ABSTRACT
[Abstract] Objective To investigate the effect of exosome in cultured in vitro H9C2 myocardial cells injury of diabetic mice and its mechanism. Methods The mouse model of diabetic myocardial injury was established by using db/db mice (n=10) and their mate mice db/+ (n=5). Serum exosomes were isolated and quantitated using the exosome isolation reagent and EXOCET Quantitation kit. The serum exosomes were labeled with PKH26 (red fluorescent cell linker) to detect the endocytosis in H9C2 cells. The expressions of exocrine associated protein and inflammatory cytokines in H9C2 cells with or without exosome stimulation were detected by Western blotting. TUNEL was used to detect apoptosis. A neutralizing antibody of Rab1a was used for blocking experiment. Results Db/db mice produced more exosomes than db/+ mice (30.95×109/ml vs. 10.45×109/ml, P<0.01). Moreover, H9C2 cells cultured in vitro could swallow more serum-exosomes derived from db/db mice. Meanwhile, serum exosome from db/ db mice, as used to interfere H9C2 cells, significantly increased the expression of inflammatory cytokines, such as 6.2 folds to IL-6 and 2.6 folds to IL-1β (P<0.01). Furthermore, the apoptosis in H9C2 cells increased compared to those from db/+ mice. Mechanism studies announced that the increased expression of Rab1a in exosomes-derived from db/db mice, and blocking the expression of Rab1a in exocrine of db/db mice with Rab1a neutralizing antibody could significantly inhibit the endocytosis and apoptosis of H9C2 cells. Conclusions Serum exosomes isolated from db/db mice may trigger inflammation and apoptosis of cardiomyocytes cultured in vitro, which may be involved in the evolution of diabetic myocardial injury. Inhibition of exosome secretion or intervention of its regulatory molecules may become a new research target for the treatment of diabetic myocardial injury.
ABSTRACT
To investigate whether transcription of hepatitis B virus (HBV) gene occurs in human sperm, total RNA was extracted from sperm of patients with chronic HBV infection (test-1), from donor sperm transfected with a plasmid containing the full-length HBV genome (test-2), and from nontransfected donor sperm (control), used as the template for reverse transcription-polymerase chain reaction (RT-PCR). Positive bands for HBV DNA were observed in the test groups but not in the control. Next, to identify the role of host genes in regulating viral gene transcription in sperm, total RNA was extracted from 2-cell embryos derived from hamster oocytes fertilized in vitro by HBV-transfected (test) or nontransfected (control) human sperm and successively subjected to SMART-PCR, suppression subtractive hybridization, T/A cloning, bacterial amplification, microarray hybridization, sequencing and the Basic Local Alignment Search Tool (BLAST) search to isolate differentially expressed genes. Twenty-nine sequences showing significant identity to five human gene families were identified, with chorionic somatomammotropin hormone 2 (CSH2), eukaryotic translation initiation factor 4 gamma 2 (EIF4G2), pterin-4 alpha-carbinolamine dehydratase 2 (PCBD2), pregnancy-specific beta-1-glycoprotein 4 (PSG4) and titin (TTN) selected to represent target genes. Using real-time quantitative RT-PCR (qRT-PCR), when CSH2 and PCBD2 (or EIF4G2, PSG4 and TTN) were silenced by RNA interference, transcriptional levels of HBV s and x genes significantly decreased (or increased) (P < 0.05). Silencing of a control gene in sperm did not significantly change transcription of HBV s and x genes (P > 0.05). This study provides the first experimental evidence that transcription of HBV genes occurs in human sperm and is regulated by host genes.