沉默囊性纤维化跨膜传导调节因子对大鼠海马神经元细胞线粒体功能及氧化应激的影响

目的探讨沉默囊性纤维化跨膜传导调节因子(cystic fibrosis transmembrane conductance regulator,CFTR)在大鼠海马神经元细胞线粒体功能及氧化应激中的作用。方法18只SD大鼠随机分为CFTR小干扰RNA(small interfering RNA,siRNA)组、阴性对照组、空白对照组各6只。分离3组海马神经元细胞,取对数生长期细胞进行实验。CFTR siRNA组神经元细胞转染CFTR siRNA,阴性对照组转染阴性对照siRNA,空白对照组不转染。转染后培养48 h,采用Western blot法检测3组神经元细胞线粒体CFTR蛋白相对表达量,JC-1荧光探针检测线粒体膜电位,荧光分光光度法检测线粒体活性氧(reactive oxygen species,ROS)和过氧化氢(hydrogen peroxide,H2O2)表达,氧电极法测定线粒体呼吸控制率(respiratory control rate,RCR),荧光素酶发光法检测线粒体三磷酸腺苷(adenosine triphosphate,ATP)浓度,高效液相色谱法检测线粒体谷胱甘肽(glutathione,GSH)和氧化型谷胱甘肽(oxidized glutathione,GSSG)表达。结果转染后培养48 h,CFTR siRNA组神经元细胞线粒体CFTR蛋白相对表达量(0.09±0.01)、线粒体膜电位[(247.16±34.28)mV]、ATP[(11.71±2.54)mol/g]、RCR(4.69±0.82)、GSH[(0.71±0.12)nmol/μg]、GSH/GSSG值(0.31±0.08)低于空白对照组[0.42±0.07、(432.18±56.04)mV、(24.18±3.64)mol/g、7.94±1.21、(1.62±0.23)nmol/μg、1.65±0.27]和阴性对照组[0.38±0.06、(434.07±52.13)mV、(22.97±3.43)mol/g、7.83±1.18、(1.57±0.21)nmol/μg、1.63±0.24](P<0.05),线粒体H2O2水平(1.73±0.11)高于空白对照组(1.00±0.02)和阴性对照组(1.08±0.06)(P<0.05);空白对照组神经元细胞线粒体CFTR蛋白相对表达量及线粒体膜电位、ATP、RCR、H2O2、GSH、GSH/GSSG值与阴性对照组比较差异均无统计学意义(P>0.05)。结论沉默大鼠神经元细胞线粒体CFTR可引起海马神经元细胞线粒体功能障碍,增强氧化应激反应。     

Sequence and genomic organization of GBV-C: A novel member of the flaviviridae associated with human non-A-E hepatitis

Recently, sequences from a novel virus, termed GB virus C (GBV-C), were identified in serum from several patients with cryptogenic hepatitis. In the present study, the nucleotide sequence of this virus has been extended to near-genome length. GBV-C encodes a putative single large polyprotein in which the structural proteins are positioned at the N-terminal end, with the nonstructural proteins located at the C-terminal end. Amino acid sequence analysis of this large polyprotein reveals the presence of protease, helicase, and replicase motifs. Sequence alignments of the polyprotein followed by phylogenetic analyses suggest that GBV-C is a member of the Flaviviridae, most closely related to the recently described GB virus A. © 1996 Wiley-Liss, Inc.     

MALAT1参与胃癌发生发展机制的研究进展

长链非编码RNA(lncRNA)是一种长度>200 nt、不能编码蛋白质,但在细胞生理活动中具有重要调控功能的RNA.研究发现,lncRNA涉及许多病理生理过程,与肿瘤的发生发展密切相关.肺腺癌转移相关转录本1(MALAT1)是lncRNA家族中的重要成员,在胃癌组织及细胞中高表达,且在胃癌的发生发展中发挥重要而复杂的...     

Validation of a quantitative RNA PCR assay for HIV-1 in human plasma

A quantitative human immunodeficiency virus type 1 (HIV-1) RNA polymerase chain reaction assay has been validated analytically and clinically in > 13,000 samples. The assay is highly reproducible with intra- and inter-assay precision of 16% and 19%, respectively. In 1,542 of 1,548 subjects with CD4⁺ counts of 0–500 cells per mm³, viral RNA levels were quantifiable and ranged from ∼ 3,000–52,200,000 copies per milliliter. Median plasma HIV-1 RNA values were inversely proportional to CD4⁺ counts from 0–400 cells per mm³. When patients were off antiretroviral therapies for ∼ 14 days prior to the initial baseline RNA PCR evaluation, the mean variance between the two baseline values was 23% (0.1 log). Of these patients, 95% had a sufficient plasma viral load to quantitate a 10-fold (1 log) diminution in viral load caused by antiviral therapy. In contrast, only 20% and 45% of these subjects had sufficient p24 and ICD p24 levels to detect a 50% diminution in circulating virus. The high precision and reproducibility of this quantitative RNA PCR assay provide an enhanced means of evaluating therapeutic drug regimens for HIV-1. © 1996 Wiley-Liss, Inc.     

