靶向VEGF发夹状RNA对宫颈癌HeLa细胞抑制作用的研究

目的:运用RNAi技术下调血管内皮生长因子(VEGF)在HeLa细胞中的表达,观察其对肿瘤细胞凋亡的影响,为人宫颈癌治疗提供理论依据。方法:设计并构建针对VEGF的携带绿色荧光蛋白(GFP)发夹状RNA(shRNA)质粒表达载体(PGPU6/GFP/Neo-shRNA),脂质体法转染HeLa细胞;荧光显微镜观察GFP的表达,并计算转染效率;RT-PCR检测HeLa细胞VEGF的表达,筛选出靶序列;再用流式细胞仪法检测细胞凋亡。结果:构建的PGPU6/GFP/Neo载体成功转入HeLa细胞;转染48h后,HeLa-shVEGF1组HeLa细胞VEGFmRNA表达的抑制率为75.0%;与HeLa组和HeLa-shNC组相比,HeLa-shVEGF1组HeLa细胞凋亡率明显增加,P<0.01。结论:本研究构建的PGPU6-shRNA表达载体携带GFP便于观察细胞的转染情况,且不影响U6启动子的转录,同时有效沉默了VEGF基因,明显增加HeLa细胞的凋亡,为未来肿瘤的治疗提供新途径。     

肺腺癌中关键lncRNA的表达及免疫相关分析

目的:通过鉴定与肺腺癌（LUAD）预后相关的长非编码RNA（lncRNA）,研究LUAD的发生机制及其预后意义,确定与LUAD预后相关的敏感性生物标志物,并对其进行免疫途径相关性分析。方法:从肿瘤基因组图谱数据库(TCGA)中获取与LUAD相关的数据,通过单因素Cox回归分析及套索算法（LASSO）筛选lncRNA,使用多因素Cox回归进行预后风险评分分析,建立预后风险模型,用计算曲线下面积（AUC）和Kaplan-Meier（K-M）生存分析方法评价模型的稳健性和准确性。利用K-M生存分析方法确定与生存状态相关的潜在生物标志物,并通过ImmLnc平台对其进行免疫途径相关性研究。结果:从49个与生存相关的lncRNAs中确定了12个预后相关生物标志物,通过K-M生存分析,MIR34AHG和PRKCA-AS1被确定为与预后相关的生物标志物(P＜0.05)。模型的3年和5年生存率的AUC分别为0.82和0.846。与MIR34AHG相关的免疫途径分别为“细胞因子受体”(P＜0.05),“抗原处理和提呈”(P＜0.05),与PRKCA-AS1相关的免疫途径为“抗原处理和提呈”(P＜0.05)。结论:通过对生物信息大数据的分析,我们确定了两个关键lncRNAs及其相关的免疫途径,为LUAD的预后评估提供了新的生物标志物。     

Immunomodulatory and anti-SARS activities of Houttuynia cordata

BACKGROUND: Severe acute respiratory syndrome (SARS) is a life-threatening form of pneumonia caused by SARS coronavirus (SARS-CoV). From late 2002 to mid 2003, it infected more than 8000 people worldwide, of which a majority of cases were found in China. Owing to the absence of definitive therapeutic Western medicines, Houttuynia cordata Thunb. (Saururaceae)(HC) was shortlisted by Chinese scientists to tackle SARS problem as it is conventionally used to treat pneumonia. AIM OF THE STUDY: The present study aimed to explore the SARS-preventing mechanisms of HC in the immunological and anti-viral aspects. RESULTS: Results showed that HC water extract could stimulate the proliferation of mouse splenic lymphocytes significantly and dose-dependently. By flow cytometry, it was revealed that HC increased the proportion of CD4(+) and CD8(+) T cells. Moreover, it caused a significant increase in the secretion of IL-2 and IL-10 by mouse splenic lymphocytes. In the anti-viral aspect, HC exhibited significant inhibitory effects on SARS-CoV 3C-like protease (3CL(pro)) and RNA-dependent RNA polymerase (RdRp). On the other hand, oral acute toxicity test demonstrated that HC was non-toxic to laboratory animals following oral administration at 16 g/kg. CONCLUSION: The results of this study provided scientific data to support the efficient and safe use of HC to combat SARS.     

A one-year survey on the use of a powder from Rosa canina lito in acute exacerbations of chronic pain

This pilot surveillance included 152 patients with acute exacerbations of chronic pain, 124 (Back group) with non-specific low back pain (NSLBP), 20 with NSLBP overridden by osteoarthritic pain (Knee-Hip group), and eight with specific LBP (included in the safety analysis). Patients were recommended the rose hip and seed powder Litozin at a dose providing up to 3 mg of galactolipid/day for up to 54 weeks. Clinical symptoms and well-being were assessed every 6 weeks. The patients also kept a diary of their pain and the requirement for rescue medication. Data were analysed by intention to treat with last observation carried forward. Only 77 patients completed the year of surveillance. Multivariate analysis suggested an appreciable overall improvement during the surveillance, irrespective of group, and this was reflected for most of the individual measures in repeated measures ANOVA. The degree and time-course of improvement echoed that seen in similar surveillances of patients receiving an aqueous extract of Harpagophytum. Multiple regression analyses indicated that percentage changes from baseline tended to be greater in patients with greater degrees of pain and disability, but were otherwise largely unrelated to the patients' characteristics. There were no serious adverse events. The rose hip and seed powder, Litozin, seems to deserve further, more definitive studies as a possible option in long-term management of NSLBP with or without osteoarthritic pain.     

