目的 研究外源性一氧化碳对大鼠肺动脉平滑肌细胞(pulmonary artery smooth muscle cells,PASMC)凋亡状况的影响,探讨低氧肺血管重建的发病机制及防治措施.方法 复合胶原酶法培养动脉平滑肌细胞,分别给予常氧、低氧、低氧合并低浓度一氧化碳处理.采用透射电镜、流式细胞仪、逆转录聚合酶链式反应(reverse transcription-polymerase chain reaction,RT-PCR)、免疫组织化学染色等方法检测肺动脉平滑肌细胞的凋亡状况及caspase-3表达水平.结果 电镜观察可见,低浓度一氧化碳处理PASMC后呈凋亡样改变,染色质浓缩成块状位于核周边,细胞核边集近似半月型,细胞器结构完整.流式细胞仪示,正常培养PASMC没有明显的亚二倍峰,低氧24 h条件下可见亚二倍峰,低浓度一氧化碳处理24 h后,PASMC亚二倍峰明显升高.RT-PCR和免疫组织化可见,低浓度一氧化碳作用后caspase-3的mRNA和蛋白水平显著升高.结论 低浓度一氧化碳可以调节caspase-3基因的表达,从而促进肺动平滑肌细胞的凋亡.

In view of the goal of postgraduate education , we point out the contradiction between clinical work and research on the basis of analysis of the status quo of academic degree education of clinical medicine .Then we analyze the major manifestations of the contradiction and causes , and then discuss ways and methods to solve the contradiction in the hope of improving the quality of academic degree education of clinical medicine .

Objective To investigate the regulatory effect of Rab1 on the expression of β-adrenergic receptors (β-ARs) in rat pulmonary microvascular endothelial cells (RPMVECs). Methods Primary cultured RPMVECs served as the study object. Three experiments were designed to detect: 1) the effect of sea water on β-ARs expression, for which a control group and sea-water treatment group were set up; 2) the effect of Rab1 WT (wild-type rat lentivirus Rab1 expression vector) and Rab1 N124 I transfer on the expressions of β-ARs and its subtypes β1-AR and β2-AR, for which Rab1 WT and Rab1 N124 I were established, and then a control group, Rab1 WT group and Rab1 N124 I group were set up; 3) the effect of sea water on the β-ARs expression in the Rab1WT-transferred RPMVECs, for which a control group, sea-water treatment (SW) group and WT/SW group (sea water treatment after Rab1WT transfection) were set up. The expressions of β-ARs and its subtypes (β1-AR and β2-AR) in RPMVECs were determined by ligand binding assay. Results 1) In the sea water experiment, the cell-surface expressions of total β-ARs were 1324.00±130.01 and 2107.00±202.36cpm/105 cells, respectively, and the difference between them was statistically significant. 2) In the experiment, the expression levels of β-ARs were 2048.00±207.64, 2833.00±203.07 and 1040.00±91.59cpm/105 cells, respectively, in control group, Rab1 WT group and Rab1 N124I group, and the expression levels of β1-AR in the aforementioned three groups were 517.78±29.65, 591.22±18.29 and 364.11±20.68cpm/105 cells, respectively, and those of total β2-AR were 1627.90±112.27, 2262.10±169.33 and 694.33±29.79cpm/105 cells, respectively. After Rab1 WT transfer, the expression of β1-AR in RPMVECs increased by 14.2% (P > 0.05), and that of β2-AR by 49.0% (P 0.05), but increased by 58% as compared with SW group (P < 0.01). Conclusion Sea water may decrease the β-ARs expression on the membrane of RPMVEC, and Rab1 seems to have an antagonistic effect, which could up-regulate the expression of β-ARs on the membrane of RPMVEC.

To investigate the effect of JAKs-STATs signal pathway on expression of S100A4 in pulmonary arterial smooth muscle cells (PASMCs), the action of S100A4 and hypoxia induced factor 1 (HIF-1) on the proliferation of hypoxic PASMCs.The results showed that S100A4 immunostaining was localized in the cytoplasm and nuclei of PASMCs exposure to hypoxia and it was predominantly expressed in rhomboid cells (R-SMCs). The mRNA and protein levels of S100A4 expression increased in PASMCs after hypoxic stimulus for 4, 8, 16 h. The immunofluorescence intensity and protein levels of S100A4 were suppressed, and the number of R-SMCs was reduced, when pretreatment with HIF-1 alpha siRNA, STAT3 siRNA, S100A4 siRNA, and S100A4 inhibitor NSC 95397. Pretreatment with HIF-1 alpha siRNA and anti-IL-6 antibodies, the levels of phospho-JAK2, -STAT3, and S100A4 were decreased, while HIF-1 alpha kept stable in hypoxic cells. Importantly, pretreatment with HIF-1 alpha siRNA, anti-IL-6 antibodies, STAT3 siRNA, and S100A4 siRNA, significantly attenuated the proliferation of PASMCs exposure to hypoxia. These data demonstrate that S100A4 is predominantly expressed in hypoxic R-SMCs, and regulated by the activation of JAK2-STAT3 signal pathway, which is dependent on hypoxia-induced HIF-1 alpha expression. These results suggest that JAK2-STAT3 and HIF-1 alpha could serve as targets for the regulation of phenotype modulation of PASMCs during the process of pulmonary vessel lesions. (C) 2012 Elsevier Ltd. All rights reserved.

