ABSTRACT
@#There are many ways to extract and culture neural stem cells in vitro, but the viability and stability of neural stem cells obtained by different methods are different. By thinking about the process of extracting and culturing neural stem cells in vitro from the cerebral cortex of SD fetal rats, we summarized extraction steps, the main points of extraction, the selection basis of culture medium, selection of inoculation density, cultural method, methods of solution changing, passage time and passage methods. At last a large number of neural stem cells with high vitality and stability have been obtained and applied to the basic research of neural stem cells.
ABSTRACT
Objective To observe the changes in cytoskeleton (CSK) and glycosaminoglycan (GAG) synthesis following passage culture of articular chondrocytes and the correlation between CSK and GAG.Methods Eight male New Zealand White rabbits (8-month-old) were sacrificed by air embolism.After the chondrocytes from their knee joints were isolated by enzymolysis method,monolayer culture was performed.The chondrocytes of primary passage (P0) and passages 1 & 2 (P1,P2) were inoculated into 24-well plates with round cover slips put at the bottoms.Cell climbing slices were fixed after attachment of chondrocytes.The CSK proteins,actin,vimentin,tubulin and vinculin were stained by immuuofluorescence antibody on P0,P1 and P2 cell climbing slices,respectively.The CSK morphology was observed by laser confocal scanning microscopy and the fluorescence intensities of CSK proteins were detected by the fluorescence intensity software.The medium was changed for each generation after cell fusion and the GAG concentrations in the supernatants were measured at 24,36,48,60 h after medium change by alcian blue method.Results The intermediate filament networks became loosen and the dense distributions surrounding the nucleus decreased;more microtubule processes formed at the cell periphery with passage.The fluorescence intensity of actin of P1 chondrocytes was significantly increased than that of P0 (P ＜ 0.05),but there were no such significant differences between P0 and P2 or between P1 and P2 (P ＞ 0.05).The fluorescence intensities of vimentin and tubulin were significantly decreased with passage respectively,and there were such significant differences between any two of P0,P1 and P2 (P ＜ 0.05).The GAG concentrations in the supernatants were significantly decreased with passage at each time point,and there were such significant differences between any two of P0,P1 and P2 (P ＜ 0.05).Conclusions Passage culture of articular chondrocytes may lead to changes in morphology and protein expression intensity of the main components of CSK,and accordingly to decreased synthesis amount of GAG,one of the extracellular matrix of chondrocytes,indicating the changed characteristics of chondrocytes after passage and a certain correlation between CSK and GAG.
ABSTRACT
Due to the increased frequency of interspecies transmission of avian influenza viruses, studies designed to identify the molecular determinants that could lead to an expansion of the host range have been increased. A variety of mouse-based mammalian-adaptation studies of avian influenza viruses have provided insight into the genetic alterations of various avian influenza subtypes that may contribute to the generation of a pandemic virus. To date, the studies have focused on avian influenza subtypes H5, H6, H7, H9, and H10 which have recently caused human infection. Although mice cannot fully reflect the course of human infection with avian influenza, these mouse studies can be a useful method for investigating potential mammalian adaptive markers against newly emerging avian influenza viruses. In addition, due to the lack of appropriate vaccines against the diverse emerging influenza viruses, the generation of mouse-adapted lethal variants could contribute to the development of effective vaccines or therapeutic agents. Within this review, we will summarize studies that have demonstrated adaptations of avian influenza viruses that result in an altered pathogenicity in mice which may suggest the potential application of mouse-lethal strains in the development of influenza vaccines and/or therapeutics in preclinical studies.
Subject(s)
Animals , Humans , Mice , Host Specificity , Influenza A virus , Influenza in Birds , Influenza Vaccines , Methods , Orthomyxoviridae , Pandemics , Serial Passage , Vaccination , Vaccines , VirulenceABSTRACT
Objective To establish a mouse lethal model of influenza B virus , which will facilitate the study on the mechanism of pathogenesis , transmission of influenza B virus , development of new vaccines and drugs against influenza B virus.Methods We obtained a mouse adaptive B/Lee/1940 virus by continuously passaging it in mice for 5 cycles.The P5 virus was propagated in MDCK cells , which was used for infecting mice .The body mass and survival rate of mice were monitored during the following 14 days after infection.At the same time,the 8 gene segments (PB2, PB1, PA, HA, NA/NB, NP, M, and NS) of P0 and P5 virus were sequenced and analyzed .Results and Conclusion Virus was detected in the lungs of mice in each generation in the process of virus passaging .The body mass of mice infected with the deadly mouse adaptive virus changed dramatically .The mortality of mice was 100%, and virus was detected in mouse lungs . Sequence analysis results indicated that the amino acid mutations occurred in PB 2 and NP.A series of experiments indicated that we had established a mouse lethal model of influenza B virus .