hnRNPB_1基因的RNA干扰抑制肺癌细胞A549增殖研究

目的 探讨特异性小分子干扰(small interference,siRNA)抑制肺癌A549细胞核内不均一核糖蛋白B1(heterogeneous nuclear ribonucleoprotein,hnRNPB1)基因的表达后,对A549细胞增殖的影响. 方法构建hnRNPB1特异性的siRNA真核细胞表达载体并转染人肺癌细胞株A549,观察重组载体分别在第1、4及6周对A549细胞hnRNPB1基因的干扰效果以及对肺癌细胞周期及凋亡的影响.结果 特异性siRNA真核表达可显著抑制肺癌细胞hnRNPB1基因的表达,使hnRNPB1 mRNA的表达减少46%～73%,蛋白表达减少77.69%～83.04%,作用时间至少可维持克隆形成后4周,同时,hnRNPB1基因表达下调抑制了A549细胞在体外的增殖,促进了肺癌细胞的凋亡.结论 特异性siRNA能特异、高效地抑制肺癌细胞株A549细胞hnRNPB1基因的表达,RNAi可能为肺癌的基因治疗提供新策略.     

超声靶向破坏微泡技术介导小鼠肝癌细胞株JNK1基因表达、细胞迁移和侵袭抑制

目的: 探讨应用超声靶向破坏微泡 (UTMD) 技术介导小鼠肝癌细胞株JNK1基因的表达、细胞迁移和侵袭抑制的作用,阐明其作用机制。方法: 构建并筛选RNA干扰效果最好的短发夹RNA(shRNA)。将小鼠肝癌细胞株Hca-F分为正常Hca-F细胞组、shRNA质粒组、脂质体组、超声微泡结合超声辐照组及脂质体结合超声微泡加超声辐照组。采用倒置荧光显微镜观察各组细胞转染率,荧光定量PCR和Western blotting 法检测JNK1基因mRNA和蛋白表达水平,CCK-8法检测各组细胞的细胞活性,应用Transwell 实验检测各组细胞的体外迁移能力。结果: 脂质体结合超声微泡加超声辐照组细胞转染率高于shRNA质粒组、脂质体组和超声微泡结合超声辐照组(均P<0.05),脂质体组和超声微泡结合超声辐照组比较差异无统计学意义(P>0.05)。脂质体结合超声微泡加超声辐照组JNK1 mRNA和蛋白表达水平低于其他各组(P<0.05);脂质体结合超声微泡加超声辐照组细胞活性和平均穿膜细胞数均低于其他各组(P<0.05)。结论: UTMD技术结合脂质体转染法可以提高小鼠肝癌细胞株JNK1 shRNA的转染效率,增强其对基因表达、细胞活力、迁移和侵袭能力的抑制。     

探讨不同实验条件对U87细胞体外增殖的影响

目的:探讨RNA干扰肝癌衍生生长因子(HDGF)后,U87细胞增殖抑制的最佳实验条件。方法:用LipofectamineTM2000将HDGF siRNA转染U87细胞后,将细胞接种于96孔板中,分别在无血清、含10%FBS和无血清Matrigel胶预处理细胞培养板条件下,采用MTS法检测细胞增殖能力。结果:HDGF表达水平下调后,U87细胞增殖能力受到抑制。在无血清、含10%FBS和无血清Matrigel胶预处理细胞培养板条件下,细胞增殖抑制率分别是18%、9%和28%。结论:在无血清Matrigel胶预先处理的培养条件下,能最大程度上反映出HDGFsiRNA对U87细胞增殖能力的抑制。     

Single-Domain Antibodies as Therapeutics for Respiratory RNA Virus Infections

Over the years, infectious diseases with high morbidity and mortality disrupted human healthcare systems and devastated economies globally. Respiratory viruses, especially emerging or re-emerging RNA viruses, including influenza and human coronavirus, are the main pathogens of acute respiratory diseases that cause epidemics or even global pandemics. Importantly, due to the rapid mutation of viruses, there are few effective drugs and vaccines for the treatment and prevention of these RNA virus infections. Of note, a class of antibodies derived from camelid and shark, named nanobody or single-domain antibody (sdAb), was characterized by smaller size, lower production costs, more accessible binding epitopes, and inhalable properties, which have advantages in the treatment of respiratory diseases compared to conventional antibodies. Currently, a number of sdAbs have been developed against various respiratory RNA viruses and demonstrated potent therapeutic efficacy in mouse models. Here, we review the current status of the development of antiviral sdAb and discuss their potential as therapeutics for respiratory RNA viral diseases.     

Identification and Characterization of Two Novel Noda-like Viruses from Rice Plants Showing the Dwarfing Symptom

Nodaviruses are small bipartite RNA viruses and are considered animal viruses. Here, we identified two novel noda-like viruses (referred to as rice-associated noda-like virus 1 (RNLV1) and rice-associated noda-like virus 2 (RNLV2)) in field-collected rice plants showing a dwarfing phenotype through RNA-seq. RNLV1 genome consists of 3335 nt RNA1 and 1769 nt RNA2, and RNLV2 genome consists of 3279 nt RNA1 and 1525 nt RNA2. Three conserved ORFs were identified in each genome of the two novel viruses, encoding an RNA-dependent RNA polymerase, an RNA silencing suppressor, and a capsid protein, respectively. The results of sequence alignment, protein domain prediction, and evolutionary analysis indicate that these two novel viruses are clearly different from the known nodaviruses, especially the CPs. We have also determined that the B2 protein encoded by the two new noda-like viruses can suppress RNA silencing in plants. Two reverse genetic systems were constructed and used to show that RNLV1 RNA1 can replicate in plant cells and RNLV1 can replicate in insect Sf9 cells. We have also found two unusual peptidase family A21 domains in the RNLV1 CP, and RNLV1 CP can self-cleave in acidic environments. These findings provide new knowledge of novel nodaviruses.