小分子双链 RNAs 联合应用抑制膀胱癌细胞增殖的实验研究

目的在膀胱癌细胞系 T24和 EJ 细胞中转染两种不同的人工合成小分子双链 RNA (dsRNA),观察联合应用与分别单独应用两种 dsRNA 对膀胱癌细胞生长的影响.方法根据对膀胱癌细胞的处理不同分为：①阴性对照组(dsControl)、dsP21-322组、dsP21-555组和 dsP21-322＋dsP21-555组;②dsControl 组、dsP21-322组、dsP53-285组和 dsP21-322＋ dsP53-285组.采用实时荧光定量聚合酶链反应(qPCR)检测各组细胞 p21 mRNA 及细胞周期依赖性激酶 CDK4、CDK6 mRNA 的表达;蛋白印迹法(Western blot)检测 P21蛋白和 CDK4、CDK6蛋白的表达;细胞增殖实验检测转染后细胞增殖能力;集落形成实验检测单个细胞克隆增殖能力.结果 qPCR 结果显示,与 dsControl 组相比,dsP21-322组、dsP2l-555组和 dsP53-285组均分别上调 T24和 EJ 细胞中p21 mRNA 的表达(P ＜0．01);而与单独转染 dsP21-322、dsP2l-555相比,同时转染 dsP21-322＋dsP21-555,差异无统计学意义(P ＞0．05).与 dsControl 组相比,dsP53-285能够上调 p53基因的表达(P ＜0．05);与单独转染 dsP53-285相比,p53 mRNA 表达在同时转染 dsP21-322和 dsP53-285组中差异无统计学意义(P ＞0．05).与单独转染 dsP21-322、dsP53-285相比,dsP21-322＋ dsP53-285能够显著增加 p21 mRNA 的表达(P ＜0．05).与 dsControl 相比,转染 dsP21-322、dsP53-285分别使 T24和 EJ 细胞中 CDK4、CDK6 mRNA 的表达下调(P ＜0．05);与单独转染 dsP21-322、dsP53-285相比,CDK4/6 mRNA 的表达在 dsP21-322＋dsP53-285组中明显被抑制(P ＜0．05).Western blot检测结果验证了组间 p21和 CDK4/6基因表达的差异.细胞增殖实验结果显示,同时转染 dsP21-322和 dsP53-285比单独转染抑制膀胱癌细胞增殖能力更明显.细胞集落形成实验中,dsP21-322＋dsP53-285组中形成的集落数量显著少于单独转染组.结论同时转染 dsP21-322和 dsP53-285能够显著抑制膀胱癌细胞的增殖.     

p21saRNA真核表达质粒的构建及其在泌尿系肿瘤细胞中的功能

目的 构建可表达具有RNA激活功能的小分子双链RNA(dsRNA)真核表达质粒,并探讨其调控前列腺癌细胞株PC-3和膀胱癌细胞株T-24中抑癌基因p21 WAF1/CIP1表达的功能.方法 构建真核表达质粒dsRNAP21-pGenesil-1,并分别转染至PC-3和T-24细胞株,通过实时定量聚合酶链反应( Real-time qPCR)和Western blot检测p21mRNA转录水平和蛋白表达的变化.结果 测序结果证实目的表达质粒dsRNAP21-pGenesil-1构建成功,将其转染入PC-3细胞和T-24细胞中,Real-time qPCR检测p21基因分别被上调4.35倍和2.83倍,Western blot进一步证明在两种细胞株中p21蛋白表达水平的增加与p21mRNA水平的上调一致,且与对照组比较,差异均有统计学意义(P＜0.05).结论 构建的dsRNAP21-pGenesil-1质粒具备在泌尿系肿瘤中上调抑癌基因p21表达的能力.     

RNA激活技术上调p21WAF1/CIP1基因表达对胆囊癌细胞增殖、侵袭与迁移能力的影响

目的利用RNA激活技术上调人胆囊癌细胞(GBC-SD)中p21基因的表达,观察其对GBS-SD细胞体外增殖、侵袭和迁移能力的影响。方法将与p21基因启动子DNA序列互补的双链RNA分子(dsRNA)转染入人胆囊癌细胞中,采用RT-PCR法和Western blot分别检测p21基因mRNA及蛋白的表达情况;MTT法检测细胞增殖活性;Transwell小室法检测RNAa后细胞侵袭、迁移能力的变化。结果 dsRNA转染GBC-SD细胞72h后能显著上调p21基因mRNA和蛋白的表达,与空白组和对照组比较,转染dsp21后,GBC-SD细胞的增殖活性明显受到抑制,细胞侵袭及迁移能力明显下降。结论 RNAa技术能有效上调p21基因的表达并抑制细胞的增殖活性,降低其侵袭及迁移能力,为胆囊癌疾病的基因治疗提供依据。     