Smad2特异性siRNA转染肝星状细胞的效率检测

[目的]提高siRNA表达载体在肝星状细胞(hepatic stellate cells,HSCs)中的转染效率。[方法]pEGFP-C1-CR4转染原代HSCs、HSC-T6细胞株,实验根据质粒DNA和阳离子脂质体的不同比例分为4组:1μg/0μL、1μg/1μL、1μg/2μL、1μg/4μL,1μg/0μL为对照组,其余3组为实验组。转染48 h后,使用荧光显微镜观察转染情况,同时流式细胞仪计算转染效率。[结果]质粒DNA和阳离子脂质体的比例为1μg/2μL和1μg/4μL时,原代HSCs转染效率为(56.55±3.12)%和(58.62±2.03)%(P>0.05),HSC-T6细胞株转染效率为(24.52±3.55)%和(25.49±2.33)%(P>0.05)。同样比例条件下,原代HSCs的转染效率显著高于HSC-T6细胞株的转染效率(P<0.05)。在实验组质粒、脂质体比例为1μg/2μL和1μg/4μL时,同种细胞的转染效率均无显著差别(P>0.05)。[结论]采用质粒DNA和阳离子脂质体的比例为1μg/2μL、1μg/4μL的条件,均能够在HSCs和HSC-T6中获得最佳转染效率,但前者更为经济。     

慢病毒携带shRNA对宫颈癌细胞中HPV16型E6表达的抑制作用

目的研究慢病毒携带的短发夹状干扰RNA(short hairpin RNA, shRNA)对宫颈癌Caski细胞中人乳头瘤病毒(human papilloma virus, HPV)16型E6表达的抑制作用.方法将靶向HPV16型E6的shRNA表达序列克隆到改建的慢病毒表达载体PLL3.7中,经限制性酶切鉴定和测序后,与3种包装质粒混合,以Lipofectamine 2000包裹后转染293FT细胞,72 h后收取含病毒的上清液并感染宫颈癌Caski细胞,感染48 h后加入G418筛选阳性克隆.每天对阳性细胞克隆进行细胞计数;提取细胞总mRNA,进行RT-PCR反应.结果与对照组相比HPV16型E6的表达降低70%,宫颈癌Caski细胞生长速度减慢50%.结论慢病毒携带的短发夹状干扰RNA能干扰宫颈癌细胞中HPV16型E6的表达并有抑制癌细胞生长的作用.     

Detection of SARS-CoV-2 RNA in a conjunctival swab sample in real-time-polymerase chain reaction positive COVID-19 patients and its association with comorbidity and severity at a designated COVID-19 hospital in Central India

Purpose:To evaluate the presence of SARS-CoV-2 RNA in the conjunctival swab sample of positive confirmed COVID-19 patients and to find out its association with comorbidity and severity of COVID-19 disease.Methods:We conducted an observational cross-sectional study at a dedicated tertiary COVID-19 hospital in central India for a period of 8 weeks from February 2021to March 2021. We included patients who tested positive for SARS-CoV-2 RNA through nasopharyngeal swab and were above 18 years of age. Swab samples have been collected within 48 h of admission. Conjunctival swab was taken from the lower fornix of both eyes and sent to microbiology laboratory for real-time- polymerase chain reaction (RT-PCR).Results:Out of 150 patients, conjunctival swab RT-PCR was positive in five patients (3.33%). Two patients had conjunctival manifestations in the form of conjunctivitis but conjunctival swab RT-PCR was negative in those patients. Among the RT-PCR positive patients, two (40%) were from mild, one (20%) was from moderate, and two (40%) were from severe category. No association could be established between conjunctival swab RT-PCR positivity and severity of the disease or associated comorbidity.Conclusion:Our study provides evidence that SARS-CoV-2 RNA could be detected in conjunctival secretions, and though the risk is relatively low, the eye may act as source of transmission. Extra caution should be taken by healthcare workers, and use of proper precautions like face shields and goggles should be encouraged.     

宫腔微生物群与女性不孕症的研究进展

宫腔微生物群与多种妇科疾病密切相关,微生物群失衡与女性不孕症有关。通过探索不孕症患者宫腔微生物组成及变化后发现,特定菌种出现或菌群构成改变会影响妊娠结局。针对微生物进行相关治疗后,通过成功纠正宫腔微生物群失衡状态可以提高临床妊娠率。为此研究者正深入研究不孕症与宫腔微生物群的相关性,试图通过相关治疗改善子宫内膜容受性,尝试将宫腔微生物状态作为妊娠结局的预测因子。因此,本文就近年来对宫腔微生物群特征、宫腔微生物群与不孕症的关系及治疗的相关性研究进行综述,旨在为进一步研究提供思路。