AAV-mediated expression of p65shRNA and bone morphogenetic protein 4 synergistically enhances chondrocyte regeneration

BACKGROUND:Adeno-associated virus(AAV)gene therapy has been proven to be reliable and safe for the treatment of osteoarthritis in recent years.However,given the complexity of osteoarthritis pathogenesis,single gene manipulation for the treatment of osteoarthritis may not produce satisfactory results.Previous studies have shown that nuclear factorκB could promote the inflammatory pathway in osteoarthritic chondrocytes,and bone morphogenetic protein 4(BMP4)could promote cartilage regeneration.OBJECTIVE:To test whether combined application of AAV-p65shRNA and AAV-BMP4 will yield the synergistic effect on chondrocytes regeneration and osteoarthritis treatment.METHODS:Viral particles containing AAV-p65-shRNA and AAV-BMP4 were prepared.Their efficacy in inhibiting inflammation in chondrocytes and promoting chondrogenesis was assessed in vitro and in vivo by transfecting AAV-p65-shRNA or AAV-BMP4 into cells.The experiments were divided into five groups:PBS group;osteoarthritis group;AAV-BMP4 group;AAV-p65shRNA group;and BMP4-p65shRNA 1:1 group.Samples were collected at 4,12,and 24 weeks postoperatively.Tissue staining,including safranin O and Alcian blue,was applied after collecting articular tissue.Then,the optimal ratio between the two types of transfected viral particles was further investigated to improve the chondrogenic potential of mixed cells in vivo.RESULTS AND CONCLUSION:The combined application of AAV-p65shRNA and AAV-BMP4 together showed a synergistic effect on cartilage regeneration and osteoarthritis treatment.Mixed cells transfected with AAV-p65shRNA and AAV-BMP4 at a 1:1 ratio produced the most extracellular matrix synthesis(P<0.05).In vivo results also revealed that the combination of the two viruses had the highest regenerative potential for osteoarthritic cartilage(P<0.05).In the present study,we also discovered that the combined therapy had the maximum effect when the two viruses were administered in equal proportions.Decreasing either p65shRNA or BMP4 transfected cells resulted in less collagen II synthesis.This implies that inhibiting inflammation by p65shRNA and promoting regeneration by BMP4 are equally important for osteoarthritis treatment.These findings provide a new strategy for the treatment of early osteoarthritis by simultaneously inhibiting cartilage inflammation and promoting cartilage repair.

基于城市公共交通移动模型的协作延迟容忍网络路由策略

如何利用有限的传输机会可靠地传送车载服务感知信息是智能交通发展的"瓶颈"问题,利用公共交通中车辆的运动规律,提出基于节点之间机会接触来进行消息的逐跳转发策略,同时结合公共交通系统自身的特点,设计了一种基于公共交通移动模型的协作延迟容忍网络(DTN)路由算法TF。首先,根据公共交通移动模型自身的特点,将公交、长途客车等节点按其运动路径进行分组,提出一种基于固定运动路径分组的DTN路由算法;然后,将出租车、行人类节点定义为自由节点,并设计了一种基于转发因子控制的DTN路由策略作为分组路由机制的补充。仿真结果表明,与Epidemic、Prophet以及SAW路由算法相比,TF路由算法具有较高的消息投递率和较低的平均延迟。

Msx homeobox gene family and craniofacial development

Vertebrate Msx genes are unlinked,homeobox-containing genes that bear homology to the Drosophila muscle segment homeobox gene.These genes are expressed at multiple sites of tissue-tissue interactions during vertebrate embryonic development.Inductive interactions mediated by the Msx genes are essential for normal craniofacial,limb and ectodermal organ morphogenesis,and are also essential to survival in mice,as manifested by the phenotypic abnormalities shown in knockout mice and in humans.This review summarizes studies on the expression,regulation,and functional analysis of Msx genes that bear relevance to craniofacial development in humans and mice.

The ubiquitin-specific protease 17 is involved in virus-triggered type I IFN signaling

Viral infection initiates a series of signaling cascades that activate the transcription factors nuclear factor kappa B and interferon regulatory factor 3, which collaborate to induce transcription of genes for type I interferons （IFNs） and other cytokines. Here we report that the deubiquitinating enzyme ubiquitin-specific protease 17 （USP17） is required for virus-induced RIG-I- and melanoma differentiation-associated protein-5 （MDA5）-mediated type I IFN signaling. Knockdown of endogenous USP17 inhibited virus-, cytoplasmic poly（I：C）- and poly（dA：dT）-induced activation of the IFN-β promoter and cellular antiviral responses. We further found that knockdown of USP17 inhibited RIG-I- and MDA5-induced but not downstream activator-induced activation of the IFN-β promoter, which was correlated with an increase in ubiquitination levels of RIG-I and MDA5. Taken together, our findings suggest that USP17 functions through deubiquitination of RIG-I and MDA5 to regulate virus-induced type I IFN signaling.

