脉冲强磁场对膀胱肿瘤BIU-87细胞生长相关基因表达的影响

目的 研究脉冲强磁场对人膀胱肿瘤BIU-87细胞生长相关基因表达的影响.方法 将体外培养的人膀胱肿瘤BIU-87细胞分为磁场组及对照组.磁场组细胞给予脉冲强磁场干预(磁场强度为8 T,频率为15 Hz),每天持续干预2 h;对照组细胞亦置于相同环境中,但未给予曝磁处理.分别于实验进行24 h、48 h及72 h后采用逆转录-多聚酶链反应(RT-PCR)技术检测各组细胞B细胞淋巴瘤/白血病基因-2(Bcl-2)、促凋亡基因(Bax)及凋亡促进因子-3(caspase-3)mRNA表达,采用流式细胞仪检测Bcl-2、Bax及caspase-3蛋白表达.结果 实验进行24 h、48 h及72 h后,发现磁场组Bcl-2 mRNA及蛋白表达在上述各时间点均较对照组明显降低,Bax mRNA及蛋白表达均较对照组显著增强(P＜0.05);caspase-3 mRNA及蛋白表达在上述各时间点组间差异均无统计学意义(P＞0.05).结论 脉冲强磁场能抑制膀胱肿瘤BIU-87细胞生长,促其凋亡,其机制可能与脉冲强磁场促进Bax mRNA及蛋白表达、抑制Bcl-2 mRNA及蛋白表达有关.

Abstract：

Objective To estimate any influence of strong pulsed magnetic fields on the expression of growth-related genes in human bladder cancer BIU-87 cells. Methods Human BIU-87 cells were cultured in vitro and randomly divided into a magnetic field group and a control group. Each group was further divided into 24 h, 48 h and 72 h sub-groups. The magnetic field group cells were exposed to an 8 T magnetic field pulsed at 15 Hz for 2 h every day. The control group cells also placed on the same environment, but not exposed to any strong, pulsed magnetic field. The expression of B cell lymphoma/leukemia gene-2 (Bcl-2) mRNA, Bax mRNA and caspase-3 mRNA was measured with RT-PCR, and flow cytometry was used to evaluate the expression of the Bcl-2, Bax and caspase-3 genes of the tumor cells in vitro. Results The expression of Bax mRNA and protein was significantly higher in the cells exposed to the magnetic field than in the control groups. The expression of Bcl-2 mRNA and protein was significantly less. The expression of caspase-3 mRNA and protein in the two groups showed no significant differences.Conclusions A strong, pulsed magnetic field can inhibit the growth of bladder tumor BIU-87 cells and promote their apoptosis. The mechanism is probably related with the magnetic field promoting Bax mRNA and protein expression and inhibiting Bcl-2 mRNA and protein expression.     

硼替佐米对内皮细胞迁移及血管新生的影响

Objective To investigate the effects of bortezomib on the migration of endothelial cells and the expression of angiogenesis-related molecules,and explore the mechanism of its antiproliferation of tumor cells.Methods Cell count kit CCK-8 was used to detect the relative proliferation activity of cells after treated by bortezomib at different concentrations for 12 h and 24 h,respectively.Transwell model was uesd to detect the migration rate of cells.Expression levels of VEGF and Annexin A2 genes were determined by realtime quantitative PCR.Annexin A2 protein was validated by Western blot.Results After treated with bertezomib at concentrations of 2.5、5.0 and 10 nmol/L for 12h,respectively,the HMEC-1 cell proliferation activity was 1.004±0.002,0.793±0.021 and 0.874 4-0.062,respectively,being no statistical difference from that of control group(1.000)P<0.05);while the migration rates of them were 0.697±0.060,0.597±0.090 and 0.874±0.062,respectively,being significantly lower than that of control group(1.000)(P<0.05 ) and so did for the expression of VEGF and Annexin A2 genes.After treated with 5 nmol/L bortezomib for 12 h.the Annexin A2 and VEGF gene relative expression level of HMEC-1 cells was 0.540±0.001 and 0.793±0.153,respectively,being of statistical difference from that of control group(1.000)P<0.05).The conspicuous downregulation of Annexin A2 protein was also confirmed by Western Blot.Conclusions Bortezomib can inhibit migration of endothelial cell HMEC-1 by downregulating the expression of VEGF and Annexin A2, displaying a new mechanism of bortezomib for inhibition of tumor proliferation.     

