Trace element load in cancer and normal lung tissue

Samples of malignant and benign human lung tissues were analysed by two complementary methods, i.e., particle induced X-ray emission (PIXE) and total reflection X-ray fluorescence (TRXRF). The concentration of trace elements of P, S, K, Ca, Ti, Cr, Mn, Fe, Cu, Zn, Se, Sr, Hg and Pb was determined in squamous cancer of lung tissue from 65 people and in the benign lung tumour tissue from 5 people. Several elements shows enhancement in cancerous lung tissue of women in comparison to men, i.e., titanium show maximum enhancement by 48% followed by Cr (20%) and Mn (36%). At the same time trace element concentration of Sr and Pb are declaimed by 30% and 20% in women population. Physical basis of used analytical methods, experimental set-up and the procedure of sample preparation are described.     

Protein Kinase C as a Therapeutic Target in Non-Small Cell Lung Cancer

Driver-directed therapeutics have revolutionized cancer treatment, presenting similar or better efficacy compared to traditional chemotherapy and substantially improving quality of life. Despite significant advances, targeted therapy is greatly limited by resistance acquisition, which emerges in nearly all patients receiving treatment. As a result, identifying the molecular modulators of resistance is of great interest. Recent work has implicated protein kinase C (PKC) isozymes as mediators of drug resistance in non-small cell lung cancer (NSCLC). Importantly, previous findings on PKC have implicated this family of enzymes in both tumor-promotive and tumor-suppressive biology in various tissues. Here, we review the biological role of PKC isozymes in NSCLC through extensive analysis of cell-line-based studies to better understand the rationale for PKC inhibition. PKC isoforms α, ε, η, ι, ζ upregulation has been reported in lung cancer, and overexpression correlates with worse prognosis in NSCLC patients. Most importantly, PKC isozymes have been established as mediators of resistance to tyrosine kinase inhibitors in NSCLC. Unfortunately, however, PKC-directed therapeutics have yielded unsatisfactory results, likely due to a lack of specific evaluation for PKC. To achieve satisfactory results in clinical trials, predictive biomarkers of PKC activity must be established and screened for prior to patient enrollment. Furthermore, tandem inhibition of PKC and molecular drivers may be a potential therapeutic strategy to prevent the emergence of resistance in NSCLC.     

Study of the structure of the air and blood capillaries of the gas exchange tissue of the avian lung by serial section three-dimensional reconstruction

We have previously reconstructed the gas exchange tissue of the adult muscovy duck, Cairina moschata using a method of manually aligning sections and tracing the contours of the components of the gas exchange tissue. This reconstruction method demonstrated that the air capillaries are comprised of an expanded globular part interconnected by narrow air channels. The blood capillaries completely surround the air capillaries forming an anastomosing meshwork of short segments. However, the resulting reconstruction was limited in scope because of the laborious process of tracing the profiles of each component through the sequence of micrographs. We have now reconstructed a larger proportion of the exchange tissue by using a cross-correlation based alignment strategy and have demonstrated that the staining intensity of each of the exchange tissue components is sufficiently different to allow them to be identified by simple filtering and thresholding. The resulting reconstructions sample a much larger proportion of the exchange tissue and demonstrate the heterogeneity of structures from different locations in the parabronchus. We have shown that a sheet-flow-type arrangement of blood capillaries surrounds the infundibulum; this represents an unexpected functional convergence with the arrangement of blood capillaries surrounding the mammalian alveoli. It is feasible, using this reconstruction strategy, to analyse the exchange tissue of a large number of avian species in order to determine structural correlates of function. The resulting reconstructions could be analysed in order to determine the basis of the functional efficiency and rigidity of the avian lung.     

Apigenin-7-Glycoside Prevents LPS-Induced Acute Lung Injury via Downregulation of Oxidative Enzyme Expression and Protein Activation through Inhibition of MAPK Phosphorylation

Apigenin-7-glycoside (AP7Glu) with multiple biological activities is a flavonoid that is currently prescribed to treat inflammatory diseases such as upper respiratory infections. Recently, several studies have shown that its anti-inflammatory activities have been strongly linked to the inhibition of secretion of pro-inflammatory proteins, such as inducible nitric oxide synthase (iNOs) and cyclooxygenase-2 (COX-2) induced through phosphorylation nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPK) pathways. Additionally, inflammation, which can decrease the activities of antioxidative enzymes (AOEs) is also observed in these studies. At the same time, flavonoids are reported to promote the activities of heme oxygenase-1 (HO-1) decreased by LPS. The purpose of this study was to assess these theories in a series of experiments on the suppressive effects of AP7Glu based on LPS-induced nitric oxide production in RAW264.7 macrophages in vitro and acute lung injury in mice in vivo. After six hours of lipopolysaccharide (LPS) stimulation, pulmonary pathological, myeloperoxidase (MPO) activity, total polymorphonuclear leukocytes (PMN) cells, cytokines in bronchoalveolar lavage fluid (BALF) and AOEs, are all affected and changed. Meanwhile, our data revealed that AP7Glu not only did significantly inhibit the LPS-enhanced inflammatory activity in lung, but also exhibited anti-inflammatory effect through the MAPK and inhibitor NF-κB (IκB) pathways.     

