三氧化二砷诱导人类胆管癌QBC939细胞凋亡的初步研究

目的研究三氧化二砷(As2O3)诱导人类胆管癌QBC939细胞凋亡。方法应用MTT法检测As2O3对胆管癌细胞的抑制作用；采用光镜、荧光显微镜观察细胞形态变化；流式细胞术检测Rhodamine 123染色并分析DNA含量及细胞周期。结果1～16μmol／L As2O3均能抑制人胆管癌细胞QBC939的生长，且抑制率具有浓度一时间依赖性。4μmol／L As2O3作用细胞后出现典型的凋亡形态特征，可检测到亚二倍体凋亡峰，Rhodamine 123荧光强度降低。结论As2O3能诱导QBC939细胞发生凋亡，其机制可能与线粒体膜电位去极化有关。     

Gastrin/CCK与胆管癌细胞bcl-2/bax蛋白表达的关系

目的 初步探讨Gastrin/CCK对胆管癌细胞bcl-2/bax蛋白表达的调节作用。方法 原位杂交和免疫组化分别检测Gastrin/CCK受体mRNA和bcl-2/bax蛋白在40例胆管癌组织标本中的表达、分析两者间关系,流式细胞术研究Gastrin/CCK及其受体拮抗剂对QBC939人胆管癌细胞系bcl-2/bax蛋白表达的影响。结果Gastrin/CCK受体 mRNA和bcl-2/bax蛋白在胆管癌组织和细胞系中均有表达;Gastrin/CCK-B受体阳性胆管癌标本bcl-2表达率高于其阴性者（P<0.01）;Gastrin-17或CCK-8S（10~（-8）mol·L~（-1）×48h）能刺激QBC939细胞bcl-2表达、同时加入L365,260或 L364,718（10~（-8）mol·L~（-1））可逆转,CCK-SS还能抑制bax表达、L364,718可逆转（P<0.01或P<0.05）。结论Gastrin/CCK可通过其受体后途径上调胆管癌细胞bcl-2表达,“CCK→CCK-A受体”信号还可能具有抑制bax表达的作用。     

WWOX表达对胆管癌QBC939细胞凋亡的影响及其机制

目的 探讨含有WW结构域的氧化还原酶基因(WWOX)表达对胆管癌QBC939细胞凋亡的影响及其作用机制.方法 用脂质体转染法将WWOX重组真核表达质粒转染QBC939细胞;采用荧光定量逆转录－聚合酶链反应(RT-PCR)和Westem blot法鉴定WWOX在QBC939细胞中的表达;流式细胞仪(FCM)法检测转染前后各细胞凋亡率的变化;JC-l染色法检测细胞线粒体膜电位(△Ψm)变化;荧光定量RT-PCR和Western blot法检测胆管癌细胞bcl-2表达的变化;将未转染和转染空质粒的细胞作为对照组接种到裸鼠皮下以检测荷瘤,TUNEL方法原位检测移植瘤的凋亡.结果 建立了稳定表达WWOX基因的QBC939/WWOX细胞株,mRNA及蛋白表达明显增加.FCM显示QBC939/WWOX组的细胞凋亡率明显增高[(1.24±0.35)％比(1.73±0.48)％比(21.40±2.35)％,P＜O.01],JC-l显示转染组的线粒体膜电位下降[(4.27±0.64)％比(4.96±0.52)％比(28.60±3.94)％,P＜O.01],bcl-2 mRNA及蛋白的表达均显著降低(P＜0.05).转染组的皮下肿瘤较对照组生长速度明显减慢(P＜0.05),TUNEL实验证实转染组的皮下肿瘤凋亡指数为(13.6±1.5)％,较对照组明显增高,差异有统计学意义(P＜0.O1).结论 WWOX基因能促进胆管癌细胞的凋亡,其机制可能与下调bcl-2的表达,激活线粒体凋亡通路有关.     

