干扰LRP16基因表达对人卵巢癌耐药SKOV3/DDP细胞耐药性的影响及机制研究

目的:探究干扰人白血病相关蛋白16(LRP16)基因表达对人卵巢癌耐药SKOV3/DDP细胞耐药性的影响及其相关机制。方法:采用Real-time PCR和蛋白免疫印迹(WB)检测LRP16在敏感组(SKOV3细胞)、耐药组(SKOV3/DDP细胞)、LRP16干扰组(SKOV3/DDP细胞-稳定转染LRP16 shRNA质粒)和NC组(即阴性对照组,SKOV3/DDP细胞-稳定转染阴性对照质粒)细胞中的表达情况;MTT试验检测LRP16对SKOV3/DDP细胞耐药性的影响;彗星试验检测LRP16对顺铂(DDP)诱导DNA损伤的影响;流式细胞术(FCM)试验检测细胞凋亡变化;WB试验检测PTEN、p-Akt和NF-κB蛋白表达水平。结果:LRP16干扰组细胞的耐药指数(RI)值(3.19±0.21)显著低于耐药组细胞(6.84±0.37)(P0.05)。DDP(25μmol/L)处理24 h后,LRP16干扰组DNA损伤的细胞百分比、细胞凋亡百分比均显著低于耐药组和NC组(P均0.05),而耐药组和NC组比较差异无统计学意义(P0.05);LRP16干扰组细胞PTEN蛋白相对表达量高于耐药组和NC组(P均0.05),而p-Akt和NF-κB相对表达量低于耐药组和NC组(P均0.05)。结论:干扰LRP16基因表达可逆转卵巢癌耐药SKOV3/DDP细胞的耐药性,PTEN/Akt/NF-κB可能是其中的关键信号通路。     

siRNA抑制S100A4表达对卵巢癌细胞CP70生物学特性的影响

目的：探讨人卵巢癌顺铂耐药细胞株CP70沉默S100A4基因后,CP70细胞对顺铂敏感性、凋亡及细胞迁移的影响。方法：设计并合成S100A4基因特异性的siRNA并转染入卵巢癌细胞CP70,48 h后应用RT-PCR和Western Blot检测在mRNA和蛋白水平siRNA对S100A4的影响,MTT法检测转染siRNA后卵巢癌细胞CP70对顺铂敏感性的变化。用流式细胞术检测顺铂（40μM）对转染S100A4 siRNA后对卵巢癌细胞CP70凋亡的影响,Transwell法观察siRNA抑制S100A4后对卵巢癌CP70迁移能力的影响。结果：与空白对照组、阴性对照组相比,S100A4siRNA转染组CP70细胞的S100A4基因和蛋白表达降低（P〈0.01）。MTT法检测顺铂敏感性发现S100A4 siRNA转染组CP70细胞顺铂敏感性增强。在顺铂刺激下,siRNA转染组细胞凋亡率高于其他各组,差异具有统计学意义（P〈0.05）。Transwell发现CP70细胞迁移能力明显下降（P〈0.05）。结论：S100A4 siRNA能够明显抑制CP70细胞S100A4的表达,从而增强细胞对顺铂的敏感性,促进细胞凋亡,减弱细胞的迁移能力。S100A4有望成为逆转卵巢癌铂类耐药的治疗靶点。     

siRNA抑制S100A4 表达对卵巢癌细胞CP70生物学特性的影响

摘要 目的：探讨人卵巢癌顺铂耐药细胞株CP70沉默S100A4 基因后,CP70细胞对顺铂敏感性、凋亡及细胞迁移的影响。方法：设计并合成S100A4基因特异性的siRNA并转染入卵巢癌细胞CP70,48 h后应用RT-PCR和Western Blot检测在mRNA和蛋白水平siRNA对S100A4的影响,MTT法检测转染 siRNA后卵巢癌细胞CP70对顺铂敏感性的变化。;用流式细胞术检测顺铂（40 μM） 对转染S100A4 siRNA后对卵巢癌细胞CP70凋亡的影响,Transwell法观察siRNA抑制S100A4后对卵巢癌CP70迁移能力的影响。 结果：与空白对照组、阴性对照组相比,S100A4 siRNA转染组CP70细胞的S100A4基因和蛋白表达降低(P<0.01)。MTT法检测顺铂敏感性发现S100A4 siRNA转染组CP70细胞顺铂敏感性增强。在顺铂刺激下,siRNA转染组细胞凋亡率高于其他各组,差异具有统计学意义（P<0.05）。Transwell发现CP70细胞迁移能力明显下降（P<0.05）。结论：S100A4 siRNA能够明显抑制CP70细胞S100A4的表达,从而增强细胞对顺铂的敏感性,促进细胞凋亡,减弱细胞的迁移能力。S100A4有望成为逆转卵巢癌铂类耐药的治疗靶点。     