人工合成小分子RNA对前列腺癌细胞周期及增殖的影响

目的 研究人工合成的dsP21-555对前列腺癌细胞株PC-3和LNCaP细胞周期和增殖的影响。方法 合成dsP21-555（实验组）和dsControl（阴性对照组）,分别转染至PC-3和LNCaP。使用Real-time PCR及Western blotting分别检测分析前列腺癌细胞转染后的p21mRNA及p21蛋白的表达情况。流式细胞术检测细胞周期分布,使用MTT实验及集落形成实验检测细胞的活力及增殖能力。结果 转染dsP21-555后PC-3和LNCaP细胞中的p21 mRNA水平分别上调至2.90倍(P<0.01)和2.05倍(P<0.01)。Western blotting实验结果符合这一趋势。流式细胞术检测显示,转染dsP21-555后,在S期和G2/M期的细胞比例下降,在G0/G1期的细胞比例则增加。MTT实验显示,与dsControl组相比,转染dsP21-555后,PC-3和LNCaP细胞的活力明显降低。集落形成实验显示,dsP21-555组的集落的数量较少,细胞增殖能力降低。结论 人工合成的dsP21-555能明显激活前列腺癌细胞中p21基因的表达,并显著抑制前列腺癌细胞周期的进展和增殖。     

Effects of p21 gene expression up-regulation by small activating RNA on poliferation, invasion and migration of gallbladder cancer cells

目的 利用RNA激活技术上调人胆囊癌细胞(GBC-SD)中p21基因的表达,观察其对GBS-SD细胞体外增殖、侵袭和迁移能力的影响.方法 将与p21基因启动子DNA序列互补的双链RNA分子(dsRNA)转染人人胆囊癌细胞中,采用RT-PCR法和Western blot分别检测p21基因mRNA及蛋白的表达情况;MTT法检测细胞增殖活性;Transwell小室法检测RNAa后细胞侵袭、迁移能力的变化.结果 dsRNA转染GBC-SD细胞72h后能显著上调p21基因mRNA和蛋白的表达,与空白组和对照组比较,转染dsp21后,GBC-SD细胞的增殖活性明显受到抑制,细胞侵袭及迁移能力明显下降.结论 RNAa技术能有效上调p21基因的表达并抑制细胞的增殖活性,降低其侵袭及迁移能力,为胆囊癌疾病的基因治疗提供依据.     

p21基因的saRNA表达质粒的构建及功能研究

目的构建可表达具有RNA激活功能的小激活RNA(saRNA)质粒表达载体,并对其激活前列腺癌细胞PC-3中p21WAF1/CIP1(p21)基因表达的功能进行相关研究。方法人工合成与p21基因启动子特定序列相应的DNA片段及同样长度的随机DNA序列,利用DNA重组技术将这些片段构建到真核小分子双链RNA(dsRNA)表达质粒pGenesil-1中,构建成能表达dsRNA片段的表达质粒载体(dsRNAp21-pGenesil-1)和随机对照dsRNA片段的表达质粒载体(dsRNACon-pGenesil-1)。采用脂质体介导方法,分别将dsRNAp21-pGenesil-1(实验组)、dsRNACon-pGenesil-1(随机对照组)及空白质粒pGenesil-1(空白对照组)转染到体外培养的PC-3细胞,通过荧光染色检测转染效率,采用RT-PCR和免疫组化技术检测各组细胞p21蛋白表达的差异。结果成功构建目的表达载体dsRNAp21-pGenesil-1及随机对照载体dsRNACon-pGenesil-1,将各组质粒转染到PC-3细胞后,实验组p21基因表达明显增强,而随机对照组和空白对照组无明显变化。结论本研究构建的dsRNA表达质粒载体能成功表达saRNA分子,并激活p21基因及蛋白的表达,为研究的激活效应提供有效工具。     

小激活RNA介导的大鼠肝细胞株BRL中诱导型一氧化氮合酶上调表达的研究

目的 应用RNA激活技术激活大鼠肝细胞株BRL中诱导型一氧化氮合酶(iNOS)基因的表达,筛选出有效的激活序列,进一步证明了该现象普遍存在于哺乳动物细胞中.方法 设计并合成了4条靶向大鼠iNOS基因启动子DNA序列互补的双链RNA分子(saRNA-1、saRNA-2、saR-NA-3及saRNA-co),脂质体法转染BRL细胞.转染后收集转染后细胞,采用逆转录-聚合酶链反应(RT-PCR)法检测iNOS的表达.结果大鼠肝细胞株BRL转染羟基荧光素(FAM)标记的saRNA后,70%以上细胞内有绿色荧光表达.转染72 h半定量RT-PCR检测显示saRNA-2转染后能够激活BRL细胞iNOS基因的表达,其他saRNA转染后未见激活现象.结论 利用RNA激活技术可成功激活大鼠肝细胞BRL细胞株iNOS基因的表达.     

