Evaluation of passage stability of H5N1(NIBRG-14) influenza virus vaccine strain in MDCK cells of serum-free suspension culture / 中国生物制品学杂志

@#Objective To evaluate the passage stability of H5N1(NIBRG-14) influenza virus vaccine strain in MDCK cells(sMDCK)of serum-free suspension culture.Methods H5N1(NIBRG-14) influenza virus working-bank vaccine strains were passed 15 consecutive times in sMDCK cells.The 8-segment nucleotide sequences(HA,NA,M,NP,NS,PA, PB1 and PB2 genes) of the main-bank,working-bank,virus of P1,P2,P3,P5,P10 and P15 generations were detected for genetic stability by second and first generation sequencing.The stability of amino acid sequences of hemagglutinin(HA)and neuraminidase(NA),the main antigens of the working-bank,P5 and P15 generation viruses,were evaluated by using peptide coverage as indicators;Influenza vaccine was prepared with working-bank,P5 and P15 generation viruses,with which the female BALB/c mice were immunized i.m.with 10 in each group,15 μg HA per mouse,and boosted 28 d later at the same dosage and route.At 28,42 and 56 d after the primary immunization,the mice were detected for the titer of neutralizing antibody in serum to evaluate the stability of immunogenicity.Results No segment insertion or deletion was detected in each generation of influenza virus,and the nucleotide sequence was completely consistent with the main-bank;Single nucleotide polymorphism(SNP) mutations did not occur in the main-bank,working-bank,P1,P2,P3,P5 and P10 generations of viruses,while the possibility of SNP mutation showed in many gene loci of P15 generation virus,with heterozygous SNP accounting for 91.62%.The coverage rate of HA and NA protein peptides of P5 and P15 generation viruses ranged from96.7% to 100%.There was no significant difference in serum neutralizing antibody titer of mice in the working-bank,P5 and P15 groups(H=2.253,2.029 and 1.408,P=0.324,0.363 and 0.495,respectively) at 28,42 and 56 d after the first immunization.Conclusion H5N1(NIBRG-14) influenza virus vaccine strain has good genetic stability in sMDCK cells,which is expected to be used in the production of sMDCK cell matrix pandemic influenza vaccine.

Preliminary analysis of isolation effect of influenza virus by recombinant MDCK cells stably expressing trypsinogen / 中国生物制品学杂志

@#ObjectiveTo investigate the isolation effect of influenza virus by recombinant MDCK cells(MTY6 cells)stably expressing trypsinogen.MethodsAccording to the virus isolation method recommended by the World Health Organization(WHO)Global Influenza Surveillance Network(GISN)and the National Influenza Centers(NICs),a total of 20 throat swab specimens containing positive nucleic acid for H1N1,H3N2 and B influenza virus were isolated simultaneously using MDCK and MTY6 cells. Guinea pig red blood cells and chicken red blood cells were used for agglutination test respectively and the agglutination effects of different types of red blood cells,the positive rate of virus and the titer of hemagglutinin isolated from different cells were statistically compared.ResultsThe agglutination effect of the same virus isolate on the two types of red blood cells was different. The complete agglutination time of guinea pig red blood cells was about 2 times that of chicken red blood cells,and the deposition shape showed a ring shape. The average hemag-glutinin titer was 23. 6 ± 1. 2times that of chicken red blood cells. Under the same conditions,3 samples were negative for both types of cells,11 samples were positive for both types of cells,and the other 6 samples were negative for MDCK cells while positive for MTY6 cells. The positive rate of MTY6 cells was 30% higher than that of MDCK cells. The isolated positive samples included 8 cases of H1N1 subtype and the hemagglutinin titer of virus isolated by MTY6 cells was significantly higher than that by MDCK cells[13. 0(1. 7,23. 0)times on average]. 2 cases of H3N2 and 2 cases of B were isolated,the hemagglutinin titer of each virus isolated by MTY6 cells was 11. 3 and 32. 0 times higher than that by MDCK cells on average respectively.ConclusionIn conclusion,guinea pig red blood cells were superior to chicken red blood cells for influenza virus detection by cell isolation. Under the same conditions,MTY6 cells were more sensitive than MDCK cells for influenza virus isolation,and had the potential to be used as a high-quality cell matrix for influenza virus isolation.

Transglutaminase 2 inhibits the proliferation of H1 subtype influenza virus in MDCK cells / 生物工程学报

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.

