Partial Alleviation of Homologous Superinfection Exclusion of SeMNPV Latently Infected Cells by G1 Phase Infection and G2/M Phase Arrest.

Viral infection can regulate the cell cycle, thereby promoting viral replication. Hijacking and altering the cell cycle are important for the virus to establish and maintain a latent infection. Previously, Spodoptera exigua multiple nucleopolyhedrovirus (SeMNPV)-latently infected P8-Se301-C1 cells, which grew more slowly than Se301 cells and interfered with homologous SeMNNPV superinfection, were established. However, the effects of latent and superinfection with baculoviruses on cell cycle progression remain unknown. In this study, the cell cycle profiles of P8-Se301-C1 cells and SeMNPV or Autographa californica multiple nucleopolyhedrovirus (AcMNPV)-infected P8-Se301-C1 cells were characterized by flow cytometry. The results showed that replication-related genes MCM4, PCNA, and BAF were down-regulated (p < 0.05) in P8-Se301-C1 cells, and the S phase of P8-Se301-C1 cells was longer than that of Se301 cells. P8-Se301-C1 cells infected with SeMNPV did not arrest in the G2/M phase or affect the expression of Cyclin B and cyclin-dependent kinase 1 (CDK1). Furthermore, when P8-Se301-C1 cells were infected with SeMNPV after synchronized treatment with hydroxyurea and nocodazole, light microscopy and qRT-PCR analysis showed that, compared with unsynchronized cells and S and G2/M phase cells, SeMNPV-infected P8-Se301-C1 cells in G1 phase induced G2/M phase arrest, and the amount of virus adsorption and intracellular viral DNA replication were significantly increased (p < 0.05). In addition, budded virus (BV) production and occlusion body (OB)-containing cells were both increased at 120 h post-infection (p < 0.05). The expression of Cyclin B and CDK1 was significantly down-regulated at 48 h post-infection (p < 0.05). Finally, the arrest of SeMNPV-infected G1 phase cells in the G2/M phase increased BV production (p < 0.05) and the number of OB-containing cells. In conclusion, G1 phase infection and G2/M arrest are favorable to SeMNPV proliferation in P8-Se301-C1 cells, thereby alleviating the homologous superinfection exclusion. The results contribute to a better understanding of the relationship between baculoviruses and insect cell cycle progression and regulation.

Research progress on the disease burden of pneumoconiosis in China / 中华劳动卫生职业病杂志

Pneumoconiosis is the largest and most serious disease among the legal occupational diseases in China, which causes long-term heavy disease burden to individuals, enterprises and society. How to scientifically and reasonably measure and reduce the health impact and economic loss caused by pneumoconiosis has become a key and difficult research topic. In recent years, with the development of global burden of disease (GBD) research, some scholars have adopted disease burden index to evaluate the disease burden of pneumoconiosis, but the research results and data are relatively independent, and there is a lack of systematic evaluation system and framework. This paper summarized the application of disease burden assessment index for pneumoconiosis, epidemiological and economic burden of pneumoconiosis, and the cost-effectiveness of reducing the burden. This paper aims to understand the present situation of pneumoconiosis disease burden in our country, discover the problems and challenges of pneumoconiosis disease burden research in our country now. It provides scientific basis for the research and application of pneumoconiosis and other occupational disease burden in China, as well as the formulation of comprehensive intervention measures, optimization of health resources allocation and reduction of disease burden.

Effect of miR-204 targeted regulation of DVL3 gene in silica-induced mouse lung epithelial cells / 中华劳动卫生职业病杂志

Objective: To construct a recombinant lentiviral vector for mouse miR-204 overexpression, and to verify the targeted regulation of miR-204 and DVL3 in silica (SiO(2)) -induced mouse lung epithelial cells (MLE-12 cells) . Methods: In October 2019, the pre-miR-204 gene was amplified from the mouse genome by the polymerase chain reaction (PCR) method. After sequencing, the amplified product was cloned into the pLenti-CMV-EGFP lentiviral vector. The positive clones were identified by PCR screening and sequencing. The miR-204 overexpressed lentiviral vector was transfected into 293T cells, and lentiviral packaging and titer determination were performed. The experiment was divided into SiO(2) control group, virus control group, and miR-204 virus group, and the expressions of miR-204 and DVL3 gene were detected by real-time PCR. Results: The miR-204 lentiviral expression vector Lv-miR-204-5p was constructed and identified correctly by PCR and sequencing, and a virus dilution with a titer of 9.57×10(8) IU/ml was obtained. The results of real-time PCR showed that the expression of miR-204 in MLE-12 cells of the miR-204 virus group was higher than that of SiO(2) control group and virus control group, and the expression of DVL3 gene was lower than that of SiO(2) control group and virus control group, the differences were statistically significant (P<0.05) . Conclusion: Overexpression of miR-204 by lentiviral vector may inhibit the expression of DVL3 gene in silica-induced mouse lung epithelial cells.

