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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.
Subject(s)
Animals , Dogs , Humans , Cell Proliferation , Influenza A Virus, H1N1 Subtype/genetics , Influenza, Human , Madin Darby Canine Kidney Cells , Protein Glutamine gamma Glutamyltransferase 2ABSTRACT
BACKGROUND: Canine kidney injury is characterized by the apoptosis and necrosis of renal tubular epithelial cells. Recent developments in mesenchymal stem cells and their exosomes research have shown great promise for the treatment of kidney injury in humans, rats and mice, but little research has been done on dogs. OBJECTIVE: To investigate the effects of canine adipose-derived mesenchymal stem cells and their exosomes on canine renal tubular epithelial cell injury induced by gentamicin in vitro. METHODS: Canine renal tubular epithelial cells were treated by 5 mmol/L gentamicin sulfate. Subsequently, canine adipose-derived mesenchymal stem cells and their conditional medium and exosomes were co-cultured with damaged canine renal tubular epithelial cells respectively. After 24 and 48 hours, the cell proliferation activity of each group was measured by cell counting kit-8 method, and the apoptosis rate of each group was detected by flow cytometry. Finally, Q-PCR was used to further reveal the effects of canine adipose-derived mesenchymal stem cell exosomes on PCNA, Bcl-2 and Bax genes in these cells. RESULTS AND CONCLUSION: Canine adipose-derived mesenchymal stem cells, their conditioned media, and exosomes could significantly promote proliferation and reduce apoptosis in damaged canine renal tubular epithelial cells (P < 0.05). Among them, canine adipose-derived mesenchymal stem cell exosomes worked best, which could significantly increase the expression of PCNA and Bcl-2 genes in damaged canine renal tubular epithelial cells (P < 0.05). These results suggest that canine adipose-derived mesenchymal stem cells can repair the canine renal tubular epithelial cell damage induced by gentamicin through their exosomes.
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Objective@#To investigate the best amount of TPCK trypsin in Madin Darby canine kidney (MDCK) cell suspension for the culture of H7N9 avian influenza virus.@*Methods@#Different concentrations of TPCK trypsin were added during the periods of cell growth and virus production. Their effects on cell growth, viability, glucose and lactate metabolism, and hemagglutination titer were monitored every 12 h. Inter-batch differences were analyzed. The amount of trypsin added in the cell growth phase was 0, 1 μg/ml, 2 μg/ml, 4 μg/ml, 6 μg/ml, 8 μg/ml, 10 μg/ml and 15 μg/ml. The amount of trypsin added during the virus production period was 0, 0.5 μg/ml, 1 μg/ml, 1.5 μg/ml, 2 μg/ml and 2.5 μg/ml. When the hemagglutination titers were same, the adding amount was further optimized at different multiplicity of infection (MOI) of 0.001, 0.005, 0.025 and 0.05.@*Results@#No significant linear effects of TPCK trypsin concentration on cell number, viability, and glucose and lactate metabolism were observed. No toxicity to cell growth was observed when TPCK trypsin concentration reached 15 μg/ml. After the inoculation of H7N9 avian influenza virus, the hemagglutination titers in the 1 μg/ml, 1.5 μg/ml, 2 μg/ml and 2.5 μg/ml TPCK trypsin groups reached the peaks at 48 h, which were 1∶26.5. At 60 h, the hemagglutination titers of the latter two groups decreased faster than those of the former two groups. When the MOI was 0.005, the hemagglutination titer of the 1.5 μg/ml group at 48 h was 26.5 higher than 26 in the 1 μg/ml group under the same condition. There were differences between different batches of TPCK trypsin.@*Conclusions@#Adding 1 μg/ml and 1.5 μg/ml of trypsin could better promote the proliferation of H7N9 avian influenza virus, and 1.5 μg/ml of trypsin had a wider range of MOI applicability.
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Objective@#To reduce the residual proteins and DNA of host cells in the preparation of H5N1 influenza A virus.@*Methods@#Core 700 was firstly used to remove residual host cell proteins, and then Capto Q was used to remove host cell DNA. Several batches of H5N1 influenza A virus cultured in Madin-Darby canine kidney (MDCK) cells were purified using this method. The efficiency of purification was evaluated using many methods including quantitative real-time PCR, hemagglutination (HA) test and single radial immunodiffusion assay. Moreover, Benzonase nuclease was used for comparison.@*Results@#Without the use of Benzonase nuclease, the overall removal rates of host cell DNA and residual proteins were 99.62% and 98.1%, and the HA antigen recovery rate was 66.96%.@*Conclusions@#This study established a purification strategy with good effect for cell-based influenza vaccines. It can efficiently remove host cell DNA and proteins and achieve a high HA recovery rate. The purification result is no worse than that of adding Benzonase nuclease, suggesting the potential of its application in actual vaccine production.
