Objective:To investigate ability of HCA587/MAGE-C2 protein combined with different adjuvants inducing antigen-specific immune response and antitumor effects in mice model.Methods:C57BL/6J mice were immunized with HCA587 protein com-bined with Freund's complete adjuvant(CFA)/Freund's incomplete adjuvant(IFA)and different doses of CpG ODN 1826(CpG),cellular and humoral immunity levels induced by different schemes were compared.ELISpot was used to evaluate frequency of IFN-γ-producing splenocytes.HCA587-specific antibodies were detected by ELISA.Intracellular cytokine staining(ICCS)analysis was mea-sured by flow cytometry.A tumor-bearing animal model was created by subcutaneously injection of B16-HCA587 tumor cells into right flank of C57BL/6J mice,which was treated with strategy with the strongest cellular and humoral immune response in immune compari-son protocol.Vernier calipers were used to measure tumor volume,and Log-rank test was used to analyze survival curve.Results:HCA587 protein combined with CFA and 50 μg CpG elicited strongest specific IFN-γ-secreting splenocytes and anti-HCA587 anti-bodies,which induced highest IFN-γ+CD4+T cells(P<0.05).In tumor treatment model,HCA587 protein combined with CFA and 50 μg CpG significantly inhibited tumor growth(P=0.026),while Log-rank test showed no significant effect on survival(P>0.05).Conclusion:HCA587 protein vaccine formulated with CFA and 50 μg CpG causes a significant cellular and humoral immune response and partial antitumor effect in mice model,providing new experimental data for preclinical research of tumor antigen protein vaccine.

Objective:To explore the involvement of serum exosomal miR-17-5p in the pathogenesis of regulatory T (Treg) cells deficiency in premature ovarian insufficiency (POI) patients.Methods:In a case-control study, 32 Chinese Han women with POI and 32 Chinese Han women with regular menstrual cycles (healthy control, HC) attending the Department of Gynecological Endocrinology, the First Affiliated Hospital of Nanjing Medical University from January 2019 to December 2020 were recruited. The relative expressional level of serum exosomal miR-17-5p was confirmed by reverse transcription quantitative polymerase chain reaction (RT-qPCR). To understand how POI-exosome affects Treg cells, peripheral blood mononuclear cells (PBMC) of healthy volunteer were transfected with miR-17-5p, and cultured with PBS as the control or with exosomes derived from POI women or HC for 72 h. Subsequently, to evaluate the functionality of the miR-17-5p upregulated in POI-exosome, native CD4 + T cells from healthy donors were sorted by magnetic beads and induced into induced Treg (iTreg) cells in vitro, and the induction efficiency, the proliferation and apoptosis rates of iTreg cells were detected after transfection with miR-17-5p mimic/inhibitor. TargetScan, miRDB, miRTarBase and miRWalk were used to predict targets gene of miR-17-5p. Results:The expression level of serum exosomal miR-17-5p was markedly up-regulated in POI compared with HC ( P<0.001). Furthermore, after culture with POI-exosome [(0.93±0.40)%], the frequency of Treg cells was significantly reduced as compared with that culture with PBS [(3.77±0.89)%, P=0.005]. Subsequently, the frequency of Treg cells in miR-17-5p mimic group [(4.30±1.91)%] was significantly lower than that in negative control group [(11.97±1.82)%, P=0.007]. In contrast, both Th1 and Th17 did not be affected (both P>0.05). The induction efficiency of iTreg cells did not be influenced by transfection with miR-17-5p (both P>0.05), however, the iTreg cells apoptosis rate in miR-17-5p mimic group [(16.31±1.27)%] was significantly higher than that in negative control group [(13.01±1.80)%, P=0.035], while apoptosis rate was significantly lower [(7.97±0.71)%] and proliferation rate was higher [(88.83±0.25)%] in miR-17-5p inhibitor group than those in negative control group [(11.42±0.23)%, P=0.001; (87.60±0.70)%, P=0.045]. Transforming growth factor beta receptor 2 was most likely targets gene of miR-17-5p. Conclusion:The exosomal miRNA profile of patients with POI differed from that of HC, and miR-17-5p, which is markedly increased in POI patients, decreased the frequency of Treg cells by inhibiting iTreg cell proliferation and promoting iTreg cells apoptosis. Thus, our study suggested that exosomal miR-17-5p promoted iTreg cells apoptosis and resulted in Treg cells deficiency in POI pathogenesis.