Transport mechanism of bergenin aross MDCK-MDRl cell monolayer model / 中国药理学通报

Aim To study the transport mechanism of bergenin passing through blood-brain barrier ( BBB ) . Methods MTT assay was used to investigate the tox-icity of bergenin on MDCK-MDRl cells. Molecular docking was used to predict the binding mode and effect ability of bergenin with P-gp. In vitro MDCK-MDRl cell monolayer model was used to analyze trans¬port characteristics of bergenin and the effect of con¬centration, time and verapamil (a P-gp inhibitor) on the transport of bergenin. Results Bergenin was non-toxic to MDCK-MDRl cells within the concentration of 5 to 40 jjunol • L . There was hydrogen-bond and hy-drophobic interaction between P-gp and bergenin, and P-gp-bergenin was more stable than P-gp-verapamil. The P value of bergenin transported from AP to BL (PappAP

Anti-Acanthamoeba castellanii activity of alkaloid-enriched extracts and lycorine from the Amaryllidaceae species

Abstract Free-living amoebae of the genus Acanthamoeba are the causative agents of granulomatous encephalitis and keratitis, severe human infections. Bioactive compounds from plants are recognized as an alternative source for the development of new drugs. The Amaryllidaceae is a botanical family able to synthesize a very specific and consistent group of biologically active isoquinoline-like alkaloids. The alkaloidal fractions from the Brazilian species Hippeastrum canastrense, H. diniz-cruziae, H. puniceum, and Crinum x amabile, along with the alkaloid lycorine, were investigated against Acanthamoeba castellanii. The in vitro assays were performed with distinct concentrations of lycorine and alkaloidal fractions, while the cell viability was evaluated by the MTT method upon MDCK cells. Chlorhexidine 0.02% was used as the positive control. The effect of alkaloid fractions was concentration dependent, and 2000 µg mL-1 of H. canastrense and H. diniz-cruziae provided a 100% inhibition. At concentrations of 250, 500, and 1000 µg mL-1, the H. diniz-cruziae alkaloidal fraction showed the lowest cytotoxic effect (5%-7%) and remarkable anti-amoebic activity, demonstrating values of IC50 285.61 µg mL-1, low cytotoxicity (5%-7%), and selectivity index (7.0). Taken together, the results are indicative of the great potential that the alkaloids from H. diniz-cruziae have as new candidates for anti-amoebicidal compounds

Acid-functionalized single-walled carbon nanotubes alter epithelial tight junctions and enhance paracellular permeability

The T(EB4)Nta, T(IBj5)Nta, and T(B362i)NtA strains were constructed by introgressing the insertionaltranslocations EB4, IBj5, and B362i from Neurospora crassa into the related species N. tetrasperma. Theprogeny from crosses of T(IBj5)Nta and T(B362i)NtA with opposite mating-type derivatives of the standard N.tetrasperma strain 85 exhibited a unique and unprecedented transmission ratio distortion (TRD) that disfavoredhomokaryons produced following alternate segregation relative to those produced following adjacent-1 segregation. The TRD was not evident among the [mat A ? mat a] dikaryons produced following either segregation. Further, crosses of the T(IBj5)Nta and T(B362i)NtA strains with the Eight spore (E) mutant showed anunusual ascus phenotype called ‘max-4’. We propose that the TRD and the max-4 phenotype are manifestations of the same Bateson-Dobzhansky-Muller incompatibility (BDMI). Since the TRD selects against 2/3 ofthe homokaryotic progeny from each introgression cross, the BDMI would have enriched for the dikaryoticprogeny in the viable ascospores, and thus, paradoxically, facilitated the introgressions.

Determination of the bioaccessibility of cadmium and arsenic in earthworms by PBET digestion in vitro / MDCK cell model with risk assessment / 药学学报

Inductively coupled plasma mass spectrometry (ICP-MS) was used to determine the content of cadmium (Cd) and arsenic (As) in earthworms. A physiologically-based extraction test (PBET) digestion in vitro /MDCK cell model was established to investigate the bioaccessibility of Cd and As in earthworms. The hazard index (HI) method and the margin of exposure (MOE) method were used to assess the risks of the total content and the bioaccessible content of Cd and As. The results showed that the total content of Cd and As in six batches of earthworms ranged from 8.319 to 33.606 mg·kg-1 and from 0.532 to 16.412 mg·kg-1, respectively. After uptake by MDCK cells, the bioaccessibility of Cd in earthworms ranged from 10.13% to 64.16%, and the bioaccessibility of As was from 2.72% to 46.57%. The results of risk assessment showed that before uptake by MDCK cells, the MOE values of As and HI values of Cd for all batches of earthworms were greater than 1, which suggests that the risks of As are acceptable but the risks of Cd are unacceptable. After transportation by MDCK cells, except for one batch of earthworms, the HI values of Cd in the other five batches were less than 1, which suggests that the risks are at a safe level. This study provides important technical support for a more objective and scientific assessment of the health risks of heavy metals in traditional Chinese medicines, and for a more scientific and reasonable standard limit of heavy metals.