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Objective To reduce the residual proteins and DNA of host cells in the preparation of H5N1 influenza A virus. Methods Core 700 was firstly used to remove residual host cell proteins, and then Capto Q was used to remove host cell DNA. Several batches of H5N1 influenza A virus cultured in Ma-din-Darby canine kidney ( MDCK) cells were purified using this method. The efficiency of purification was evaluated using many methods including quantitative real-time PCR, hemagglutination ( HA) test and single radial immunodiffusion assay. Moreover, Benzonase nuclease was used for comparison. Results Without the use of Benzonase nuclease, the overall removal rates of host cell DNA and residual proteins were 99. 62％ and 98. 1％, and the HA antigen recovery rate was 66. 96％. Conclusions This study established a purification strategy with good effect for cell-based influenza vaccines. It can efficiently remove host cell DNA and proteins and achieve a high HA recovery rate. The purification result is no worse than that of adding Benzonase nuclease, suggesting the potential of its application in actual vaccine production.
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Fluorescein isothiocyanate-labeled insulin (FITC-insulin) has been widely used for bioanalytical appli-cations. Due to the high cost of commercial FITC-insulin and tedious labeling procedures described in the literature, there is still a need to develop a cost effective, reliable and quick labeling method for insulin. The purpose of the present work was to develop a quick and affordable method for FITC labeling of human insulin and to determine the effect of different conjugations of FITC to human insulin on its permeability through the MDCK cell monolayer. FITC labeling of insulin gives mono-, di-or tri-conjugates depending on the reaction time and the molar ratio of FITC:insulin. Mono-conjugate with unlabeled insulin, mixture of di-and tri-conjugate, and tri-conjugate with very little amount of di-conjugate were synthesized in less than 4 h. Degree of conjugation had an effect on the permeability of insulin through the MDCK cell monolayer. Mono-conjugate had higher permeability than the unlabeled insulin due to increase in partition coefficient. However, tri-conjugate showed lower permeability than the unlabeled insulin due to the increase in molecular weight.
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OBJECTIVE: To establish a cell model stably expressing mouse organic anion transporter1( OAT1) in MDCK cells, for the purpose of screening potent OAT1 inhibitors in vitro. METHODS: Recombinant plasmid pcDNA3.1(+) -OAT1 was constructed and transfected into MDCK cells using Lipofectamine™ 2000 reagent. After the process of G418 screening, cells were collected for further validation. Cells were harvested, and the quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) was carried out to test the OAT1 mRNA expression in MDCK-OAT1 cells. The function of the stably transfected cells were validated by the uptake activity of (6-Carboxyfluorescein, 6-CFL),a substrate of OAT1. The inhibitors of OAT1 were selected according to their inhibition activity towards the uptake of 6-CFL into the OAT1-over expressing cells in comparison with the typical inhibitor of OAT1,probenecid. RESULTS: The pcDNA3.1(+) -OAT1 was well conducted. The mRNA expression of OAT1 was significantly higher than that in mock cells; MDCK-OAT1 cells had a significantly high mRNA expression comparing with the mock cells, being 4 862 fold of that in mock cells. The uptake-ability of 6-CFL in MDCK-OAT1 and MDCK-mock cells was obviously different, with a 14.9 fold increase in comparison with mock cells. In the presence of probenecid and several monomers from Chinese herbs, fluorescence values in cell lysates were reduced to varying degrees, and results showed that rhein, luteolin, chrysin and quercetin could significantly inhibited the 6-CFL uptake mediated by hOAT1,with a reduction of more than 80% of the control. CONCLUSION: The aim to establish a cell model which could stably express OAT1 is achieved. Further study could be done using this cell model, for the screening of potential inhibitors of OAT1 from monomers of Chinese herbs,and then could be used as a tool in the research of herb-drug interaction.
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Aim To screen a more suitable transfection recep-tor,and improve the efficiency of constructing cell lines highly expressing human peptide transporters 1 (hPepT1 ).Methods The recombinant plasmid pcDNA3.1 (+)-hPepT1 was transfect-ed into MDCK cells and HeLa cells by LipofectamineTM 2000 transfection reagent,respectively.The monoclonal cells were se-lected and cultured.Expression of hPepT1 mRNA and protein were determined by qRT-PCR and Western blot,respectively. The uptake capacity of Glysar in transfected cells was examined. Results Compared with wild type cells,the expression of hPepT1 and the uptake of Glysar in transfected MDCK cells and HeLa cells significantly increased (P <0.05).Although the up-take of Glysar in HeLa cells was higher than that of MDCK cells,on the contrary,the expression of hPepT1 and the uptake of Glysar in MDCK-hPepT1 cells was higher than that of HeLa-hPepT1 cells.Conclusion MDCK cells may serve as a more suitable transfected receptor for the construction of a cellular model with high expression of hPepT1 ,which would make the construction of a cell model highly expressing hPepT1 more effi-cient.