小干扰RNA抑制雄激素受体基因表达对人膀胱癌T24细胞增殖及凋亡的影响

目的 观察小干扰RNA技术(siRNA)抑制雄激素受体(AR)基因的表达对人膀胱癌T24细胞增殖及凋亡的影响.方法 化学合成针对AR的siRNA,用脂质体转染T24细胞.采用实时荧光定量逆转录-聚合酶链反应(RT-PCR)和Western blot检测AR mRNA和蛋白的变化.转染细胞48 h后用噻唑蓝(MTT)比色法检测细胞体外增殖活性的变化,流式细胞术检测细胞的凋亡状况.结果 成功的转染T24细胞,实时荧光定量RT-PCR筛选出一条AR mRNA抑制效率最高的siRNA,行Western blot检测可抑制AR蛋白表达至70.3%.转染细胞48 h后,T24细胞增殖活性抑制率达到(25.9±5.4)%,细胞凋亡率达到21.61%.结论 应用siRNA干扰技术能有效的抑制AR基因的表达,同时可抑制人膀胱癌T24细胞的体外增殖活性及促进其凋亡的发生.     

小分子干扰RNA沉默人结直肠癌HT-29细胞TCF21基因对Kiss-1基因表达的影响

目的 观察小于扰RNA(siRNA)沉默结直肠癌HT-29细胞TCF21基因对Kiss-1基因表达的影响,以及细胞增殖、侵袭及迁移能力等生物学行为的变化.方法 利用脂质体lipofectamineTM 2000将siRNA稳定转染结直肠癌HT-29细胞,实验分为干扰组、阴性对照组、空白对照组;应用实时定量逆转录聚合酶链反应(RT-qPCR)与Western blot技术检测TCF21与Kiss-1基因mRNA与蛋白的表达水平,分别采用细胞计数试剂盒(CCK-8)法和Transwell小室法检测干扰前后细胞增殖、侵袭与迁移能力的变化.结果 siRNA干扰后成功沉默了HT-29细胞TCF21基因的表达,Kiss-1基因mRNA的表达量为0.58 ±0.02,较对照组的1.00±0.00明显降低(P＜0.05),蛋白表达量为0.3491±0.0009,与对照组比较(0.8485 ±0.0016)亦出现明显下降(P＜0.05),同时细胞增殖、侵袭及迁移能力均明显增强(P＜0.01).结论 沉默TCF21基因的表达可以下调结直肠癌细胞Kiss-1基因的表达水平,并导致相应生物学行为的变化,因此TC F21基因可能是Kiss-1基因的上游调控因子.     

RNAi表达载体对膀胱癌细胞Livin基因表达的抑制作用

目的观察RNA干扰（RNAi）表达载体对膀胱癌BIU-87细胞Livin基因的抑制作用。方法构建针对Livin基因的RNAi质粒表达载体，脂质体法转染膀胱癌BIU-87细胞株，应用逆转录-聚合酶链反应（RT-PCR）、Western blot检测其对Livin mRNA及蛋白表达的影响。结果构建的表达质粒在膀胱癌BIU-87细胞株中均抑制了Livin mRNA及蛋白表达。与空白载体组细胞作对照，前者产生的小干扰RNA（siRNA）在对Livin mRNA的抑制率24、48h分别为（81．28±9．21）％、（55．88±6．27）％，蛋白抑制率24h为（64．75±7．55）％。结论RNAi表达载体可以有效地抑制膀胱癌细胞Livin基因的表达。     

Deficiency of TBL1XR1 causes asthenozoospermia

Transducin (β)-like 1 X-linked receptor 1 (TBL1XR1) is an evolutionarily conserved protein related to spermatozoa. To clarify its role and mechanism of action in spermatozoa, qRT-PCR was used to analyse the expression of TBL1XR1 in human spermatozoa and mouse testes. The mice were established as an animal model by injecting the mice testes with small interfering RNA against TBL1XR1 or control siRNA. Our results indicated that deficiency of TBL1XR1 in mice reduced the motility of spermatozoa and disrupted the histone-to-protamine transition. We also found the decreased expression of TBL1XR1 in the spermatozoa of human patients with asthenozoospermia (AZ) compared with that in the spermatozoa of healthy males. Moreover, we carried out chromatin immunoprecipitation analyses and found that genes downstream of TBL1XR1 were related to sperm motility. Thus, TBL1XR1 might be related to sperm motility and might function through its downstream genes. Our data highlight the role of TBL1XR1 involved in spermatozoa and provide new molecular insights into the intricate systems required for male fertility.     

