Effect of tissue inhibitor of metalloproteinases-l siRNA on endothelial cells injury induced by septic serum北大核心CSCD

Objective: To investigate the effect and possible mechanism of tissue inhibitor of Metalloproteinases-l (TIMP-1) siRNA on human umbilical vein endothelial cells injury induced by serum of septic patient. Methods: Serum samples were separately collected from septic patients and healthy controls. Human umbilical vein endothelial cells (HUVECs) were randomly divided into blank group (normal culture cells), control group (culture medium with 10% control serum), septic group (culture medium with 10% septic serum), negative control group (negative siRNA + 10% septic serum), and TIMP-1 siRNA group (TIMP-1 siRNA + 10% septic serum). The survival rate of endothelial cells was detected by MTT assay. The levels of matrix Metalloproteinase-9 (MMP-9) and TIMP-1 in supernatant of culture medium were measured by enzyme-linked immunosorbent assay (ELISA). The levels of MMP-9, TIMP-1 and Thrombomoduline (TM) in endothelial cells were examined by Western blot. Results: Compared with control group, the cell survival rate of septic group decreased 12 hours after the addition of serum (P<0.05) and reached minimum 48 hours later. The levels of MMP-9 and TIMP-1 in supernatant of culture medium of septic group significantly increased (P<0.01). The levels of MMP-9 and TIMP-1 increased in the septic group (P<0.01), while the level of TM reduced at the same time in septic group (P<0.01). Compared with septic group, the cell survival rate of TIMP-1 siRNA group decreased (P<0.05). The level of MMP-9 in supernatant of culture medium of TIMP-1 siRNA group increased (P<0.05), while the level of TIMP-1 decreased (P<0.05). The level of MMP-9 increased in TIMP-1 siRNA group (P<0.01), whereas the levels of TIMP-1 and TM reduced in TIMP-1 siRNA group (P<0.01). Conclusions: TIMP-1 plays a protective role in endothelial cells injury induced by septic serum. © 2018 Chinese Medical Association. All rights reserved.     

柯萨奇病毒B4 2B基因siRNA达载体的构建及其对柯萨奇病毒B4抑制作用的研究

目的构建针对柯萨奇病毒B4 2B基因的siRNA表达载体并检测该载体在体外培养细胞中对柯萨奇病毒B4的抑制作用.方法选择柯萨奇病毒B4的2B基因区21bp的基因片段作为靶序列,合成含有靶序列的DNA片段并克隆到pGCsi-U6/Neo/GFP/siNeGative载体中,构建pGCsi-U6/Neo/GFP/2B,使其可表达具有发夹结构的双链siR-NA,在转染该载体后用柯萨奇病毒B4攻击Hela细胞,检测该载体对病毒感染细胞的保护效应.结果通过酶切、电泳、序列分析及荧光显微镜观察证明载体构建成功,而且pGCsi-U6/Neo/GFP/2B转染组的柯萨奇病毒B4的滴度及TCID50都明显低于pGCsi-U6/Neo/GFP/siNeGative转染组.结论成功构建了柯萨奇病毒2B基因的siRNA表达载体,而且该载体在体外培养细胞中对柯萨奇病毒的复制具有抑制作用.     

靶向人乳头瘤病毒16型-E6的siRNA对宫颈癌肿瘤生长的影响

目的 研究靶向HPV16-E6的小分子干扰RNA抑制宫颈癌裸鼠移植瘤的生长.方法 建立裸鼠宫颈癌模型,HPV16-E6 siRNA瘤体内多点注射观察肿瘤的生长情况,使用HE染色方法观察肿瘤组织细胞在pSiRNA或pCON治疗后的病理学改变;免疫组化采用SP法.结果 靶向HPV16-E6的小分子干扰RNA能显著抑制宫颈癌裸鼠移植瘤的生长.结论 利用宫颈癌裸鼠移植瘤模型,证明靶向HPV16-E6的siRNA能够显著抑制宫颈癌裸鼠移植瘤的生长.     

Polymersome carriers: From self-assembly to siRNA and protein therapeutics

Polymersomes are polymer-based vesicular shells that form upon hydration of amphiphilic block copolymers. These high molecular weight amphiphiles impart physicochemical properties that allow polymersomes to stably encapsulate or integrate a broad range of active molecules. This robustness together with recently described mechanisms for controlled breakdown of degradable polymersomes as well as escape from endolysosomes suggests that polymersomes might be usefully viewed as having structure/property/function relationships somewhere between lipid vesicles and viral capsids. Here we summarize the assembly and development of controlled release polymersomes to encapsulate therapeutics ranging from small molecule anti-cancer drugs to siRNA and therapeutic proteins.     

携带cdc2-siRNA的重组腺相关病毒对C型尼曼-皮克病小鼠行为学的影响

目的观察携带cdc2～小干扰RNA（cdc2-siRNA）的重组腺相关病毒（rAAV）小脑注射对C型尼曼-皮克病（NPC）小鼠行为学的影响。方法2周龄npc^-/-小鼠经小脑注射携带cdc2-siRNA的rAAV，动态测量小鼠体重，并进行衣架悬挂实验及足印实验评估小鼠4～8周的运动能力。结果（1）cdc2-siRNA组小鼠体重减轻明显延缓；（2）衣架悬挂实验显示cdc2-siRNA组小鼠运动缺陷明显改善；（3）足印实验显示cdc2-siRNA组相对步距[（75．1±8．6）％]与对照病毒组及非手术组相比明显增加。结论小脑注射携带cdc2-siRNA的rAAV改善了npc^-/-小鼠的行为学，有可能为NPC的治疗提供新的有效途径。     