Canine adipose-derived mesenchymal stem cells and their exosomes in the repair of gentamicin-induced renal tubular epithelial cell injury / 中国组织工程研究

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.

Cloning and analyzing of MDCK cells for H5N1 influenza vaccine production / 中华微生物学和免疫学杂志

Objective To screen a Madin-Darby canine kidney (MDCK) cell line for H5N1 influ-enza virus isolation and to evaluate its safety in vaccine production. Methods MDCK cells were cloned by the method of limiting dilution. Hemagglutination test was used to screen MDCK cells that were suitable for H5N1 influenza virus production. Tests for analyzing the characteristics, extraneous agents, endogenous agents and tumorigenicity of MDCK cells were performed according to Chinese Pharmacopeia Volume Ⅲ. Results A total of 108 MDCK cell lines were obtained and three of them were selected after hemagglutina-tion test. G1 cells were chosen following further screening with tumorigenicity test and receptor abundance analysis. The average number of chromosomes of the MDCK-G1 cells was 78±4. No bacteria, fungi or myco-plasma contamination was detected. In experimental group, each nude mouse was injected with 1×107/ml viable cells to observe their tumorigenicity. Twelve weeks after cell injection, no node was found at injection sites or in gross anatomy. There was no significant difference between the experimental and negative control groups. The result of the tumorigenicity test was negative. No node formation was found after injecting nude mice with cell lysate or cellular DNA collected from equivalent amount of cells. It was indicated that the MDCK-G1 cells were of low-oncogenic potential. Conclusions The MDCK-G1 cell line could be used as a substrate to produce H5N1 influenza virus vaccine.

Optimization of the amount of TPCK trypsin adding to MDCK cell suspension for culturing H7N9 avian influenza virus / 中华微生物学和免疫学杂志

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.

Purification method for cell-cultured influenza virus H5N1 / 中华微生物学和免疫学杂志

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.

Purification method for cell-cultured influenza virus H5N1 / 中华微生物学和免疫学杂志

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.

FITC labeling of human insulin and transport of FITC-insulin conjugates through MDCK cell monolayer / 药物分析学报

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.

Study on transport mechanism of brucine and strychnine in MDCK-MDR1 cell monolayer model / 中草药

Objective: To study the bi-direction transport behavior of brucine and strychnine in the MDCK-MDR1 cell monolayer model. Methods: MTT method was employed to confirm the safe concentration of brucine and strychnine towards MDCK-MDR1 cells. The effects of transport time, drug concentration, and P-glycoprotein inhibitor verapamil on cumulative absorption concentration (Ccum) and apparent permeability coefficient (Papp) of brucine and strychnine in MDCK-MDR1 monolayer cells were studied. Results: The Papp value of brucine and strychnine was larger than 1 × 10-5 cm/s and the ratio of Papp(BL→AP) vs Papp(AP→BL) was less than 2. Brucine/ strychnine combined with verapamil decreased the ratio of Papp(BL→AP) vs Papp(AP→BL). Conclusion: The absorption of brucine and strychnine in MDCK-MDR1 cell monolayer model was well and the passive transference was its main intestinal absorption mechanism. The P-gp inhibitor verapamil has a significant inhibitory effect on brucine and strychnine absorption. Brucine and strychnine may be a substrate of P-glycoprotein.

