重组TRAIL蛋白对胰腺癌细胞SW1990增殖、凋亡的影响及吉西他滨的干预作用

目的 探讨重组肿瘤坏死因子相关凋亡诱导配体(TRAIL)蛋白的抗胰腺癌作用及吉西他滨的干预作用.方法 分别采用不同浓度TRAIL 和(或)吉西他滨处理人胰腺癌细胞株SW1990并分为TRAIL组、吉西他们滨组、联合组.采用MTT法检测各组生长抑制率(IR),流式细胞仪检测细胞凋亡率,Heochst 33342染色法检测凋亡细胞形态,Western blot检测凋亡相关蛋白Smac/DIABLO、Caspase-3表达.结果 TRAIL组、吉西他滨组及联合组均出现细胞增殖抑制和凋亡;联合组IR、凋亡率及凋亡相关蛋白表达均显著高于其他两组(P＜0.05).结论 TRAIL可通过调节细胞凋亡相关蛋白表达抑制SW1990细胞增殖及诱导凋亡;其与吉西他滨联用可为胰腺癌的靶向治疗提供新思路.     

Survivin启动子驱动Trail诱导肝癌细胞凋亡的实验研究

目的了解Survivin启动子在HepG2肝癌细胞与3T3细胞中诱导Trail蛋白表达能力的差异,并评价其驱动Trail诱导肝癌细胞凋亡的能力。方法以pGL3-surp340质粒为模板,扩增Survivin340启动子,构建重组质粒pcDNA3.1/SurP/Trail。将重组质粒分别转染HepG2肝癌细胞和3T3细胞,通过Western Blot检测两种细胞中Trail蛋白表达的差异。利用流式细胞术检测HepG2肝癌细胞及3T3细胞转染重组质粒pcDNA3.1/SurP/Trail后细胞凋亡的情况。结果成功构建了重组质粒pcDNA3.1/SurP/Trail,转染HepG2和3T3两种细胞后,通过Western Blot检测,HepG2肝癌细胞Trail蛋白表达的相对灰度值为0.49±0.43,较3T3细胞Trail蛋白表达(0.15±0.37)明显增强(P0.05)。利用流式细胞术检测,HepG2肝癌细胞转染重组质粒组的凋亡率(%)为11.59±0.74,空白质粒组为3.45±0.52,未转染组为2.67±0.34;3T3细胞重组质粒组的凋亡率(%)为3.26±0.24,空白质粒组3.16±0.15,未转染组2.83±0.27。HepG2细胞转染重组质粒组凋亡率明显高于其他对照组(P0.05)。结论含Survivin基因启动子的重组质粒pcDNA3.1/SurP/Trail在肿瘤细胞中能增强Trail蛋白的表达,并能诱导HepG2肝癌细胞凋亡。     

人胰腺癌吉西他滨耐药细胞株APE1/Ref-1表达升高

背景:吉西他滨是中晚期胰腺癌的主要化疗药物,但由于胰腺癌对吉西他滨存在高度先天性和获得性耐药,吉西他滨不能明显改善胰腺癌患者的预后。目的:探讨DNA损伤修复及其碱基切除修复途径中的关键酶人脱嘌呤/脱嘧啶核酸内切酶1/氧化还原因子-1(APE1/Ref-1)表达与胰腺癌吉西他滨耐药之间的关系。方法:应用前期研究建立的人胰腺癌吉西他滨耐药细胞株SW1990-0.5(耐药指数9.32)及其亲本细胞株SW1990,以彗星实验评估两者经吉西他滨作用后的DNA损伤程度,real-time PCR和蛋白质印迹法检测APE1/Ref-1 mRNA和蛋白表达。结果:经吉西他滨作用24 h,SW1990-0.5和SW1990细胞的彗星实验OTM值分别为0.32±0.13和26.96±6.83,相对于SW1990细胞,SW1990-0.5细胞的APE1/Ref-1 mRNA表达量为2.48±0.49,两者APE1/Ref-1蛋白相对表达量分别为1.57±0.08和0.84±0.06,组间差异均有统计学意义(P均0.05)。结论:DNA损伤修复可能与胰腺癌吉西他滨耐药相关,APE1/Ref-1表达上调可能是通过修复DNA损伤参与胰腺癌对吉西他滨耐药。     