沉默MDR1基因表达逆转肺癌A549/DDP细胞对顺铂的耐药性

本研究以A549/DDP细胞为实验对象,利用shRNA(short hairpin RNA)沉默MDR1基因,逆转人肺癌A549/DDP细胞对顺铂的耐药性。构建3种靶向MDR1基因重组干扰载体,稳定转染A549/DDP细胞,qRT-PCR检测MDR1 mRNA表达水平,Western blotting检测MDR1蛋白表达水平,MTT法检测细胞对顺铂的敏感性。结果显示成功构建了3种靶向MDR1的重组表达载体p2.1-1、p2.1-2和p2.1-3。3种干扰表达载体均能有效沉默A549/DDP细胞MDR1基因表达,其中p2.1-3对MDR1沉默效果最好,对mRNA和蛋白的沉默效率分别为51.47%和53.24%。转染p2.1-3的细胞对顺铂的IC50由(72.08±7.00)μmol/L降至(31.89±3.39)μmol/L,逆转率达到(67.60±5.70)%。这些结果表明靶向MDR1的重组干扰载体均能够有效抑制MDR1表达,其中p2.1-3干扰效果最佳并且能逆转A549/DDP细胞对顺铂的耐药性。     

卵巢癌组织中dbpA的表达及其对卵巢癌细胞增殖的影响

目的:探讨dbp A蛋白在卵巢癌组织中的表达及其对卵巢癌细胞增殖的影响。方法:通过荧光定量和蛋白质免疫印迹方法检测临床卵巢癌组织和正常癌旁组织中dbp A的表达量;设计合成针对dbp A基因的双链小干扰RNA转染人卵巢癌细胞系SKOV3和A2780细胞,用荧光定量和蛋白质免疫印迹方法检测细胞中dbp A的表达量,MTT法和克隆形成试验检测细胞的增殖能力和克隆形成能力,流式细胞术检测各组细胞周期和细胞凋亡的变化。结果:dbp A在卵巢癌组织、SKOV3和A2780细胞中表达较癌旁正常卵巢组织显著升高。沉默dbp A后,SKOV3和A2780细胞中dbp A蛋白表达量显著降低,SKOV3和A2780细胞增殖能力和克隆形成能力显著下降(P0.05),G0/G1期细胞百分比显著增加(P0.01),S期细胞百分比明显减少(P0.05),细胞凋亡率显著升高(P0.01)。结论:dbp A在卵巢癌组织和卵巢癌细胞SKOV3和A2780中过表达,沉默dbp A基因后可抑制SKOV3和A2780细胞的增殖能力和克隆形成能力。     

LRP16影响宫颈癌Siha细胞对化疗药物的敏感性

目的:探讨抑制LRP16的表达对宫颈癌Siha细胞的化疗药物敏感性的影响。方法:将抑制LRP16表达的小干扰RNA:negativecontrol-si RNA(NC)、si RNA-374(si374)转染入Siha宫颈鳞癌细胞系中,通过顺铂(DDP)和紫杉醇(TAX)的处理后,采用CCK-8检测不同浓度紫杉醇、顺铂作用宫颈癌细胞系Siha48 h后,计算出细胞被抑制一半时顺铂、紫杉醇的药物浓度(IC50);使用Hoechst33342染色观察细胞凋亡,采用流式细胞仪检测顺铂IC50作用Siha细胞48小时后的细胞凋亡情况,紫杉醇IC50作用Siha细胞之后的细胞周期分布情况。结果:CCK-8检测转染的Siha细胞增殖活性受到抑制,Hoechst33342染色观察转染的Siha细胞凋亡明显增加,流式细胞仪检测凋亡显示,si374+顺铂的早期凋亡率22.15±2.24,NC+顺铂12.45±2.72,流式细胞仪检测周期显示G2/M(%),si374+紫杉醇29.94±1.87,NC+紫杉醇17.66±2.32。结论:LRP16基因表达下调之后,抑制Siha细胞的增殖、促进其凋亡,使细胞周期滞留于G2/M期,从而提高Siha细胞的化疗敏感性。     

