An indirect treatment comparison of the efficacy of patisiran and tafamidis for the treatment of hereditary transthyretin-mediated amyloidosis with polyneuropathy

Background: Hereditary transthyretin-mediated amyloidosis (hATTR amyloidosis) is a progressive, life-threatening disease. Until recently, tafamidis was the only approved pharmacotherapy. Patisiran significantly improved polyneuropathy and quality of life (QoL) in the phase III APOLLO trial. In the absence of direct comparisons, this analysis aimed to evaluate the comparative efficacy of tafamidis and patisiran in hATTR amyloidosis with polyneuropathy.

Research design and methods: Randomized controlled trial evidence for tafamidis was identified by systematic literature review. Indirect treatment comparisons were performed using the standard pairwise Bucher method for endpoints used in both APOLLO and the tafamidis Fx-005 trial: change from baseline in Neuropathy Impairment Score-lower limbs (NIS-LL), Norfolk QoL-Diabetic Neuropathy questionnaire (QoL-DN), NIS-LL response, and mBMI vs. placebo. Inter-trial population differences were assessed by sensitivity analysis.

Results: The base-case analysis (FAP Stage 1 APOLLO patients vs. intent-to-treat Fx-005 population) suggested patisiran had a greater treatment effect vs. tafamidis for all endpoints, with significant improvements in mean change in NIS-LL (–5.49) and QoL-DN (–13.10) from baseline to Month 18. Similar trends were observed in all sensitivity analyses.

Conclusions: In the absence of direct comparisons, this analysis suggests patisiran has a greater treatment effect than tafamidis in patients with hATTR amyloidosis with polyneuropathy.     

转录因子NF-KB对血管成形术后血管狭窄和新生内膜形成的影响

目的探讨核转录因子NF-KB的反义和诱骗性寡核苷酸对大鼠颈动脉球囊损伤后血管增殖反应及相应细胞因子、细胞外信号调节激酶的影响。方法SD大鼠随机分为7组．每组分为6个时相点（6h，1，3，5，7，14d），每个时相点3只大鼠。制作血管球囊损伤模型.相应时间点处死动物，使用病理形态学、逆转录聚合酶链反应、免疫组化、Western blot等方法测定血管病理形态改变、单核细胞趋化因子（Monocytes chemotactic protein-1，MCP-1）的mRNA和蛋白质表达、NF-KB p65和细胞外信号调节激酶（Extra-cellular signal regulated kinase，ERK2）的蛋白质含量。结果 模型组、正义组、Scramble组血管内膜／中膜在d 5增加，14d达到高峰。反义组、诱骗组、反义组＋诱骗组干预后，血管内膜／中膜明显减小（P〈0．05），反义组＋诱骗组较反义组、诱骗组治疗效果不明显。MCP-1 mRNA表达在血管损伤后6h即可检测到，3、5、7d后持续表达增加，14d后表达降低，免疫组化显示MCP-1蛋白质表达在6个时相点均为阳性染色，14d达到高峰；反义组、诱骗组、反义组＋诱骗组治疗后，与模型组、正义组、Scramble组相比，MCP-1 mRNA表达和蛋白合成在各时相点均降低（P〈0．05）．Western blot检测核蛋白抽提物显示核因子NF-KB p65在血管损伤后6h有一定蛋白表达，1d后蛋白表达明显增加，至7d达高峰，14d后蛋白表达略降低。ERK2在血管损伤后1d蛋白表达开始增加，d7达到峰值．14d后与d7相比尤明显差异。反义组、诱骗组、反义组＋诱骗组干预后，各时相点蛋白质表达均较模型组、正义组、Scramble组减弱（P〈0．05）。结论转录因子NF-KB调控MCP-1、ERK2基因表达和蛋白质合成；球囊损伤后血管壁细胞增殖在不同时相点有动态变化；脂质体介导局部转染反义、诱骗性寡核苷酸可抑制NF-KB激活对下游基因的调控，两者联合作用效果较单独应用未表现出更明显的作用。     

Altered Regulation of mRNA and miRNA Expression in Epithelial and Stromal Tissue of Keratoconus Corneas

PurposeEvaluation of mRNA and microRNA (miRNA) expression in epithelium and stroma of patients with keratoconus.MethodsThe epithelium and stroma of eight corneas of eight patients with keratoconus and eight corneas of eight non-keratoconus healthy controls were studied separately. RNA was extracted, and mRNA and miRNA analyses were performed using microarrays. Differentially expressed mRNAs and miRNAs in epithelial and stromal keratoconus samples compared to healthy controls were identified. Selected genes and miRNAs were further validated using RT-qPCR.ResultsWe discovered 170 epithelial and 1498 stromal deregulated protein-coding mRNAs in KC samples. In addition, in epithelial samples 180 miRNAs and in stromal samples 379 miRNAs were significantly deregulated more than twofold compared to controls. Pathway analysis revealed enrichment of metabolic and axon guidance pathways for epithelial cells and enrichment of metabolic, mitogen-activated protein kinase (MAPK), and focal adhesion pathways for stromal cells.ConclusionsThis study demonstrates significant differences in the expression and regulation of mRNAs and miRNAs in the epithelium and stroma of Patients with KC. Also, in addition to the well-known target candidates, we were able to identify further genes and miRNAs that may be associated with keratoconus. Signaling pathways influencing metabolic changes and cell contacts are affected in epithelial and stromal cells of patients with keratoconus.     

