Solid tumor cells show a resistance to immunologic effector cells in vitro[1]. The resistance may be one reason why these tumors withstand immunotherapeutic approaches in humans. Dendritic cells play an important role in the immune response to tumor-associated antigens in humans. Dendritic cells in the periphery capture and process antigens,express lymphocyte costimulatory molecules, migrate to lympoid organs, and secrete cytokines to initiate immune response.

RNA interference (RNAi) is a recently observed process by which double-stranded RNA (dsRNA) directs sequence-specific degradation of messenger RNA (mRNA) in animal and plant cells. In several model systems, RNAi had been developed into a useful tool for the investigation of gene function. In order to study the effectiveness of RNAi in mammalian cells, we introduced chemically synthetic 21-nucleotide small interference RNA (siRNA) duplexes into 293T/GFP cells, which were transduced by enhanced green fluorescence protein (EGFP) gene, by means of TransIT-TKO, Oligofectamine reagent, Lipofectamine 2000 respectively. The results demonstrated that EGFP expression was significantly and specifically inhibited by the corresponding dsRNA, but not by unrelated dsRNA. In three different vectors, Lipofectamine 2000 demonstrated the highest transfection efficiency with a 48h exposure. The decrease in EGFP fluorescence intensity was approximately 80%. Although TransIT-TKO and Oligofectamine displayed similar trends, the transfections were inefficient, and often toxic. The results also exhibited that siRNA inhibited the EGFP gene expression in a dose and time-dependent manner. Therefore, we concluded that the Lipofectamine 2000 was a better transfection reagent for RNAi. RNAi pathway seems operative in mammalian embryo cells. RNAi may be developed into a potential tool for gene therapy.

目的：探讨采用体外化学合成的针对表皮生长因子受体（EGFR）设计的双链RNA（double-stranded RNA， dsRNA），是否能诱导肺非小细胞肺癌（NSCLC）细胞出现序列特异性基因沉默，及抑制EGFR基因表达后NSCLC细胞的生物学特征。方法：体外合成EGFR序列特异性dsRNA（dsRNA-EGFR），结合脂质体2000转染肺腺癌SPC2A21细胞，用荧光显微镜及流式细胞仪测定转染细胞的EGFR受体数量;适时定量聚合酶链反应检测其EGFR基因表达水平。采用集落形成试验检测SPC2A21细胞的增殖和集落形成能力。建立裸鼠肿瘤模型，计算肿瘤抑制率。结果：dsRNA2EGFR可使EGFR在蛋白水平表达下降7113%、基因水平表达下降5010%， SPC2A21细胞集落形成下降6618%，并显著抑制在体肿瘤生长，肿瘤抑制率为7510%。结论：dsRNA2EGFR可序列特异性下调NSCLC细胞EGFR在基因及蛋白水平的表达，有效抑制肿瘤生长。

Recently identified suppressors of cytokine signaling (SOCS) have been proposed as negative regulators of cytokine signaling, which have distinct mechanisms of inhibiting JAK-STAT pathway. In this study, using cultures of rat primary pulmonary vascular smooth muscle cells (PASMC), we found that hypoxia induced strongly STAT3 phosphorylation by up to four-fold. At the same time, mRNA for the endogenous cytokine signaling repressor SOCS3, but not SOCS1, was markedly induced in PASMC early as 2 h following hypoxic stimulation. Furthermore, forced expression of SOCS3 gene suppressed tyrosine phosphorylation of STAT3 and transcription of c-myc gene by more than 70% and 60% in PASMC under hypoxic conditions, respectively. Additionally, we showed here that hypoxia enhanced nearly two-fold increase of PASMC proliferation and overexpression SOCS3 gene downregulated hypoxia-induced PASMC proliferation by about 50%. The finding suggest that STAT3-dependent pathway is involved in the activation and proliferation of PASMC stimulated by hypoxia, and SOCS3 is a rapidly hypoxiainducible gene and acts to inhibit activation of cellular signaling pathway in a classical negative feedback loop.