Phylogeny of forkhead genes in three spiralians and their expression in Pacific oyster Crassostrea gigas

The Fox genes encode a group of transcription factors that contain a forkhead domain, which forms a structure known as a winged helix. These transcription factors play a crucial role in several key biological processes, including development. High-degree identity in the canonical forkhead domain has been used to divide Fox proteins into 23 families （FoxA to FoxS）. We surveyed the genome of three spiralians, the oyster Crassostrea gigas, the limpet Lottia gigantea, and the annelid Capitella teleta. We identified 25 C. gigas fox genes, 21 L. gigantea fox genes, and 25 C. teleta fox genes. The C. gigas fox and L. giganteafox genes represented 19 of the 23 families, whereas FoxI, QI, R, and S were missing. The majority of the Fox families were observed within the C. teletafox genes, with the exception of FoxR and S. In addition, thefoxAB-like gene,foxY-like gene, andfoxH gene were also present in the three genomes. The conserved FoxC-FoxL 1 cluster, observed in mammals, was also found in C. gigas. The diversity of temporal expression patterns observed across the developmental process implies the C. gigasfox genes exert a wide range of functions. Further functional studies are required to gain insight into the evolution of Fox genes in bilaterians.

Characterization of flounder (Paralichthys olivaceus) FoxD5 and its function in regulating myogenic regulatory factor

As one member of winged helix domain transcription factors, FoxD5 was reported to be a trunk organizer. Recent study showed that zebrafish foxd5 is expressed in the somites. To further understand the function of FoxD5 in fish muscle development, the FoxD5 gene was isolated from flounder. Its expression pattern was analyzed by in situ hybridization, while its function in regulating myogenic regulatory factor, MyoD, was analyzed by ectopic expression. It showed that flounder FoxD5 was firstly expressed in the tailbud, adaxial cells, and neural plate of the head. In flounder embryo, FoxD5 is expressed not only in forebrain but also in somite cells that will form muscle in the future. When flounder FoxD5 was over-expressed in zebrafish by microinjection, the expression of zebrafish MyoD in the somites was reduced, suggesting that FoxD5 is involved in myogenesis by regulating the expression of MyoD.

Mechanistic basis and clinical relevance of the role of transforming growth factor-β in cancer

Transforming growth factor-β(TGF-β) is a key factor in cancer development and progression. TGF-β can suppress tumorigenesis by inhibiting cell cycle progression and stimulating apoptosis in early stages of cancer progression. However, TGF-β can modulate cancer-related processes, such as cell invasion, distant metastasis, and microenvironment modification that may be used by cancer cells to their advantage in late stages. Corresponding mechanisms include angiogenesis promotion, anti-tumor immunity suppression, and epithelial-to-mesenchymal transition(EMT) induction. The correlation between TGF-β expression and cancer prognosis has also been extensively investigated. Results suggest that TGF-β pathway can be targeted to treat cancer; as such, the feasibility of this treatment is investigated in clinical trials.

Preliminary evidence that overexpression of nuclearfactor for IL6 expression (NF-IL6) in NIH3T3 cells may berelated to malignant transformation

NF-IL6 is a member of c/EBP family and has multiple functions in regulation of cellular gene expression. We have constructed NF-IL6 expression plasmids and trans fected the NIH3T3 cells with them. The sense NF-IL6 transfectants showed significantly increased tumorigenicity, and the stable integration of NF-IL6 cDNA into cellular DNA and its expression were demonstrated. Our results suggest that NF-IL6 may be related to tumorigenesis.

Effect of transforming growth factor beta and bone morphogenetic proteins on rat hepatic stellate cell proliferation and transdifferentiation

AIM: To explore different roles of TGF-β (transforming growth factor beta) and bone morphogenetic proteins (BMPs)in hepatic stellate cell proliferation and trans-differentiation.METHODS: Hepatic stellate cells were isolated from male Sprague-Dawley rats. Sub-cultured hepatic stellate cells were employed for cell proliferation assay with WST-1 reagent and Western blot analysis with antibody against smooth muscle alpha actin (SMA).RESULTS: The results indicated that TGF-β1 significantly inhibited cell proliferation at concentration as low as 0.1 ng/ml, but both BMP-2 and BMP-4 did not affect cell proliferation at concentration as high as 10 ng/ml. The effect on hepatic stellate cell trans-differentiation was similar between TGFβ1 and BMPs. However, BMPs was more potent at transdifferentiation of hepatic stellate cells than TGF-β1. In addition, we observed that TGF-β1 transient reduced the abundance of SMA in hepatic stellate cells.CONCLUSION: TGF-β may be more important in regulation of hepatic stellate cell proliferation while BMPs may be the major cytokines regulating hepatic stellate cell transdifferentiation.