Alpha1-antitrypsin deficiency. 1: epidemiology of alpha1-antitrypsin deficiency

The protein and molecular characteristics of variants of the alpha1-antitrypsin (AAT) gene are described, and available data on the genetic epidemiology of AAT deficiency are presented.     

手术创伤中腹膜组织Sp1、COL1A1和TIMP-1的表达

目的　探讨人手术创伤腹膜组织中核转录因子Sp1激活 ,COL1A1和TIMP 1表达变化与腹膜纤维化之间的关系。方法　采用凝胶电泳迁移率改变分析法 (EMSA)检测手术创伤后不同时间的腹膜组织核转录因子Sp1的表达水平 ,WesternBlot检测COL1A1和TIMP 1蛋白表达 ,Masson染色观察腹膜组织中胶原纤维的变化。结果　Sp1在手术创伤后 0 .5h被活化 ,随着手术时间延长Sp1活性逐渐增强 ,至创伤后 4h时达高峰 ,同时创伤腹膜组织中的COL1A1和TIMP 1蛋白表达水平逐渐升高 ,存在差异显著性 (P <0 .0 1)。在手术创伤期内随手术时间的延长腹膜组织中胶原纤维增加。结论　核转录因子Sp1活化导致Ⅰ型胶原合成增加 ,细胞外基质降解减少 ,从而启动腹膜纤维化进程。     

Evaluation of the influence of polymorphic variants CYP1A1 2B, CYP1B1 2, CYP3A4 1B, GSTM1 0, and GSTT1 0 in prostate cancer

Background/objectiveGenetic polymorphisms in cytochrome P-450 (CYPs) and glutathione S-transferase (GSTs) genes can influence the appearance of tumors by the formation of new enzymes with altered activities. In the present study, 5 polymorphic variants were examined in 154 patients with prostate carcinoma and in 154 controls.Materials and methodsDNA analysis was carried out through PCR-based methods. The statistical methods used were odds ratio and confidence interval (95% CI), χ², Fisher, and Mann-Whitney.ResultsThe study showed absence of association for CYP1A1*2B, CYP1B1*2, GSTM1*0, and GSTT1*0. The statistical analysis implied a positive association of variant CYP3A4*1B for prostate cancer. The combined analysis of CYP1A1*2B, CYP1B1*2, and CYP3A4*1B genotypes showed positive association. The analysis of histopathologic parameters detected statistically significant differences for Gleason score and biochemistry recurrence risk. The presence of the GSTT1*0 genotype in red meat consumers increased the risk for this disease.ConclusionSome polymorphic variants analyzed can influence the development and the progression of prostate cancer.     

Genetic polymorphisms of CYP1A1, CYP2E1, GSTM1, and GSTT1 associated with head and neck cancer

BACKGROUND: Alcohol intake and tobacco smoke, in addition to other environmental and genetic factors, have been associated with head and neck cancer. We evaluated the role of metabolic enzyme polymorphisms on the risk of head and neck cancer in a hospital-based case-control study. METHODS: CYP1A1MspI, CYP2E1PstI, GSTM1, and GSTT1polymorphisms were evaluated in 103 histologically confirmed head and neck cancer cases and 102 controls by means of polymerase chain reaction-restriction fragment length polymorphism methods. RESULTS: GSTM1null increased the risk of head and neck cancer (odds ratio [OR], 2.2; 95% confidence interval [95% CI], 1.24-3.79), oral cancer (OR, 2.8; 95% CI, 1.28-5.98), and pharyngeal cancer (OR, 2.2; 95% CI, 1.08-4.63). CYP2E1PstI polymorphism indicated a risk for oral cancer (OR, 3.6; 95% CI, 1.29-11.56). The joint effect of GSTM1 null and CYP1A1 polymorphism increased the risk of head and neck cancer (OR, 2.4; 95% CI, 1.13-5.10). CONCLUSIONS: GSTM1 null alone or associated with CYP1A1 increased the risk of head and neck cancer; the CYP2E1PstI mutated allele increased the risk for only oral cancer.     

