The culture of suspended Vero cells is facing difficulties such as low cell viability and long doubling time. To investigate the main reasons for the slow growth and low viability of suspended Vero cells, this study conducted transcriptomic analysis of suspended Vero cells (Vero-XF) and adherent Vero cells (Vero-AD) to screen the differentially expressed genes (DEGs) affecting the growth of suspended cells. In addition, epidermal growth factor (EGF) was supplemented to the culture system to improve the growth of Vero-XF. The results showed that compared with the Vero-AD group, the Vero-XF group had 7 376 significant DEGs. Kyoto encyclopedia of genes and genomes enrichment analysis revealed that the DEGs were mainly enriched in the autophagy and mitophagy pathways. Eleven DEGs were selected and verified by quantitative real-time PCR, which showed up-regulated expression of ATG9B, WIPI2, LAMP2, OPTN, Rab7a, and DEPTOR and down-regulated expression of ATG4D, being consistent with the results of transcriptomic analysis. In addition, the Vero-XF group showed significantly up-regulated expression of ATG101, ATG2A, and STX17 and insignificant change in the expression of NBR1, compared with the Vero-AD group. The protein levels of LC3 and P62 in Vero-XF and Vero-AD were determined by Western blotting, which showed up-regulated expression of LC3Ⅱ/Ⅰ and down-regulated expression of P62 in Vero-XF, indicating a higher level of autophagy. Finally, the exogenous supplementation of EGF at 10, 20, and 30 μg/L in the culture system reduced the autophagy level of Vero-XF by 22.35%, 48.15%, and 71.29%, increased the specific growth rate by 15.48%, 33.33%, and 57.14%, and decreased the apoptosis rate by 2.84%, 15.46%, and 16.23%, respectively. The results of this study preliminarily reveal that the activation of autophagy is one of the reasons for the slow growth of Vero-XF, which provides reference for the subsequent culture of suspended Vero cells.
Animals ; Vero Cells ; Autophagy/genetics* ; Chlorocebus aethiops ; Epidermal Growth Factor/pharmacology* ; Gene Expression Profiling ; Transcriptome ; Cell Survival

Transglutaminase 2 (TGM2) is a ubiquitous multifunctional protein, which is related to the adhesion of different cells and tumor formation. Previous studies found that TGM2 is involved in the interaction between host cells and viruses, but the effect of TGM2 on the proliferation of influenza virus in cells has not been reported. To explore the effect of TGM2 during H1N1 subtype influenza virus infection, a stable MDCK cell line with TGM2 overexpression and a knockout cell line were constructed. The mRNA and protein expression levels of NP and NS1 as well as the virus titer were measured at 48 hours after pot-infection with H1N1 subtype influenza virus. The results showed that overexpression of TGM2 effectively inhibited the expression of NP and NS1 genes of H1N1 subtype influenza virus, while knockout of TGM2 up-regulated the expression of the NP and NS1 genes, and the expression of the NP at protein level was consistent with that at mRNA level. Virus proliferation curve showed that the titer of H1N1 subtype influenza virus decreased significantly upon TGM2 overexpression. On the contrary, the virus titer in TGM2 knockout cells reached the peak at 48 h, which further proved that TGM2 was involved in the inhibition of H1N1 subtype influenza virus proliferation in MDCK cells. By analyzing the expression of genes downstream of influenza virus response signaling pathway, we found that TGM2 may inhibit the proliferation of H1N1 subtype influenza virus by promoting the activation of JAK-STAT molecular pathway and inhibiting RIG-1 signaling pathway. The above findings are of great significance for revealing the mechanism underlying the interactions between host cells and virus and establishing a genetically engineering cell line for high-yield influenza vaccine production of influenza virus.
Animals ; Cell Proliferation ; Dogs ; Humans ; Influenza A Virus, H1N1 Subtype/genetics* ; Influenza, Human ; Madin Darby Canine Kidney Cells ; Protein Glutamine gamma Glutamyltransferase 2

Objective To research the molecular biology characteristics and transient expression in BHK-21 cells of E geue segment from Japanese encephalitis virus(JEV) and construct an eukaryotic expression vector pEGFP-C1-JEV.Methods E gene segment of JEV was amplified by RT-PCR,construct the recombinant vector pEGFP-C1-JEV,which could express EGFP label proteins.Transfect pEGFP-C1-JEV vector into BHK-21 via LipofectAMINETM 2000,to observe expressing of EGFP label protein and transcription of aim gene,and to check up localization and antigenicity of expressed E protein by Immunohistochemistry and Western blot.Results It showed that the recombinant plasmid pEGFP-C1-JEV was successfully constructed and transfected to BHK-21 cells,the normal expression of green fluorescent protein expression rate was higher.RT-PCR showed that gene transcription in BHK-21 and normal expression,expression protein was mainly distributed in the cytoplasm of BHK-21 cells and the envelope in,and can with guinea pig anti-JEV antibody binding.Conclusion pEGFP-C1-JEV vector in BHK-21 cells was normal expression and there were no effect on cell growth and morphology.Meanwhile,on eukaryotic antigens was good antigenicity.This research as a base foundation for E protein gene of JEV eukaryotic expression and function in vitro and applied research.