Effect and mechanism of Mahuang Tang against influenza A/H1N1 virus / 中国中药杂志

To study the effect and underlying mechanism of Mahuang Tang against influenza A virus ， the influenza virus-infected Madin-Darby canine kidney(MDCK) cells were used as the carrier in this study to detect the median tissue culture-infective dose(TCID₅₀) of influenza A virus strains(A/PR8/34) on MDCK cells with cytopathic effect(CPE) assay. Blocking influenza virus invading host cells and anti-influenza virus biosynthesis were used as two different administration methods， and then the methyl thiazolyl tetrazolium(MTT) assay was utilized to determine the antiviral effective rate(ER)， median efficacious concentration(EC₅₀) and therapeutic index(TI) of Mahuang Tang. The quantitative Real-time polymerase chain reaction(RT-PCR) was used to measure virus load and the mRNA expression levels of TLR4， TLR7， MyD88 and TRAF6 in MDCK cells at 24， 48 h after the treatment. The experiment results indicated that TCID₅₀ of A/PR8/34 for MDCK cells was 1×10-4.32/mL. The EC₅₀ values of two different treatment methods were 4.92，1.59 g·L⁻¹ respectively， the TI values were 12.53， 38.78 respectively， and when the concentration of Mahuang Tang was 5.00 g·L⁻¹， ER values were 50.21%， 98.41% respectively， showing that Mahuang Tang can block influenza virus into the host cells and significantly inhibit their biosynthesis. Meanwhile， as compared with the virus group， the virus load was significantly inhibited in Mahuang Tang groups， and Mahuang Tang high and middle doses had the significant effect on decreasing the mRNA expression of TLR4， TLR7，MyD88 and TRAF6 at 24， 48 h after the treatment. It can be demonstrated that the mechanisms of Mahuang Tang against influenza A virus are related to the inhibition of influenza virus replication and the mRNA expression of correlative genes in TLR4 and TLR7 signaling pathways.

The aminoglycoside antibiotic gentamicin is able to alter metabolic activity and morphology of MDCK-C11 cells: a cell model of intercalated cells

It is well known that the aminoglycoside antibiotic gentamicin is capable of causing damage to kidney cells. Given the known involvement of Ca2+ in the nephrotoxic action of gentamicin, the purpose of this study was to establish a relationship between the concentration of intracellular Ca2+ ([Ca2+]i) and cellular cytotoxicity using MDCK-C11 cells, a clone that has several properties that resemble those of intercalated cells of the distal nephron. Changes in [Ca2+]i was determined using fluorescence microscopy. Cell viability was evaluated by the neutral red method, and cell cytotoxicity by the MTT method. The [Ca2+]i gradually increased when cells were exposed to 0.1 mM gentamicin for 10, 20, and 30 min. The presence of extracellular Ca2+ was found to be necessary to stimulate the increase in [Ca2+]i induced by gentamicin, since this stimulus disappeared by using 1.8 mM EGTA (a Ca2+ chelator). Morphological changes were observed with scanning electron microscopy in epithelial cells exposed to the antibiotic. Furthermore, with the MTT method, a decrease in metabolic activity induced by gentamicin was observed, which indicates a cytotoxic effect. In conclusion, gentamicin was able to alter [Ca2+]i, change the morphology of MDCK-C11 cells, and promote cytotoxicity.

Mechanism underlying bergapten-mediated regulation of vincristine transport in MDCK-MDR1 cells / 中草药·英文版

Objectives: To investigate the effects of bergapten of Angelicae Dahuricae Radix on the transport of vincristine and its possible mechanism. Methods: The apparent permeability coefficient (Papp) for the transport of vincristine through the membrane of MDCK-MDR1 cells was used as an indicator of the effect of bergapten on vincristine transport. Molecular docking was employed to predict the binding force between bergapten and P-glycoprotein (P-gp). The effects of bergapten on P-gp function and P-gp ATPase activity were determined by rhodamine 123 (Rho123) accumulation and activity analysis, respectively. 1,6-Diphenyl-1,3,5-hexatriene (DPH) was used to study the effects of bergapten on membrane fluidity, and Western blotting and quantitative real-time PCR assays were performed to analyze the effect of bergapten on the protein and mRNA expression of P-gp, respectively. These experiments clarified the effects of bergapten on the transport of vincristine and allowed exploration of the possible mechanism underlying the effects of bergapten. Results: The results showed that bergapten could inhibit the transport of vincristine in MDCK-MDR1 cells, and the binding force between bergapten and P-gp was weaker. Bergapten could reduce the accumulation of Rh123 in MDCK-MDR1 cells, increase the membrane fluidity, and upregulate P-gp protein and mRNA expression but it had no effect on P-gp ATPase activity. Conclusions: Overall, we concluded that the possible mechanism through which bergapten inhibits vincristine transport was related to the bergapten-mediated upregulation of P-gp protein and mRNA expression, membrane fluidity or P-gp enzyme activity.

