Effect of bioreactor scale-up culture of micro-carrier Vero cells after extra-tank trypsinization on virus-producing ability of rabies virus CTN-1Ⅴ strain / 中国生物制品学杂志

@#Objective To investigate the effect of amplification culture of micro-carrier Vero cells from 30 L bioreactor to 300 L bioreactor after extra-tank trypsinization on the virus-producing ability of rabies virus(RABV)CTN-1Ⅴ strain.Methods The 140-passage of Vero cells were cultured at 37 ℃ for 72-120 h,then amplified by passaging at a cell density ratio of 1∶4 into the 10 × cell factory. After incubation at 37 ℃ for 72-120 h,the monolayer cells were detached and inoculated into the 30 L bioreactor with micro-carriers 7-10 g/L,culture temperature 37 ℃,pH 7. 0-7. 4,dissolved oxygen 30%-80%,stirring speed 10-50 r/min,and continuous perfusion culture 72-120 h. Total three batches of microcarrier Vero cells were cultured,which were amplified to the 300 L bioreactor after extra-tank trypsinization,with microcarrier5-8 g/L,culture temperature 37 ℃,pH 7. 0-7. 4,dissolved oxygen 30%-80%,stirring speed 30-80 r/min,and perfusion culture 72-120 h. RABV CTN-1Ⅴ was inoculated at the MOI of 0. 05,and the virus solution was harvested every 24 h and detected for the virus titer and antigen content. Results The cell density was about 1 × 10~7 cells/mL after culture for 96 h in the 30 L bioreactor,and was about 7. 4 × 10~6 cells/mL after culture for 96 h in the 300 L bioreactor. At 96 h after virus inoculation,the virus harvest solution reached the peak potency,with the average virus titer of 6. 8 lgLD_(50)/mL and the average antigen content of 2. 58 IU/mL. Conclusion The scale-up culture process of micro-carrier Vero cells after extra-tank trypsinization from 30 L bioreactor to 300 L bioreactor is stable and feasible,with no significant effect on the virus-producing ability of RABV CTN-1Ⅴ strain,which provides a reference for the large-scale production of inactivated RABV vaccine

Development of large-scale amplification process of rabies virus in 150 L bioreactor / 中国生物制品学杂志

@#Objective To develop a large-scale culture process for rabies virus(RABV)in 150 L bioreactor,and lay a foundation for the further development of a larger-scale and high-density microcarrier reactor process.Methods Vero cells and RABV strain CTN-1V were cultured in 30 L(model:C30-2)and 150 L(model:VESSEL FERMENTER 300L)bioreactors by perfused culture with 20 g/L Cytodex-1 microcarrier and DO 20%-60%,at culture temperature 36-38 ℃ and pH 7.0-7.4.During the culture process,the cell density and virus titer were measured.The virus culture media was harvested for consecutive 13 d and detected for the sterility,mycoplasma,and the residues of antigen,host cell protein(HCP),bovine serum albumin(BSA)and DNA.Results The density of cultured cells in 30 L and 150 L bioreactors all reached above 1.2 ×10~7cell/mL.There was no significant difference in cell density at different time points during the culture(t = 0.225-2.173,P = 0.096-0.833).The highest virus titer(8.5 lgLD_(50)/mL)was found in the both bioreactors 6 d after infection with no significant difference(t = 1.000,P = 0.374).The residues of antigen,HCP,BSA and DNA in the virus suspension from the two bioreactors were basically the same.Conclusion 150 L bioreactor can be used for the large-scale culture of RABV,and the harvested virus conformed to the relevant standards in Chinese Pharmacopoeia(Volume Ⅲ,2020 edition).