卵巢癌中HMGB1、BRCA1和p62蛋白的表达与其化疗敏感性的相关性

为了探讨卵巢癌中HMGB1、BRCA1和p62蛋白的表达与化疗敏感性的相关性,培养卵巢癌顺铂化疗耐药细胞ES-2和敏感细胞SKVO3,顺铂100 mg/m2环境培养5 d,分别采用Western blotting法和RT-PCR方法检测两种细胞中的HMGB1、BRCA1和p62蛋白表达情况,采用流式细胞术测算细胞凋亡率。敏感组细胞SKOV3和耐药组细胞ES-2中,BRCA1蛋白表达率分别为(38.08±22.56)%和(45.65±22.42)%,HMGB1蛋白表达率分别为(75.13±16.45)%和(83.08±24.22)%,p62蛋白表达率分别为(52.31±25.13)%和(37.26±21.09)%;顺铂处理后,敏感组SKOV3细胞中BRCA1蛋白表达量显著提高(p0.05),耐药组ES-2细胞中p62蛋白相对表达量高于敏感组SKOV3细胞(p0.01)。卵巢癌化疗后HMGB1下调与BRCA1、p62上调共存;卵巢癌中BRCA1蛋白的不同表达与化疗敏感性相关,其水平变化有可能作为一种新的肿瘤标志物,动态观察卵巢癌病情进展,为临床治疗提供客观指标。     

TGF-β1经DNMT1调控肺癌A549细胞对顺铂的敏感性

转化生长因子-β1(transforming growth factor-beta 1,TGF-β1)与肿瘤的发生、发展以及凋亡关系密切,DNA甲基化关键酶DNMTs(DNA methyltransferases)在肿瘤发生及耐药中发挥重要作用,SPARC(secreted protein acidic and rich in cysteine)常因异常甲基化而表达下调。为探究肺癌对顺铂耐受的分子机制,该研究以肺腺癌A549细胞为研究对象,通过外源TGF-β1作用A549细胞,利用RT-PCR检测TGF-β1作用后DNMTs和SPARC m RNA水平的变化以及A549细胞增殖能力和对顺铂敏感性的影响。结果显示:5 ng/m L、10 ng/m L TGF-β1作用24 h后,A549细胞DNMT1 m RNA表达均显著下调(P0.01、P0.001),SPARC m RNA表达均显著上调(P0.001、P0.001);5 ng/m L、10 ng/m L TGF-β1作用后的A549细胞对顺铂的IC50均显著低于对照组[(12.34±0.36)μmol/L、(10.93±0.69)μmol/L,对照组为(21.54±1.21)μmol/L;P0.01、P0.01];5 ng/m L、10 ng/m L TGF-β1作用后的A549细胞在顺铂环境中,其克隆数显著低于空白对照;15μmol/L顺铂作用24 h时,5 ng/m L、10 ng/m L TGF-β1组细胞凋亡分数均显著高于空白对照(P0.05、P0.01)。结果提示:TGF-β1可下调A549细胞DNMT1的表达,进而上调抑癌基因SPARC并增加其对顺铂的敏感性,成功逆转肺腺癌A549细胞的恶性表型。该研究为进一步阐明肺癌对顺铂的耐受机制提供了新的思路。     