RNA干扰抑制FAK基因表达对人胰腺癌细胞凋亡及吉西他滨敏感性的影响

目的 探讨RNA干扰技术抑制黏着斑激酶（FAK）基因表达增强人胰腺癌PANC-1细胞对化疗药物敏感性及凋亡能力的研究.方法 针对FAK mRNA序列设计合成短发夹状干扰RNA（siRNA）的DNA模板,构建pRNAT-FAK重组表达载体,转染人胰腺癌PANC-1细胞;通过RT-PCR分析其对PANC-1细胞内源性FAK表达的影响;激光共聚焦显微镜检测PANC-1细胞凋亡的形态学改变;用四甲基偶氮唑蓝法观察PANC-1细胞对化疗药物吉西他滨敏感性的改变;采用分光光度计检测 Caspase 活性.结果 成功构建 pRNAT-FAK重组质粒,并成功转染PANC-1细胞;RT-PCR证实重组质粒在mRNA显著抑制FAK基因表达（P＜0.01）;吉西他滨组及吉西他滨加pRNAT-FAK组细胞则检测出明显的细胞凋亡;四甲基偶氮唑蓝结果证明在和吉西他滨联合作用下,pRNAT-FAK组PANC-1细胞的生长抑制率明显增高（P＜0.01）;Caspase 3活性明显高于空白对照绀和阴性对照组.结论 pRNAT-FAK可抑制FAK在人胰腺癌PANC-1细胞中的表达,并增强PANC-1细胞对吉西他滨敏感性.     

血管紧张素转化酶Ⅱ对吉西他滨治疗胰腺癌细胞株敏感性的影响

目的探讨血管紧张素转化酶Ⅱ（ACE2）在胰腺癌细胞化学治疗中的作用。方法构建质粒,通过脂质体转染技术将质粒DNA转入胰腺癌细胞株SW1990并建立稳定高表达ACE2的胰腺癌细胞克隆。设实验组（转染ACE2表达质粒）、阴性对照组（转染GFP对照质粒）及未转染组,Western印迹法检测转染后各组细胞ACE2蛋白的表达。采用化学治疗药物吉西他滨作用不同处理组细胞,应用MTT法检测细胞增殖变化,流式细胞仪和TUNEL法检测细胞的凋亡变化。结果50μmol／L吉西他滨干预细胞24h后,实验组、阴性对照组和未转染组的细胞增殖抑制率分别为（51．2±4．8）％、（24．2±3．3）％和（21．3±2．6）％,3组间差异有统计学意义（P〈0．05）;流式细胞计数检测实验组细胞凋亡较阴性对照组及未转染组明显增加[（31．2±3．8）％、（17．64±2．3）％、（15．9±1．7）％,P〈0．05],TUNEL标记分析发现典型凋亡特征。结论稳定高表达ACE2基因可明显增强SW1990对吉西他滨的治疗敏感性,两者联合应用在胰腺癌治疗中具有潜在的价值。     

