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Objective: To establish an assay using 9-color flow cytometry immunophenotyping to detect activation and apoptosis of human TCR Vβ lymphocyte subpopulations in peripheral blood samples., Methods: We used 5 antibodies (CD3, CD4, CD8, CD95, CD69), phospholipids binding proteins Annexin V, TCR Vβ Repertoire Kit and nucleus dye DAPI to establish a 9-color flow cytometry assay. Peripheral blood samples were taken from eight healthy people for test of antibodies and determination of optimal PMT and staining method (single-stained vs stained with all but one antibody)., Results: Appropriate detecting voltage, antibody concentration and compensation methods were determined. The distribution of TCR Vβ subgroup in our samples was consistent with the TCR Vβ Repertoire Kit instruction and other published literature., Conclusion: We have established a effective easy using 9-color flow cytometry immunophenotyping to detect human TCR Vβ lymphocyte subpopulations in peripheral blood samples.

Objective: To evaluate the relationship between receptor-mediated endocytosis, autophagosome formation and apoptosis by observing the morphological changes of mouse peritoneal macrophages in the course of receptor-mediated endocytotic activities, autophagy and apoptosis., Methods: Mouse peritoneal macrophages were incubated with horseradish peroxidase-labeled concanavalin A (ConA-HRP), and morphological examinations were performed at different time points after the incubation., Results: After the incubation of the macrophages with ConA-HRP, ConA-HRP was observed to enter the vesicles by way of receptor-mediated endocytosis. Three kinds of endosomes were observed, namely vesicles, tubes and double-membrane sheets. The double-membrane sheets enveloped a portion of the cytoplasm and organelles, thus giving rise to vesicles, or the autophagosomes, which later fused with lysosome, followed by the apoptosis of the macrophages., Conclusion: Receptor-mediated endocytosis of ConA-HRP is associated with autophagy and apoptosis.

Cisplatin (DDP) resistance in patients suffering from ovarian cancer is a considerable hurdle to successful treatment. The present study aimed to identify a possible long non-coding RNA (lncRNA)-microRNA (miRNA)-mRNA axis participating in ovarian cancer DDP-resistance based on the critical roles of non-coding RNAs, including lncRNAs and miRNAs, in carcinogenesis. According to online data and experimental results, lncRNA HAND2-AS1 expression was significantly downregulated within ovarian carcinoma, especially within recurrent and DDP-resistant ovarian carcinoma. The expression of HAND2-AS1 was also shown to be markedly inhibited in SKOV3/DDP (DDP) cells with resistance to DDP. In SKOV3/DDP cells, HAND2-AS1 overexpression inhibited cell viability and promoted cell apoptosis upon DDP treatment through the Bcl-2/caspase-3 apoptotic signaling. It was hypothesized that PTEN mRNA expression was also markedly inhibited in SKOV3/DDP ovarian cancer cells, while HAND2-AS1 overexpression rescued PTEN proteins and blocked PI3K/AKT signaling activation. Moreover, miR-106a was found to bind directly to PTEN 3′ UTR and HAND2-AS1. Upon DDP treatment, miR-106a overexpression in SKOV3/DDP cells promoted cell viability. It inhibited cell apoptosis through the Bcl-2/caspase-3 apoptotic signaling pathway and downregulated the protein levels of PTEN and upregulated PI3K/AKT signaling activity. Furthermore, miR-106a overexpression partially reversed the effect of HAND2-AS1 overexpression upon PTEN proteins and SKOV3/DDP cell proliferation upon DDP treatment. In conclusion, a lncRNA HAND2-AS1/miR-106a/PTEN axis that re-sensitizes DDP-resistant SKOV3/DDP cells to DDP treatment has been established. [ABSTRACT FROM AUTHOR]

Background: Serum leptin levels are augmented in obese infertile men and in men with azoospermia. They also correlate inversely with sperm concentration, motility and normal forms. The mechanisms underlying the adverse effects of excess leptin on male reproductive function remain unclear. The present study aimed to evaluate the effects of exogenous leptin on sperm parameters in mice and to explore the underlying mechanisms. Methods: We treated normal adult male mice with saline, 0.1, 0.5 or 3 mg/kg leptin daily for 2 weeks. After treatment, serum leptin levels, serum testosterone levels, sperm parameters and testicular cell apoptosis were evaluated. Blood testis barrier integrity and the expression of tight junction-associated proteins in testes were also assessed. We further verified the direct effects of leptin on tight junction-associated proteins in Sertoli cells and the possible leptin signaling pathways involved in this process. Results: After treatment, there were no significant differences in body weights, reproductive organ weights, serum leptin levels and serum testosterone levels between leptin-treated mice and control mice. Administration of 3 mg/kg leptin reduced sperm concentration, motility and progressive motility while increasing the percentage of abnormal sperm and testicular cell apoptosis. Mice treated with 3 mg/kg leptin also had impaired blood testis barrier integrity, which was related to decreased tight junction-associated proteins in testes. Leptin directly reduced tight junction-associated proteins in Sertoli cells, JAK2/STAT, PI3K and ERK pathways were suggested to be involved in this process. Conclusions: Exogenous leptin negatively affects sperm parameters and impairs blood testis barrier integrity in mice. Leptin reduced tight junction-associated proteins in Sertoli cells, indicating that leptin has a direct role in impairing blood testis barrier integrity. Given the function of blood testis barrier in maintaining normal spermatogenesis, leptin-induced blood testis barrier impairment may be one of the mechanisms contributing to male subfertility and infertility. [ABSTRACT FROM AUTHOR]