bcl-2 mRNA、baX mRNA表达在三氧化二砷诱导肝癌细胞凋亡中的意义

目的 分析三氧化二砷(As2O3)诱导肝癌细胞凋亡中凋亡相关基因bcl—2 mRNA、bax mRNA表达的意义。方法 采用电镜、流式细胞仪、电泳及半定量RT—PCR方法检测不同剂量三氧化二砷诱导人肝癌细胞株bel—7402后凋亡的出现及bcl-2mRNA、bax mRNA的表达。结果 0．5にmol/L As2O3处理肝癌细胞未见凋亡，8μmol/L As2O3诱导肝癌细胞出现明显凋亡。对照组和药物组未检测到bcl-2 mRNA的表达，随浓度增加，bax mRNA的表达上调。结论 As2O3通过诱导凋亡抑制肝癌细胞株bel-7402的增殖，凋亡诱导基因bax mRNA的表达上调与此过程有关。bcl—2mR—NA表达与此过程无关。     

多肽K237对PC-3M细胞增殖及bax、bcl-2 mRNA表达的影响

目的:研究多肽K237对体外培养的雄激素非依赖性前列腺癌PC-3M细胞的抑制作用,及其可能的作用机制。方法:将培养的PC-3M细胞分为4组:实验组(分别以50、100、200μmol/L的多肽K237处理48h)和对照组(K237浓度为0μmol/L),采用MTT法观察多肽K237对前列腺癌PC-3M细胞增殖的影响,用RT-PCR法检测bax、bcl-2mRNA表达的变化。结果:不同浓度的多肽K237处理48h后,PC-3M细胞形状变圆,体积变小,胞质透亮度下降,部分细胞脱落悬浮于培养液中。在50、100、200μmol/L的多肽K237作用48h后,MTT法检测的细胞生长抑制率分别为(12.6±0.95)%、(17.8±0.99)%、(27.2±1.12)%。RT-PCR结果显示:50、100、200μmol/L实验组和对照组的bax/β-actin值分别为0.919±0.071、0.971±0.083、0.992±0.102,(0.889±0.06),bcl-2/β-actin值分别为0.896±0.085、0.791±0.084、0.764±0.702,0.922±0.097,3组中上述两项指标均较对照组有明显变化(P均<0.01),其中,baxmRNA表达水平上调,而bcl-2mRNA表达水平下调,上述作用呈现剂量效应关系。结论:多肽K237可能通过影响bax、bcl-2mRNA的表达来诱导PC-3M细胞凋亡,从而抑制前列腺癌细胞的增殖。     

Gastrin、CCK对胆管癌细胞bcl-2、baX基因表达的调控

目的探讨Gastrin、CCK对胆管癌细胞bcl-2和bax基因表达的调控作用.方法用免疫组织化学、原位杂交、流式细胞术、逆转录-聚合酶链反应(RT-PCR)等技术,研究了Gastrin17肽(G-17)和CCK-8S肽(CCK-8 Sulfated)及CCK-A受体拮抗剂L364,718(L18)和Gastrin/CCK-B受体拮抗剂L365,260(L60)对QBC939胆管癌细胞bcl-2和bax mRNA、蛋白含量的影响.结果在正常情况下胆管癌细胞bcl-2和bax mRNA表达均为阳性,bax mRNA强于bcl-2 mRNA;G-17(1×10-10 mol/L、48 h)、CCK-8S(1×10-10mol/L)明显增强bcl-2 mRNA表达,而对bax mRNA表达无影响;同时加入L18和/或L60(1×10-10 mol/L)可明显抑制G-17和CCK-8S对bcl-2mRNA的促表达作用.经G-17或CCK-8S(1×10-8 mol/L)作用48 h,胆管癌细胞bcl-2蛋白表达明显增加,而bax蛋白表达无显著变化;L60或L18(1×10-8 mol/L)能明显抑制G-17和CCK-8S对bcl-2蛋白表达的促进作用.结论 Gastrin和CCK对bcl-2基因表达有上调作用,提示Gastrin和CCK对胆管癌细胞凋亡过程有抑制作用.     