BACKGROUND:Industrialization of new-born bovine serum and abundant resource of bovine lendon enable industrialization of medical collagen sponge.OBJECTIVE:To prepare collagen sponge with new-born bovine tendons by inoculating Veto cells,primary embryo skin cell of Tianzhu White Yak and lamb testicle cell of Minxian black fur sheep of the tissue scaffold of collagen sponge.and observe the biocompatibility of collagen sponge with three different cells.DESIGN,TIME AND SETTING:Repetitive measurement was performed at the Key Laboratory of State Nationalities Afrairs Committee,College of Life Sciences and Engineering,Northwest University for Nationalities from February to May 2006.MATERIALS:Tendons of new-born bovine within 24 hours were digested by glacial acetic acid and pepsinum firstly and then salting-out,dialysis and vacum freeze drying were performed to prepare collagen sponge.f2 passage of embryo skin cells of Tianzhu Whit Yak and f2 passage of lamb testicle cells of Minxian black fur sheep were prepared by out laboratory;Vero cells were provided by Union Medical University.METHODS:In a 6-hole plate,the Vero cell,embryo skin cells of Tianzhu White Yak and lamb testicle cells of Minxian Black Fur Sheep were inoculated into the collagen sponge pretreated by ultraviolet and sterilized by ozone.and incubated in 5%CO2 at 37℃.In addition,cells only inoculated ia a culture plate served as control. MAIN OUTCOME MEASURES:Inverted phase contrast microscope was used to observe cell growth condition in the collagen sponge and 6-hole plate at 5,12,24 and 72 hours.In addition,Coomassie brilliant blue as well as HE staining were conducted at 11 days after culture to identify the culture.RESULTS:Five hours after inoculation,cell adherence and expansion was observed at the bottom of culture plate.and some of the cells showed division.On the surface of collagen sponge.a round cell arrangement was observed.After inoculation of 48-72 hours,monolayer was found at the bottom of the plate.On the 11th day of culture.Coomassie brilliant blue and HE staining of three kinds of cells showed there were lager amount of cells well grew in the holes of collagen sponge,and the collagen sponge turned to be eminent,transparent and tenacious.CONCLUSION:The collagen sponge made from new-born bovine tendons exhibit good biocompatibility with three kinds of cells from different animals and tissues,and can be served as culture seaffoId of skin cells,tenal ceils.and testicle cells.

BACKGROUND:It is confirmed that collagen sponge prepared from human tendon,bovine tendon,rat tail,pig skin and newborn bovine tendon have good cytocompatibility.OBJECTIVE:To extract collagen from newborn bovine skin,prepare the collagen sponge for biomedical application,and observe the biocompatibility and cytocompatibility of collagen sponge with lamb fibroblasts.DESIGN,TIME AND SETTING:Controlled study was performed in the Key Laboratory of Bioengineering of State Ethical Committee,Life Science and Engineering College,Northwest University for Nationalities from May 2006 to February 2007.MATERIALS:Newborn Galiba bovine within 24 hours and black fur lamb kidney fibroblasts were used.METHODS:Newborn bovine skin was harvested to prepare the collagen sponge with a series of procedures,including depilation,pepsin+glacial acetic acid,salting-out,dialysis and freeze drying.The obtained collagen sponge was inoculated with fibroblast suspension,which were divided into collagen sponge group,negative control group (saline) and positive control group (rubber bung leaching liquor).MAIN OUTCOME MEASURES:Inverted phase contrast microscope and JVC digital camera system were used to observe the cell morphology and growth.Acridine orange dyeing was used to observe the proliferation of cell in collagen sponge at 20 and 35 days of culture.Hematoxylin-eosin staining was used to observe the growth of cells in collagen sponge at 65 days of culture.RESULTS:The cells of positive control group were not adhesive and all died three days later.Those of collagen sponge group and negative control group were normal and adhesive.With the prolong of culture time,the sponge pore decreased gradually,sponge appearance became eminent and transparent,the cell increased in number but decreased in morphology.Acridine orange dyeing at 20 and 35 days of culture showed that a large amount of cells appeared in the co-culture of collagen sponge with lamb kidney fibroblast,and pack of cell clumps grew.Abundant blue nuclei and newborn red collagen fiber were found by hematoxylin-eosin staining.CONCLUSION:The collagen sponge from newborn bovine skin has a good biocompatibility with lamb kidney fibroblast cell of black fur,and no cytotoxicity appears.