CⅡTA反义RNA抑制皮肤成纤维细胞表面MHCⅡ类抗原表达

目的探讨用纳米载体介导主要组织相容性复合物(majorhistocompatibilitycomplex,MHC)Ⅱ类抗原转录激活因子(MHCⅡtransactivator,CⅡTA)的反义RNA,抑制皮肤成纤维细胞表面MHCⅡ类抗原表达的可行性。方法将CⅡTA反义RNA(pDarⅡ质粒)稳定转染人类原代皮肤成纤维细胞(pDarⅡ-D),流式细胞仪检测pDarⅡ-D表面经典的MHCⅡ(HLA-DR、-DP、-DQ)类抗原表达,RT-PCR检测CⅡTA及经典的MHCⅡ类分子的mRNA水平。体外混合淋巴细胞反应检测pDarⅡ-D组刺激外周血T细胞反应的能力。结果与正义链组比较,在重组人干扰素(IFN)-γ诱导下,pDarⅡ-D组HLA-DR及-DP抗原诱导性表达分别降低了95.63%、87.89%;同时CⅡTA及经典的MHCⅡ类分子的mRNA含量明显减少(P<0.01);pDarⅡ-D组刺激T细胞分泌IL-2mRNA水平降低(P<0.05)。结论CⅡTA反义片段抑制了自身mRNA含量,从而阻止了其调控的MHCⅡ类分子的相应表达。     

Therapeutic application of sequence-specific binding molecules for novel genome editing tools

Genome editing has been expected to widely increase the available treatment options for various diseases and permit pharmaceutical interventions in previously untreatable conditions. The availability of genome editing tools was dramatically increased by the development of the CRISPR-Cas9 system. However, a number of issues limit the use of the CRISPR-Cas9 system and other gene-editing tools in the clinical treatment of diseases. This review summarized the history and types of genome editing tools and limitations of their use. In addition, the study addressed several next-generation technologies aiming to overcome the limitations of current gene therapy protocols in an effort to accelerate the clinical development of potential treatment options. This review has provided an extensive foundation of the current state of genome editing technology and its clinical development. This review also indicate that the study additionally highlighted the need for multidisciplinary approaches to overcome current bottlenecks in the development of genome editing.     

XIAP反义寡核苷酸对喉癌Hep-2细胞化疗增敏作用的研究

目的 通过X染色体连锁的凋亡抑制蛋白(XIAP)反义寡核苷酸(antisense oligonucleotides,ASODN)转染喉癌Hep-2细胞,探讨其对喉癌Hep-2细胞增殖、凋亡及化疗敏感性的影响.方法 体外培养人喉鳞状细胞癌Hep-2细胞,应用脂质体法进行XIAP ASODN基因转染,荧光显微镜下观察计算转...     

Gallium‐68‐labelled NOTA‐oligonucleotides: an optimized method for their preparation

One of the most essential aspects to the success of radiopharmaceuticals is an easy and reliable radiolabelling protocol to obtain pure and stable products. In this study, we optimized the bioconjugation and gallium‐68 (⁶⁸Ga) radiolabelling conditions for a single‐stranded 40‐mer DNA oligonucleotide, in order to obtain highly pure and stable radiolabelled oligonucleotides. Quantitative bioconjugation was obtained for a disulfide‐functionalized oligonucleotide conjugated to the macrocylic bifunctional chelator MMA‐NOTA (maleimido‐mono‐amide (1,4,7‐triazanonane‐1,4,7‐triyl)triacetic acid). Next, this NOTA‐oligonucleotide bioconjugate was radiolabelled at room temperature with purified and pre‐concentrated ⁶⁸Ga with quantitative levels of radioactive incorporation and high radiochemical and chemical purity. In addition, high chelate stability was observed in physiological‐like conditions (37 °C, PBS and serum), in the presence of a transchelator (EDTA) and transferrin. A specific activity of 51.1 MBq/nmol was reached using a 1470‐fold molar excess bioconjugate over ⁶⁸Ga. This study presents a fast, straightforward and reliable protocol for the preparation of ⁶⁸Ga‐radiolabelled DNA oligonucleotides under mild reaction conditions and without the use of organic solvents. The methodology herein developed will be applied to the preparation of oligonucleotidic sequences (aptamers) targeting the human epidermal growth factor receptor 2 (HER2) for cancer imaging.     

Oral delivery of RNase P ribozymes by Salmonella inhibits viral infection in mice

Safe, effective, and tissue-specific delivery is a central issue for the therapeutic application of nucleic-acid-based gene interfering agents, such as ribozymes and siRNAs. In this study, we constructed a functional RNase P-based ribozyme (M1GS RNA) that targets the overlapping mRNA region of M80.5 and protease, two murine cytomegalovirus (MCMV) proteins essential for viral replication. In addition, a novel attenuated strain of Salmonella, which exhibited efficient gene transfer activity and little cytotoxicity and pathogenicity in mice, was constructed and used for delivery of anti-MCMV ribozyme. In MCMV-infected macrophages treated with the constructed attenuated Salmonella strain carrying the functional M1GS RNA construct, we observed an 80-85% reduction in the expression of M80.5/protease and a 2,500-fold reduction in viral growth. Oral inoculation of the attenuated Salmonella strain in mice efficiently delivered antiviral M1GS RNA into spleens and livers, leading to substantial expression of the ribozyme without causing significant adverse effects in the animals. Furthermore, the MCMV-infected mice that were treated orally with Salmonella carrying the functional M1GS sequence displayed reduced viral gene expression, decreased viral titers, and improved survival compared to the untreated mice or mice treated with Salmonella containing control ribozyme sequences. Our results provide direct evidence that oral delivery of M1GS RNA by Salmonella-based vectors effectively inhibits viral gene expression and replication in mice. Moreover, this study demonstrates the utility of Salmonella-mediated oral delivery of RNase P ribozyme for gene-targeting applications in vivo.     

胰腺癌MiR-224的表达与细胞增殖和凋亡

目的:分析miR-224在胰腺癌组织中的表达,探讨其在胰腺癌细胞增殖、细胞周期及凋亡中的意义.方法:采用TagMan MGB探针法定量分析40例原发性胰腺癌及对应的癌旁组织MiR-224的表达;利用反义技术降低胰腺癌细胞(Aspc-1和Bxpc-3)中miR-224的表达;采用MTT比色法检测细胞增殖的改变,利用流式细胞技术检测胰腺癌细胞周期和凋亡情况.结果:在40例胰腺癌病例中,43%(17/40)的胰腺癌组织miR-224表达明显高于癌旁组织(P<0.05);反义miR-224转染胰腺癌细胞Aspc-1和Bxpc-3后,miR-224的表达明显降低,Aspc-1和Bxpc-3胰腺癌细胞生长受到明显抑制,其生长主要停滞在G0/G1期,而S期和G2/M期细胞的比例下降;另外降低miR-224的表达,Aspc-1和Bxpc-3胰腺癌细胞早期凋亡明显增加.结论:miR-224在胰腺癌组织中表达上调,降低其表达能明显抑制Aspc-1和Bxpc-3细胞的生长和诱导细胞早期凋亡增加,miR-224有可能成为胰腺癌基因表达调控的新靶点.