Objective The aim of this study is to investigate whether ambroxol inhibits inflammatory responses in a murine model of lipopolysaccharide-induced acute lung injury (ALI). Methods Mice (n=295) were first intratracheally instilled with lipopolysaccharide (LPS) to induce ALI and then received an intraperitoneal (ip) injection of either normal saline (NS), ambroxol (30 or 90mg/kg per day) or dexamethasone (2.5 or 5mg/kg per day) for 7 days. Metabolism (n=10, each), lung morphology (n=5, each) and wet-to-dry lung weight ratio (n=10, each) were studied. The levels of tumor necrosis factor (TNF-), interleukin-6 (IL-6) and transforming growth factor (TGF-1) and the protein concentration (n=5 or 7, each) in bronchoalveolar lavage (BAL) were measured. Results Mice with LPS-induced ALI that were treated with ambroxol at a dosage of 90mg/kg per day significantly gained weight compared to the control and dexamethasone-treated groups. Ambroxol and dexamethasone significantly reduced the lung hemorrhage, edema, exudation, neutrophil infiltration and total lung injury histology score at 24 and 48h. In addition, ambroxol and dexamethasone significantly attenuated the lung wet-to-dry weight ratio at 24 and 48h (p<0.05). Compared to the control group, TNF, IL-6 and TGF-1 levels in the BAL in both ambroxol-and dexamethasone-treated groups were significantly reduced at 24 and 48 h. The protein in BAL, an index of vascular permeability, was also significantly decreased in the ambroxol-and dexamethasone-treated groups (p<0.05). Conclusion Ambroxol inhibited proinflammatory cytokines, reduced lung inflammation and accelerated recovery from LPS-induced ALI.

A murine model of lipopolysaccharide (LPS)-induced acute lung injury (ALI) was used to evaluate whether aquaporin-1 (AQP1) is involved in lung inflammation and lung edema formation. Swiss strain mice (n=122) had LPS (5mg/kg) instilled intratracheally (IT), and were then treated with either 0.9% saline or dexamethasone (5mg/kg/day). Mice were euthanized at 2 days and 7 days after treatment. Inflammatory cytokines (TNF-α, IL-6), protein concentration in bronchoalveolar lavage (BAL) fluid, lung wet-to-dry weight ratio, histology, immunohistochemistry, and AQP1 Western blot were performed. Lung wet-to-dry weight ratio and lung vascular permeability were also measured in the AQP1 knockout mice (n=9) that received IT LPS (5mg/kg) at 2 days. Intratracheal instillation of LPS produced a severe lung injury at 2 days, characterized by elevation of TNF-α, IL-6 in the BAL fluid, and by histological changes consistent with increased lung vascular permeability and neutrophil infiltration. AQP1-immunoreactivity in the pulmonary capillary endothelium was reduced at 2 days and 7 days. Administration of dexamethasone improved LPS-induced ALI and retained expression of AQP1. However, depletion of AQP1 did not affect lung edema formation, lung vascular permeability, or lung histology. The results suggest that although AQP1 expression is decreased after lung injury, depletion of AQP1 does not alter lung inflammation and lung edema induced by LPS.

The incidence of the pulmonary complications in patients with acute pancreatitis is up to 60%, from mild hypoxaemia without clinical or radiological abnormalities to severe acute lung injury. Although a number of reviews has described clinical evidence of pancreatitis-associated lung injury (PALI), there is still a lack of information on potential interorgan signalling involved in the development of PALI. The present review discusses the potential factors that have been considered as candidates responsible for interorgan signalling coupling pancreatitis to lung injury. Inflammatory mediators produced from the pancreas, leucocyte system and extrapancreatic organs may directly or indirectly contribute to the high concentration in the circulation, activate circulating leucocytes, initiate the inflammatory responses of pulmonary resident cells or induce acute lung injury. Circulating leucocytes have been suggested to play an important role in interorgan signalling during the development of PALI, by producing the inflammatory mediators, reactive oxygen species and proteinases, interacting with endothelial cells, communicating with pulmonary cells and compromising the lung tissue. Extrapancreatic organ fluid, e.g. liver, PAAF and gut, may also contribute to the interorgan signalling between the pancreas and lungs. There is still a great need to clarify the specificity, sensitivity and repeatability of these candidates for interorgan signalling in PALI.

