Objective To clone the fragment of hepatitis C virus(HCV) core gene and express it in E.coli.Methods The fragment of HCV core gene(approximate 366bp) was amplified by PCR and inserted into the pMD18-T vector.The cloned HCV core gene,which was confirmed by the digestion with EcoRⅠ/BamHⅠ,was subcloned into the expression vector pBV220 to construct recombinant plasmid pBV220/HCV-C.The expressed gene product was identified by SDS-PAGE and Western blotting.Results The fragment of HCV core gene was expressed successfully after temperature induction and a protein of 14 000 u was resulted.Conclusion Expression of the HCV-C gene in E.coli was achieved,which may be helpful for further studies on characterizations of HCV-C gene.

Objective To investigate the mineralized capacities of ectomesenchymal stem cells (EMSC) from facial process of Sprague Dawley(SD) rat embryo of different age in vitro and the expression of p75 neurotrophin receptor(p75NTR) in this process.Methods The stem cell surface antigens of EMSC from 12.5 d,15.5 d and 18.5 d SD rat embryonic facial process were tested by flow cytometry technology.E12.5 d EMSC,E15.5 d EMSC and E18.5 d EMSC were incubated under mineralization induction and analysed by alkaline phosphatase(ALP) staining on day 7(d7) and alizarin red staining on day 21(d21).Expression changes of Runt-related transcription factor-2(RUNX2),collagen Ⅰ (Col Ⅰ) and p75NTR in each group were measured using Western blotting and real time(RT)-PCR on day 0(d0),day 7(d7),day 14(d14) and day 21(d21).Results The expression of the special substances CD29,CD146 and p75NTR in E12.5 d EMSC,E15.5 d EMSC and E18.5 d EMSC were positive,and the expression of CD45 was negative.The expression level of p75NTR in E18.5 d EMSC(84.04％) was much higher than that of E12.5 d EMSC (22.53％) and E15.5 d EMSC(81.43％).The mineralized capacities of E18.5 d EMSC was stronger than that of E12.5 d EMSC and E15.5 d EMSC.The higher expression of RUNX2,Col Ⅰ in E18.5 d EMSC(RUNX2:1.92±0.20,Col Ⅰ:1.85±0.66) was found compared with E12.5 d EMSC(RUNX2:0.38±0.02,Col Ⅰ:0.33±0.94)and E15.5 d EMSC(RUNX2:0.72±0.22,Col Ⅰ:0.64±0.07)(P＜0.05),and p75NTR in the E18.5 d EMSC experimental group(E12.5 d:0.79±0.23,E15.5 d:0.84±0.29,E18.5 d:1.35±0.22) was significantly higher than the in control group(E12.5 d:0.42±0.12,E15.5 d:0.43±0.13,E18.5 d:0.48±0.15)(P＜0.05).RTPCR further proved the results of the Western blotting.Conclusions p75NTR participated in the mineralization differentiation of EMSC.E18.5 d EMSC had a higher expression of p75NTR and stronger mineralization capacity and was the ideal engineering seed cells.

AIM:This study aim to investigate the effects of lipopolysaccharide(LPS)-induced inflammation on the aging phenotype of hematopoietic stem/progenitor cells(HSPCs)in the bone marrow(BM)and spleen of mice.METHODS:(1)Young(2-month old)wild-type(WT)mice were treated with LPS to establish an actue inflammation model.The percentage of HSPCs in the BM and spleen of mice after LPS stimulation,as well as the ratio of mature cells in peripheral blood(PB)and spleen,were analyzed using flow cytometry.The proliferation of HSPCs in the BM and spleen was evaluated by examining the expression of the proliferation marker Ki67.In addition,changes in CD45 expression on HSPCs in the spleen of mice following LPS exposure were investigated by flow cytometry.(2)The percentage of HSPCs in BM and mature cells in PB and spleen of both young(2-month old)and old(24-month old)WT mice were analyzed by flow cytometry.(3)The transcriptome changes of hematopoietic stem cells(HSCs)after LPS stimulation was performed by an in silico analysis.RESULTS:(1)Mice exposed to LPS exhibited a significant increase in the percentage of HSPCs in BM and a marked elevation in the percentages of myeloid cells in PB and spleen compared to the mice in control group(P<0.05).(2)LPS exposure resulted in increased spleen weight and cell counts(P<0.05),along with a higher per-centage of HSPCs in the spleen compared to controls(P<0.05).(3)LPS stimulation promoted the proliferation of HSPCs in the BM and spleen(P<0.05).(4)The expression of CD45 was reduced on HSPCs from spleen of mice after LPS stimu-lation(P<0.01).(5)In comparison to young mice,aged mice showed an increase in spleen weight and a higher percent-age of HSPCs in the spleen(P<0.05).(6)Aged mice,in comparison to young mice,demonstrated a significantly higher percentage of HSPCs in the BM and myeloid skewing in the PB and spleen(P<0.01).(7)The silico analysis revealed up-regualtion of reactive oxygen species(ROS)and apoptosis signaling in HSPCs following LPS stimulation.CONCLU-SION:Young HSPCs stimulated by LPS exhibited an increase in cell number,a bias towards myeloid differentiation,en-hanced extramedullary hematopoiesis,and elevated levels of ROS and apoptosis,all of which collectively manifested the aging phenotype of HSPCs.