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The occasional influenza pandemics and the seasonal influenza epidemics have destroyed millions of lives since the last century. It is therefore necessary to understand the virus replication patterns as this provides essential information on the virus infectivity, pathogenicity and spread patterns. This study aimed to investigate the replication of avian influenza A virus H5N1 (A/Chicken/Malaysia/5858/2004) in MDCK cells. In this study, the TCID50 (50% tissue culture infectious dose) of AIV H5N1 was first determined. The MDCK cells were then infected with AIV H5N1 at TCID50 for 0-48 h. The CPE (cytopathic effect) was observed and cell death was determined hourly. The virus-infected cells and media were subsequently collected for gene analysis. The results showed that the TCID50 of AIV H5N1 was 10-9 dilution. The CPE percentage showed a strong and positive correlation with the infection period (r = 1.0, n = 9, p 0.05) and infected cell (r = 0.73, n = 9, p < 0.05) were also positively correlated with the infection period. In conclusion, although CPE started to be observed in the early time points of infection, however, the M2 gene was only amplified from the infected media and cells after 48 h and 24 h, respectively. This signifies that AIV H5N1 used in this study is pathogenic and it is able to cause severe cytopathology to host cells even at low virus load.
Subject(s)
Influenza, Human , Influenza A Virus, H5N1 SubtypeABSTRACT
Objective To explore the most suitable condition of influenza virus which was culturing on plateau region,and im-prove the effect of the separation of influenza virus.Methods The original specimens were respective inoculated in MDCK cells,by comparing CO2 percent concentration (5%,4.8%,4.6%,4.4%,4.2%),TPCK-pancreatic enzyme dosage (1,2,3, 4,5μg/m and 6,8,10μg/ml),inoculation amount (100,200,300,400,500μl),incubation time (48,72,96,120,144 h),inoc-ulation methods (adsorption,direct inoculated),and culture vessel(cells,6 orifice)that were influenced on influenza virus, the best culture conditions was determined.Hemagglutinatio (HA)method was used to detect the virus titers.Results Through the comparison,the HA titer of virus was in the highest titer with 6 orifice plate culture vessel,4.4% CO2 and 4.6% CO2 ,2μg/ml TPCK-pancreatic enzyme,300μl and 400μl inoculation amount,72h~96h of incubation time.Conclusion The optimal condition of the influenza virus cultured with the MDCK cell has been established in the laboratory on plateau region.
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Objective: To modify the structure of natural monomeric compound 3-O-caffeoylquinic acid from medicinal plant Pandanus tectorius, and evaluate the anti-influenza virus activity of the derivatives. Methods: Applying 3-O-caffeoylquinic acid as material, the target compounds were prepared by three routes and evaluated for their anti-influenza virus effects by MDCK cells in vitro. Results: Thirteen caffeoylquinic acid derivatives B1-B13 were synthesized. The structures of the target compounds were identified by spectrum. Pharmacological results showed that the compounds B4, B6, and B10 had the different potencial inhibition on the replication of influenza virus in cells. Conclusion: The new compounds B2 and B4-B13 which show the potential biological activity of anti-influenza virus, have not been reported in any literatures and are worth studying further.
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Objective:To study the nephrotoxicity induced by triptolide ( TP) on MDCK cell model and investigate its effect on oxidative stress. Methods:Aristolochic acid was chosen as the positive control. After the MDCK cells were incubated with 0. 5, 5, 50 and 500 nmol·L-1 TP for 24h, MTT method was used to observe the cell inhibiting rate and lactate dehydrogenase (LDH) release test was used to detect the cell membrane damage caused by TP. The cell morphology was observed under an inverted microscope. After the MDCK cells were incubated with 500 nmol·L-1 TP respectively for 30min, 1h, 2h, 4h and 6h, the level of reactive oxygen species ( ROS) was detected using 2′,7′-dichlorodihydro-fluorescein diacetate ( DCFH-DA) as the fluorescent probe. Results:Compared with those of the negative control group, the cell inhibiting rates and the relative LDH release rates in TP-treated group were increased sig-nificantly(P<0. 01). The cells in TP-treated group were creased, turned into the round shape and began to shed off. After the MDCK cells were incubated with TP for 30min, the level of ROS reached the highest value, and then began to decrease (P<0. 01). Conclu-sion:TP can induce the toxic effects on MDCK cells and the mechanism may be related to oxidative stress.