鹿茸口服液促进RNA和蛋白质合成作用的研究

目的：研究鹿茸口服液对青年及老年小鼠RNA和蛋白质合成的作用。方法：观察口服鹿茸口服液对小鼠肝RNA和蛋白质含量及^3H-Uridine和^3H-Leucine掺入的影响。结果：鹿茸口服液可明显增加老年小鼠肝RNA和蛋白质含量及[^3H]Uridine和[^3H]Leucine掺入RNA和蛋白质，作用明显强于青年小鼠。结论：鹿茸口服液对老年小鼠RNA和蛋白质合成有明显促进作用。     

mRNA差异显示法筛选和克隆胃癌相关基因

目的 :筛选和克隆胃印戒细胞癌与正常胃黏膜细胞差异表达基因片段。方法 :采用mRNA差异显示法 (mRNAdifferentialdisplayPCR ,DD PCR) ,对比胃印戒细胞癌组织与正常胃黏膜组织mRNA的表达差异 ,克隆胃印戒细胞癌差异片段 ,经过RNA的斑点杂交、测序以及序列分析和同源性比较。结果 :胃印戒细胞癌与正常胃黏膜中存在明显的基因表达差异 ,发现与胃癌相关的差异表达条带 16条 ,其中 7条为缺失表达条带 ,9条为过度表达条带。对其中 4条差异表达条带进行克隆、测序 ,经序列分析及同源性比较和RNA的斑点杂交 ,表明确认其中 1条为GenBank/BLAST中尚未收录的片段。结论 :胃印戒细胞癌的mRNA差异显示证明 ,该EST序列可能在胃癌的发生发展中具有一定作用     

肝素钠细菌内毒素检查法的建立

目的: 建立肝素钠原料药的细菌内毒素检查方法.方法: 通过干扰评价试验,证明肝素钠对细菌内毒素检查有抑制作用,使用稀释剂(I)适当补充二价阳离子,对样品作适当稀释,以消除其干扰抑制作用.结果: 补充适量的二价阳离子可以消除干扰作用.结论: 肝素钠原料药可以建立细菌内毒素检查法.     

RNA干扰及其技术的应用

RNA干扰(RNAi)是细胞内由双链RNA诱导降解与其配对的特定mRNA的过程。细胞内双链RNA在酶的作用下,形成20-25碱基大小的小干扰RNA(si RNAs),由si RNAs进一步掺入多组分核酸酶并使其激活,从而精确降解与si RNAs序列相同的mRNA,抑制该基因在细胞内的翻译表达。介绍了RNA干扰的分子机制、制备方法、以及RNA干扰技术在功能基因组学、微生物学、基因治疗和信号转导等研究领域里的应用。     

激光捕获显微切割结合RNA线性扩增技术在膀胱移行细胞癌相关基因研究中的应用

目的当对有间质细胞混杂的膀胱移行上皮细胞与膀胱移行细胞癌进行基因差异表达分析时,激光捕获显微切割技术(lasercap-turemicrodissection,LCM)是不可缺少的,然而从LCM所得的细胞中获取足够RNA量相当困难。本文首次将LCM与RNA线性扩增结合用于膀胱癌相关基因研究,目的是确定一条切实可行的技术路线,将膀胱移行上皮细胞从膀胱粘膜中分离出来,将膀胱癌细胞从肿瘤间质细胞中分离出来,并对其进行RNA体外线性扩增,以备进一步研究。方法采用LCM技术分别从正常膀胱粘膜及膀胱癌组织冰冻切片中获取膀胱移行上皮细胞及膀胱癌细胞,提取RNA,并对微量RNA进行体外线性扩增。然后用RT-PCR验证扩增前、后RNA中β-actin基因表达水平。结果对照实验I证实经LCM后RNA完整性较好;经对照实验Ⅱ初步确定设定条件下LCMshooting次数与可获得RNA量间对应关系。从膀胱粘膜种捕获膀胱移行上皮细胞25万shootings;从膀胱癌组织中获取癌细胞20万shootings。产物RNA扩增后获得片段大小为0.5-2.5kh的aRNA,且β-actin基因表达表达完整。结论LCM结合RNA体外线性扩增技术能成功地获取较为单一的研究目的细胞,扩增后RNA完整性较好,能用于进一步研究中。     

A Unique Robust Dual-Promoter-Driven and Dual-Reporter-Expressing SARS-CoV-2 Replicon: Construction and Characterization

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2, SARS2) remains a great global health threat and demands identification of more effective and SARS2-targeted antiviral drugs, even with successful development of anti-SARS2 vaccines. Viral replicons have proven to be a rapid, safe, and readily scalable platform for high-throughput screening, identification, and evaluation of antiviral drugs against positive-stranded RNA viruses. In the study, we report a unique robust HIV long terminal repeat (LTR)/T7 dual-promoter-driven and dual-reporter firefly luciferase (fLuc) and green fluorescent protein (GFP)-expressing SARS2 replicon. The genomic organization of the replicon was designed with quite a few features that were to ensure the replication fidelity of the replicon, to maximize the expression of the full-length replicon, and to offer the monitoring flexibility of the replicon replication. We showed the success of the construction of the replicon and expression of reporter genes fLuc and GFP and SARS structural N from the replicon DNA or the RNA that was in vitro transcribed from the replicon DNA. We also showed detection of the negative-stranded genomic RNA (gRNA) and subgenomic RNA (sgRNA) intermediates, a hallmark of replication of positive-stranded RNA viruses from the replicon. Lastly, we showed that expression of the reporter genes, N gene, gRNA, and sgRNA from the replicon was sensitive to inhibition by Remdesivir. Taken together, our results support use of the replicon for identification of anti-SARS2 drugs and development of new anti-SARS strategies targeted at the step of virus replication.     

Human Retrovirus Genomic RNA Packaging

Two non-covalently linked copies of the retrovirus genome are specifically recruited to the site of virus particle assembly and packaged into released particles. Retroviral RNA packaging requires RNA export of the unspliced genomic RNA from the nucleus, translocation of the genome to virus assembly sites, and specific interaction with Gag, the main viral structural protein. While some aspects of the RNA packaging process are understood, many others remain poorly understood. In this review, we provide an update on recent advancements in understanding the mechanism of RNA packaging for retroviruses that cause disease in humans, i.e., HIV-1, HIV-2, and HTLV-1, as well as advances in the understanding of the details of genomic RNA nuclear export, genome translocation to virus assembly sites, and genomic RNA dimerization.     

改进Trizol法提取白色念珠菌总RNA初探

目的:比较Trizol法、改进Trizol法和超声粉碎法提取白色念珠菌总RNA产量、纯度和完整性的差异。方法:对生长至静止期的白色念珠菌标准株ATCC10231分别采用Trizol法、改进Trizol法和超声粉碎法提取总RNA,用紫外分光光度计测量其A260,A280的值,并进行琼脂糖凝胶电泳,对总RNA的产量、纯度和完整性进行比较。结果:三种方法提取白色念珠菌总RNA产量分别为1.904μg、7.208μg和5.000μg,A260/A280比值分别为1.896、1.415和1.612。电泳结果显示改进Trizol法提取的总RNA呈现28 s rRNA,18 s rRNA和5 s rRNA三条清晰的条带,而Trizol法和超声粉碎法提取的总RNA均有降解。结论:改进Trizol法提取的白色念珠菌总RNA产量及纯度均高于Trizol法和超声粉碎法,且完整性更好。该法优于Trizol法和超声粉碎法。