Chemical vectors for gene delivery: a current review on polymers, peptides and lipids containing histidine or imidazole as nucleic acids carriers

DNA/cationic lipid (lipoplexes), DNA/cationic polymer (polyplexes) and DNA/cationic polymer/cationic lipid (lipopolyplexes) electrostatic complexes are proposed as non-viral nucleic acids delivery systems. These DNA-nanoparticles are taken up by the cells through endocytosis processes, but the low capacity of DNA to escape from endosomes is regarded as the major limitations of their transfection efficiency. Here, we present a current report on a particular class of carriers including the polymers, peptides and lipids, which is based on the exploitation of the imidazole ring as an endosome destabilization device to favour the nucleic acids delivery in the cytosol. The imidazole ring of histidine is a weak base that has the ability to acquire a cationic charge when the pH of the environment drops bellow 6. As it has been demonstrated for poly(histidine), this phenomena can induce membrane fusion and/or membrane permeation in an acidic medium. Moreover, the accumulation of histidine residues inside acidic vesicles can induce a proton sponge effect, which increases their osmolarity and their swelling. The proof of concept has been shown with polylysine partially substituted with histidine residues that has caused a dramatic increase by 3–4.5 orders of magnitude of the transfection efficiency of DNA/polylysine polyplexes. Then, several histidine-rich polymers and peptides as well as lipids with imidazole, imidazolinium or imidazolium polar head have been reported to be efficient carriers to deliver nucleic acids including genes, mRNA or SiRNA in vitro and in vivo. More remarkable, histidylated carriers are often weakly cytotoxic, making them promising chemical vectors for nucleic acids delivery.This article is part of a themed section on Vector Design and Drug Delivery. For a list of all articles in this section see the end of this paper, or visit: http://www3.interscience.wiley.com/journal/121548564/issueyear?year=2009     

Environmental risk factors for prevention and molecular intervention of cervical cancer

Cervical cancer (CC) is potentially the most preventable and treatable cancer in human but it is a leading cause for cancer morbidity and mortality in women around the world. Therefore, more innovative prevention and treatment protocols need to be developed and implemented. With better understanding of the etiology of the disease, specific prevention protocols that involve life-style modifications to minimize the impact of environmental risk factors can be developed. It may be necessary to implement unique modification protocols for different countries. In addition, antiviral vaccine is a highly promising prevention approach. With respect to therapy, the development of more specific protocols that have fewer side effects is needed. With the availability of sophisticated molecular techniques, a new generation of targeted approach that has the potential to generate outstanding efficacy is being tested. Using the siRNA technology against the expression of human papillomavirus oncogenes, specific biological pathways that are essential to the growth and survival of the CC cells can be interrupted. Another promising approach is the molecular intervention of the estrogen pathway by blocking the expression of estrogen receptors. These molecular techniques may work by reactivating endogenous regulatory processes, e.g., the core apoptotic machinery, that can cause self-destruction of the CC cells, thus providing potentially effective molecular therapy. These topics are discussed in this review.     

siRNA逆转survivin介导的T24/AMD细胞多药耐药的研究

目的:研究siRNA(small interfering RNA)逆转survivin介导的膀胱癌多药耐药性。方法:设计针对survivin基因的siRNA,转染膀胱癌T24/ADM细胞,采用RT-PCR检测mRNA表达,免疫印迹法检测蛋白表达,流式细胞仪检测细胞内阿霉素累积量。结果:siRNA能明显逆转T24/ADM细胞的多药耐药,使survivin基因的mRNA及蛋白表达水平下调,细胞内阿霉素累积量显著增加(P<0.05)。结论:siRNA通过抑制survivin基因表达从而逆转膀胱癌的多药耐药。     

TrkAsiRNA表达载体构建及对TrkA基因表达的抑制

目的　构建具有特异阻断TrkA基因功能的siRNA表达系统 ,为前列腺癌基因治疗提供新的方法。方法　根据Genbank提供的TrkA基因mRNA序列 ,应用设计软件设计特异性的短链寡核苷酸 ,化学合成后经退火形成双链DNA片段 ,克隆到pSlincerTM2 1 U6载体中 ,用BamHⅠ和HindⅢ双酶切和序列测定对重组体进行鉴定 ,最后将构建的表达载体转染前列腺癌细胞株 ,观察对TrkA基因表达的影响。结果　酶切和序列测定表明 ,成功构建了siRNA表达载体 ,能够抑制细胞内TrkA基因表达。结论　本研究构建的siRNA表达载体 ,具有阻断TrkA基因的表达的功能 ,为前列腺癌基因治疗提供新的有效的方法。     

干扰Livin对HEC-1-A细胞化疗增敏作用的研究

目的 研究Livin在提高人子宫内膜癌HEC-1-A细胞对顺铂化疗敏感性方面的作用及可能机制。方法 将Livin的小分子干扰RNA(Livin-siRNA)转染HEC-1-A细胞,qRT-PCR及Western blot法检测转染siRNA后Livin mRNA及蛋白表达情况。CCK-8法检测转染siRNA并暴露于顺铂后,细胞增殖能力的变化。Western blot法检测干扰Livin后,相关蛋白Caspase-3、Caspase-7 、Caspase-9、Smac的表达情况。结果 抑制Livin表达并暴露于顺铂后,细胞增殖能力明显减弱(P<0.01),Caspase-3、Caspase-7 、Caspase-9、Smac蛋白的相对表达量显著增加(P<0.01)。结论 干扰Livin可显著抑制顺铂诱导下的HEC-1-A细胞的增殖能力,其化疗增敏作用是通过解除对Caspase的抑制和减少Smac降解两条途径来实现的。