CO-TRANSFECTION OF RAT BONE MARROW MESENCHYMAL STEM CELLS WITH HUMAN BMP2 AND VEGF165 GENES

Objective To explore the feasibility and efficacy of lentivirus-mediated co-transfection of rat bone marrow mesenchymal stem cells （MSCs） with human vascular endothelial growth factor 165 （hVEGFI65） gene and human bone morphogenetic protein 2 （hBMP2） gene. Methods The hVEGF165 and hBMP2 cDNAs were obtained from human osteosarcoma cell line MG63 and cloned into lentiviral expression vectors designed to co-express the copepod green fluorescent protein （copGFP）. The expression lentivector and packaging Plasmid Mix were co-transferred to 293TN cells, which produced the lentivirus carrying hVEGF165 （Lv-VEGF） or hBMP2 （ Lv-BMP） , respectively. MSCs of Wistar rats were co-transfected with Lv-BMP and Lv-VEGF （BMP ＋ VEGF group）, or each alone （BMP group and VEGF group）, or with no virus （ Control group）. The mRNA and protein expressions of hVEGF165 and hBMP2 genes in each group were detected by real-time PCR and enzyme linked immunosorbent assay （ELISA）. Results Lentiviral expression vectors carrying hVEGF165 or hBMP2 were correctly constructed and confirmed by restriction endonucleses analysis and DNA sequencing analysis. A transfer efficiency up to 90% was archieved in all the transfected groups detected by the fraction of fluorescent cells using fluorescent microscopy. From the results generated by real-time PCR and ELISA, VEGF165 and BMP2 genes were co-expressed in BMP ＋ VEGF group. No significant difference of BMP2 expression was detected between BMP ＋ VEGF and BMP groups （ P 〉 0. 05）. Similarly, there was no significant difference of VEGF165 expression between BMP ＋ VEGF and VEGF groups （ P 〉 0. 05）. Conclusion VEGF165 and BMP2 genes were successfully co-expressed in MSCs by lentivirus-mediated co-transfection, which provided a further foundation for the combined gene therapy of bone regeneration.

Identification and expression of amphioxus AmphiSmad1/5/8 and AmphiSmad4

Smad family proteins are identified as intracellular signal mediators of the TGF-β superfamily.In this study,we identified two novel members of the Smad family,termed as AmphiSmad1/5/8 and AmphiSmad4,from Chinese amphioxus.Both AmphiSmad1/5/8 and AmphiSmad4 showed a typical domain structure of Smad proteins consisting of conserved MH1 and MH2 domains.Phylogenetic analysis placed AmphiSmad1/5/8 in the Smad1,5 and 8 subgroup of the R-Smad subfamily,and AmphiSmad4 in the Co-Smad subfamily.The spatial and temporal gene expression patterns of AmphiSmad1/5/8 and AmphiSmad4 showed that they may be involved in the embryonic development of notochord,myotome and alimentary canal,and may help to establish the specification of dorsal-ventral axis of amphioxus.Moreover,AmphiSmad1/5/8 and AmphiSmad4 showed extensive distribution in all adult tissues examined,suggesting that these two genes may play important roles in the morphogenesis of a variety of tissues especially notochord and gonad.

Foxn4:A multi-faceted transcriptional regulator of cell fates in vertebrate development

Vertebrate development culminates in the generation of proper proportions of a large variety of different cell types and subtypes essential for tissue,organ and system functions in the right place at the right time.Foxn4,a member of the forkhead box/winged-helix transcription factor superfamily,is expressed in mitotic progenitors and/or postmitotic precursors in both neural(e.g.,retina and spinal cord)and non-neural tissues(e.g.,atrioventricular canal and proximal airway).During development of the central nervous system,Foxn4 is required to specify the amacrine and horizontal cell fates from multipotent retinal progenitors while suppressing the alternative photoreceptor cell fates through activating Dll4-Notch signaling.Moreover,it activates Dll4-Notch signaling to drive commitment of p2 progenitors to the V2b and V2c interneuron fates during spinal cord neurogenesis.In development of non-neural tissues,Foxn4 plays an essential role in the specification of the atrioventricular canal and is indirectly required for patterning the distal airway during lung development.In this review,we highlight current understanding of the structure,expression and developmental functions of Foxn4 with an emphasis on its cell-autonomous and non-cell-autonomous roles in different tissues and animal model systems.