siRNA沉默APE1／Ref-1基因增强人胰腺癌细胞株对吉西他滨的敏感性

背景：人脱嘌呤脱嘧啶核酸内切酶／氧化还原因子-1（APE1／Ref-1）基因与肿瘤的化放疗抵抗和预后密切相关．是肿瘤基因治疗的理想靶点。目的：以RNA干扰技术靶向沉默人胰腺癌细胞株APE1／Ref-1基因,观察该方法对细胞增殖和凋亡的影响及其能否增强胰腺癌细胞对吉西他滨的敏感性。方法：将靶向APE1／Ref-1基因的小干扰RNA（siRNA）转染人胰腺癌细胞株SW1990,半定量逆转录聚合酶链反应（RT-PCR）和蛋白质印迹法检测APEl／Ref-1基因和蛋白表达,CCK-8检测细胞增殖情况,流式细胞仪和Hoechst33258染色检测细胞凋亡情况。结果：转染APE1siRNA后．SW1990细胞APE1／Ref-1mRNA和蛋白表达显著减低,蛋白表达抑制率为55．4％±3．6％;24h、48h和72h时细胞增殖抑制率分别为41．7％±2．8％、24．8％±3．7％和21．3％±9．8％：吉西他滨组、si-APE1组和联合组均可见明显细胞凋亡．联合组早期凋亡率显著高于两者单用和空白对照组（19．8％±3．5％对7．7％±1．1％、8．4％±1．0％和2．7％±1．4％．P〈0．05）,凋亡核形态学变化最为明显。结论：沉默APE1／ReG1基因能抑制SW1990细胞增殖,促进细胞凋亡,显著提高细胞对吉西他滨的敏感性。RNAi沉默APE1／Ref-1基因联合吉西他滨化疗可能成为胰腺癌治疗的新的选择。     

过表达RGC-32对胰腺癌BxPC-3细胞增殖、凋亡的影响

目的探讨RGC-32基因在胰腺癌Bx PC-3细胞增殖、凋亡中的作用。方法常规培养胰腺癌Bx PC-3细胞,构建RGC-32表达质粒pc DNA3.1/myc-His C-RGC-32,并瞬时转染Bx PC-3细胞作为实验组;将转染空质粒pc DNA3.1/myc-His C的Bx PC-3细胞作为对照组;应用实时定量PCR及Western blotting确定转染效率。采用细胞增殖试剂盒CCK-8(cell counting kit-8)法检测转染前后细胞增殖、流式细胞术检测细胞凋亡的变化。结果定量PCR及Western blotting检测结果均显示转染实验组较对照组RGC-32mRNA及蛋白表达显著上调(P0.05)。细胞增殖实验表明实验组较对照组细胞存活率显著性下降(P0.05);细胞凋亡实验表明实验组较对照组细胞凋亡率无显著性下降(P0.05)。结论在胰腺癌Bx PC-3细胞中,过表达RGC-32可抑制细胞增殖,但对细胞凋亡无明显作用。     

人胰腺癌吉西他滨耐药细胞株的建立及其耐药机制初步探讨

背景:吉西他滨是胰腺癌的一线化疗药物,但由于存在原发性和获得性耐药,其改善胰腺癌患者预后的作用并不明显。因此,探讨吉西他滨获得性耐药机制具有重要临床意义。目的:建立人胰腺癌吉西他滨耐药细胞株,初步探讨胰腺癌对吉西他滨的耐药机制。方法:在体外以0.5μmol/L吉西他滨持续刺激人胰腺癌细胞株SW1990,获得耐药细胞株SW1990-0.5。以CCK-8实验检测SW1990-0.5细胞株的耐药指数,细胞群体倍增实验和划痕实验分别检测亲本和耐药细胞株在体外的生长和侵袭能力,流式细胞术检测细胞周期和细胞凋亡,real-time PCR检测多药耐药相关基因MDR-1、MRP-1、BRCP和吉西他滨代谢相关酶基因dC K、RRM1、RRM2表达。结果:SW1990-0.5细胞株的耐药指数为9.32。与亲本细胞株相比,耐药细胞株体外增殖能力减弱,体外侵袭能力无明显变化;经吉西他滨作用后,耐药细胞株细胞周期无明显变化,但细胞凋亡率显著降低,MRP-1、BRCP、dC K mRNA表达降低,MDR-1、RRM1、RRM2 mRNA表达无明显变化。结论:成功建立了稳定的人胰腺癌吉西他滨耐药细胞株SW1990-0.5;胰腺癌对吉西他滨获得性耐药可能与拮抗细胞凋亡和dC K表达下调有关。     