Objective To investigate the the mechanism of S100A4/Mtsl in pulmonary arterial smooth muscle cells(PASMCs) under hypoxia.Methods PASMCs were divided into normoxic group,hypoxic group and an AG490 plus hypoxic group(AG490).The immunofluorescence method was used to detect the expression of the S100A4/Mtsl in normoxic group and hypoxic group.Western blot was performed to examine the expression of S100A4/Mtsl protein,phospho-JAK2 protein and phospho-STAT3 protein in normoxic group,hypoxic group and an AG490 plus hypoxic group respectively.Results Immunofluorescence assay showed few expression of the S100A4/Mtsl in cytoplasm of PASMC in normoxic group,and significant expression in cytoplasm and nucleus of PASMC in hypoxic group;S100A4/Mtsl protein,phospho-JAK2 protein,phospho-STAT3 protein in 8 h hypoxic group was significantly higher than that in normoxic group(P<0.05) and they declined significantly in AG490 group as compared to 8 h hypoxic group.Conclusion S100A4/Mtsl gene plays important role in PAMSC proliferation induced by hpoxia,and could be blocked by AG490.

Objective To identify the clinical features,diagnosis and treatment of interstitial pneumonia related to epidermal growth factor receptor tyrosine kinase inhibitors(EGFR-TKIs) which were used in advanced non-small cell lung cancer(NSCLC).Methods Retrospective analysis was carried out in 4 cases with NSCLC who developed interstitial pneumonia after EGFR-TKIs therapy in our department from March 2007 to September 2011.Their clinical features,diagnosis,treatment and outcome were analyzed.Results The 4 patients had previously received chemotherapy until disease progression or unacceptable toxicity and then turned to receive gefitinib or erlotinib orally.However,within 30 d after the treatment,the patients suffered from dyspnea,cough and hypoxemia and developed interstitial pneumonia.Three patients died of respiratory failure due to rapid disease progression,which reflected the poor prognosis of the interstitial pneumonia.Conclusion EGFRTKIs-related interstitial pneumonia is with poor prognosis and high mortality.Timely chest CT scanning,pulmonary function testing,blood gas analysis and other tests should be performed as soon as dyspnea,cough and hypoxemia are seen.Relevant treatment such as discontinuation of EGFR-TKIs immediately,use of corticosteroids,oxygen therapy and respiration supporting when necessary,can probably improve the prognosis and survival of patients.

Previous studies have demonstrated that Rab1 is involved in the export of angiotensin II (Ang II) type 1 receptor (AT1R) to the cell surface in endothelial cells and cardiomyocytes. The aim of this study was to evaluate whether the modification of Rab1-mediated endoplasmic reticulum (ER) to the Golgi body transport alters the cell surface expression and function of endogenous AT1R and AT1R-mediated phenotypic modulation in primary cultures of pulmonary artery smooth muscle cells (PASMCs). Lentiviral expression of wild-type Rab1 (Rab1WT) significantly increased cell surface expression of endogenous AT1R. However, Rab1 siRNA had the opposite effect, and attenuated downregulation of the expression of PASMCs phenotype markers, α smooth muscle actin (α-SMA) and vimentin (VIM) in rat pulmonary artery smooth muscle cells (RPASMCs) during hypoxia. Analysis of the subcellular localization of AT1R revealed that Rab1 regulated AT1R transport from the ER to the Golgi in PASMCs. Consistent with their effects on AT1R export, Rab1 modified the AT1R-mediated cell growth and the phosphorylation of signal transducing activator of transcription 3 (STAT3) during hypoxia. We found that hypoxia promoted Rab1 expression and strongly correlated with the repressed expression of PASMC phenotype markers in RPASMCs. These data strongly indicate that Rab1 modulates PASMCs function by manipulating AT1R traffic from the ER to the Golgi and provide the first evidence implicating ER-to-Golgi transport as a regulatory step for the control of RPASMCs growth.