Objective:To explore the involvement of serum exosomal miR-17-5p in the pathogenesis of regulatory T (Treg) cells deficiency in premature ovarian insufficiency (POI) patients.Methods:In a case-control study, 32 Chinese Han women with POI and 32 Chinese Han women with regular menstrual cycles (healthy control, HC) attending the Department of Gynecological Endocrinology, the First Affiliated Hospital of Nanjing Medical University from January 2019 to December 2020 were recruited. The relative expressional level of serum exosomal miR-17-5p was confirmed by reverse transcription quantitative polymerase chain reaction (RT-qPCR). To understand how POI-exosome affects Treg cells, peripheral blood mononuclear cells (PBMC) of healthy volunteer were transfected with miR-17-5p, and cultured with PBS as the control or with exosomes derived from POI women or HC for 72 h. Subsequently, to evaluate the functionality of the miR-17-5p upregulated in POI-exosome, native CD4 + T cells from healthy donors were sorted by magnetic beads and induced into induced Treg (iTreg) cells in vitro, and the induction efficiency, the proliferation and apoptosis rates of iTreg cells were detected after transfection with miR-17-5p mimic/inhibitor. TargetScan, miRDB, miRTarBase and miRWalk were used to predict targets gene of miR-17-5p. Results:The expression level of serum exosomal miR-17-5p was markedly up-regulated in POI compared with HC ( P<0.001). Furthermore, after culture with POI-exosome [(0.93±0.40)%], the frequency of Treg cells was significantly reduced as compared with that culture with PBS [(3.77±0.89)%, P=0.005]. Subsequently, the frequency of Treg cells in miR-17-5p mimic group [(4.30±1.91)%] was significantly lower than that in negative control group [(11.97±1.82)%, P=0.007]. In contrast, both Th1 and Th17 did not be affected (both P>0.05). The induction efficiency of iTreg cells did not be influenced by transfection with miR-17-5p (both P>0.05), however, the iTreg cells apoptosis rate in miR-17-5p mimic group [(16.31±1.27)%] was significantly higher than that in negative control group [(13.01±1.80)%, P=0.035], while apoptosis rate was significantly lower [(7.97±0.71)%] and proliferation rate was higher [(88.83±0.25)%] in miR-17-5p inhibitor group than those in negative control group [(11.42±0.23)%, P=0.001; (87.60±0.70)%, P=0.045]. Transforming growth factor beta receptor 2 was most likely targets gene of miR-17-5p. Conclusion:The exosomal miRNA profile of patients with POI differed from that of HC, and miR-17-5p, which is markedly increased in POI patients, decreased the frequency of Treg cells by inhibiting iTreg cell proliferation and promoting iTreg cells apoptosis. Thus, our study suggested that exosomal miR-17-5p promoted iTreg cells apoptosis and resulted in Treg cells deficiency in POI pathogenesis.

Objective To explore the relationships of insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein 3 (IGFBP-3) with growth of very low birth weight (VLBW) infants in the early postnatal stage. Methods According to the individual gestational age and birth weight, 32 cases of VLBW infants were divided into small for gestational age (SGA) group and appropriate for gestational age (AGA) group. After birth, all the infants were given the same nutritional intake. The body weight, body length, head circumference and body mass index (BMI) were monitored at different time points (d0, d7, d14 and d28 after birth). Serum IGF-1 and IGFBP-3 levels were measured by radiommunoassay, and IGF-1/IGFBP-3 molar ratio was calculated. Results There was no signiifcant difference of body weight, body length, head circumference and BMI between two groups at d0, d7, d14 after birth. Body weight and BMI in SGA group were less than those in AGA group at d28 after birth (P<0.05). The levels of IGF-1, IGFBP-3 and IGF-1/IGFBP-3 in SGA group and IGF-1/IGFBP-3 in AGA group did not change with age after birth. The levels of IGF-1 and IGFBP-3 were increased in AGA group after birth. The level of IGF-1 in AGA group at d14 and d28 after birth was higher than that in AGA group at d0 after birth (P<0.05). The level of IGFBP-3 in AGA group at d28 after birth was higher than that in AGA group at d0 after birth (P<0.05). The levels of IGF-1 and IGFBP-3 in SGA group at d28 after birth were lower than those in AGA group at d28 after birth (P<0.05). Conclusions Serum IGF-1 and IGFBP-3 levels in SGA group are lower than those in AGA group. Low levels of IGF-1 and IGFBP-3 may result in growth retardation.