阻断PI3K/Akt通路增强缺氧环境胰腺癌细胞化疗敏感性的实验研究

目的研究LY294002联合吉西他滨是否能增强缺氧环境下胰腺癌细胞株PANC-1的化疗敏感性。方法缺氧条件下培养胰腺癌细胞株PANC-1,运用MTT检测LY294002联合吉西他滨对胰腺癌细胞株细胞PANC-1生长的影响,运用Western blot检测AKT及磷酸化AKT蛋白表达的水平。结果 MTT检测在缺氧环境下LY294002联合吉西他滨作用的胰腺癌细胞PANC-1与单纯吉西他滨作用组相比细胞生存率显著下降(P=0.003),且具有时间依赖性。Western blot检测显示在缺氧环境下,LY294002联合吉西他滨作用的胰腺癌细胞PANC-1磷酸化AKT蛋白水平与单纯吉西他滨作用组相比显著降低(P=0.002)。结论阻断PI3K/Akt通路能增强缺氧环境下胰腺癌细胞化疗敏感性,为胰腺癌的治疗方法及逆转耐药提供了新的实验依据。     

PUMA在吉西他滨诱导胰腺癌细胞凋亡过程中的作用

目的 研究吉西他滨体外对人胰腺癌AsPC-1细胞生长的作用机制.方法 用脂质体转染法将p53正向凋亡调控因子(PUMA)反义核酸(反义PUMA cNA)的真核表达载体pcDNA3.1-PUMAAS和空载体pcDNA3.1-导入AsPC-1细胞,G418筛选阳性细胞,获得稳定转染的阳性克隆.将转染载体的AsPC-1阳性克隆细胞和未转染载体的AsPC-1细胞分别暴露于浓度1、5、10和15 μmol/ml的吉西他滨中作用72 h.RT-PCR和Western blotting检测不同组细胞经吉西他滨作用72 h后的PUMA表达;MTT检测细胞生长抑制,流式细胞仪(FCM)、Hoechst 33258荧光染色法和TUNEL法检测细胞凋亡.结果 吉西他滨促进AsPC-1细胞凋亡,抑制细胞生长,并有明显的剂量依赖性,在细胞凋亡的同时伴有PUMA表达的上调;当细胞转染PUMA反义核酸抑制PUMA表达后,受吉西他滨作用的细胞出现PUMA蛋白表达明显降低,同时伴有细胞凋亡的抑制及细胞的明显增殖.结论 吉西他滨促进体外AsPC-1细胞凋亡,并抑制其生长,其诱导凋亡与上调PUMA有关.     

H9N2亚型禽流感病毒A/Chicken/Gansu/2/99株基因组的分子特征

目的测定H9N2亚型禽流感病毒A/Chicken/Gansu/2/99(CK/GS/2/99)分离株的基因组序列并与参考毒株进行同源性分析,阐明该毒株的遗传变异及分子特征。方法采集发病鸡泄殖腔样品,经鸡胚尿囊腔接种分离病毒,采用RT-PCR方法对CK/GS/2/99株的8个分节段基因进行扩增,分别将其克隆到pGEM-T easy载体后进行序列测定与同源性分析。结果CK/GS/2/99株PB2、PB1、PA、HA、NP、NA、M、NS基因的开放阅读框分别由2280、2274、2151、1683、1497、1401、759、864个碱基组成,分别编码759、757、716、560、498、466、252/97、231/122个氨基酸残基。该毒株HA上HA1和HA2裂解位点序列为PARSSR↓GLF,具有典型的低致病性禽流感病毒的特征。同源性分析显示,CK/GS/2/99株与1998-2002年间大部分中国大陆分离株遗传关系较近,尤其与CK/NX/4/99和CK/HB/31/00株遗传关系密切。结论CK/GS/2/99与大部分流行于中国内陆的H9N2亚型毒株均来源于共同的祖先毒株CK/BJ/1/94,这为了解中国H9N2亚型禽流感病毒的分子流行病学提供了资料。     