Screening and identification of Hantaan virus Vero cell adaptation strains / 中国生物制品学杂志

@#Objective To study the adaptability of Hantaan virus(HTNV) PS-6 strain in Vero cells after passing through the brain of suckling mice,and to screen out a high titer HTNV strain adaptable to Vero cells.Methods HTNV PS-6 strain was cultured continuously in the brain of suckling mice,and then continued to pass on Vero cells for 10 times.The cytopathic effect(CPE) was observed for each generation of the virus,and the virus titer was determined by plaque method.After the virus titer increased on Vero cells,the monoclonal virus was screened by plaque cloning and purification and the titer of monoclonal virus was measured.After infecting Vero cells at MOIs of 0.01,0.05,and 0.1,the growth curve of the virus was drawn.The LD_(50) of the virus was calculated by Reed-Muench method,the specificity of the virus was initially identified by Western blot,and the size and structure of the virus before and after screening were observed by transmission electron microscopy.The nucleotide sequences of the whole genome and amino acid sequences were compared with those of HTNV PS-6 strain.The strain adaptive to Vero cells,V-PS-6,was injected intraperitoneally into 10 female BALB/c mice,which were dissected when dying.The tissues of heart,liver,spleen,kidney,brain,lung,small intestine and muscle were taken and analyzed by immunohistochemistry.Results The passage strain in mouse brain had good adaptability to Vero cells,with an initial virus titer of 1.3 lgPFU/mL,and the virus titer gradually increased with the increase of passage times and stabilized at 7.5-7.9 lgPFU/mL.The plaque boundary of the adapted strain V-PS-6 was clear,round and visible to naked eyes,the size was like a needle tip,the LD_(50) was 10~(-7.8),and the specific protein band was found at the relative molecular mass of about 50 000,consistent with that of the pre-adapted virus strain HTNV PS-6.The diameter of V-PS-6 strain was80-210 nm,with no significant difference in structure between V-PS-6 strain and HTNV PS-6 strain under electron microscope,and the homology of nucleotide sequences and amino acid sequences was 99.39% and 98.85%,respectively.The VPS-6 strain was obviously distributed in kidney,liver,small intestine,muscle of hind leg and heart of mice.Conclusion A HTNV strain well adaptive to Vero cells with high titer was successfully screened and had good genetic stability,laying a foundation for the research and development of HTNV Vero cell vaccine and the study of related mechanisms.

Identification of peak 2 protein on right side of main peak of SEC-HPLC in lyophilized human rabies vaccine (Vero cells) stock solution(without human blood albumin) / 中国生物制品学杂志

@#Objective To identify the peak 2 protein on the right side of the main peak of size exclusion chromatograghy-high performance liquid chromatography(SEC-HPLC)in freeze-dried human rabies vaccine(Vero cell)stock solution(without human blood albumin),so as to determine the protein composition of the peak and ensure the vaccine quality.Methods The lyophilized human rabies vaccine(Vero cells)stock solution(without human blood albumin)was analyzed by SECHPLC,of which the peak 2 on the right side of the main peak was collected and concentrated by ultrafiltration,then reduced by dithiothreitol(DTT),alkylated by iodoacetamide (IAM) and hydrolyzed by Trypsin. The products were analyzed by nano LC-MS/MS.Results Four proteins were successfully identified in the right peak 2 of SEC-HPLC main peak in lyophilized human rabies vaccine(Vero cell)stock solution(without human blood albumin),and the number of matching peptides for a single protein ranged from 59 to 79. The number of single protein-matching characteristic peptides ranged from 6 to 12. The detection times of single protein-matching characteristic peptide segments ranged from 20 to 36 times. The sequence coverage of the identified proteins ranged from 37. 40% to 64. 31%. A total of 280 peptides participated in the statistics,and the mass spectrum deviation was less than 0. 15 Da.Conclusion The peak 2 on the right side of SEC-HPLC main peak of lyophilized human rabies vaccine(Vero cell)stock solution(without human blood albumin)is derived from rabies strain 4a GV,which is the vaccine particle dissociation.

Primer aislamiento y caracterización de la cepa prototipo del virus SARS-CoV-2 a inicios de la pandemia de la COVID-19 en el Perú