Regulatory effects of hydrogen sulfide on alveolar epithelial cell endoplasmic reticulum stress in rats with acute lung injury

BACKGROUND: The present study was undertaken to examine the regulatory effect of hydrogen sulfide(H2S) on endoplasmic reticulum stress in alveolar epithelial cells of rats with acute lung injury(ALI) induced by oleic acid(OA).METHODS: Seventy-two male Sprague Dawley(SD) rats were divided into control group, oleic acid-induced ALI group(OA group), oleic acid-induced ALI with sodium hydrosulfide(Na HS) pretreatment group(OA+Na HS group), and sodium hydrosulfide treatment group(Na HS group). Rats of each group were further subdivided into 3 subgroups. Index of quantitative assessment of histological lung injury(IQA), wet/dry weight ratio(W/D) and H2 S level of lung tissues were measured. The expressions of endoplasmic reticulum stress markers including glucose-regulated protein 78(GRP78) and α-subunit of eukaryotic translation initiation factor-2(el F2α) in lung tissues were measured by immunohistochemical staining and Western blotting.RESULTS: The IQA score and W/D ratio of lung tissues at the three time points significantly increased in rats injected with OA, but significantly decreased in other rats injected with OA and Na HS. The level of H2 S in lung tissue at the three time points significantly decreased in rats injected with OA, but significantly increased in other rats injected with both OA and Na HS. GRP78 and el F2α decreased in rats injected with OA, but increased in other rats injected with both OA and Na HS, especially at 4-hour and 6-hour time points.CONCLUSION: The results suggested that H2 S could promote alveolar epithelial cell endoplasmic reticulum stress in rats with ALI.     

硼替佐米对内皮细胞迁移及血管新生的影响

Objective To investigate the effects of bortezomib on the migration of endothelial cells and the expression of angiogenesis-related molecules,and explore the mechanism of its antiproliferation of tumor cells.Methods Cell count kit CCK-8 was used to detect the relative proliferation activity of cells after treated by bortezomib at different concentrations for 12 h and 24 h,respectively.Transwell model was uesd to detect the migration rate of cells.Expression levels of VEGF and Annexin A2 genes were determined by realtime quantitative PCR.Annexin A2 protein was validated by Western blot.Results After treated with bertezomib at concentrations of 2.5、5.0 and 10 nmol/L for 12h,respectively,the HMEC-1 cell proliferation activity was 1.004±0.002,0.793±0.021 and 0.874 4-0.062,respectively,being no statistical difference from that of control group(1.000)P<0.05);while the migration rates of them were 0.697±0.060,0.597±0.090 and 0.874±0.062,respectively,being significantly lower than that of control group(1.000)(P<0.05 ) and so did for the expression of VEGF and Annexin A2 genes.After treated with 5 nmol/L bortezomib for 12 h.the Annexin A2 and VEGF gene relative expression level of HMEC-1 cells was 0.540±0.001 and 0.793±0.153,respectively,being of statistical difference from that of control group(1.000)P<0.05).The conspicuous downregulation of Annexin A2 protein was also confirmed by Western Blot.Conclusions Bortezomib can inhibit migration of endothelial cell HMEC-1 by downregulating the expression of VEGF and Annexin A2, displaying a new mechanism of bortezomib for inhibition of tumor proliferation.     

Protective effects of ulinastatin on intestinal barrier damaged after cardiopulmonary resuscitation in rats北大核心CSCD