金属卟啉化合物的光动力学作用对人膀胱癌细胞增殖与凋亡的影响及其机制

目的探讨金属卟啉的光动力学作用对人膀胱癌细胞增殖和凋亡基因bcl-2、bax表达的影响及其诱导细胞凋亡的机制。方法合成水溶性锰卟啉化合物后,分别采用噻唑蓝(MTT)比色法、膜联蛋白/碘化丙锭流式细胞术、免疫组织化学和逆转录聚合酶链反应(RT-PCR)检测锰卟啉的光动力学作用对人膀胱癌BI U-87细胞的生长活性、凋亡率和bcl-2、bax蛋白及相应mRNA表达水平变化的影响。结果1μmol/L锰卟啉光动力作用BI U-87细胞30min后,MTT法和流式细胞术结果显示细胞生长抑制率和凋亡率分别为(62.39±9.17)%、(19.15±5.49)%,免疫组化和RT-PCR结果表明bax蛋白及相应mRNA表达水平分别为(46.4±5.67)%、(0.63±0.13),均分别明显高于对照组(2.26±0.04)%、(3.95±0.84)%、(32.7±4.52)%、(0.29±0.02)(P<0.05);而bcl-2蛋白及其mRNA表达水平分别为(31.5±2.59)%、(0.44±0.02),均明显低于对照组(54.8±6.21)%、(0.73±0.07)。结论锰卟啉的光动力作用可以通过抑制bcl-2的表达、促进bax的表达来诱导BI U-87细胞发生凋亡。     

胰腺癌细胞凋亡与bcl-2,bax基因的表达

目的探讨胰腺癌组织中的bcl-2和bax的表达及bcl-2/bax比值与胰腺癌细胞凋亡的关系.方法对30例胰腺癌标本应用免疫组化SP方法测定胰腺癌切片中的凋亡抑制基因bcl-2和凋亡促进基因bax的表达;同时应用TUNEL法测定胰腺癌细胞原位凋亡情况.结果高分化腺癌的凋亡指数(AI)为(5.70±1.21)%;中低分化腺癌为(6.80±2.01)%.AI与bcl-2/bax比值呈负相关关系(r=- 0.5583,P<0.05 ).结论 bcl-2和bax在胰腺癌细胞凋亡调节过程中起着重要作用.     

外源性IL-6对胆管癌细胞的抗凋亡作用及其对Bcl-2 mRNA的调控

目的观察外源性IL-6对胆管癌细胞系QBC939凋亡及对Bcl-2 mRNA表达的影响。方法用MTT法检测外源性IL-6对胆管癌细胞QBC939的促增殖作用;通过annexin V/FITC和PI染色、流式细胞分析检测外源性IL-6对QBC939细胞凋亡的影响,RT-PCR测定Bcl-2 mRNA的表达。结果外源性IL-6能刺激QBC939细胞增殖,且QBC939细胞增殖与IL-6浓度呈正相关(r=1);外源性IL-6处理后,凋亡率显著低于对照组(P0.05),外源性IL-6处理细胞后Bcl-2 mRNA显著高于对照组(P0.05)。结论外源性IL-6在一定剂量范围内促进QBC939细胞生长,并抑制QBC939细胞凋亡;而此作用可能与上调Bcl-2 mRNA表达有关。     

反义DNMT3b基因真核表达载体转染对人胆管癌细胞DNMT3b基因表达的影响

目的观察反义DNA甲基转移酶3b(DNMT3b)基因真核表达载体转染对人胆管癌QBC939细胞中DNMT3b基因表达的影响。方法构建反义DNMT3b基因真核表达载体,通过脂质体转染到人胆管癌细胞株QBC939中,G418筛选得到稳定转染细胞株,PCR鉴定外源性neoR基因在转染细胞中的表达以确定转染是否成功。半定量RTPCR观察转染前后DNMT3b基因mRNA水平的变化,流式细胞术检测转染前后DNMT3b蛋白的变化。结果反义DNMT3b基因真核表达载体转染能使人胆管癌QBC939细胞中DNMT3b基因的mRNA水平从0.956±0.053降低至0.209±0.023,差异有统计学意义(P<0.01);流式细胞术检测结果显示,DNMT3b蛋白水平从(75.38±3.22)%降低到(29.87±3.46)%,差异有统计学意义(P<0.01)。结论反义DNMT3b基因转染能降低人胆管癌QBC939细胞中DNMT3b基因的表达水平,为研究DNMT3b基因功能及其在胆管癌细胞中的作用提供了方法和实验依据。     