Introducción: Actualmente los contagios por el virus del SARS-CoV-2 supera los 600 millones de casos en el mundo. Objetivo: Aislar y caracterizar el virus SARS-CoV-2 causante de la COVID-19 a inicios de la pandemia en el Perú. Materiales y métodos: Se realizó el aislamiento viral a partir de 20 muestras de hisopado nasal y faríngeo positivas a SARS-CoV-2 por RT-PCR. El aislamiento se realizó en las líneas celulares Vero ATCC CCL-81 y Vero E6, evaluando el efecto citopático, la presencia del virus por RT-PCR, inmunofluorescencia indirecta (IFI) y posterior identificación por secuenciación genómica. Posteriormente, uno de los aislamientos de mayor circulación fue seleccionado y denominado cepa prototipo (PE/B.1.1/28549/2020), realizándose 10 pasajes sucesivos en células Vero ATCC CCL-81 para evaluar la dinámica de mutaciones. Resultados: Se observaron 11 aislamientos de virus por efecto citopático confirmándose por RT-PCR e IFI, de los cuales 6 fueron secuenciados identificándose los linajes B.1, B.1.1, B.1.1.1 y B.1.205, según el comité Pango de los genomas. La cepa prototipo corresponde a la variante B.1.1 y el análisis de las secuencias de los pasajes sucesivos mostró mutaciones a nivel de la proteína de la espiga (S) del virus, sin variación en la identidad del linaje. Conclusiones: Se aislaron 4 linajes en la línea celular Vero ATCC CCL-81. Los subcultivos en la misma línea celular muestran mutaciones en la proteína de la espiga, lo que indica mayor adaptabilidad a la célula hospedera y variación de la patogenicidad in vitro, comportamiento que le permite tener más éxito de supervivencia.

Introduction: Currently, infections caused by the SARS-CoV-2 virus exceed 600 million cases in the world. Objective: Isolation and characterization of the SARS-CoV-2 virus causing COVID-19 at the beginning of the pandemic in Peru. Materials and methods: Twenty nasal and pharyngeal swab samples were isolated from SARS-CoV-2 using two cell lines, Vero ATCC CCL-81 and Vero E-6; virus identification was performed by RT-PCR and the onset of cytopathic effect (CPE) was evaluated by indirect immunofluorescence and subsequent identification by genomic sequencing. One of the most widely circulating isolates were selected and named the prototype strain (PE/B.1.1/28549/2020). Then 10 successive passages were performed on Vero ATCC CCL-81 cells to assess mutation dynamics. Results: We detected 11 virus isolates by cytopathic effect, and subsequently confirmed by RT-PCR and indirect immunofluorescence. Of these, six were sequenced and identified as the lineages B.1, B.1.1, B.1.1.1, and B.1.205 according to the Pango lineage nomenclature. The prototype strain corresponded to lineage B.1.1. The analysis of the strains from the successive passages showed mutations mainly at in the spike (S) protein of the virus without variation in the identity of the lineage. Conclusions: Four lineages were isolated in the Vero ATCC CCL-81 cell line. Subcultures in the same cell line show mutations in the spike protein indicating greater adaptability to the host cell and variation in pathogenicity in vitro, a behavior that allows it to have more survival success.

Optimization of culture conditions of SARS-CoV-2 four Omicron variants in Vero cells / 中国生物制品学杂志

@#Objective To optimize the culture conditions of four vaccine candidates of severe acute respiratory symptom coronavirus 2(SARS-CoV-2) Omicron variants BA.1,BA.1.1,BA.2 and BA.5 in Vero cells.Methods The harvest time(24,48,72 and 96 h) and MOI(0.01,0.001,0.0001 and 0.000 01) of four Omicron variants cultured in Vero cells were optimized by using cytopathic effect(CPE),viral nucleic acid copy number and viral titer as evaluation indexes.Results The optimum harvest time of the four Omicron variants BA.1,BA.1.1,BA.2 and BA.5 in Vero cells was 72 h,and the optimum MOI was 0.001～0.000 01,0.001～0.000 01,0.01～0.000 01 and 0.01～0.000 01,respectively.Conclusion The culture conditions of four Omicron variants in Vero cells were optimized,which laid a foundation of the development of SARS-CoV-2 Omicron variant inactivated vaccine based on Vero cells.

