RESUMEN
AIM: To establish the transgenic cell strains expressing recombinant adenovirus vector of human Oncostain M(hOSM)gene which is supposed to be used as feeder layer cells for the proliferation of umbilical cord blood CD34(+) hematopoietic stem/progenitor cell (HSPC) and compare its migration capacity before and after amplification in vitro. METHODS: Establish the transgenic cell strains expressing recombinant adenovirus vector of hOSM gene, and the objective gene was detected by RT-PCR and ELISA. The purity of umbilical cord blood CD34(+) HSPC separated by magnetic-activated cell sorting (MACS) was detected by the FCM. After culturing with feeder layer cells, detect the rate of proliferation by flow cytometry (FCM). To compare the homing ability of HSPC after amplification in vitro, detect the spontaneous migration rate and migration rate induced by SDF-1 using transmembrane migration assay (Transwell experiment). RESULTS: The green fluorescence was observed by fluorescence microscope in the transgenic cell strains, and the objective gene was confirmed by RT-PCR and ELISA.The purity of umbilical cord blood CD34(+) HSPC separated by MACS could reach(96.8 ± 2.28)%. After culturing with feeder layer cells for 7 days, the CD34(+) cells were 15.73 times in group containing hOSM more than in group without hOSM. The expression rate of adhension molecules on the surface of CD34(+) cells were also higher in the group containing hOSM than without hOSM. After using Transwell assys to detect the homing ability of culturing cells, the induction migration rate of stem cells clturing on transgenic cell strains was (40.68 ± 1.35)%, significantly higher than the control, which reveals a better homing ability. CONCLUSION: Recombinant adenovirus vector of hOSM gene as feeder layer cells can effectively proliferate umbilical cord blood CD34(+) HSPC in vitro and delay it differentiate, what's more, the stem cells retain a high homing ability after culturing on transgenic cell strains in vitro.
Asunto(s)
Sangre Fetal/citología , Sangre Fetal/metabolismo , Células Madre Hematopoyéticas/metabolismo , Oncostatina M/genética , Oncostatina M/metabolismo , Células Madre/metabolismo , Adenoviridae/genética , Antígenos CD34/inmunología , Técnicas de Cultivo de Célula/métodos , Proliferación Celular , Separación Celular/métodos , Células Cultivadas , Técnicas de Cocultivo/métodos , Femenino , Técnicas de Transferencia de Gen/instrumentación , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Receptores CXCR4/metabolismoRESUMEN
BACKGROUND & OBJECTIVE: S100A13 is involved in tumor formation, and is highly expressed in human thyroid gland. This study was to investigate the effect of exogenous S100A13 overexpression on the proliferation of human thyroid cancer cell line TT. METHODS: The eukaryotic expression plasmid pCDNA3.1/NT-GFP-S100A13 and empty vector pCDNA3.1/NT-GFP were transfected into TT cells. The cells were selected by G418. The expression of green fluorescent protein (GFP) was observed under laser scanning microscope, and the expression of S100A13 mRNA and protein was detected by real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. The effects of S100A13 on cell proliferation and cell cycle progression were measured by cell growth curve and flow cytometry. RESULTS: TT-S100A13-GFP and TT-GFP cells, which separately expressed S100A13 and pCDNA3.1/NT-GFP, were constructed successfully. TT-S100A13-GFP cells grew faster than TT-GFP and TT cells [(2.30+/-0.24) x 10(5) vs. (1.40+/-0.25) x 10(5) and (1.50+/-0.22) x 10(5) at the 7th day of cell culture, P<0.05]; both S phase proportion and G2/M phase proportion were significantly higher in TT-S100A13-GFP cells than in TT-GFP and TT cells [(6.47+/-0.14)% vs. (5.86+/-0.23)% and (5.99+/-0.28)% at S phase, P<0.05; (50.27+/-0.66)% vs. (39.39+/-0.23)% and (39.64+/-0.64)% at G2/M phase, P<0.05]. CONCLUSION: Exogenous S100A13 gene overexpression could accelerate cell proliferation, and promote cell cycle progression of TT cells from G0/G1 phase to S and G2/M phase.