Objective To investigate inhibitory action of T0901317,a liver X receptor-α activator,on expression of MPO and TNF-α in rats with acute lung injury(ALI).Methods 72 male Wistar rats were randomly divided into three group: control group(C group),LPS group(L group),T0901317 group(LT group).The rats were killed at 1h,2h,4h,8h after treatment.The lung tissue was collected,myeloperoxidase activity,lung histopathologic changes and expression of TNF-α were observed.Results In rats with ALI,myeloperoxidase activity rised compared with control group(P<0.05),and lung injury is more severe than that in rats of control group.TNF-α increased markedly in lung homogenate and blood serum at the same period(P<0.05).However,The expression of TNF-α and myeloperoxidase activity in rat lung tissues in LT group were significantly declined.Conclusion T0901317 has played an anti-inflammatory effect through suppressing the expression of TNF-α and myeloperoxidase activity in rat lung with acute lung injury.

It is known that Rab1 regulates the expression and function of beta-adrenoceptors (beta-ARs) in many cells. However, the effect of these changes in rat pulmonary microvascular endothelial cells (RPMVECs) is not known. In the present study, we investigated the role of Rab1, a Ras-like GTPase that coordinates protein transport from the endoplasmic reticulum (ER) to the Golgi body and regulates the cell-surface targeting and function of endogenous beta-ARs in RPMVECs in the presence of lipopolysaccharide (LPS).We found that lentivirus-driven expression of wild-type Rab1 (Rab1WT) in RPMVECs strongly enhanced the amount of beta-ARs on the cell surface, whereas the dominant-negative mutant Rab1N1241 significantly attenuated beta-ARs expression on the cell surface. In addition, LPS stimulation significantly reduced beta-ARs expression on the cell surface in RPMVECs; however, this effect was reversed by overexpression of wild-type Rab1WT. Fluorescent microscopy analysis demonstrated that expression of Rab1N1241 and Rab1 small interfering RNA (siRNA) significantly induced the accumulation of green fluorescent protein (GFP)-tagged beta(2)-AR in the ER. Consistent with their effects on P-ARs export, Rabl WT and Rab1N1241 differentially modified the 3-AR-mediated activation of extracellular signal-regulated kinase1/2 (ERK1/2). Importantly, over-expression of Rab1WT markedly reduced LPS-induced hyperpermeability of RPMVECs by increasing the expression of beta 2-AR on the cell surface. These data reveal that P-ARs function in RPMVECs could be modulated by manipulating P-ARs traffic from the ER to the Golgi body. We propose the ER-to-Golgi transport as a regulatory site for control of permeability of RPMVECs. (C) 2010 Elsevier Ltd. All rights reserved.

目的 研究bcl-2、bax凋亡相关基因在低氧处理大鼠肺动脉中的表达,探讨低氧肺动脉高压的发病机制及防治措施.方法 36只Wistar大鼠随机分为3组,分别为正常对照组、低氧肺动脉高压组、低浓度一氧化碳(CO)组,每组12只,采用原位细胞凋亡、原位杂交、免疫组化等方法检测肺动脉的凋亡状况.结果 ①原位凋亡检测示,正常对照组、低氧肺动脉高压组、低浓度CO组的凋亡率分别为8.1%,37.6%,76.5%,三者间差异显著;②原位杂交显示,正常对照组bax、bcl-2杂交呈弱阳性表达.低氧肺动脉高压组bax杂交呈弱阳性表达,低浓度CO组呈强阳性表达,但bcl-2杂交可见低氧肺动脉高压组呈强阳性表达,低浓度CO组呈弱阳性表达;③免疫组织化结果表明,低浓度CO组bcl-2、bax蛋白均有升高,以bax为著.低氧肺动脉高压组虽然也有表达,但程度较弱,且bcl-2表达处于优势.结论 低浓度CO可以调节bcl-2、bax凋亡基因的表达,从而促进肺动脉组织的凋亡.