P2Y2 receptors and GRK2 are involved in oscillatory fluid flow induced ERK1/2 responses in chondrocytes

Mechanical loading is an important factor regulating cartilage metabolism maintained by chondrocytes. However, some of its underlying mechanisms remain poorly understood. In this study, we employed a chondrogenic cell line ATDC5 to investigate roles of P2Y₂ and GRK2 in chondrocyte mechanotransduction. We first confirmed the expression of chondrocyte markers in differentiated ATDC5 cells. We then exposed both differentiated and undifferentiated ATDC5 cells to oscillatory fluid flow, and found that differentiated ATDC5 cells responded to oscillatory fluid flow by increasing COX‐2 and aggrecan expressions. More importantly, fluid flow induced ERK1/2 response in differentiated cells was increased more than 10 times compared to those in undifferentiated cells. Furthermore, we found that P2Y₂ mRNA and protein levels in differentiated ATDC5 cells were significantly higher than those in undifferentiated cells. In contrast, GRK2 protein levels in differentiated cells were significantly lower than those in undifferentiated cells. Finally, overexpressions of P2Y₂ and GRK2 in differentiated ATDC5 cells result in a 34% increase and a 21% decrease of the ERK1/2 phosphorylation, respectively, in response to oscillatory fluid flow, suggesting important roles of P2Y₂ and GRK2 in chondrocyte mechanotransduction. © 2010 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:828–833     

环氧化酶2及其抑制剂与前列腺癌

环氧化酶2在前列腺癌细胞中呈高表达,通过促炎症反应、抑制细胞凋亡、刺激血管生成和氧化损伤等作用参与前列腺癌的发生和发展,选择性环氧化酶2抑制剂可以通过多种途径抑制前列腺癌的生长。应用选择性环氧化酶2抑制剂治疗前列腺癌是当前研究的热点,具有重要价值。     

Multidomain synthetic peptide B2A2 synergistically enhances BMP-2 in vitro.

A multidomain, synthetic peptide designated B2A2 synergizes the activity of BMP-2. B2A2 interacts with BMP receptor isoforms, potentiating the action of BMP-2 in activating alkaline phosphatase and triggering Smad and MAPK signaling. B2A2's design permits its delivery as a local surface coating as well as a soluble co-factor, thus broadening potential bioengineering applications. INTRODUCTION: BMP-2 induces osteogenic differentiation and accelerates bone repair. Although BMP-2 inhibitors have been discovered, no BMP-2 mimetics or enhancers that function in the physiological range have yet been found. Here we report that a synthetic peptide designated B2A2, consisting of (1) a BMP receptor-targeting sequence, (2) a hydrophobic spacer, and (3) a heparin-binding sequence, is a positive modulator of recombinant BMP-2. MATERIALS AND METHODS: Cultures of mesenchymal cell lines C2C12 and C3H10T1/2 were given B2A2, recombinant BMP-2, or both. Alkaline phosphatase (ALP) activity was assayed by conversion of paranitrophenol phosphate (PNPP). Signaling through Smad and MAP kinase pathways was monitored by Western blot. Receptor binding was assessed by incubating immobilized B2A2 with soluble recombinant receptor-Fc chimeras and detecting bound receptor by anti-Fc antibody ELISA. Surface coating of medical device materials was done by first dip-coating with silyl-heparin, followed by B2A2. RESULTS AND CONCLUSIONS: Treatment of cells with B2A2 alone marginally increased ALP activity. However, B2A2 plus BMP-2 resulted in 5- to 40-fold augmentation of ALP compared with BMP-2 alone in C3H10T1/2 or C2C12 cells, respectively. This synergistic enhancement was observed over a broad concentration range (4-1000 ng/ml BMP-2). B2A2 interacted directly with BMP receptor isoforms (preferentially to BMPR-Ib and ActivinR-II). In cells, B2A2 + BMP-2 led to a repression of MAP kinase and an increase of Smad activation, consistent with known activation pathways of BMP-2. B2A2 was ineffective when paired with other cytokine/growth factors (basic fibroblast growth factor [FGF-2], TGF-beta1, vascular endothelial growth factor [VEGF]). Simultaneous co-administration was not strictly required. Pulse-chase experiments revealed that temporal separations up to 1 h were still effective. B2A2 was also effective when delivered in a polystyrene- or stainless steel-coated surface through a heparin platform (silyl-heparin) while BMP-2 was added exogenously in solution. These results suggest that B2A2 might promote aggregation of receptor subunits, enabling BMP-2 to activate signaling pathways at effectively lower concentrations. Synthetic multidomain constructs like B2A2 may be useful to accelerate bone repair/deposition through augmentation of endogenous levels of BMP-2 or through local BMP-2 contained in artificial or engineered matrices.     