Lung cancer has emerged as a leading cause of cancer death in the world. Non-small cell lung cancer (NSCLC) accounts for 75–80% of all lung cancers. Current therapies are ineffective, thus new approaches are needed to improve the therapeutic ratio. Double stranded RNA (dsRNA) -mediated RNA interference (RNAi) has shown promise in gene silencing, the potential of which in developing new methods for the therapy of NSCLC needs to be tested. We report here RNAi induced effective silencing of the epidermal growth factor receptor (EGFR) gene, which is over expressed in NSCLC. NSCLC cell lines A549 and SPC-A1 were transfected with sequence- specific dsRNA as well as various controls. Immune fluorescent labeling and flow cytometry were used to monitor the reduction in the production of EGFR protein. Quantitative reverse-transcriptase PCR was used to detect the level of EGFR mRNA. Cell count, colony assay, scratch assay, MTT assay in vitro and tumor growth assay in athymic nude mice in vivo were used to assess the functional effects of EGFR silencing on tumor cell growth and proliferation. Our data showed transfection of NSCLC cells with dsRNA resulted in sequence specific silencing of EGFR with 71.31% and 71.78% decreases in EGFR protein production and 37.04% and 54.92% in mRNA transcription in A549 and SPC-A1 cells respectively. The decrease in EGFR protein production caused significant growth inhibition, i.e.: reducing the total cell numbers by 85.0% and 78.3%, and colony forming numbers by 63.3% and 66.8%. These effects greatly retarded the migration of NSCLC cells by more than 80% both at 24 h and at 48h, and enhanced chemo-sensitivity to cisplatin by four-fold in A549 cells and seven-fold in SPC-A1. Furthermore, dsRNA specific for EGFR inhibited tumor growth in vivo both in size by 75.06% and in weight by 73.08%. Our data demonstrate a new therapeutic effect of sequence specific suppression of EGFR gene expression by RNAi, enabling inhibition of tumor proliferation and growth. However, in vivo use of dsRNA for gene transfer to tumor cells would be limited because dsRNA would be quickly degraded once delivered in vivo. We thus tested a new bovine lentiviral vector and showed lentivector-mediated RNAi effects were efficient and specific. Combining RNAi with this gene delivery system may enable us to develop RNAi for silencing EGFR into an effective therapy for NSCLC.

目的：探讨RNA干扰（RNAi）技术抑制非小细胞肺癌（NSCLC）细胞株A549细胞表皮生长因子受体（EGFR）表达后，对A549细胞生物学特性的影响。方法：体外化学合成EGFR 序列特异性双链RNA（dsRNA），用Lipofectamine 2000 转染A549细胞，采用Western blot技术和流式细胞仪检测EGFR的表达，并观察A549细胞周期、集落形成、化疗敏感性等生物学特性的改变。结果：序列特异性dsRNA-EGFR可显著抑制A549细胞EGFR表达;dsRNA-EGFR组细胞生长抑制率为85.0%，集落形成抑制率63.3%;细胞周期分析结果表明，dsRNA-EGFR组G0～G1期细胞数较对照组增加了12.7%，进入S期的细胞数较对照组减少了6.6%。转染dsRNA-EGFR后，可将A549细胞对顺铂的敏感性提高约4倍。结论：dsRNA-EGFR可有效抑制A549细胞EGFR的表达，并将更多的细胞阻滞在G0～G1期，抑制细胞增生，显著增加细胞对顺铂的敏感性。RNAi可能为NSCLC的基因治疗提供新策略。

Objective: This study examined whether continuous hemofiltration favorably affects cardiopulmonary variables, lung inflammation, and lung fluid balance in a canine model of oleic acid induced acute lung injury. Methods: Eleven pentobarbital-anesthetized dogs were randomly divided into a control (mechanical ventilation, MV) group (n=6) and a MV plus hemofiltration (HF) group (n=5). All animals received an intravenous injection of oleic acid (0.09ml/kg) to induce acute lung injury. Continuous arterialvenous hemofiltration (blood flow 100 ml/min, ultrafiltration rate at 50–65 ml kg−1 h−1) was started after establishment of oleic acid induced acute lung injury and continued for 4 h. Hemodynamics, lung mechanics, gas exchange, lung fluid balance, lung histology, and the level of plasma cytokines were assessed. Results: After 240 min of HF treatment there was a significant increase in cardiac output, reduction in pulmonary arterial pressure, and improvement in both oxygenation and lung mechanics. Also, in the HF group the lung wet-to-dry weight ratio was significantly reduced. Histologically, HF reduced edema and inflammatory cell infiltration in the lung. There was also a significantly greater decrease in plasma IL-6 and IL-8 levels in the HF group than in group receiving MV alone. Conclusions: In a canine model of acute lung injury continuous HF improved cardiopulmonary function, reduced pulmonary edema, decreased lung permeability and inflammation, and decreased the plasma concentration of proinflammatory cytokines.