苦参素对人肝癌细胞顺铂敏感性的影响

[目的]观察苦参素注射液对人肝癌细胞株HepG2/DDP顺铂敏感性的影响,并探讨其机制。[方法]取人耐顺铂肝癌细胞株HepG2/DDP培养,每孔分装1.5×10^(6)个细胞。对照组加入2 mg/L顺铂,低、中、高剂量组分别加入2 mg/L顺铂+0.75 mg/mL苦参素注射液、2 mg/L顺铂+1.5 mg/mL苦参素注射液、2 mg/L顺铂+3 mg/mL苦参素注射液,均继续培养72 h。[结果]培养72 h后,对照组细胞贴壁生长状态良好,3剂量组均可导致细胞株形态学改变;3剂量组可提高增殖抑制率和细胞凋亡率(P<0.05),且高剂量组效果最佳[(25.55±3.24)%、(32.93±3.62)%、(39.32±4.15)%;(26.95±1.58)%、(34.36±3.07)%、(42.46±4.47)%](P<0.05);3剂量组均可降低p-JAK2/JAK2、p-STAT3/STAT3水平(P<0.05),且高剂量组效果最佳[(0.143±0.007)、(0.143±0.010)](P<0.05)。[结论]苦参素注射液可增强人肝癌细胞株HepG2/DDP顺铂化疗敏感性,抑制细胞增殖并促进细胞凋亡,且3 mg/mL苦参素注射液的效果更佳,推测与控制JAK2、STAT3蛋白磷酸化水平有关。     

肝素结合表皮生长因子抑制剂CRM197 对裸鼠卵巢癌移植瘤 的抑制作用

目的:研究CRM197对裸鼠卵巢癌移植瘤生长的抑制作用探讨其对逆转卵巢癌组织耐药作用及其在卵巢癌治疗中的可行性.方法:用人卵巢癌亲本细胞A2780、耐紫杉醇细胞A2780/Taxol及耐顺铂细胞A2780/DDP分别建立裸鼠卵巢癌移植瘤模型,观察注射CRM197组与阴性对照组裸鼠肿瘤生长情况,免疫组化法测定各组裸鼠肿瘤组织中HB-EGF及P-gp表达情况.结果:HB-EGF及P-gp在各肿瘤组织中不同程度的表达,在注射CRM197的裸鼠肿瘤组织中表达明显降低(P＜0.05),差异具有统计学意义.结论:CRM197通过抑制肝素结合表皮生长因子的信号传导通路激活抑制了裸鼠卵巢癌移植瘤的生长,使裸鼠卵巢癌移植瘤HB-EGF及P-gp表达明显降低.     

六种鲟鱼消化酶活性的比较研究

测定了两个生长阶段 6种鲟鱼幼鱼胃、肠道和肝脏中蛋白酶、脂肪酶、淀粉酶活性。幼鲟消化酶活性在两个生长阶段变化不明显。 6种鲟鱼不同消化器官蛋白酶活性以肠道为最高 ,肝脏为最低 ,肝脏中的蛋白酶活性明显低于胃、肠道 (P <0 0 1)。不同消化器官脂肪酶活性 ,以肠道为最高 ,且肠道脂肪酶活性显著高于胃、肝脏 (P <0 0 1) ,胃中的脂肪酶活性与肝脏中的脂肪酶活性差异不明显 (P >0 0 5 )。不同消化器官淀粉酶活性 ,以肠道为最高 ,且明显高于胃、肝脏 (P <0 0 1)。幼鲟在第一阶段 ,肝脏中没有淀粉酶活性 ,其活性出现在第二阶段 ,且在此生长阶段 ,肝脏中的淀粉酶活性达到胃中的水平 (P >0 0 5 )。对 6种鲟鱼而言 ,除个别存在较大差异外 ,3种消化酶活性大体上都没有明显差异     

Characterization of monoclonal lipid A antibodies with synthetic lipid A analogues

Abstract The specificity of monoclonal antibodies directed against the Salmonella minnesota R595 lipid A and the structural requirements of lipid A epitopes were studied with chemically synthesized lipid A analogues by enzyme-linked immunosorbent assay and its inhibition test. The results suggest that lipid A has specific and common epitopes, in which the specificities are derived from the chemical and conformational structures of the backbone and/or acyl groups.     

马铃薯蛋白酶A抑制剂的纯化与性质

采用硫酸铵沉淀、Sephadex G-50和CM柱层析,从马铃薯汁中纯化蛋白酶A（protease A,PrA）抑制剂,并对其部分性质进行研究。蛋白酶A抑制剂为单亚基,相对分子质量为16．71kDa。热稳定性良好,抑制最佳pH范围为5—5．5,最佳反应时间为1．5h,对PrA抑制类型为竞争和非竞争性混合抑制作用。     

A study of the possible association between adenosine A2A receptor gene polymorphisms and attention‐deficit hyperactivity disorder traits