Development and verification of qPCR method for detection of residual host DNA in human rabies vaccine(Vero cells) / 中国生物制品学杂志

@#Objective To develop and verify a qPCR method for the qualitative and quantitative analysis of residual host DNA in human rabies vaccine(Vero cells) stock solution.Methods The qPCR standard curve was established by using the Vero cell DNA quantitative national standard,and the residual host DNA was extracted using magnetic beads.The specificity,repeatability,intermediate precision,accuracy and durability of the method were verified,and the linear range and limit of quantification were determined.The residual DNA of three batches of human rabies vaccine(Vero cells) stock solution was quantitatively analyzed and the fragment size was qualitatively analyzed by using this method.Results The correlation coefficients(R~2) of Vero cell DNA quantitative national standard amplification standard curve were all more than 0.99 by qPCR,and the quantitative range was 0.3 pg/mL～30 ng/mL.The method showed good specificity and repeatability.In the verification of intermediate precision,accuracy and durability,the relative standard deviations(RSD)of detection results of the samples were all less than 10%.The residual DNA content of Vero cells in three batches of stock solution was 0.20～0.77 ng/dose,which met the relevant standard of Chinese Pharmacopoeia(Volume Ⅲ,2020edition).The residual DNA fragments greater than 154 bp accounted for 52%～63%.Conclusion The developed qPCR method for the detection of residual DNA in human rabies vaccine(Vero cells) stock solution had good specificity,repeatability,intermediate precision and durability,and qualitatively and quantitatively analyzed the residual DNA rapidly and accurately,which was of great significance for improving the detection and control of residual DNA content in the production process and final product of human rabies vaccine(Vero cells).

Development and verification of a quantitative real-time PCR method for determination of host cell DNA residues in inactivated SARS-CoV-2 vaccine(Vero cells) / 中国生物制品学杂志

@#Objective To develop and verify a quantitative real-time PCR method for determination of the content of host cell DNA residues in severe acute respiratory symptom coronavirus 2(SARS-CoV-2) inactivated vaccine(Vero cells),in order to better control the safety of products.Methods DNA was extracted from inactivated SARS-CoV-2 vaccine(Vero cells) bulk by magnetic bead separation method,and the DNA residues of host cells were quantitatively analyzed by probetype PCR.The linear range,repeatability,intermediate precision,quantitative limit,specificity,durability and accuracy of the developed method were verified,and the host cell DNA re sidues of 5 batches of inactivated SARS-CoV-2 vaccine(Vero cells)were determined by this method.Results DNA standard curve showed good linearity in the range of 300～0.003 pg/μL(each R~2> 0.99);Relative standard deviations(RSD) of repeatability and intermediate precision verification were less than 20%;The quantitative limit was 0.001 pg/μL;Sample dilution and purified liquid dilution had no interference to detection;The results of 60 min incubation at 53,55,57 ℃ and 56,60,64 min incubation at 55 ℃ showed no significant difference;The recoveries of accuracy verification were 79%～83%,RSD <5%.This method had good adaptability in detecting DNA residues in the bulk of inactivated SARS-CoV-2 vaccine(Vero cells).Conclusion The quantitative realtime PCR method for determination of host cell DNA residues in inactivated SARS-CoV-2 vaccine(Vero cells) has been successfully developed,of which the linearity and range,repeatability,intermediate precision,quantitative limit,specificity,durability and accuracy meet the acceptable standards,and are suitable for the detection and quality control of host cell DNA residues in inactivated SARS-CoV-2 vaccine(Vero cells).

Long-term infection passaging of Human Adenovirus 36 in monkey kidney cells

ABSTRACT Human Adenovirus 36 (HAdV-36) has been related to diverse effects on metabolism and may attenuate the lipid accumulation in kidneys with increased adiposity. Some of these effects would be related to viral persistence. However, until now, a model of persistent in vitro infection by HAdV-36 is unknown. In this study, we examined the cells of the Vero lineage to explore their permissiveness to long-term HAdV-36 infection. HAdV-36 was productively replicated in Vero cells and maintained long-term infection for up to 35 cell passages. A subculture was obtained from the cells that survived the primary infection at a low MOI (0.5). The production of the extracellular infectious virus with titers ranging from 104 to 106 TCID50/mL and DNA-bearing cells was detected. In long-term infected cells, the intracellular distribution of viral antigen was demonstrated by performing immunolocalization (IFI) and expression of cell-viral antigen in 50% of cells by flow cytometry, using anti-HAdV-36 hyperimmune rabbit serum. Furthermore, E1a and E4orf1 genes in long-term infected passages showed a decreasing trend. Our preliminary results reveal that renal epithelial monkey cells are permissive for the productive infection of HAdV-36. Vero cell culture long-term infection might be a promising model for addressing the fundamental aspects of the HAdV-36 biology that cannot reveal broadly-used cultures, which do not maintain long-term infection in primary or transformed cells.

