot技术检测不同来源的5种人NSCLC细胞系(H358、H460、H1299、A549、SK-MES-1)NRP1的基础表达水平,MTT方法检测经不同剂量X射线照射后肺癌细胞的存活情况,初步筛选5种细胞中辐射敏感和辐射抵抗的2种细胞;采用克隆形成实验及流式细胞术分别检测细胞存活分数及凋亡率的变化,分析2种肺癌细胞的放射敏感性,采用Western blot检测2种细胞受X射线作用后 NRP1 的表达变化,通过克隆形成实验检测靶向抑制NRP1对A549细胞放射敏感性的影响.结果 5种肿瘤细胞NRP1表达量由高到低依次为:A549>SK-MES-1>H358>H460>H1299;X射线对肿瘤细胞的抑制能力呈剂量依赖性增强,其抑制率由高到低依次为:H460>H1299>H358>SK-MES-1>A549,因此选取A549细胞和 H460细胞做后续研究.克隆形成实验显示,A549在2 Gy照射下的存活分数(SF₂)是0.887,而H460细胞为0.313.A549细胞受照后的凋亡率为对照组的122.54%,明显低于H460细胞的238.88%.辐射可以诱导A549细胞NRP1的表达上调达40%,同时靶向抑制NRP1则可以增强A549细胞的放射敏感性.结论 在NSCLC细胞中A549细胞具有较强的辐射抗性,且辐射抗性的形成与其NRP1表达上调有关.     

Matrix-Metallo-Proteinases and their tissue inhibitors in radiation-induced lung injury

PURPOSE: Remodeling of extracellular matrix (ECM) after lung damage depends on collagen degrading Matrix-Metallo-Proteinases (MMP) and their endogenous inhibitors (Tissue-Inhibitors of Metallo-Proteinases, TIMP). Transforming growth factor (TGF)-beta1 has been implicated in the pathogenesis of radiation-induced lung fibrosis upon its effects on fibroblast proliferation and collagen synthesis. Lung cancer patients have often elevated TGF-beta1 plasma levels as a result of increased TGF-beta1 expression in their tumours. On this background, we investigated the effect of irradiation on the MMP/TIMP system in the lung tissue of normal and transgenic TGF-beta1 mice, in which TGF-beta1 is overexpressed in the liver resulting in high TGF-beta1 plasma levels. MATERIAL AND METHODS: Transgenic (TG) and wild-type (WT) mice underwent thoracic irradiation with 12 Gy or sham-irradiation. For each study group (TG 12 Gy; TG 0 Gy; WT 12 Gy; WT 0 Gy) 8 mice were sacrificed at 4 and 8 weeks after (sham-) irradiation. The TGF-beta1, TIMP-1/-2/-3 expression in the lung tissue was quantified by Western blot; the MMP-2 and MMP-9 activity was analysed by zymography. The cellular origin of the MMP and TIMP was localised by immunohistochemistry. RESULTS: Irradiation had no influence on the TIMP-1/-2/-3, but increased significantly the MMP-2 /-9 expression. In the lung tissue of TG mice the TIMP-1/-2/-3 expression was elevated, the MMP-9 activity was decreased. The immunhistochemical study showed that parenchymal and inflammatory cells express these MMP/TIMP. CONCLUSION: Our results provide evidence that the overexpression of MMP-2 and MMP-9 is involved in the inflammatory response of radiation-induced lung injury. MMP-2 and MMP-9 are known to degrade collagen IV of basement membranes, therefore affecting the structural integrity of lung tissue. In contrast, in lung tissue of TG mice the TIMP-1/-2/-3 expression was up-regulated and the MMP-9 activity was diminished, thereby decreasing possibly the ECM degradation leading to lung fibrosis.     

Dcr3 inhibit p53-dependent apoptosis in γ-irradiated lung cancer cells

Purpose:?To identify genes responsible for the radiosensitivity, we investigated the role of the differential gene expression profiles by comparing radioresistant H1299 with radiosensitive H460 lung cancer cell lines.

Materials and methods:?mRNA profiles of lung cancer cell lines were assessed using microarray, and subsequent validation was performed with qRT-PCR (Quantitative real time-polymerase chain reaction). The expression levels of differentially expressed genes were determined by Western blot and the radioresistance of lung cancer cell lines was measured by clonogenic assay.

Results:?From the differentially expressed apoptosis-related genes between H1299 and H460, we found Dcr3 (Decoy receptor 3, also known as TNFRSF6B; Tumour necrosis factor receptor super family member 6B) expression was significantly (P?=?4.38 × 10^?7) higher in H1299 cells than H460 cells. Moreover, the Dcr3 mRNA expression level in the radioresistant cell lines (H1299, A549, DLD1, MB231, MB157) was increased in comparison to the radiosensitive cell lines (ME180, Caski, U87MG, MCF7, H460). Overexpression of Dcr3 increased the survival rate of radiosensitive H460, MCF7, and U87MG cells, and knockdown of Dcr3 abolished the radioresistance of A549 cells. The survival rate of p53 (Tumour protein 53)-deficient H1299 after gamma-irradiation was not affected by the suppression of Dcr3 expression. However, when we introduced p53 into H1299 cells, siDcr3 (siRNA of Dcr3) suppressed the radioresistance of H1299 cells by inducing p53-dependent Fas (Fas receptor, also known as TNFRSF6; Tumour necrosis factor receptor super family member 6)-mediated apoptosis pathway.

