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Objective To generate hemogenic endothelial cells(HECs)from human induced pluripotent stem cells (hiPSCs)in vitro in order to learn more about the mechanism by which the vascular niche affects HECs production and self -renewal.Methods hiPSCs with reporter gene runx1c were differentiated to hematopoietic cells by spinEB method.The CD34 positive cells were sorted by magnetic-activated cell sorting(MACS)at day 10 after hematopoietic differentiation. Afterwards,these CD34 positive cells were co-cultured with DLL4 overexpressed vascular niche cells VeraVec to further differentiate to HECs.The HECs derived from the hiPSCs were characterized by FACS.Results We first established an hiPSCs single cell culture method for spinEB differentiation.Single cell cultured hiPSCs with reporter gene runx 1c were differentiated to form embryonic bodies(EBs)by spinEB method.The HECs were enriched from the day 10.Meanwhile, we cultured the E4ORF1 transfected human umbilical vein endothelial cell(HUVEC)line(VeraVec)and examined the expression of NOTCH signaling pathway related genes.According to the results, VeraVec had a high expression level of NOTCH ligand DLL4 at both mRNA and protein levels.And the CD34 positive HECs were co-cultured with DLL4 overexpressed VeraVec cells,which promoted the expression of tdTomato during hematopoitic differentiation and increased HSCs production.Conclusion A method of inducing hiPSCs differentiation by spinEB has been established, which can enrich HECs.This model can be applied to study the mechanism by which the vascular niche promotes hematopoietic differentiation from hPSCs.The generated functional HSCs are of great social and military values for HSCs transplantation and battlefield radiation injury treatment.
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Objective To evaluate the cytotoxicity of natural killer(NK)-92 cell lines against various human hepatocellular carcinoma cells(HCCs)and to explore the potential mechanism.Methods We established a culture method of NK-92 cell lines in vitro.Lactate debydrogenase(LDH)cytotoxicity assays and cytokine release assays were performed to determine whether NK-92 cell lines could recognize and kill HCCs in vitro.At the same time,Nu/Nu mices were housed. Subcutaneous(sc)xenografts HepG2 models of human hepatocellular carcinoma were established.1×107NK-92 cells were intravenously(iv)injected through the tail vein on days 2,9,16,23 while the control group was injected with PBS in the same way.Tumor size, tumor volume, tumor mass and mouse survival status were closely observed in experimental and control groups.Mice were euthanized when tumor-bearing time reached 28 days.Xenograft tissues were taken for general observation.Sections were cut and processed for HE staining and immunofluorescence staining.The expression of glypican-3(GPC3)protein in xenografts tissue was clearly defined.Results NK-92 cell lines that were chronically cultured in vitro and maintained typical phenotypic characteristics of NK cells with good cellular activity.Enhanced cytotoxicity and IFN-γ production of NK-92 cell lines were identified by LDH and ELISA,indicating that NK-92 cell lines could recognize and kill different kinds of HCCs.In addition,NK-92 cell lines efficiently suppressed the growth of HCC xenografts in vivo.Tumor volume in experimental group was significantly reduced compared with control group and there was low a GPC 3 expression in experimental group through immunofluorescence and immunohistochemistry results, pointing to the possibility that the cytotoxicity of NK cells was correlated with GPC3 +HCCs.Conclusion NK cells provide a promising means of therapeutic intervention for HCCs.NK-92 cell lines could eliminate HCC cells in vitro and in vivo.The cytotoxicity of NK-92 cell lines may work by killing the GPC3-positive cells in the liver cancer tissue.In addition to the anti-tumor effect, NK cells also have cytotoxicity on pathogens such as bacteria and viruses.
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Objective To explore the in vivo exposure levels of cigarette smoking ( CS) by measuring the biomar-kers nicotine and cotinine. Methods One hundred and sixty male SD rats were divided into 15 cigarette exposure groups (10, 20 and 30 nonfilter cigarettes/day, for 2, 4, 6, 8 and 12 weeks) and a control group (without CS exposure). The rats were sacrificed at different time?points. The concentration of plasma nicotine and cotinine were measured by GC?MS/MS. Results The CS?exposed rats displayed decreased locomotor activity, ataxic gait, irregular respiration, nasal noise, and salivation after smoking exposure for 3 weeks. Rats in the CS exposure groups had lower body weight, and the reduction of body weight was time and dose related (P<0. 01). The retention time of nicotine was 7. 5 to 8. 5 min. The concentra?tion of plasma nicotine in the CS exposure groups was higher than control group (155 ± 56. 65) ng/mL. The retention time of cotinine was 11. 5 to 12. 5 min, the concentrations of plasma cotinine in CS exposure groups were higher than control group (340 ± 41. 97) ng/mL, the increase of plasma cotinine in CS groups was time?related (P<0. 05), and the exposure concentration and duration had synergistic effect on the level of plasma cotinine ( P<0. 05 ) . Conclusions CS exposurecauses structural damages in male rats. The plasma concentration of cotinine can effectively reflect the in vivo exposure lev?els of cigarette smoking, and well presents a dose?response relationship.