Freeze-dried rabies vaccine followed by death due to septic shock： a case report / 上海预防医学

ObjectiveTo analyze a case of death after inoculation of a freeze-dried rabies vaccine for human use so as to provide reference for the vaccination of the rabies vaccine and the process of investigation and diagnosis involving adverse events following immunization （AEFI） in the future. MethodsData on vaccination， clinical symptoms， treatments， investigation and diagnosis were collected and analyzed. ResultsRabies post-exposure prophylaxis and vaccination were in line with the protocols. On the 3rd day after the inoculation of the rabies vaccine， the patient developed fever， weakness， headache， dizziness， diarrhea and other symptoms. The white blood cell count and neutrophil count increased progressively. At about 17：00 on the same day， the patient suffered a sudden cardiac arrest and died clinically. Autopsy was not carried out. ConclusionThe cause of sepsis /septic shock of the patient is unknown. It is necessary to formulate detailed rabies immunization procedures as well as norms and expert consensus in the field of investigation and diagnosis of AEFI.

Freeze-dried rabies vaccine followed by death due to septic shock： a case report / 上海预防医学

ObjectiveTo analyze a case of death after inoculation of a freeze-dried rabies vaccine for human use so as to provide reference for the vaccination of the rabies vaccine and the process of investigation and diagnosis involving adverse events following immunization （AEFI） in the future. MethodsData on vaccination， clinical symptoms， treatments， investigation and diagnosis were collected and analyzed. ResultsRabies post-exposure prophylaxis and vaccination were in line with the protocols. On the 3rd day after the inoculation of the rabies vaccine， the patient developed fever， weakness， headache， dizziness， diarrhea and other symptoms. The white blood cell count and neutrophil count increased progressively. At about 17：00 on the same day， the patient suffered a sudden cardiac arrest and died clinically. Autopsy was not carried out. ConclusionThe cause of sepsis /septic shock of the patient is unknown. It is necessary to formulate detailed rabies immunization procedures as well as norms and expert consensus in the field of investigation and diagnosis of AEFI.

Suspension culture of Vero cells for the production of adenovirus type 5

Virulência e multiplicação de isolados de Toxoplasma gondii da região central do Rio Grande do Sul / Virulence and multiplication of Toxoplasma gondii isolates from the central region of Rio Grande do Sul, Brazil

O protozoário Toxoplasma gondii possui a capacidade de infectar diversas espécies animais, geralmente causando distúrbios reprodutivos. Quatro diferentes isolados de T. gondii - Pains #1 (P1), Pains #2 (P2), Santa Flora #1 (SF1) e Santa Flora #306 (SF306) - foram avaliados, após prévia genotipagem. A capacidade de multiplicação e virulência destes isolados foi analisada in vitro (células Vero) e in vivo (camundongos), sendo realizadas 3 passagens para cada isolado em cada modo avaliado, sendo sempre inoculada a dose de 1x104 taquizoítos em todas as passagens. Os camundongos eram observados diariamente, quanto à presença de sinais clínicos e ocorrência de mortalidade após inoculação dos taquizoítos. Os isolados SF1 e SF306, foram os que apresentaram maior multiplicação média do número total de taquizoítos em cada uma das 3 diferentes passagens realizadas para cada um dos isolados tanto in vitro quanto in vivo. Os primeiros sinais clínicos observados nos camundongos ocorreram entre os dias 5 a 11, após inoculação, com mortalidade acontecendo entre os dias 6 a 15, após inoculação. Assim, a multiplicação parasitária in vitro é semelhante à multiplicação in vivo destes isolados de T. gondii; diferentes isolados com o mesmo genótipo apresentam comportamento de virulência semelhante, caracterizando o isolado SF1 como mais virulento para camundongos.(AU)