Ectopic bone formation of human bone morphogenetic protein-2 gene transfected goat bone marrow-derived mesenchymal stem cells in nude mice / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>To evaluate the osteogenic potential of bone morphogenetic protein (BMP)-2 gene transfected goat bone marrow-derived mesenchymal stem cells (MSCs).</p><p><b>METHODS</b>Goat bone marrow-derived MSCs were transfected by Adv-human bone morphogenetic protein (hBMP)-2 gene (Group 1), Adv-beta gal transfected MSCs (Group 2) and uninfected MSCs (Group 3). Western blot analysis, alkaline phosphatase staining, Von Kossa staining and transmission electron microscopy were adopted to determine the phenotype of MSCs. Then the cells were injected into thigh muscles of the nude mice. Radiographical and histological evaluations were performed at different intervals.</p><p><b>RESULTS</b>Only Adv-hBMP-2 transfected MSCs produced hBMP-2. These cells were positive for alkaline phosphatase staining at the 12th day and were positive for Von Kossa staining at the 16th day after gene transfer. Electron microscopic observation showed that there were more rough endoplasmic reticulum, mitochondria and lysosomes in Adv-hBMP-2 transfected MSCs compared to MSCs of other two groups. At the 3rd and 6th weeks after cell injection, ectopic bones were observed in muscles of nude mice of Group 1. Only fibrous tissue or a little bone was found in other two groups.</p><p><b>CONCLUSIONS</b>BMP-2 gene transfected MSCs can differentiate into osteoblasts in vitro and induce bone formation in vivo.</p>

Osteogenic potential of hBMP-2 gene transduced human adipose-derived stem cells / 中华创伤杂志

Objective To investigate the ostengenie potential of adipose-derived stem cells(AD- SCs)when exposed to adenovirns containing hBMP-2 cDNA(Adv-hBMP-2)and offer a choice of cell source for gene therapy and tissue engineering.Methods Human adipose tissues were obtained from patients who received orthopaedic surgery or liposuction.ADSCs were obtained by digesting the adipose tissues.Firstly,flowcytometric analysis was performed for the confirmation of mesenchymal stem cell ori- gin and the surface markers including CD34,CD44,CD68,CD71,CD90,and CD105.The ADSCs were transfected by Adv-hBMP-2 and the effects were tested in vitro,lmmunoprecipitation and Western blotting and ELISA were performed for confirming BMP gone transduction and its stable expression.The transform of ADSCs was assessed by extracellular ALP staining,intracellular ALP spectrophotometry,von Kossa staining and RT-PCR.In the in vivo experiment ADSC-Adv-hBMP-2 cells were injected into the hind limb of nude mice and analyzed radiographically and histologically.Results ADSCs were successfully isolated from human adipose tissues.The isolated ADSCs expressed CD44,CD71,CD90 and CD105 and CD34 and CD68 were absent.The result confirmed the mesenchymal stem cell origin of the cells.West- ern blotting and ELISA confirmed successful and persistent hBMP-2 production by ADSC-Adv-hBMP-2 cells.Extracellular ALP staining,intracellular ALP spectrophotometry,yon Kossa staining and RT-PCR revealed that ADSCs treated with Adv-hBMP-2 had a tendency of transfering into osteoblast.X-ray and H&E sections from hind limb of nude mice injected with ADSC-Adv-hBMP-2 cells confirmed bone forma- tion at 2 weeks.Conclusions Liposuction aspirates contain abundant ADSCs that can be transduced with hBMP-2 gene,and the tranduced ADSCs differentiate into the osteoblast.ADSCs may be an ideal source of mesenchyme-lineage stem cells for gone therapy and tissue engineering.