Objective To investigate the protective effects of Ulinastatin on intestinal barrier damaged after cardiopulmonary resuscitation (CPR) in rats in order to illustrate the possible mechanism. Methods Twenty-one male SD rats were divided into three groups randomly (random number) including control group (sham group, n = 7), cardiopulmonary resuscitation group (CPR group, n = 7) and ulinastatin group (UTI group, n = 7). The rats were anesthetized with pentobarbital sodium (45 - 60 mg/kg) by intraperitoneal injection. The rats of sham group were only treated with endotracheal intubation. Ulinastatin (100 000 U/kg) were injected via caudal vein 2 hours prior to CPR, and cardiac arrest was made in rats and cardiopulmonary resuscitation was carried out in the UTI group, while equivalent volume of sterile saline was used instead in the CPR group. Blood and ileum samples were obtained at 48 hour after restoration of spontaneous circulation (ROSC). The levels of tumor necrosis factor alpha (TNF-α) and interleukin -1β (IL-1β) were assayed by ELISA (enzyme-linked immunosorbent assay), the protein levels of caspase-3 were determined by western blot, the intestinal mucosa were stained by terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) and ileac mucosa were observed under transmission electron microscope. Data were processed with SPSS 17.0 software. Results The plasma levels of TNF-α and IL-1β were dramatically higher in CPR group than those in other two groups (CPR vs. sham, P < 0.01 ; CPR vs. UTI, P < 0.05). Moreover, the tight junctions between cells obviously broadened and loosened in the CPR group were found under electron microscope, however, this phenomenon was not obvious in the UTI group. A large number of apoptotic cells were observed by TUNEL assay in the CPR group, but a small number of apoptotic cells were observed in the UTI group. The protein levels of caspase-3 in the UTI group were higher than those in sham group, but lower than those in CPR group (both P < 0.05). Conclusions Ulinastatin has protective effects on the intestinal barrier damaged after cardiopulmonary resuscitation in rats by decreasing the proinflammatory mediators in the blood, reducing the expression of caspase-3 and then reducing the numbers of apoptotic intestinal cells.     

Bystander tumoricidal effect and cell apoptosis of mouse mammary carcioma cell transduced with HSV-tk gene

To apply gene therapy to breast carcinoma wepropose a model for in vitro gene transfer of the herpessimplex thymidine kinase (HSV-tk) gene using Stkvector-producing cells. In this study the HSV-tk gene wastransferred to mouse mammary carcinoma cell line 4Tlwith the method of infection. The HSV-tk gene in thegenome of transduced cells (4Tl/tk) was demonstratedby the method of DNA-PCR. Using a modified XTT     

The protective effect of rosiglitazone on the rats with high altitude pulmonary edema北大核心CSCD

Objective To investigate the protective effect of rosiglitazone on the rats with high altitude pulmonary edema- Methods Thirty-six SD rats were randomly (random number) divided into 6 groups (n - 6 each): control group ( Control), hypobaric hypoxia model group ( HH), rosiglitazone groups (RSG) which were administered with 3 different doses [RSG-L: 5 mg/ (kg • d), RSG-M: 10 mg/ (kg • d), RSG-H: 20 mg/ (kg • d)], dexamethasone group [Dex, 4 mg/ (kg • d)] . Rats were injected intraperitoneally with different doses of rosiglitazone (RSG), dexamethasone ( Dex) or vehiclc (Control and HH) for 3 days before placed in simulated altitude of 6 000 m hypobaric hypoxia animal chamber where the temperature and pressure were constant. After 72 h in the chamber, each rat was anesthetized. The water content of lung was determined with wet/dry weight ratio. Bronchoalveolar lavage fluid was measured by bradford method. The contents of GSH was measured by micro-ezymcd labeled method. The contents of MDA was measured by TBA method. The enzymatic activities of SOD was measured by WST-1 method. The changes of the TNF-A, IL-6 and IL-10 in serum were determined by ELJSA. Light microscope was used to observe the pathological changes of lung tissue. Results Compared with Control group, the wet/dry weight ratio of lung (5.08 ±0.24) and total protein content of BALF (351.06 ± 44. 55) jig/mL increased significantly (P <0.01) in HH group. There were red blood cells in the alveolar and interstitium, pink fluid exudation in the alveolar, the alveolar septum enhancement, and a large number of inflammatory cell infiltration; the SOD activity (10.65 ±0.94) U/mgprot and the content of CSH (1. 63 ±0. 20) jimol/gprot in lung tissue were significantly decreased ( P < 0. 01), the contents of MDA (2. 15 ±0.18) nmol/mgprot increased significantly (P <0.01), TNF-A (56.92 ±2.87) pg/mL and IL-6 (217. 80 ±48. 01) pg/mL levels in serum were significantly increased ( P < 0. 01 ), and IL-10 (76. 85 ± 16.72) pg/mL level decreased (P <0.05). Compared with the HH group, the wet/dry ratio of lung and total protein content of BALF in different doses of rosiglitazone group significandy decreased (P <0. 01), the pathological changes of the lung tissue was significantly improved, SOD activity and the content of GSH in lung tissue was significantly increased (P < 0.01 ), the content of MDA decreased ( P < 0. 01), The levels of TNF-A and IL-6 in serum were significantly decreased (P < 0. 01 ), while the IL-10 level was significantly increased (P<0. 01) . Conclusion Rosiglitazone could protect the high altitude pulmonary edema by alleviating the oxidative stress and inflammatory response.     