The protozoan Toxoplasma gondii has the ability to infect several animal species, usually causing reproductive disorders. Four different isolates of T. gondii - Pains #1 (P1), Pains #2 (P2), Santa Flora #1 (SF1) and Santa Flora #306 (SF306) were evaluated with prior genotyping. Their virulence and multiplication capacity were analyzed in vitro (Vero cells) and in vivo (mice). For each isolate, three passages were performed with dose inoculation 1x104 tachyzoites at each passage. The mice were daily observed to verify of clinical signs and occurrence of mortality after tachyzoites inoculation. The SF1 isolate and SF306 isolate, presented the highest multiplication of the total tachyzoites number in each of the 3 different passages performed in vitro and in vivo. The initial clinical signs in mice were observed between 5 and 11 days, and occurring mortality between 6 and 15 days, after inoculation. Thus, the parasitic multiplication of theses isolates is similar in vitro and in vivo; different isolates within the same genotype have a similar virulence and the SF1 isolate is the most virulent for mice.(AU)

Effect of Siji Kangbingdu Mixture on Inhibition of the Proliferation of Coxsackievirus A16 / 医药导报

Objective To verify the antiviral effects of Siji Kangbingdu mixture (SJKBDM) against coxsackievirus A16 (CoxA16).Methods Vero cells and 5-day-old suckling mice, injected with 75/50 and 1×106 TCID50 CoxA16, were used as evaluation models.The preventive influences of SJKBDM against CoxA16 in Vero cells were assessed in the models.The effects of SJKBDM on the mortality, survival time, change rate of body weight, and clinical symptom scores of suckling mice were observed.Results ①The half maximal inhibitory concentration of SJKBDM on Vero cells was 9.59 mg·mL-1.②Toxic effects were not observed from 32.3 g·kg-1 single dose or continuous intraperitoneal injectin of SJKBDM in suckling BALB/c mice.③The SJKBDM had significant inhibitory effect against CoxA16 virus.Doses higher than 1.22 mg·mL-1 could significantly improve the Vero cell survival rate, and the SJKBDM inhibition of 75/50 TCID50 CoxA16 induced pathological changes in Vero cells.④The SJKBDM significantly improved clinical symptoms of mice with CoxA16 viral infection, especially with crude drug doses of higher than 1.62 g·kg-1.The survival rate and other indicators were comparable or slightly higher compared with ribavirin, and the clinical score was higher than that of ribavirin.Conclusion The SJKBDM has significant inhibitory effect on CoxA16 cell proliferation, significantly decreases death rate, and improves clinical symptoms of mice infected with CoxA16 virus.

PKH26 combined with Hoechst 33258 to trace the iRhom2 gene and its mutant proteins of Uncv mice in Vero cells / 中国比较医学杂志

Objective To determine the localization of iRhom2 and its mutant proteins of Uncv mice in Vero cells by PKH26 combined with Hoechst 33258 staining.Methods The cell membrane was stained with PKH26, and the nuclei were stained with Hoechst 33258 dye, and observed by laser scanning confocal microscopy.Results It was found that wild iRhom2 was distributed in the cytoplasm, and its iRhom2mut was present both in cytoplasm and cell nuclei.Conclusions The results of our study suggest that a deletion in N-terminal of iRhom2 affects its subcellular localization.

Antiviral effect of Reduning Injection against coxsackievirus A16 / 中草药

Objective: To verify the antiviral effects of Reduning Injection against coxsackievirus A16 (CoxA16) in vivo and in vitro. Methods: Vero cells and 5-day-old suckling mice, injected with 75/50 and 106 TCID50 CoxA16, were used as evaluation models. The preventive influences of Reduning Injection against CoxA16 in Vero cells were assessed in the models. The effects of Reduning Injection on the mortality, survival time, change rate of body weight, and clinical symptom scores of suckling mice were observed. Results: In Vero cells, cytopathic effects induced by 75 and 50 TCID50 CoxA16 were obviously relieved by crude drug5.0 mg/mL Reduing Injection respectively. Meanwhile, mice death caused by CoxA16 was markedly rescued, survival time was prolonged, and growth inhibition was recovered by Reduing Injection. Meanwhile, the clinical symptom induced by virus was also improved. Conclusion: The endogenous and exogenous antivirus effects of Reduning Injection on CoxA16 are proven.

Safety and Efficacy of Prunus africana and Warburgia ugandensis Against Induced Asthma in BALB/c Mice.