The adenosine A_2A receptor (ADORA2A) is linked to the dopamine neurotransmitter system and is also implicated in the regulation of alertness, suggesting a potential association with attention‐deficit hyperactivity disorder (ADHD) traits. Furthermore, animal studies suggest that the ADORA2A may influence ADHD‐like behavior. For that reason, the ADORA2A gene emerges as a promising candidate for studying the etiology of ADHD traits. The aim of this study was to examine the relationship between ADORA2A gene polymorphisms and ADHD traits in a large population‐based sample. This study was based on the Child and Adolescent Twin Study in Sweden (CATSS), and included 1747 twins. Attention‐deficit hyperactivity disorder traits were assessed through parental reports, and samples of DNA were collected. Associations between six single nucleotide polymorphisms (SNPs) and ADHD traits were examined, and results suggested a nominal association between ADHD traits and three of these SNPs: rs3761422, rs5751876 and rs35320474. For one of the SNPs, rs35320474, results remained significant after correction for multiple comparisons. These results indicate the possibility that the ADORA2A gene may be involved in ADHD traits. However, more studies replicating the present results are warranted before this association can be confirmed .     

Activin A和Lefty A对人胚胎干细胞生长的影响

目的探讨TGF-β/Activin/Nodal信号通路的相关因子Activin A和Lefty A在一定浓度范围内,对人胚胎干细胞(hESC)自我更新的影响。方法在hES3细胞株的无滋养层无血清培养体系中加入1-100ng/ml的Activin A和Lefty A。7天后,通过碱性磷酸酶染色法对hES3细胞的自我更新状态进行评估。结果 Activin A在浓度为1,3,10,30和100ng/ml时,与阴性对照(SR培养基)组相比,未分化克隆的比率从7.7%分别提高到了18.5%,46.8%,61.4%,64.4%和79.1%,差异有统计学意义(P<0.01)。Lefty A组在浓度为1,3,10,30和100ng/ml时,与阴性对照(MCM培养基)组相比,未分化克隆的比率从80.5%分别降低到了72.4%,74.6%,72.2%,69.5%和65.3%,在浓度为100ng/ml时,差异有统计学意义(P<0.05)。结论较低浓度的Activin A即能有效维持hESC的自我更新,而较高浓度的Lefty A能诱导hESC分化。该结果进一步揭示了TGF-β/Activin/Nodal信号通路及其相关因子对hESC自我更新和分化的作用特点,有待对其机制进行深入研究。     

Quantitative detection of bisphenol A and bisphenol A diglycidyl ether metabolites in human plasma by liquid chromatography–electrospray mass spectrometry

Due to the ubiquity of epoxy resin compounds and their potential role in increasing the risk for reproductive dysfunction and cancer, the need for an assessment of human exposure is urgent. Therefore, we developed a method for measuring bisphenol A (BPA) and bisphenol A diglycidyl ether (BADGE) metabolites in human blood samples using high-performance liquid chromatography–electrospray ionization mass spectrometry (LC–MS). Human blood samples were processed using enzymatic deconjugation of the glucuronides followed by a novel sample preparation procedure using a solid-phase-cartridge column. This selective analytical method permits rapid detection of the metabolites, free BPA and a hydrolysis product of BADGE (BADGE-4OH) with detection limits in the low nanogram per milliliter range (0.1 ng ml⁻¹ of BPA and 0.5 ng ml⁻¹ of BADGE-4OH). The sample extraction was achieved by Oasis HLB column on gradient elution. The recoveries of BPA and BADGE-4OH added to human plasma samples were above 70.0% with a standard deviation of less than 5.0%. This selective, sensitive and accurate method will assist in elucidating potential associations between human exposure to epoxy-based compounds and adverse health effects.     

The detection of the sialoglycoprotease gene and assay for sialoglycoprotease activity among isolates of Pasteurella haemolytica A1 strains, serotypes A13, A14, T15 and A16

Abstract Polymerase chain reaction (PCR) using specific primers to the sialoglycoprotease gene ( gcp ) of Pasteurella haemolytica biotype A, serotype 1 amplified a 1-kb fragment from each of P. haemolytica serotypes A7, A13, A14 and A16, but not T15; which was confirmed by Southern blot hybridization analysis. Using a sialoglycoprotease (Gcp) activity assay, Gcp activity was found in serotypes A13, A14 and A16. Inclusion of these three serotypes confirms that all recognized A biotypes are positive for both gcp gene and activity, with the exception of serotype A11 (which has a different genetic organization and exhibits no Gcp activity). Furthermore, all recognized T biotypes are negative for both the gene and Gcp activity.     