Effects of melatonin on microglia activation in rats with acute intracerebral hemorrhag北大核心CSCD

Objective: To investigate the effects of melatonin on activation of microglia and the changes of superoxide dismutase (SOD) and malondialdehyde (MDA) after intracerebral hemorrhage (ICH) in rats. Methods: One hundred and thirty male SD rats were randomly divided into four groups: normal group, sham-operated group, intracerebral hemorrhage model group (Model group) and melatonin intervention group (MT group). Each group was further divided into 5 subgroups respectively at 12 h, 1 d, 2 d, 4 d and 7 d after modeling. The ICH models were made in SD rats by using Rosenberg methods. Melatonin in dose of 10 mg/kg in solution of 1 mg/mL was given intraperitoneally to rats of MT group. The morphology of microglia was observed under electron microscope. OX42-positive cells were detected by immunohistochemical (ABC) methods. The contents of MDA or the activity of SOD was measured respectively by thiobarbituric acid or xanthine oxidation method. Results: Electron microscope showed that the activation of microglia cell displayed in ameboid shape and swelling of neuron in hemisphere cortex in model group at 2 d after ICH, the activation of microglia cell of the cortex was insignificant in MT group. OX42-positive microglia cell in large amount surrounded the hematoma at 12 h after ICH, peaked at 1 d and lasted for 7 d. OX42-positive microglia cells in MT group were significantly fewer than that in model group at different intervals after ICH (P < 0. 05). The content of MDA in model group increased significantly after ICH, and higher than that in normal group at 7 d, [(0. 875 + 0. 098) nmol/mg prot vs. (0. 725 ± 0. 061) nmol/mg prot, P<0. 05, whereas the activity of SOD changes in the opposite direction, (70.46 ±3. 12) U/mg prot vs. (85. 86 ±4. 95)/U/mg prot, P <0. 05. Compared with model group, the content of MDA were lower and the activity of SOD were higher in MT group after ICH (P < 0. 05). Conclusion: Melatonin provides a protective effect on the damage of nerve cell after ICH. The mechanism might be associated with melatonin by reducing the level of oxidative stress in brain tissue and attenuating the activation of microglia after ICH.     

Relationship between exon deletion frequency of CDKN2／P16 and pathological type,metastasis,sex in osteosarcoma

Objective:To study on the CDKN2/P16 gene i primary osteosarcoma.Method:By using molecular biological methods that inclued genome DNA extraction from paraffined tissue and PCR-SSCP analysis technique,we studied alternations of CDKN2/P16 gene in 25 primary osteosareomas.Results:(1)The deletions frequency in differentiation degree of osteosarcomas was ①bone brood cell,16.7%;②cartilage brood cell,12.5%;③Fiber brood cell:20%,(P&;gt;0.05).(2)The deletion frequency in male patients was 17.6,female patients 12.5%,(P&;gt;0.05).(3)In early metastatic osteosarcomas the deletion rate was 33.3%,which was significantly higher than that of the control group with the rate of 10.5%(P&;lt;0.05).(4)The deletion rate was 16% and the mutations were not found.Conclusion:(1)The deletion rate was 16% and the mutations were not found.This suggests that the deletions of CDKN2/P16 gene were closely related to the genesis of primary osteosarcoma and that the main type of the alternation of CDKN2/P16 gene was deletion.(2) In early metastatic osteosaarcomas the deletion rate was 33.3%,which was significantly higher than that of the control group with the rate of 10.5%.This indicates to great extend that the deletions of CDKN2/P16 gene were closely related to the metastatic ability.(3)The deletions frequency had no significant relationship with differentiation degree of osteosarcomas,so was with the sex of the patient.     