Aim: In-Vitro and In-Vivo safety and anti-asthmatic activity of stem bark extracts of Prunus africana and Warburgia ugandensis against induced asthma in BALB/c mice. Methodology: Cytotoxicity on Vero E6 cells were investigated using MTT assay. Acute toxicity was determined by administering single oral gavages of extracts to five groups of BALB/c at 500, 889.56, 1581.64, 2812.15 and 5000mg/kg body weight doses. Efficacy against induced asthma was determined by assaying heart blood serum for ovalbumin specific immunoglobulin E (IgE) antibodies and quantification of eosinophil proportion in Bronchoalveolar lavage fluid (BALF). Eight sensitized groups were used, 2 were controls, 3 were treated with P. africana extract and 3 with W. ugandensis; each treatment group received one dose concentration of 125, 250 or 500mg/kg body weight of either plant extracts. Results: P. africana CC50 was 104.08μg/ml while W. ugandensis had CC50 > 250 μg/ml. In acute toxicity, mortality and signs of toxicity were recorded within 24 hours and the mice monitored for 14 days. There was 20%, 60% and 100% mortality within 24 hours for mice that received P. africana extracts at 1581.64, 2812.15 and 5000mg/kg body weight respectively. Lethal dose (LD50) for P. africana was 2201.207mg/kg body weight. W. ugandensis extracts had no mortality recorded and the LD50 was >5000mg/kg body weight. Treatment with P. africana extracts at 500mg/kg body weight reduced the IgE and BALF Eosinophil to 0.100±0.0001 and 2.80±0.20 respectively which were significantly different from positive controls P<0.05. W. ugandensis extracts at the same concentration reduced the IgE and BALF eosinophils to 0.134±0.00016 and 3.80±0.20 respectively and were significantly different from positive controls P<0.05. Conclusion: The results attested that P. africana and W. ugandensis stem bark extracts have anti-asthmatic property though there is need for further validation of anti-asthmatic chemical compounds to augment the findings.

The influence of serum substituents on serum-free Vero cell conditioned culture media manufactured from Dulbecco's modified Eagle medium in mouse embryo culture

OBJECTIVE: This study was conducted to examine the influences of supplementation of the serum substituents and available period of serum-free Vero cell conditioned media (SF-VCM) manufactured from Dulbecco's modified Eagle medium cultured with Vero cells for in vitro development of mouse preimplantation embryos. METHODS: A total of 1,099 two-cell embryos collected from imprinting control region mice were cultured in SF-VCM with 10% and 20% human follicular fluid (hFF), serum substitute supplement (SSS), and serum protein substitute (SPS). Development of embryos was observed every 24 hours. Results between different groups were analyzed by chi-square test, and considered statistically significant when P-value was less than 0.05. RESULTS: The rates of embryonic development cultured in SF-VCM supplemented with serum substituents were significantly higher compare with serum-free group (P < 0.05). The rates of embryonic development after 48 hours (morula< or =) and 96 hours (blastocyst< or =) were significantly higher in 20% SSS and 10% SPS than in 20% hFF supplementation (P < 0.05). And the rates of embryonic development after 96 hours (hatching blastocyst< or =) were significantly higher in 10% SPS (94.5%) than in 20% SSS (82.6%) and 20% hFF supplementation (68.5%). The rates of embryonic development according to storage period of the SF-VCM supplemented with 10% SPS showed no significant difference between control, 2 weeks and 4 weeks group. However developmental rate in 6 weeks storage group was significantly lower than other groups. CONCLUSION: The rate of embryonic development after 96 hours (hatching blastocyst< or =) was significantly higher in SF-VCM supplemented with 10% SPS. And storage period of media up to 4 weeks did not affect on embryonic development.

Effects of microRNA hsa-mir-634 on the proliferation and apoptosis of Vero cells / 中华皮肤科杂志