A1/A2 polymorphism of GpIIIa gene and a risk of aneurysmal subarachnoid haemorrhage

Platelet glycoproteins are involved in pathophysiology of cerebrovascular diseases. The aim of this study was to investigate the association between the GpIIIa gene A1/A2 polymorphism and a risk of aneurysmal subarachnoid haemorrhage (SAH) in a Polish population. In a case-control study we genotyped 288 Caucasian patients with aneurysmal SAH and 457 age-, gender- and race-matched controls. The GpIIIa A1/A2 polymorphism was genotyped with RFLP technique. No difference was found in the distribution of the polymorphism between the cases and controls (cases: A1A1—201 (69.8%), A1A2—83 (28.8%) and A2A2—4 (1.4%) vs. controls: A1A1—323 (70.7%); A1A2—128 (28.0%); A2A2—6 (1.3%), P > 0.05. In a multivariate analysis female gender (OR = 1.950; 95%CI: 1.308-2.907), hypertension (OR = 4.774; 95%CI: 3.048-7.478) and smoking (OR = 2.034; 95%CI: 1.366-3.030), but not GpIIIa A1/A2 polymorphism, were independent risk factors for aneurysmal SAH. The GpIIIa A1/A2 polymorphism is not a risk factor of aneurysmal SAH in a Polish population.     

Structural properties of trimers and tetramers of ribonuclease A

Ribonuclease A aggregates (dimers, trimers, tetramers, pentamers) can be obtained by lyophilization from 40% acetic acid solutions. Each aggregate forms two conformational isomers distinguishable by different basic net charge. The crystal structure of the two dimers has recently been determined; the structure of the higher oligomers is unknown. The results of the study of the two trimeric and tetrameric conformers can be summarized as follows: (1) RNase A trimers and tetramers form by a 3D domain-swapping mechanism. N-terminal and C-terminal types of domain swapping could coexist; (2) the secondary structures of the trimeric and tetrameric conformers do not show significant differences if compared with the secondary structure of monomeric RNase A or its two dimers; (3) a different exposure of tyrosine residues indicates that in the aggregates they have different microenvironments; (4) the two trimeric and tetrameric conformers show different susceptibility to digestion by subtilisin; (5) dimers, trimers, and tetramers of RNase A show unwinding activity on double-helical poly(dA-dT) x poly(dA-dT), that increases as a function of the size of the oligomers; (6) the less basic conformers are more stable than the more basic ones, and a low concentration in solution of trimers and tetramers favors their stability, which is definitely increased by the interaction of the aggregates with poly(dA-dT) x poly(dA-dT); (7) the products of thermal dissociation of the two trimers indicate that their structures could be remarkably different. The dissociation products of the two tetramers allow the proposal of two models for their putative structures.     

A surface plasmon resonance-based assay for small molecule inhibitors of human cyclophilin A

A simple protocol for generating a highly stable and active surface plasmon resonance (SPR) sensor surface of recombinant human hexahistidine cyclophilin A (His-CypA) is described. The sensor surface was sensitive and stable enough to allow, for the first time, the screening and ranking of several novel small-molecule (Mr approximately 250-500 Da) ligands in a competition binding assay with cyclosporin A (CsA). It also allowed us to accurately determine the kinetic rate constants for the interaction between His-CypA and CsA. His-CypA was first captured on a Ni2+-nitrilotriacetic acid (NTA) sensor chip and was then briefly covalently stabilized, coupling via primary amines. The significant baseline drift observed due to dissociation of weakly bound His-CypA from the Ni2+-NTA moiety was eliminated, resulting in a surface that was stable for at least 36 h. In addition, immobilized protein activity levels were high, typically between 85 and 95%, assayed by the interaction between His-CypA and CsA. The mean equilibrium dissociation constant for CsA (K(dCsA)) binding to the immobilized His-CypA was 23+/-6 nM, with on and off rates of 0.53+/-0.1 microM(-1) s(-1) and 1.2+/-0.1 (x 10(-2)) s(-1), respectively. These values agree well with the values for the corresponding binding constants determined from steady-state and kinetic fluorescence titrations in solution.