RNA干扰技术抑制类风湿关节炎成纤维细胞COX-2合成

目的 体外合成针对人环氧化酶-2（hCOX-2）的小分子干扰RNA（siRNA）,并转染人人滑膜成纤维细胞,通过比较不同siRNA抑制COX-2 mRNA表达、COX-2蛋白合成以及下游产物PGE2水平,旨在确定特异性阻断人滑膜成纤维细胞COX-2的siRNA.方法 分离、培养和传代人类风湿关节炎（类风关）滑膜成纤维细胞.设计合成4条针对hCOX-2 mRNA siRNA（1#-4#siRNA）,1条随机序列siRNA.设立1#-4#siRNA和随机序列siRNA组（NC）及未转染对照组（CTL组）.应用Lipofect AMINE 2000将上述siRNA分别转染人滑膜成纤维细胞,4 h后各培养孔加入终浓度为100 nmol/L的佛波酯.转染36 h、48 h后,各组提取蛋白质和总RNA,应用RT-PCR检测hCOX-2 mRNA表达水平,通过Western Blot检测hCOX-2蛋白表达水平.采用ELISA方法检测各组上清液PGE2的水平.结果 转染36 h,PCR结果显示,CTL、NC、1#siRNA、2#RNA、3#RNA、4#siRNA、阳性对照HeLa细胞组hCOX-2 mRNA电泳条带密度值分别为1、0.72、0.3、0.25、0.4、0.04、2.1.Western Blot结果提示上述各组hCOX-2蛋白密度值分别为1、1.04、0.52、0.39、0.9、0和2.48.转染48 h后,各组hCOX-2蛋白条带密度值分别为0.05、0.52、0.51、0.9和0.15.转染24 h、48 h和72 h后,4#siRNA组培养上清液PGE2的水平较其他各组低.结论 4#siRNA能有效抑制人滑膜成纤维细胞COX-2 mRNA表达和COX-2蛋白的合成,且上清液中PGE2水平最低,证实4#siRNA能特异性阻断COX-2在滑膜成纤维细胞表达.     

人喉癌Hep-2细胞膜钾离子通道与RNA编辑酶1的相关性

目的研究人喉癌细胞系Hep-2细胞膜钾离子通道特性及其与RNA编辑酶1(RNA-dependent adenosinedeaminase1,ADAR1)的相关性.方法以Hep-2细胞为研究对象,采用穿孔膜片钳全细胞记录法研究钾离子通道特性,用逆转录聚合酶链反应检测四乙胺(tetraethylammonium,TEA)阻断钾离子通道前后Hep-2细胞ADAR1 mRNA的表达.结果 Hep-2细胞膜的静息膜电位为(-29.8±1.9)mV,在钳制电压-40 mV,阶跃电压在-80～ 80 mV,记录到一种跨膜电流,该跨膜电流具有电压依赖、外向整流特性,该电流在延迟25ms后最大激活,800 ms内不失活,可被阻断剂TEA阻断.Hep-2细胞钾离子通道被TEA阻断前后ADAR1mRNA相对表达量存在显著性差异.结论 Hep-2细胞膜上存在延迟整流钾离子通道,此钾离子通道可能和ADAR1 mRNA的表达密切相关.     

反向重复序列法构建神经病靶标酯酶RNA干涉表达质粒

目的构建基于通用真核表达载体的人神经病靶标酯酶双链RNA的稳定表达载体。方法在正义和反义引物两端分别加上EcoRⅠ和BamHⅠ的识别位点扩增得到神经病靶标酯酶活力域序列,构建含有目标基因的倒置重复序列的双链RNA表达载体pcDNA.NTE.dsRNA,酶切分析鉴定重组载体。脂质体法转染到Hela细胞中瞬时表达重组载体,酶活力测定其对细胞内神经病靶标酯酶活力的影响。结果成功构建了NTE双链RNA稳定表达载体pcDNA.NTE.dsRNA,表达该载体细胞内NTE活力明显下降至对照细胞的20%左右。结论采用反向重复序列法将靶标基因的编码序列正反向插入到通用真核细胞表达载体中,是构建哺乳细胞基因双链RNA稳定表达载体的有效方法。     

乳腺癌细胞DNA及RNA定量分析与鉴别诊断

目的研究乳腺癌基因诊断的准确率。方法用流式细胞光度分析技术对84例乳腺癌细胞 DNA、RNA含量进行定量分析。结果DNA异倍率为71．43％;RNA增高率为85．71％;用“标准DI”和“标准RI”为指标判断乳腺癌的准确率分别为78．57％和83．33％;且双指标同时应用时判断的准确率 92．86％,高于任一单指标。结论“标准DI”和“标准RI”是判断乳腺肿瘤良恶性生物学性质的理想指标。