Validation of Tuba1a as Appropriate Internal Control for Normalization of Gene Expression Analysis during Mouse Lung Development

The expression ratio between the analysed gene and an internal control gene is the most widely used normalization method for quantitative RT-PCR (qRT-PCR) expression analysis. The ideal reference gene for a specific experiment is the one whose expression is not affected by the different experimental conditions tested. In this study, we validate the applicability of five commonly used reference genes during different stages of mouse lung development. The stability of expression of five different reference genes (Tuba1a, Actb Gapdh, Rn18S and Hist4h4) was calculated within five experimental groups using the statistical algorithm of geNorm software. Overall, Tuba1a showed the least variability in expression among the different stages of lung development, while Hist4h4 and Rn18S showed the maximum variability in their expression. Expression analysis of two lung specific markers, surfactant protein C (SftpC) and Clara cell-specific 10 kDA protein (Scgb1a1), normalized to each of the five reference genes tested here, confirmed our results and showed that incorrect reference gene choice can lead to artefacts. Moreover, a combination of two internal controls for normalization of expression analysis during lung development will increase the accuracy and reliability of results.     

当归挥发油对人肺腺癌GLC-82细胞增殖及凋亡的影响

目的: 探讨当归挥发油对人肺腺癌GLC-82细胞增殖及凋亡的作用。方法: MTT比色法检测当归挥发油对GLC-82细胞的细胞毒作用;流式细胞术检测GLC-82细胞凋亡;吖啶橙/溴化乙锭（AO/BE）双荧光染色法观察GLC 82细胞凋亡。结果: 当归挥发油在浓度6.25～200 μg/mL范围内对GLC-82细胞的增殖具有浓度依赖性抑制作用,对GLC-82细胞的IC50为113.03 μg/mL。流式细胞术检测结果表明,当归挥发油可诱导GLC-82细胞凋亡、坏死,且细胞凋亡、坏死率随药物浓度增高而升高。AO/BE双荧光染色法结果显示,用药组随着药物浓度的升高,细胞结构固缩加重,凋亡、坏死细胞增多。结论: 当归挥发油具有诱导GLC-82细胞凋亡的作用。     

KDR基因遗传变异对贝伐珠单抗一线治疗晚期非小细胞肺癌患者临床疗效的影响

目的: 本研究旨在探讨激酶插入区受体（KDR）基因遗传变异对贝伐珠单抗联合化疗治疗晚期非小细胞肺癌（NSCLC）患者疗效的影响。方法:本研究纳入135例一线接受贝伐珠单抗治疗的NSCLC患者,收集外周血及活检癌组织标本分别用来进行基因分型及表达测定。多态性位点的基因型和其他变量的相关性通过logistic回归模型进行分析。基因型和预后的单变量分析用Kaplan-Meier生存分析方法,并通过Cox风险比例模型对其他变量进行校正。结果:在KDR的标记多态性位点中,只发现了V297I位点的临床意义。V297I位点位于该基因的编码区,在研究人群的基因分布频率为：CC型99例（73.33%）,CT型33例（24.44%）,TT型3例（2.23%）,最小等位基因频率为0.14,三种基因型分布频率符合哈迪温伯格平衡（P=0.898）。后期比较将TT型和CT型患者合并,对不同基因型患者进行疗效分析发现：CT/TT基因型患者和野生型CC型患者的客观缓解率（ORR）分别为41.67%和47.47%（P=0.549）。CT/TT和CC基因型患者的中位无进展生存期（mPFS）分别为6.2和8.6个月,差异具有统计学意义（P=0.003）。在总生存期（OS）方面,两种基因型患者的中位总生存期（mOS）分别为18.9和21.5个月,差异具有统计学意义（P=0.017）。对无进展生存期（PFS）构建多变量的Cox模型校正之后发现CT/TT基因型对PFS的影响仍然具有统计学意义（OR=1.95,P=0.019）。另外,进一步在68例癌组织标本的表达分析中发现,CT/TT基因型患者相对于野生型的CC型患者,癌组织中KDR的表达明显较高,且差异具有统计学意义（P<0.01）。结论:在接受贝伐珠单抗治疗的非小细胞肺癌患者中,KDR基因V297I位点可能通过影响KDR基因的表达从而影响贝伐珠单抗一线治疗非小细胞肺癌患者的疗效。     