Research on the Ignition Height and Reaction Flame Temperature of PTFE/Al/Si/CuO with Different Mass Ratios of PTFE/Si

Recent studies have shown that the energy release capacity of Polytetrafluoroethylene (PTFE)/Al with Si, and CuO, respectively, is higher than that of PTFE/Al. PTFE/Al/Si/CuO reactive materials with four proportions of PTFE/Si were designed by the molding–sintering process to study the influence of different PTFE/Si mass ratios on energy release. A drop hammer was selected for igniting the specimens, and the high-speed camera and spectrometer systems were used to record the energy release process and the flame spectrum, respectively. The ignition height of the reactive material was obtained by fitting the relationship between the flame duration and the drop height. It was found that the ignition height of PTFE/Al/Si/CuO containing 20% PTFE/Si is 48.27 cm, which is the lowest compared to the ignition height of other Si/PTFE ratios of PTFE/Al/Si/CuO; the flame temperature was calculated from the flame spectrum. It was found that flame temperature changes little for the same reactive material at different drop heights. Compared with the flame temperature of PTFE/Al/Si/CuO with four mass ratios, it was found that the flame temperature of PTFE/Al/Si/CuO with 20% PTFE/Si is the highest, which is 2589 K. The results show that PTFE/Al/Si/CuO containing 20% PTFE/Si is easier to be ignited and has a stronger temperature destruction effect.     

黄连温胆汤调控PI3K/Akt/mTOR信号通路干预ApoE-/-小鼠动脉粥样硬化的作用机制

目的观察黄连温胆汤对载脂蛋白E(ApoE)^-/-小鼠动脉粥样硬化的干预效应,从磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物西罗莫司靶蛋白(mTOR)信号通路探讨其机制。方法采用高脂饲料饲养ApoE^-/-小鼠4周构建动脉粥样硬化模型。采用全自动生化分析仪检测小鼠血脂水平;采用苏木精-伊红(HE)染色、油红O染色、Masson染色观察主动脉斑块情况;采用免疫组化法检测小鼠主动脉根部PI3K、Akt、mTOR蛋白表达水平。结果与模型组比较,黄连温胆汤高剂量组总胆固醇(TC)、三酰甘油(TG)、低密度脂蛋白胆固醇(LDL-C)均降低(P<0.05)。HE染色显示:模型组主动脉根部斑块明显,呈易损斑块特征,各给药组斑块有不同程度减轻;油红O和Masson染色显示:与模型组比较,雷帕霉素组、黄连温胆汤高剂量组主动脉根部斑块面积减小,胶原纤维含量升高(P<0.05),易损斑块趋于稳定。蛋白免疫组化结果显示:黄连温胆汤中剂量组、黄连温胆汤高剂量组小鼠主动脉PI3K、Akt、mTOR蛋白表达水平降低(P<0.05)。结论黄连温胆汤具有降脂和抗动脉粥样硬化的作用,其机制可能与抑制PI3K/Akt/mTOR信号通路,诱导巨噬细胞自噬有关。     