白介素6对过氧化氢诱导的肺泡上皮细胞凋亡的影响

目的:研究白介素6(IL-6)在过氧化氢诱导的肺泡上皮细胞凋亡中的作用及其机制。方法:以人Ⅱ型肺泡上皮细胞A549细胞株为实验对象,MTT法检测IL-6对A549细胞增殖活性的影响,TUNEL检测法检测IL-6对A549细胞凋亡率的影响,Westernblot观察caspase-3和caspase-9蛋白量的变化。结果:IL-6能够增加A549细胞的增殖活性,减少其凋亡。与模型组相比,IL-6干预组的caspase-3和caspase-9的蛋白表达量明显降低,有统计学意义。结论:IL-6能够抑制过氧化氢诱导的肺泡上皮细胞凋亡,并且与caspase-3和caspase-9蛋白的表达量下降有关。提示IL-6对肺泡上皮细胞的保护作用可能是通过抑制细胞凋亡相关蛋白来实现的。     

Hypocapnic alkalosis enhances oxidant-induced apoptosis of human alveolar epithelial type II cells

Apoptosis of alveolar epithelial type II (AEC-II) cells induced by reactive oxygen species (ROS) contributes to extensive alveolar damage during acute lung injury. Hypercapnic acidosis and hypocapnic alkalosis are known to modulate ROS-mediated lung damage. This study assessed the effects of acid-base balance disturbances on hydrogen peroxide (H2O2)-induced apoptosis of the AEC-II-like human cell line A549, which was cultured under different conditions of pH and CO2 tension (normal pH and CO2, hypercapnic acidosis, metabolic acidosis, hypocapnic alkalosis and metabolic alkalosis). H2O2-induced apoptosis was assessed by a dye-uptake bioassay and induction of caspase activity, which were quantified using analytical digital photomicroscopy. Acidosis or alkalosis of the culture medium alone did not induce A549 cell apoptosis. Hypocapnic alkalosis significantly increased H2O2-induced apoptosis and caspase activation of A549 cells. Metabolic alkalosis non-significantly increased H2O2-induced A549 cell apoptosis and caspase activation. These data suggest that hypocapnic alkalosis intensifies oxidative-induced apoptosis of alveolar epithelial cells.     

HMGB1基因沉默对阿霉素诱导K562/A02细胞凋亡增敏作用的研究

目的 探讨高迁移率族蛋白1(HMGB1)基因沉默对白血病细胞耐药逆转的作用.方法 将HMGB1基因特异性干扰RNA(HMGB1 siRNA)导入K562/A02细胞中,通过Western blot和RTPCR方法检测HMGB1基因在转染前后的表达;WST8法检测阿霉素(ADM)对转染前后K562/A02细胞的半数抑制浓度(IC50);流式细胞术检测凋亡细胞百分率;Western blot检测线粒体促凋亡蛋白Smac/DIABLO的释放;采用caspase活性定量检测试剂盒分析caspase-3的活性.结果 ①与未经处理的K562/A02细胞组相比,转染HMGB1 siRNA的K562/A02细胞组HMGB1 mRNA和蛋白水平分别下降86%和71%;②HMGB1基因沉默使K562/A02细胞对ADM的药物敏感性增强,其IC50值从转染前的(4.83±0.08)μg/ml降低到(1.33±0.10)μg/ml,并在ADM浓度为1μg/ml和5μg/ml时,细胞凋亡百分率分别增加27%、32%;③HMGB1基因沉默可促进ADM所致Smac/DIABLO从线粒体向胞浆释放,并增加caspase-3的活性.结论 HMGB1基因沉默能明显增加K562/A02细胞对ADM的敏感性,逆转K562/A02细胞对ADM耐药.     

RNA干扰敲除Smac基因的表达促进人肺癌细胞的生长及增强顺铂耐药性

目的 探讨RNA干扰敲除Smac基因的表达对肺癌细胞的生长及顺铂(cDDP)耐药性的影响.方法 通过转染含有以人Smac基因为靶基因的慢病毒载体系统,即含有小分子干扰RNA序列的pGC-FU载体,分别在A549和95D细胞中实施Smac基因敲除.通过转染含有Smac全长编码序列的pGC-FU载体来实现Smac的高表达.细胞生长、细胞周期及凋亡采用四甲基偶氮唑盐(MTT)法、克隆形成实验及流式细胞仪测定.药物耐药用10 μg/ml顺铂检测.结果 Smac下调表达促进A549和95D细胞中肺癌细胞的生长及增强顺铂耐药性;Smac高表达抑制A549细胞生长并且增强其对顺铂的敏感性.结论 Smac抑制肺癌细胞生长并且增强其对顺铂的敏感性.     