早期静脉注射特布他林对大鼠急性肺损伤的影响

目的观察早期静脉注射特布他林注射液对大鼠急性肺损伤（ALI）的影响。方法将40只SD大鼠随机分为4组：生理盐水对照组（A组）、ALI组（B组）、ALI＋特布他林治疗组（C组）、ALI＋特布他林＋哇巴因干预组（D组）,每组10只。各组大鼠分别以10％水合氯醛3．5uL·g-1腹腔麻醉。麻醉成功后,A组大鼠采用尾静脉注射生理盐水5mg·kg-1,1h内注完;B组大鼠采用尾静脉注射脂多糖（LPS）5mg·kg-1（融于1mL生理盐水中）,分4次给药,1h内注完;C、D2组大鼠采用尾静脉注射LPS5mg·kg-1（融于1mL生理盐水中）,分4次给药,1h内注射完后,再注射硫酸特布他林注射液5．0uL·g-1体质量,1h内注射完后。然后将大鼠头部抬高45°,光源照射颈部直视下行气管插管,插管成功后,A、B、C3组气管内滴注林格氏液1mL·kg-1,D组气管内滴注含哇巴因10-3mol·L-1的林格氏液1mL·kg-1通过建立LPS诱导的大鼠急性肺损伤模型,对各组大鼠肺组织病理学评分,观察各组大鼠的肺湿干重比（W／D）、肺组织Na＋-K＋-ATP酶的活性,并测定各组大鼠肺泡灌洗液中的细胞数、中性粒细胞计数及蛋白含量。结果A组肺组织病理学评分值、W／D值、细胞计数、中性粒细胞百分比、蛋白含量均明显低于B、C、D3组（均P〈0．05）,B、D2组上述指标均明显高于C组（均P〈0．05）,B组上述指标与D组比较差异无统计学意义（P〉0．05）。A组肺组织Na＋-K＋-ATP酶的活性明显高于B、C、D3组（P〈0．05）,B、D2组均明显低于C组（均P〈0．05）,B组与D组比较差异无统计学意义（P〉0．05）。结论早期静脉注射特布他林可通过上调肺组织Na＋K＋-ATP酶的活性来促进肺泡上皮细胞对液体重吸收,从而控制大鼠急性肺损伤的进展。     

IL-13和乙醇对人肺成纤维细胞增殖的影响

目的研究乙醇和（或）IL-13对人肺成纤维细胞（HFL-1）的促增殖作用。方法培养HFL-1,通过MTT法检测乙醇和（或）IL-13对HFL-1增殖的影响,并对结果进行分析。结果①25、50、100、200 mmol·L^-1乙醇对HFL-1的增殖无影响（P〉0.05）。②10、20、50μg·L^-1的IL-13对乙醇有促增殖作用,且有浓度依赖性（P〈0.05）。③不同浓度的乙醇（25、50、100、200 mmol·L^-1）和10μg·L^-1IL-13共同刺激HFL-1后增殖效果与10μg·L^-1IL-13单独刺激的效果差异无统计学意义（P〉0.05）;除50 mmol·L^-1外,其余浓度的乙醇（25、100和200 mmol·L^-1）和20μg·L^-1IL-13共同刺激HFL-1后增殖效果高于20μg·L^-1IL-13单独刺激的效果（P〈0.05）;不同浓度的乙醇（25、50、100和200 mmol·L^-1）和50μg·L^-1IL-13共同刺激HFL-1后增殖效果明显高于50μg·L^-1IL-13单独刺激的效果（P〈0.05）,且具有浓度依赖性。结论单独低浓度乙醇（25、50、100和200 mmol·L^-1）不会影响HFL-1的增殖,但与一定浓度IL-13共同作用下将对HFL-1的增殖有显著影响。     

重组肺癌抑癌基因1蛋白的表达、纯化及其多克隆抗体的制备

目的表达、纯化重组人肺癌抑癌基因1(Tumorsuppressor in lung cancer1,TSLC1)蛋白,并制备其多克隆抗体。方法采用RT-PCR法扩增TSLC1基因全长编码区序列,克隆入原核表达质粒pQE30,转化大肠杆菌M15,IPTG诱导表达,表达的重组蛋白经Ni2+-NTA亲和层析纯化后,免疫家兔,ProteinA亲和层析纯化抗血清,并经Westernblot分析其反应原性。结果重组表达质粒pQE30-TSLC1经双酶切及测序鉴定证明构建正确。重组TSLC1蛋白的表达量约占菌体总蛋白的14%,主要以包涵体形式存在。纯化的重组蛋白纯度为93.4%,可与小鼠抗His-Tag单克隆抗体发生特异性反应。以其制备的多克隆抗体具有良好的抗原识别特异性。结论已成功制备TSLC1多克隆抗体,为深入研究TSLC1分子的生物学活性奠定了基础。