ABSTRACT
Objective To explore the stability of resistant phenotypes and changes of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) gene system on four Shigella strains in the absence of antibiotics.Methods Four clinical isolated Shigella strains that resistant to different antibiotics were consecutive passaged for 90 times without antibiotics.Agar dilution method was used to determine the minimum inhibitory concentration of Shigella strains.After sequence analysis with PCR,CRISPR Finder and Clustal X 2.1 were applied to identify the changes of CRISPR loci in the Shigella strains.Results After the consecutive transfer of 90 generations,sensitivity to certain antibiotics of four Shigella strains with different drug resistant spectrums increased.Mel-sf1998024/zz resistance to ampicillin,cephalexin,cefotaxime,chloramphenicol decreased,mel-s2014026/sx resistance to norfloxacin,trimethoprim decreased,mel-sf2004004/sx drug resistance to ampicillin,cefuroxime,cefotaxime,chloramphenicol,trimethoprim decreased and mel-sf2013004/bj resistance to chloramphenicol decreased.The spacer of which matched gene codes Cas and its upstream repeat in 3'end of CRISPR3 got lost in mel-sf1998024/zz and mel-sf2013004/bj.Conclusions Shigella strains could reduce or lose their resistance to some antibiotics after consecutive transfers,without the interference of antibiotics.CRISPR3 locus had dynamic spacers in Shigella strains while CRISPR3 locus and cas genes might have been co-evolved.
ABSTRACT
Objective To explore the stability of resistant phenotypes and changes of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) gene system on four Shigella strains in the absence of antibiotics.Methods Four clinical isolated Shigella strains that resistant to different antibiotics were consecutive passaged for 90 times without antibiotics.Agar dilution method was used to determine the minimum inhibitory concentration of Shigella strains.After sequence analysis with PCR,CRISPR Finder and Clustal X 2.1 were applied to identify the changes of CRISPR loci in the Shigella strains.Results After the consecutive transfer of 90 generations,sensitivity to certain antibiotics of four Shigella strains with different drug resistant spectrums increased.Mel-sf1998024/zz resistance to ampicillin,cephalexin,cefotaxime,chloramphenicol decreased,mel-s2014026/sx resistance to norfloxacin,trimethoprim decreased,mel-sf2004004/sx drug resistance to ampicillin,cefuroxime,cefotaxime,chloramphenicol,trimethoprim decreased and mel-sf2013004/bj resistance to chloramphenicol decreased.The spacer of which matched gene codes Cas and its upstream repeat in 3'end of CRISPR3 got lost in mel-sf1998024/zz and mel-sf2013004/bj.Conclusions Shigella strains could reduce or lose their resistance to some antibiotics after consecutive transfers,without the interference of antibiotics.CRISPR3 locus had dynamic spacers in Shigella strains while CRISPR3 locus and cas genes might have been co-evolved.