人端粒酶逆转录酶基因沉默对肺腺癌细胞侵袭、增殖、凋亡的影响

目的:探讨靶向人端粒酶逆转录酶(hTERT)基因的小干扰RNA(siRNA)转染对肺腺癌细胞株A549生物学行为的影响。方法:构建靶向hTERT的siRNA表达质粒转染A549细胞,通过Western blot检测蛋白的表达水平,确定有效后,通过Transwell小室检测细胞体外侵袭能力,MTT法检测细胞增殖活性,流式细胞术测定转染后肿瘤细胞的细胞周期和凋亡率。结果:成功构建靶向hTERT的siRNA表达质粒。实验组siRNA转染后的细胞侵袭能力较对照组减弱(P<0.01),生长抑制率明显高于阴性对照组(P<0.01),且表现出时间依赖性。流式细胞术测定显示,实验组siRNA转染48h后,肿瘤细胞凋亡率显著增加(P<0.01)。结论:靶向hTERT的siRNA转染后可以显著降低A549细胞侵袭能力,抑制增殖,诱导细胞凋亡的发生。     

人HMGN5基因shRNA慢病毒载体构建与RNAi效率的鉴定

目的构建人HMGN5基因的shRNA慢病毒载体并鉴定在肺癌A549和H1299细胞上的沉默效率。方法设计HMGN5基因特异性siRNA靶点,构建于慢病毒pLL-3.7载体,并筛选获得有效的shRNA慢病毒载体,在293T细胞包装成病毒颗粒,将其感染肺癌A549和H1299细胞,应用Real-time PCR方法从mRNA水平上检测HMGN5的沉默效率。结果构建的慢病毒载体shRNA的PCR鉴定和测序正确,包装病毒后滴度达到5×108TU/ml。shRNA慢病毒颗粒感染A549和H1299细胞后HMGN5基因的mRNA表达量较阴性对照载体慢病毒感染组分别下降了71.7%和50.7%。结论成功构建了HMGN5基因的shRNA慢病毒表达载体,在分子水平能够有效沉默靶基因,为探讨HMGN5在肿瘤基因治疗中的作用奠定了基础。     

多发性骨髓瘤特异APE1siRNA表达载体的构建及鉴定

目的构建多发性骨髓瘤(MM)特异的 APE1siRNA 表达载体 pSilencer K-IE-IgP-APE1siRNA,观察其对 MM 细胞 APE1蛋白表达的特异性敲除作用。方法设计合成的 APE1siRNAcDNA 序列与线性化 pSilencer2.0-U6母本质粒连接后,用 BamH Ⅰ、EcoR Ⅰ双酶切,插入 IgP 寡核苷酸片段;随后用 EcoR Ⅰ酶切 pSilencer IgP-APE1siRNA,将线性化载体与回收 IEcDNA 片段用 T4 DNA 连接酶连接;再用 Xho Ⅰ酶切,与回收 Kappa cDNA 片段连接,克隆出 MM 特异表达载体 pSilencer K-IE-IgPAPE1siRNA,每次连接后均经酶切鉴定和测序确认。用脂质体转染法将重组质粒转染 KM3、HOS 和MDA-231细胞,Western blot 分析其对 KM3细胞 APE1的特异性敲除作用。结果 pSilencer K-IE-IgP-APE1siRNA 能特异且有效地敲除 KM3细胞 APE1蛋白表达,转染 siRNA 后培养2d 的 KM_3细胞 APE1相对表达水平为0.118±0.047,而单独脂质体转染组,APE1相对表达水平为0.988±0.029,基因缄默效率为90%。结论成功构建了针对 MM 的 APE1siRNA 表达载体。     

