ABSTRACT
Objective To observe the effects of different collagenase digestions on isolating human umbilical cord mesenchymal stromal cells (MSC) from Wharton’s jelly, to exam their differentiation ability and to investigate their passage effect on the immune phenotype. Methods Human umbilical cord samples were digested by collagenaseⅠorⅡorⅣfor 4-18 hours then were passed through sieves . Cells were collected by centrifugation then inoculated in DMEM/F12 medium at concentration within range of 4.8×103-1×104/cm2 to compare the effect of different digestions on MSC. Von kossa staining and tetracycline fluorescence was used to label the osteogenic differentiation capacity of MSC. Also RT-PCR was employed to identify the differentiate capacity of MSC into myocardial-like cells. The immunophenotype of MSCs were detected by flow cytometry after subculture. Results Using collagenaseⅠdigestion, the number of MSCs isolated from human umbilical cord in Wharton’s jelly and their vitality were much higher while the period to show cell extension and primary culture time were shorter than those using collagenaseⅡorⅣdigestions. The analysis of surface marker revealed that the expression of positive markers include CD29, CD44, CD73, CD90 and CD105 did not change with passages while the negative markers such as CD31, CD34 and HLA-DR increased significantly with passages;Differential experiments induced in vitro show that human umbilical cord MSC in wharton’s jelly had the ability to differentiate into osteoblasts and myocardial-like cells. Con?clusion The human umbilical cord MSC in Wharton’s jelly was successfully isolated by collagenaseⅠdigestion. This meth?od was simple with a high success rate while cell loss and damage were minimum. This makes large-scale cultivation possi? ble. Negative markers increased with cell passages. This phenomenon revealed that MSC showed directional differentiation.
ABSTRACT
Objective To understand the functions of ORF27 protein of the Thai isolated Helicoverpa armigera nucleopolyhedrovirus(HaNPV-Th) and explain the possible molecular mechanism of HaNPV-Th in passage effect.Methods ORF27 gene was cloned and amplified by polymerase chain reaction(PCR) from HaNPV-Th genome.The physical-chemical properties,subcellular localization,signal peptide,transmembrane structure and functional motifs were analyzed by the software of ProtParam,TMpred,SignalP and ScanProsite.The truncated ORF27 gene which lacked parts of the amino-terminal was expressed by bacterial expression system.Results The full length sequence of ORF27 was 768bp long and encoded 255aa with a molecular weight of 29.5ku.Several functional motifs on ORF27 were predicted by bioinformatics software.The truncated ORF27 recombinant expressed 35ku target protein mainly in the form of inclusion body.Conclusion ORF27 protein of HaNPV-Th is presumed to possess extensive biological activities.It may regulate various cellular signaling pathways,apoptosis and cell cycle,which are involved in passage effect.