Objective To investigate the expression of heme oxygenase (HO) in pulmonary arteries of rats with hypoxic pulmonary hypertension (HPH). Methods A total of 60 rats were employed and equally divided into 5 groups (n=12 in each group) , i. e. control (group Ⅰ), HPH (group Ⅱ), hemin (group Ⅲ), tin protoporphyrin (SnPP) (group Ⅳ), and low concentration CO group (group Ⅴ). Of them, the rats from mid-dle 3 groups were treated with hypoxia under normal pressure for 7 h in every day except Sunday, consecutively for 3 weeks. Hemin and tin protoporphyrin (SnPP) were given half an hour before hypoxia while low concentra-tion CO at 2 h after hypoxia. HO-1 activity of pulmonary arteries tissues was tested with ultraviolet spectropho-tometry. The expression of HO was detected with reverse transcriptionpolymerase chain reaction (RT-PCR), immunohistochemical staining and in situ hybridization. Results ①The bilirubin production increased signifi-cantly in group [(396.28±69.62) pmol·mg-1·h-1], group [(668.56±189.29) pmol·mg-1·h-1], group [(267.38±88.59) pmol·mg-1·h-1]and group [(589.32±132.35) pmol·mg-1·h-1], as com-pared with those in group [(125.62±31.52) pmol·mg-1·h-1]. ②The levels of HO-1 mRNA expression of pulmonary arteries in group Ⅱ, group Ⅲ, group Ⅳ and group Ⅴ were significantly higher than those in group Ⅰ. The most significant HO-1 mRNA expression was found in group Ⅴ (P <0.01). The levels of HO-1 mRNA expression of pulmonary arteries in group Ⅲ and group Ⅴ were higher than those in group Ⅱ, but it was lower in group Ⅳ than that in group Ⅱ. ③The strong positive granules of HO-1 were observed in pulmonary arteries intima and media in group Ⅱ, group Ⅲ and group Ⅳ, while group Ⅲ and group Ⅳ involved more pos-itive granules of HO-1 as compared with those in group Ⅱ. ④The staining was deepened in three layers of the pulmonary arteries in group Ⅱ, while the staining was deepened more in group Ⅲ and group Ⅳ. No significant change was found in staining of HO-2 in different groups. Conclusion CO can up-regulate the expression of HO-1, and this may play important roles in the genesis and development of pulmonary hypertension.

Objective To investigate the effects of hypoxia on the lung and pulmonary arteries of rats and the therapeutic effects of carbon monoxide in low concentration.Methods Thirty-six Wistar rats were employed and randomized into 3 groups:control group,hypoxic pulmonary artery hypertension(HPAH) group and carbon monoxide in low concentration(CO) group.The mean right ventricle pressure(mRVP) and mean pulmonary artery pressure(mPAP) were examined and the whole blood viscosity,plasma viscosity,hematocrit,erythrocyte rigidity index,right ventricular hypertrophy index [RV/(LV+S)] were assessed.Pathological changes in the heart,lung and pulmonary artery were observed.Results The mPAP,mPVP and [RV/(LV+S)] in HPAH group were remarkably higher than that in control group(P<0.01),and that in CO group were higher than that in control group(P<0.05),but lower than that in HPAH group(P<0.01).The whole blood viscosity decreased with attenuated erythrocyte aggregation,and erythrocyte rigidity index declined slightly in CO group,with no significant difference compared with that in control group(P>0.05),but were significantly lower than that in HPAH group(P<0.05).Pathological observation revealed that there were thickening of pulmonary smooth muscle and narrowing of arterial lumen in PHAP group compared with control group,while those pathological changes were milder in CO group.Ultrastructural changes,such as alveolar-interstitial thickening,fibrosis,vacuolazation of type II alveolar epithelial cells,endothelial cell hypertrophy and heteromorphism of nuclei,were found by transmission electron microscopy in HPAH group,while only occasional thickening of alveolar interstitium with insignificant fibrosis in CO group.Conclusion Hypoxia can damage the lung tissue in rats,and low concentration of CO may provide a protective effect on the lung tissue of rats with hypoxic pulmonary hypertension.

博士学位论文反映了博士生所接受的教育水平和独立研究能力,是博士生培养质量的基本标志.近几年来,我国研究生招生规模不断扩大,一定程度上造成了博士生教育水平的下降和学术质量的滑坡.为了深入了解和评估笔者所在研究所博士生教育质量状况,配合国家和军队管理部门的相关要求,随机抽取笔者所在研究所(来自UMI 博士论文全文中国集团)与美国、加拿大(来自Proquest Information and Learning)20年来部分呼吸专业的临床医学博士论文进行对比分析,寻找问题并提出相应措施,将工作中心转移到重视素质、提高质量上来.

现在国际社会呈现多元化的格局,经济建设与和平发展是当今社会的主题.但是,人类社会发展也同时面临着严重的挑战,恐怖主义盛行,局部战争不断,"天下还不太平".在高科技局部战争中,战场的环境变化,比如高原低氧、高寒、深海等条件,对军事医学,尤其是对内科学与野战内科学的医疗救护体系和救护技术带来很多问题和严重挑战[1,2].

目的探讨野战内科疾病的调查研究方法,促进本学科的建设与发展。方法创建野战内科疾病调查的基本方法和资料收集方法,包括伤(病)员症状自评方法、病历、医嘱和护理记录表等。结果采用量表方法能保证资料收集的稳定性、简便性和经济性。结论在野战内科疾病的调查研究中采用量表方法可提高工作效率。