MKK6 p38α信号通路在机械牵张诱导肺泡上皮细胞表达晚期糖基化终末产物受体中的作用

目的 探讨p38α及其上游激酶丝裂素活化蛋白激酶6(MKK6)在机械牵张诱导肺泡上皮细胞(A549)表达晚期糖基化终末产物受体(RAGE)中的作用.方法 应用阳离子脂质体DOTAP介导的基因转染方法将p38α显性无活性突变体p38α(AF)+绿色荧光表达载体pGFP及MKK6b的组成性活性突变体MKK6b(E)+pGFP分别导入A549细胞,以质粒pcDNA3+pGFP作为阴性对照,以pGFP作为空白对照.应用蛋白质免疫印迹法(Western blotting)检测转染基因.建立体外细胞牵张应变模型,观察各组细胞在施加20%牵张应变时p38激酶活性变化及RAGE的蛋白和mRNA表达变化.结果 Western blotting检测结果显示,外源基因成功转染A549细胞,并在细胞内表达.在A549细胞牵张应变模型中观察到,MKK6b(E)转染细胞内p38激酶活性明显升高,而p38α(AF)转染细胞内p38激酶活性显著下降;MKK6b(E)基因转染可促进20%牵张应变诱导A549细胞RAGE的蛋白和mRNA表达,而p38α(AF)基因转染则抑制牵引应变诱导的RAGE蛋白和mRNA表达.结论 牵张应变通过MKK6 p38α信号通路调节A549细胞RAGE的表达.     

Silencing of X-linked inhibitor of apoptosis decreases resistance to cisplatin and paclitaxel but not gemcitabine in non-small cell lung cancer

Non-small cell lung cancer (NSCLC) is a highly lethal malignancy that often becomes resistant to chemotherapy. The effect of silencing the X-linked inhibitor of apoptosis gene (XIAP) on resistance to cisplatin, paclitaxel and gemcitabine was studied in the NSCLC cell lines A549 and H460. Transfection of these cells with small interfering RNA (siRNA) for XIAP blocked overexpression of the gene, suppressed cell proliferation, increased apoptosis and increased the cells' sensitivity to cisplatin and paclitaxel by preventing the binding of XIAP to caspase3 and increasing the activity of this enzyme. There was no significant difference in resistance to gemcitabine between XIAP-silenced cells and non-transfected cells. Changes in chemoresistance were independent of the activity of caspase-9. Silencing XIAP with siRNA can decrease chemoresistance in NSCLC and may have a potential role in the treatment of this disease.     

Role of interleukin-8 and growth-regulated oncogene-alpha in the chemotactic migration of all-trans retinoic acid-treated promyelocytic leukemic cells toward alveolar epithelial cells

OBJECTIVE: Although all-trans retinoic acid (ATRA) can treat acute promyelocytic leukemia (APL), it also causes retinoic acid syndrome with presentations similar to acute respiratory distress syndrome. We investigated the role of interleukin (IL)-8 and growth-regulated oncogene (GRO)-alpha in the chemotactic transmigration of ATRA-treated NB4 (ATRA-NB4) APL cells toward A549 alveolar epithelial cells. DESIGN: An in vitro human cell culture study. SETTING: University hospital research laboratories. SUBJECTS: NB4 and A549 cells. INTERVENTIONS: NB4 and A549 cells were separately cultured with ATRA and/or dexamethasone for 1-3 days. NB4 or ATRA-NB4 cells were then placed in an upper insert and co-incubated with A549 cells or their conditioned medium located in a lower plate. MEASUREMENTS AND MAIN RESULTS: ATRA stimulated NB4 cells to transmigrate toward the A549 cells in a time- and dose-dependent manner. Replacement of A459 condition medium by its original medium abrogated this transmigration. Only A549 cells constitutively secreted GRO-alpha, and both A549 and NB4 cells constitutively secreted IL-8, which was enhanced by ATRA. Exogenous administration of IL-8 or GRO-alpha also promoted the ATRA-NB4 transmigration. The binding assay demonstrated that ATRA-NB4 cells bound IL-8, but not GRO-alpha, more avidly. Pretreatment with antibodies directed against IL-8 and GRO-alpha receptors reduced ATRA-NB4 transmigration by about 60%. Dexamethasone did not suppress their IL-8 secretion and transmigration in ATRA-NB4 cells, but when applied to A549 cells, IL-8 secretion was suppressed but not GRO-alpha secretion, and there was attenuation of ATRA-NB4 transmigration. CONCLUSIONS: IL-8 and GRO-alpha secreted from alveolar epithelial cells play an important role in the cell-cell interaction involved in the chemotactic transmigration of ATRA-treated APL cells toward alveolar epithelial cells.