Conclusion:?Characterisation of gene expression profiles in two lung cancer cell lines revealed that Dcr3 expression and p53-dependent apoptosis signalling pathway regulate cellular response to ionising radiation.     

miR-30a-5p和NUAK1在非小细胞肺癌中的表达及对A549细胞增殖、迁移和侵袭的影响

 目的 研究miR-30a-5p和靶基因NUAK1在非小细胞肺癌中的表达及对A549细胞增殖、迁移侵袭的影响。方法 收集2016-09至2019-03在保定市第一医院行手术切除治疗的非小细胞肺癌患者癌组织和癌旁组织标本64例,QPCR和Western blot分别检测非小细胞肺癌组织样本和细胞中miR-30a-5p和NUAK1的表达;TargetScan网站预测miR-30a-5p与NUAK1间的靶向关系,双荧光素酶报告基因实验和Western blot进行验证;MTT和Transwell实验检测miR-30a-5p及联合过表达NUAK1对A549细胞增殖、迁移和侵袭的影响。结果 非小细胞肺癌组织中miR-30a-5p的相对表达量(0.33±0.14)低于癌旁组织(1.00±0.21),差异有统计学意义(t=21.237,P＜0.05);非小细胞肺癌组织中NUAK1 mRNA表达(4.13±1.87)和蛋白表达(3.41±1.62)均高于癌旁组织(1.00±0.17、1.00±0.16),差异有统计学意义(t=13.335、11.844,P＜0.05)。非小细胞肺癌细胞系H460、H1299、A549中miR-30a-5p表达量(0.35±0.03、0.51±0.05、0.28±0.03)低于人正常肺上皮细胞BEAS-2B(1.00±0.11),差异有统计学意义(F=77.100,P＜0.05);H460、H1299、A549细胞中NUAK1 mRNA相对表达量(2.98±0.30、2.39±0.24、3.42±0.36)和蛋白相对表达量(3.06±0.31、2.27±0.23、4.12±0.41)均显著高于BEAS-2B细胞(1.00±0.09、1.00±0.11),差异有统计学意义(F=46.660、63.070,P＜0.05)。结论 miR-30a-5p可通过靶向NUAK1抑制非小细胞肺癌细胞A549增殖、迁移和侵袭,提示miR-30a-5p和NUAK1可能成为一种新的临床诊断和治疗非小细胞肺癌的靶点。     

塞来昔布对非小细胞肺癌移植瘤的辐射增敏实验研究

目的 建立裸鼠非小细胞肺癌H460细胞动物模型,观察环氧化酶2选择性抑制剂塞来昔布对H460放疗的增敏作用,并对其作用机理进行初步探讨。 方法 将40只荷瘤鼠用完全随机法分成4组,分别为空白对照组、放疗组、塞来昔布组、塞来昔布+放疗组,每组10只。采用灌肠法给予塞来昔布16 mg·kg-1·d-1,给药2 h后进行放疗,放疗分割剂量为5 Gy/次,2次/周,4周后处死所有荷瘤鼠,解剖瘤块称重。采用免疫组化方法分析毛细血管扩张性共济失调症突变蛋白（ATM）和表皮生长因子受体（EGFR）表达的变化。 结果 空白对照组、塞来昔布组、放疗组、塞来昔布+放疗组肿瘤瘤重分别为（133.62±12.37）、（130.37±12.59）、（81.17±8.29）、（35.51±4.23）mg,塞来昔布+放疗组与放疗组比较差异有统计学意义（t=5.41,P < 0.01）。塞来昔布+放疗组的ATM和EGFR表达水平明显降低,与放疗组比较差异有统计学意义（t=4.23和3.17,P均 < 0.01）。 结论 塞来昔布可能通过降低ATM和EGFR的表达水平,增强H460细胞的放疗敏感性,将来可能具有较好的临床应用价值。     

酪氨酸代谢物对肺癌细胞增殖、周期及化疗药物敏感性的影响

目的 探讨4种酪氨酸代谢物对肺癌细胞增殖、周期及化疗药物敏感性的影响.方法 不同浓度的酪氨酸代谢物——酪氨酸、4-羟苯丙酮酸(HPPA)、尿黑酸(HGA)、延胡索酸(FA)分别处理A549细胞,通过高内涵实时监测肺癌细胞的增殖,流式细胞仪检测肺癌细胞周期的变化及对化疗药物的敏感性,Western印迹检测凋亡相关蛋白Bcl-2、Bax的表达.结果 4种酪氨酸代谢物对A549细胞的增殖及周期均无影响.HGA可降低吉非替尼(Gefitinib)诱导的A549细胞凋亡,抑制Bax表达,同时上调Bcl-2的表达水平;FA则可增加其诱导的A549细胞凋亡,促进Bax表达,同时下调Bcl-2的表达水平.酪氨酸和HPPA对Gefitinib诱导的细胞凋亡均无明显影响.结论HGA与FA可调节肺癌细胞化疗药物的敏感性,揭示了酪氨酸代谢通路对肺癌细胞的调控作用,为肿瘤代谢异常的研究奠定了基础.