Nuclear Factor Erythroid-2-related Factor 2 Gene Polymorphisms in Vitiligo

BackgroundOxidative stress is now one of the accepted theories of vitiligo development. Nuclear factor erythroid-2-related factor 2 (Nrf2) regulates the expression of antioxidant proteins.ObjectiveThis work aimed to evaluate the association of Nrf2 gene polymorphisms with the susceptibility to vitiligo among a sample of Egyptian patients with vitiligo.MethodsThis case-control study included 100 patients with vitiligo and 50 healthy matched volunteers serving as a control group. Genotyping was carried out by real-time polymerase chain reaction.ResultsThe frequencies of TT, CT, and combined (TT+CT) genotypes and the T allele of Nrf2 (rs35652124) were significantly increased in the studied patients with vitiligo relative to the healthy controls (p<0.001, p=0.012, p<0.001 and p<0.001, respectively). There was a nonsignificant difference between patients and controls regarding Nrf2 (rs6721961) genotypes. However, the T allele of Nrf2 (rs6721961) was significantly predominant in the studied patients compared to in the controls (p=0.029). Among the studied criteria, the T allele of Nrf2 (rs6721961) was predominant in patients with a marginal type of repigmentation (p=0.022), while the G allele of the same single-nucleotide polymorphism was associated with a higher body mass index value (p=0.034). One hundred percent of patients with vitiligo with the Nrf2 (rs6721961) GT genotype had a progressive disease course (p=0.015).ConclusionNrf2 (−617 T/G) and (−653 T/C) polymorphism might play a role in patient susceptibility to vitiligo and modify the clinical presentation of the disease.     

CO2-Laser in neurosurgery

Summary New technical tools in surgery are only beneficial if they offer real improvements compared to the traditional techniques or if they open absolute new indications in surgery. Therefore we have tried to find absolute indications for the use of the indifferent laser system.Due to the specific wavelength the CO₂-Laser is entirely absorbed at the surface. This specific performance allows one to use the CO₂-Laser as the most precise cutting and as a completely new vaporizing instrument. It has become an irreplaceable tool in surgery of central lesions and brain stem tumours and also for spinal cord tumours. In addition the CO₂-Laser is adaptable to the operating microscope.A gain of the same importance in the meantime is the Nd-YAG Laser, which works specially for coagulation, volume coagulation and endoscopic use.It is certain that laser surgery will evolve for beyond our imagination.     

Role of spinal cyclooxygenase-2 and prostaglandin E2 in fentanyl-induced hyperalgesia in rats

Background

Accumulated evidence suggests that spinal cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) may be implicated in the development of opioid-induced hyperalgesia.