Timing Characteristics Of Morphology And Epithelial Mesenchymal Features Of Neural Crest Stem Cells In Rats

Objective:Neural crest stem cells?NCSCs?,a kind of cell with multi-directional differentiating potential during embryonic development,which can be isolated from neural crest by epithelial-mesenchymal transition?EMT?and differentiate into central and peripheral neurons and glial cells.At present,a large number of researches focus on NCSCs,but the research on morphological and biological functions of NCSCs at different developmental stages is still scarce.Therefore,we observed the morphology and in vitro value-added mode of NCSCs at different developmental stages,in order to provide research dependency for NCSCs selection.Further study on the characteristics of epithelial mesenchyme of rat NCSCs with different days,select E-cadherin/N-cadherin which is common in EMT as target gene and use immunofluorescence,Western blot and qRT-PCR technology to detect the expression of E-cadherin/N-cadherin in NCSCs of different days,to explore the role of EMT in the migration of NCSCs.Methods:1.Isolation and culture of NCSCsThe healthy,mature and unpregnant Wistar rats were selected.The male and female rats were caged in the midnight at ratio of 4:1 and the female vagina was smeared at8:00 the next day.Rats whose sperm were observed with microscope were confirmed to be mated,when to be considered as day 0 of pregnancy?embryonic day0,E0?.The Wistar pregnant rats at E8.5,E9.5,E11 and E13 were selected to extract NCSCs and culture passage.2.Cell climbing and cell HE staining.3.Detection of E-cadherin/N-cadherin in rat NCSCs by immunofluorescence,Western blot and qRT-PCR.4.Statistical analysisData are expressed as mean±standard deviation,using SPSS software to analyze experimental data.The relative expression level of target gene mRNA was analyzed by 2^-??ctmethod.On the experimental data,observe the change trend of the target gene expression of cells in following days.Results:1.Cultured different days of NCSCsNCSCs were successfully cultured on DMEM/F12 complete medium at 8.5,9.5,11and 13 days.2.Different days of NCSCs growth rate and HE staining resultsThe adherence,gathering,balling speed of small number of days NCSCS cells is faster,and the passaging ability of slightly stronger,can be passed to about 4generations.On the contrary,large number of days NCSCs grow slower than small number of cells,and the ability of passage is weaker,whose expression of neural crest stem cells is obviously weakened when they are passaged to the third generation,which made it difficult to gather into clusters to form neurospheres.HE staining showed that NCSCs have large nucleus and few cytoplasmic cytoplasm.In vitro culture we can find,after cells adherent,these cells get close to each other,arranged closely,perform as a colony-like growth,and eventually form a neurosphere suspension.As the number of days of primary cells increase,the number of nucleus slightly decrease and cytoplasm plasma increased gradually.3.ThechangesofE-cadherin/N-cadherinexpressioninNCSCswith Immunofluorescence,Western blot and qRT-PCR detection over time.The expression of E-cadherin decreases and the expression of N-cadherin increases with the number of NCSCs increasing.The expression of E-cadherin in NCSCs in the8.5 days is more obvious,while the 9.5th day,the 11th day and the 13th day's expression of E-cadherin significantly decrease.On the contrary,8.5 days expression of N-cadherin is not obvious,9.5 days,11 days,13 days are more obvious.Conclusion:?1?With the increase of the number of primary cells,there are some differences in cell morphology and growth rate of NCSCs.The smaller the day number,the larger the nucleus,the smaller the cytoplasm,the faster the cell growth,and the stronger the passage ability.?2?The decrease of E-cadherin and the increase of N-cadherin in the growth of NCSCs suggest that EMT plays an important role in the migration of NCSCs.And NCSCs may undergo EMT from 8.5 to 9.5 days after embryogenesis,leading to cell migration.