IgA肾病患者C1GALT1C1基因的体细胞突变筛查

目的 探讨IgA肾病（IgAN）患者β1,3半乳糖转移酶的分子伴侣Cosme编码基因C1GALT1C1基因体细胞突变情况。方法 27例IgA肾病患者及19例正常健康对照作为研究对象。提取研究对象外周血基因组DNA,扩增C1GALT1C1基因的编码区,采用PCR产物直接测序的方法进行突变筛查。然后,分离其中15例IgA肾病患者及7例健康男性对照的外周血B淋巴细胞,提取DNA。对C1GALT1C1基因编码区进行扩增,PCR产物进行克隆,各挑选平均8～10个克隆进行体细胞突变筛查。结果 46例个体全血基因组DNA的PCR扩增产物测序发现,2例患者及1例健康对照存在外显子T393A变异,次等位基因频率（MAF）为6．9%[SNP数据库（dbSNP）报告为9．5%]。B淋巴细胞DNA序列分析显示,在22例个体（15例IgA肾病患者,7例健康对照）送检的总共202个克隆中,未发现新的突变和多态性位点。结论 C1GALT1C1基因编码区T393A多态位点在本研究人群中为唯一发现的多态性位点,其次等位基因频率（MAF）较既往报道略低。本研究尚未发现IgA肾病患者B淋巴细胞存在体细胞突变。     

The HLA-DPB1--associated component of the IDDM1 and its relationship to the major loci HLA-DQB1, -DQA1, and -DRB1

The major histocompatibility complex (MHC) HLA region on chromosome 6p21 contains the major locus of type 1 diabetes (IDDM1). Common allelic variants at the class II HLA-DRB1, -DQA1, and -DQB1 loci account for the major part of IDDM1. Previous studies suggested that other MHC loci are likely to contribute to IDDM1, but determination of their relative contributions and identities is difficult because of strong linkage disequilibrium between MHC loci. One prime candidate is the polymorphic HLA-DPB1 locus, which (with the DPA1 locus) encodes the third class II antigen-presenting molecule. However, the results obtained in previous studies appear to be contradictory. Therefore, we have analyzed 408 white European families (200 from Sardinia and 208 from the U.K.) using a combination of association tests designed to directly compare the effect of DPB1 variation on the relative predisposition of DR-DQ haplotypes, taking into account linkage disequilibrium between DPB1 and the DRB1, DQA1, and DQB1 loci. In these populations, the overall contribution of DPB1 to IDDM1 is small. The main component of the DPB1 contribution to IDDM1 in these populations appears to be the protection associated with DPB1*0402 on DR4-negative haplotypes. We suggest that the HLA-DP molecule itself contributes to IDDM1.     

Potential influence of IL1B haplotype and IL1A-IL1B-IL1RN extended haplotype on hand osteoarthritis risk

OBJECTIVE: To assess osteoarthritis (OA) association with the human interleukin-1 (IL-1) region. DESIGN: Sixty-four European-descent cases with radiographic hand OA and 48 European-descent controls were genotyped at nine single nucleotide polymorphism (SNP), one variable-number-of-tandem-repeat (VNTR), and one microsatellite marker extending across loci for IL-1alpha (IL1A), IL-1beta (IL1B), and IL-1 receptor antagonist (IL1RN). The genotype data were used to reconstruct individual locus haplotypes, and then locus haplotypes were used as superalleles for extended haplotype reconstruction. RESULTS: Nine different extended IL1A-IL1B-IL1RN haplotypes occurred at a frequency 0.05 or greater in either cases or controls. Only two IL1A-IL1B-IL1RN extended haplotypes were consistent with previously described extended risk haplotypes and totaled n=9 in cases and n=3 in controls [odds ratio (OR) 2.1, Haldane's chi(2) 1.67, one-sided P 0.1]. Our prior report showed hand OA association with homozygous IL1B rs1143633 minor allele genotype. All except one extended risk haplotype copy also had the IL1B rs1143633 minor allele. The rs1143633 genotype association was explained by one common six-SNP IL1B haplotype bearing rs1143633 minor allele and also risk alleles at rs1143634, rs1143627, and rs16944, component markers of the previously described extended risk haplotypes. The IL1B haplotype bearing all three risk alleles was found in 16 haplotype-homozygous hand OA cases and in four haplotype-homozygous controls and conferred OR 3.4 among homozygotes (nominal P value 0.006). CONCLUSION: Our evidence broadly supports the genetic association of OA phenotypes with an IL-1 region extended risk haplotype and specifically IL1B genotype. The extended risk haplotype previously associated with hip OA appears to be less frequent and has weaker genetic effect in hand OA. Hand OA risk is conferred by homozygous state for the IL1B haplotype characteristic of the extended risk haplotype.