Effects of Hydrophobic Structure of Amphiphilic Polymers on the Activity of P-glycoprotein in MDCK-MDR1 Cell / 中国药学杂志

OBJECTIVE: To determine the effects of the amphiphilic block polymers, which have the same hydrophilic block with the different hydrophobic block, on the function of P-glycoprotein(P-gp). METHODS: The three different micelles were prepared by film dispersion method. The particle sizes and distributions were measured by dynamic light scattering. Critical micelle concentrations(CMC) were detected by fluorescence probe technique with the pyrene. Rhodamine 123, a specific probe substrate of P-gp, was applied to determine the effects of polymers on the function of P-gp using uptake and efflux method. RESULTS: The particle sizes of mPEG-PCL, mPEG-PDLLA, mPEG-PLGA were (55.9±0.2), (53.7±1.1) and (61.6±0.6)nm. The CMC values were 2.08, 5.42 and 26.4 μg·mL-1. R123 accumulation in Madin-Darby canine kidney/multidrug resistance 1(MDCK-MDR1)cell detected by uptake assay increased to a maximum in the presence of polymers at concentrations of 250 μg·mL-1 for mPEG-PCL, 1~25 μg·mL-1 for mPEG-PDLLA and mPEG-PLGA. In efflux assay, mPEG-PCL, mPEG-PDLLA, mPEG-PLGA decreased the percentage of efflux of R123 at concentrations above the CMC, below/at the CMC or below the CMC respectively, showed the similar RESULTS with uptake assay. CONCLUSION: The mPEG-PCL, mPEG-PDLLA, mPEG-PLGA polymers might have a potential to inhibit the efflux activity of P-gp, which was likely related to the structures of hydrophobic segments, concentrations and existing states of the polymers.

Effects of microemulsion on transport and mechanism of puerarin in MDCK-MDR1 cells / 中草药

Objective To explore the effect of microemulsion on the transport and mechanism of puerarin in blood brain barrier (BBB) cell model MDCK-MDR1. Methods MTT assay was used to evaluate the cytotoxicity of puerarin microemulsion and solution, and determine the appropriate concentration of administration. The bilateral transport characteristics of puerarin solution-microemulsion was investigated in MDCK-MDR1 monolayer. Immunohistochemical staining was used to study the expression of tight junction proteins, and the changes in cell membrane fluidity was studied by fluorescence bleaching recovery, and the changes of membrane potential was measured by anion probe combined with flow cytometry. The mechanism of the effect of microemulsion on puerarin transport was clarified. Results The MDCK-MDR1 showed no significant toxicity when the mass concentration of puerarin solution ranged from 50 to 300 μg/mL and the microemulsion dilution was over 500 times. The Papp value in absorption direction of puerarin solution on MDCK-MDR1 monolayer was 1.04 × 10-6 cm/s, and the Papp value of excretion direction was 1.05 × 10-6 cm/s. The Papp value of puerarin in microemulsion was significantly increased compared with that in solution (P < 0.05). Microemulsification could reduce the expression of Claudin-1, Occludin, ZO-1, and F-actin in MDCK-MDR1, promote cell membrane flow, and decrease cell membrane potential. Conclusion Microemulsion can promote the bilateral transport of puerarin in the BBB cell model MDCK-MDR1. The mechanism is closely connected with the opening of tight junctions, increasing the cell membrane fluidity, making the cell depolarizing and reducing membrane potential, and increasing the permeation of paracellular.

Study on the Transport Mechanism of Punicalagin in MDCK Cells Monolayer Model / 中国药师

Objective:To investigate the transport mechanism of punicalagin in MDCK monolayer model .Methods:The safe con-centration of punicalagin in MDCK cells was determined by CCK8 assay.Millicell -ERS was used to measure cell monolayer TEER value to determine the integrity of the cell monolayer .The effects of direction , drug concentration , time, P-gp inhibitor and EDTA-Na2 on the absorption and transport of punicalagin were studied systematically .And then the drug concentration was analyzed by HPLC to calculate the apparent permeability coefficient (Papp) and efflux ratio(ER).Results: Punicalagin transport in MDCK cells was time and concentration dependent .Punicalagin showed poor absorption in MDCK cells .Papp from apical to basolateral side ( AP-BL) within the concentration range of 100-300μg· ml-1 was (6.13 ±0.12) ×10 -7 cm· s-1 , (6.96 ±0.26) ×10 -7 cm· s-1 and (5.94 ±0.10) ×10 -7 cm· s-1 , respectively .P-gp inhibitor and EDTA-Na2 could significantly increase the transport of punicalagin in AP-BL direc-tion, while the transport decreased at 4℃.Conclusion:The transport mechanism of punicalagin might be passive diffusion as the dom-inating process involving active transportation .Punicalagin is one of P-gp substrates with exocytosis and absorbed via the paracellular route.