ABSTRACT
Objective To observe the effects of long-term low dose arsenic exposure through drinking water on learning ability of different generations of C3H and Balb/c mice.Methods Mice (C3H and Balb/c) were exposed to arsenic at 0 mg/L (control) and 85 mg/L (20 female mice and 10 male mice per group).The control group and F1,F2,F3 and F4 mice were selected and divided into 5 experimental groups,8 mice in each group.Their offsprings were detected by the Morris water maze test (the average escape latency of 1 to 5 days) and spatial probe test (the times of through target area on the sixth day).Statistical analysis was performed with SPSS 18.0 software.Results The average escape latencies of 1 to 5 days in C3H control group were (48.09 ± 2.63),(46.09 ± 3.27),(42.72 ± 3.29),(39.31 ± 2.69) and (36.75 ± 3.92) s,F1 were (49.59 ± 3.29),(47.34 ± 3.01),(44.28 ± 6.58),(44.50 ±1.67) and (42.16 ± 2.27) s,F2 were (51.41 ± 0.78),(48.88 ± 1.45),(45.54 ± 1.46),(43.94 ± 1.69) and (42.22 ± 3.27) s,F3 were (50.91 ± 4.20),(49.78 ± 5.18),(48.03 3.45),(46.16 ± 4.42) and (44.06 ± 1.04) s,F4 were (52.66 ± 4.60),(52.38 ± 5.78),(49.06 ± 1.22),(47.69 ± 2.34) and (46.47 ± 1.56) s.The average escape latencies of Balb/c control group were (50.91 ± 2.84),(47.03 ± 4.22),(45.56 ± 4.53),(39.72 ± 5.90) and (36.22 ± 4.85) s,F1 were (50.47 ±3.20),(48.25 ± 6.53),(47.13 ± 1.25),(43.72 ± 4.27) and (40.66 ± 4.52) s,F2 were (51.31 ± 4.73),(48.88 ± 1.53),(46.56 ± 1.43),(44.25 ± 1.16) and (41.20 ± 3.79) s,F3 were (51.72 ± 3.54),(50.78 ± 4.45),(45.03 ± 3.56),(41.19 ±5.63) and (42.81 ± 6.29) s,F4 were (53.34 ± 4.60),(52.34 ± 2.77),(48.72 ± 5.92),(46.97 ± 7.38) and (44.94 ± 1.75) s.On the fourth and fifth days of F1,F2,F3 and F4 generations of C3H,the escape latencies between generations were significantly different (all P ＜ 0.05).The times of through target area in the sixth day of the C3H control group and F1,F2,F3 and F4 mice were 2.25,1.75,1.63,1.50 and 1.38,Balb/c were 2.13,1.75,1.63,1.38 and 1.13.Conclusion Arsenic accumulation due to serial passage of C3H and Balb/c through long-term low doses arsenic exposure through drinking water has resulted in decreased learning and memory ability.
ABSTRACT
BACKGROUND AND OBJECTIVES: Adipose-derived mesenchymal stem cells (ADSCs) are promising candidates in regenerative medicine. The need for in vitro propagation to obtain therapeutic quantities of the cells imposes a risk of impaired functionality due to cellular senescence. The aim of the study was to analyze in vitro senescence of previously cryopreserved human ADSCs subjected to serial passages in cell culture. METHODS AND RESULTS: ADSC cultures from 8 donors were cultivated until proliferation arrest was reached. A gradual decline of ADSC fitness was observed by altered cell morphology, loss of proliferative, clonogenic and differentiation abilities and increased β-galactosidase expression all of which occurred in a donor-specific manner. Relative telomere length (RTL) analysis revealed that only three tested cultures encountered replicative senescence. The presence of two ADSC subsets with significantly different RTL and cell size was discovered. The heterogeneity of ADSC cultures was supported by the intermittent nature of aging seen in tested samples. CONCLUSION: We conclude that the onset of in vitro senescence of ADSCs is a strongly donor-specific process which is complicated by the intricate dynamics of cell subsets present in ADSC population. This complexity needs to be carefully considered when elaborating protocols for personalized cellular therapy.
Subject(s)
Humans , Aging , Cellular Senescence , Cell Culture Techniques , Cell Size , Mesenchymal Stem Cells , Population Characteristics , Regenerative Medicine , Serial Passage , Telomere , Tissue DonorsABSTRACT
Objective To understand the sensitivity of cytopathogenic effect (CPE) in MadinDarby canine kidney cells(MDCK) that cultured influenza A pharyngeal swab specimens of patients for one,two and three passages. Methods Influenza A pharyngeal swab specimens of patients were inoculated in MDCK for three blind passages. The presence of CPE of every passage was observed by inverted microscope. Results Of the 279 influenza A pharyngeal swab specimens of patients tested by colloidal gold, the presence of CPE in MDCK for one,two and three passages was 65.9%(184/279),91.4%(255/279) and 96.4%(269/279), respectively. Two hundred and seventy-one of 279specimens were identified as influenza A by multiplex reverse transcription-polymerase chain reaction (RT-PCR). Conclusion The positive separation rate can reach more than 95% by inoculating influenza A pharyngeal swab specimens of patients in MDCK for three blind passages.