The effect of Connexin43 downregulation on biological functions of HUVEC / 药学学报

Connexin43 has been shown to play a pivotal role in wound healing process. Wound repair is enhanced by acute downregulation of connexin43, by increasing proliferation and migration of keratinocyte and fibroblast. Angiogenesis is also a central feature of wound repair, but little is known about the effects of connexin43 modulation on functions of endothelial cells. We used connexin43 specific small interference RNA (siRNA) to reduce the expression of connexin43 in human umbilical vein endothelial cell (HUVEC), and investigated the effects of connexin43 downregulation on intercellular communication, viability, proliferation, migration and angiogenic activity of HUVEC. Treatment of siRNA markedly reduced the expression of connexin43 by -80% in HUVEC (P < 0.05), and decreased the intercellular communication by -65% (P < 0.05). The viability, proliferation, migration and angiogenic activity of HUVEC decreased significantly (P < 0.05), compared with that of the normal cells. The results suggest that temporally downregulation of connexin43 expression at early stage of wound to inhibit the abnormal angiogenesis characterized with leaky and inflamed blood vessels, maybe a prerequisite for coordinated normal healing process.

Expression of human long-acting FSH in CHO cell and its bioactivity in vivo / 生物工程学报

Follicle-stimulating hormone (FSH) is a pituitary glycoprotein hormone that is essential for the development of ovarian follicles and testicular seminiferous tubules. The relatively short half-life of FSH in vivo requires daily injections for more than 10 days that is inconvenient and possibly contribute to the stress perceived by the patients. The goal of the present study was to increase FSH glycosylation, in order to develop a long-acting recombinant FSH. The cDNA of native alpha and beta subunit of human FSH was linked by a sequence with two N-linked glycosylation sites, and the resulted DNA was inserted into pcDNA3.1 vector to generate a recombinant vector of pcDNA3.1-FSH. The pcDNA3.1-FSH was linearized and transfected into CHO-K1, positive transformants were selected by G418 and confirmed by PCR and Western blotting. A single chain recombinant FSH was expressed, with molecular weight of about 49 kDa. The recombinant FSH expression level in CHO-K1 cell strain in serum-free culture was 3 mg/L. Single injection of this recombinant FSH could induce folliculogenesis and ovulation in rats, the efficacy was similar with the commercially available FSH preparation (Folltropin-V) administrated 8 times consecutively. The results suggested a long-acting FSH was produced successfully.

Ribozyme riboswitch based gene expression regulation systems for gene therapy applications: progress and challenges / 药学学报

Robust and efficient control of therapeutic gene expression is needed for timing and dosing of gene therapy drugs in clinical applications. Ribozyme riboswitch provides a promising building block for ligand-controlled gene-regulatory system, based on its property that exhibits tunable gene regulation, design modularity, and target specificity. Ribozyme riboswitch can be used in various gene delivery vectors. In recent years, there have been breakthroughs in extending ribozyme riboswitch's application from gene-expression control to cellular function and fate control. High throughput screening platforms were established, that allow not only rapid optimization of ribozyme riboswitch in a microbial host, but also straightforward transfer of selected devices exhibiting desired activities to mammalian cell lines in a predictable manner. Mathematical models were employed successfully to explore the performance of ribozyme riboswitch quantitively and its rational design predictably. However, to progress toward gene therapy relevant applications, both precision rational design of regulatory circuits and the biocompatibility of regulatory ligand are still of crucial importance.

Recent progress of the aptamer-based antiviral drugs / 药学学报

Aptamers are capable of binding a wide range of biomolecular targets with high affinity and specificity. It has been widely developed for diagnostic and therapeutic purposes. Because of unique three dimensional structures and cell-membrane penetration, aptamers inhibit virus infection not only through binding specific target, such as the viral envelope, genomic site, enzyme, or other viral components, but also can be connected to each other or with siRNA jointly achieve antiviral activity. Taking human immunodeficiency virus and hepatitis C virus as examples, this paper reviewed the effects and mechanisms of aptamers on disturbing viral infection and replication steps. It may provide an insight to the development of aptamer-based new antiviral drugs.

Engineering and screening of artificial riboswitch as a novel gene control element / 生物工程学报

Various artificial riboswitches have been constructed by utilization of designed aptamers or by modification of natural riboswitch systems, because they can regulate gene expression in a highly efficient, precise and fast way, and promise to supply simple cis-acting, modular, and non-immunogenic system for use in future gene therapy applications. In this review, we present an overview of currently available technologies to design and select engineered riboswitches, and discuss some possible technologies that would allow them highly responsive to non-natural ligands, and dynamic control of gene expression in mammalian cells. Though how to bring custom-designed riboswitches as a novel and versatile tool box to gene control system is still a great challenge, the combination of structure-activity relationship information, computer based molecular design, in vitro selection, and high-through screening will serve as powerful tools for further development of riboswitch based gene regulatory systems.