Objective To investigate the function and possible action mechanisms of microRNA hsa-mir-634 in Vero cells.Methods The binding sites for hsa-mir-634 in the 3' UTR of cyclin D1 (CCND1) were predicated by bioinformatics methods.Then,the 3'UTR sequence of CCND1 containing the binding sites for hsamir-634 was amplified by PCR.Site-directed mutagenesis was used to create mutations in the binding sites.The wild and mutant 3' UTR sequences of the CCND1 gene were ligated into the psi-CHECK2 vector separately to construct dual-luciferase reporter vectors,including CHECK2-CCND1 wild,CHECK2-CCND1 mut 1,CHECK2-CCND1 mut 2 and CHECK2-CCND1 mut 3.Then,293T cells were transfected with the four constructed plasmids,and luciferase activity was measured 48 hours after the transfection.Vero cells were transfected with hsa-mir-634 mimics and negative control separately,and harvested after additional culture for different durations; the Vero cells remaining untreated served as the blank control.Subsequently,fluorescence-based quantitative PCR and Western blot were performed to detect the mRNA and protein expressions of CCND1 respectively in,3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt (MTS) assay to evaluate the proliferation of,and flow cytometry to detect the apoptosis in,Vero cells.Results The binding sites for hsa-mir-634 in the 3'UTR of CCND1 were successfully predicated.Sequencing results showed the successful construction of dual-luciferase reporter vectors.As the luciferase assay revealed,the overexpression of hsa-mir-634 could significantly inhibit the CCND1 3'UTR-mediated luciferase activity.Compared with the negative control,the hsamir-634 mimics markedly decreased the protein expression of CCND1,but had no obvious effect on the mRNA expression of CCND1 in Vero cells.The proliferation of Vero cells transfected with hsa-mir-634 mimics was significantly restrained compared with those transfected with the negative control,and the strongest restraining effect was observed on day 4 after the transfection.In addition,the overexpression of hsa-mir-634 also induced the apoptosis of Vero cells,with the apoptosis rate being 8.03％,7.96％ and 17.33％ in the blank control group,negative control group and mimics group respectively.Conclusion Hsa-mir-634 may regulate the proliferation and apoptosis of Vero cells via influencing the expression of CCND1.

Effects of herpes simplex virus 2 latency-associated transcript open reading frame 3 on the apoptosis in Vero cells / 中华皮肤科杂志

Objective To explore the effects of herpes simplex virus 2 (HSV-2) latency-associated transcript open reading frame 3 (LAT ORF3) gene on Vero cells against cisplatin-induced apoptosis.Methods Recombinant plasmid enhanced green fluorescent protein-open reading frame 3 (named pEGFP-ORF3) was constructed and transfected into Vero cells; then,reverse transcription (RT)-PCR was performed to detect the expression of the target gene.Cisplatin of 3 mg/L was selected to induce the apoptosis in Vero cells.Cultured Vero cells were transfected with empty plasmid and induced by cisplatin (pEGFP-C2 group),transfected with recombinant plasmid pEGFP-ORF3 and induced by cisplatin (pEGFP-ORF3 group),only induced by cisplatin (cisplatin-induced control group),or remained untreated (normal control group).Subsequently,fluorescence microscopy was conducted to observe apoptotic bodies,Giemsa stain to observe the morphology of cell nuclei,methyl thiazolyl tetrazolium (MTT) assay to evaluate cell proliferation,and flow cytometry to assess cell apoptosis.Data were assessed by using SPSS 13.0 software,and statistical analysis was carried out by one-way ANOVA and t test.Results HSV-2 333 LAT ORF3 gene was successfully cloned.The eukaryotic expression plasmid for LAT ORF3 was constructed,and the expression of LAT ORF3 gene in Vero cells was confirmed by RT-PCR.Giemsa stain showed blue-staining nuclei and pale cytoplasm in recombinant plasmid-transfected and cisplatin-induced Vero cells with a normal shape.The value of cell proliferation (absorbance at 490 nm) by MTT assay was 2.56 ± 0.21 in pEGFP-ORF3 group,similar to that in the normal control group (2.66 ± 0.13,P ＞ 0.05),but significantly higher than cisplatin-induced control group (1.65 ± 0.11,P ＜ 0.05) and pEGFP-C2 group (1.56 ± 0.18,P ＜ 0.05).As far as the apoptosis rate was concerned,no significant difference was observed between pEGFP-ORF3 group and normal control group (4.03％ ± 1.04％ vs.2.13％ ± 0.09％,P ＞ 0.05),but pEGFP-ORF3 group was statistically lower than pEGFP-C2 group (19.45％ ± 2.05％,P ＜ 0.05).Conclusion The transfected HSV-2 LAT ORF3 gene could protect Vero cells from cisplatin-induced apoptosis.