Shock-Induced Energy Release Performances of PTFE/Al/Oxide

In recent years, polytetrafluoroethylene (PTFE)/aluminum (Al) energetic materials with high-energy density have attracted extensive attention and have broad application prospects, but the low-energy release efficiency restricts their application. In this paper, oxide, bismuth trioxide (Bi₂O₃) or molybdenum trioxide (MoO₃) are introduced into PTFE/Al to improve the chemical reaction performance of energetic materials. The pressurization characteristics of PTFE/Al/oxide as pressure generators are compared and analyzed. The experiments show that the significantly optimized quasi-static pressure peak, impulse, and energy release efficiency (0.162 MPa, 10.177 s·kPa, and 0.74) are achieved for PTFE/Al by adding 30 wt.% Bi₂O₃. On the other hand, the optimal parameter obtained by adding 10% MoO₃ is 0.147 MPa, 9.184 s·kPa, and 0.68. Further, the mechanism of enhancing the energy release performance of PTFE/Al through oxide is revealed. The mechanism analysis shows that the shock-induced energy release performance of PTFE/Al energetic material is affected by the intensity of the shock wave and the chemical reaction extent of the material under the corresponding intensity. The oxide to PTFE/Al increases the intensity of the shock wave in the material, but the chemical reaction extent of the material decreases under the corresponding intensity.     

Fish oil alleviates liver injury induced by intestinal ischemia/reperfusion via AMPK/SIRT-1/autophagy pathway

AIM To evaluate whether fish oil(FO) can protect liver injury induced by intestinal ischemia/reperfusion(I/R) via the AMPK/SIRT-1/autophagy pathway.METHODS Ischemia in wistar rats was induced by superior mesenteric artery occlusion for 60 min and reperfusion for 240 min. One milliliter per day of FO emulsion or normal saline was administered by intraperitoneal injection for 5 consecutive days to each animal. Animals were sacrificed at the end of reperfusion. Blood andtissue samples were collected for analyses. AMPK, SIRT-1, and Beclin-1 expression was determined in lipopolysaccharide(LPS)-stimulated HepG2 cells with or without FO emulsion treatment.RESULTS Intestinal I/R induced significant liver morphological changes and increased serum alanine aminotransferase and aspartate aminotransferase levels. Expression of p-AMPK/AMPK, SIRT-1, and autophagy markers was decreased whereas tumor necrosis factor-α(TNF-α) and malonaldehyde(MDA) were increased. FO emulsion blocked the changes of the above indicators effectively. Besides, in LPS-stimulated HepG2 cells, small interfering RNA(siRNA) targeting AMPK impaired the FO induced increase of p-AMPK, SIRT-1, and Beclin-1 and decrease of TNF-α and MDA. SIRT-1 siRNA impaired the increase of SIRT-1 and Beclin-1 and the decrease of TNF-α and MDA.CONCLUSION Our study indicates that FO may protect the liver against intestinal I/R induced injury through the AMPK/SIRT-1/autophagy pathway.     

Prevalence and risk factors of osteopenia/osteoporosis in Turkish HIV/AIDS patients

BackgroundRecent studies showed a high frequency of low bone mineral density (BMD) in HIV-infected patients and no reports have been issued in Turkey. Our aim was to evaluate BMD and risk factors for osteopenia/osteoporosis in HIV-infected patients that attended an outpatient clinic in Istanbul, Turkey.MethodIn order to determine the prevalence of BMD, 126 HIV-infected patients had been studied with dual energy X-ray absorptiometry (DEXA). The association between BMD and age, gender, body mass index (BMI), habits, 25(OH)vitamin D, HIV RNA, CD4 lymphocyte nadir, using and duration of highly active antiretroviral treatment (HAART) were investigated by using multivariate analysis.ResultsMedian age was 40.1 years (range, 20–70); 84% were male; 35.7% patients had AIDS, 63.5% were treated with HAART. Osteopenia and osteoporosis were diagnosed in 53.9% and 23.8%, respectively. Mean plasma HIV RNA was 5.2 (SD 1.0) log₁₀ copies/mL and CD4 lymphocyte nadir was 313.8 (SD 226.2)/mm³. Factors associated with bone loss were high viral load (p = 0.034), using (p = 0.033) and duration of HAART (p = 0.008). No correlation had been seen between sex and osteopenia/osteoporosis (p = 0.794). However, males showed higher rates of osteoporosis than females (p = 0.042).ConclusionsOur results show a very high prevalence of bone mass reduction in Turkish HIV-infected patients. This study supports the importance of both HIV and antiretroviral therapy in low BMD.