BMC Molecular and Cell Biology最新文献

Background: Cryopreservation is a crucial procedure for safeguarding cells or other biological constructs, showcasing considerable potential for applications in tissue engineering and regenerative medicine.

Aims: This study aimed to evaluate the effectiveness of different cryopreservation conditions on human cells viability.

Methods: A set of cryopreserved data from Department of Tissue Engineering and Regenerative Medicine (DTERM) cell bank were analyse for cells attachment after 24 h being revived. The revived cells were analysed based on different cryopreservation conditions which includes cell types (skin keratinocytes and fibroblasts, respiratory epithelial, bone marrow mesenchymal stem cell (MSC); cryo mediums (FBS + 10% DMSO; commercial medium); storage durations (0 to > 24 months) and locations (tank 1-2; box 1-5), and revival methods (direct; indirect methods). Human dermal fibroblasts (HDF) were then cultured, cryopreserved in different cryo mediums (HPL + 10% DMSO; FBS + 10% DMSO; Cryostor) and stored for 1 and 3 months. The HDFs were revived using either direct or indirect method and cell number, viability and protein expression analysis were compared.

Results: In the analysis cell cryopreserved data; fibroblast cells; FBS + 10% DMSO cryo medium; storage duration of 0-6 months; direct cell revival; storage in vapor phase of cryo tank; had the highest number of vials with optimal cell attachment after 24 h revived. HDFs cryopreserved in FBS + 10% DMSO for 1 and 3 months with both revival methods, showed optimal live cell numbers and viability above 80%, higher than other cryo medium groups. Morphologically, the fibroblasts were able to retain their phenotype with positive expression of Ki67 and Col-1. HDFs cryopreserved in FBS + 10% DMSO at 3 months showed significantly higher expression of Ki67 (97.3% ± 4.62) with the indirect revival method, while Col-1 expression (100%) was significantly higher at both 1 and 3 months compared to other groups.

Conclusion: In conclusion, fibroblasts were able to retain their characteristics after various cryopreservation conditions with a slight decrease in viability that may be due to the thermal-cycling effect. However, further investigation on the longer cryopreservation periods should be conducted for other types of cells and cryo mediums to achieve optimal cryopreservation outcomes.

Background: Hepatitis B virus (HBV) infection poses a substantial threat to human health, impacting not only infected individuals but also potentially exerting adverse effects on the health of their offspring. The underlying mechanisms driving this phenomenon remain elusive. This study aims to shed light on this issue by examining alterations in paternally imprinted genes within sperm.

Methods: A cohort of 35 individuals with normal semen analysis, comprising 17 hepatitis B surface antigen (HBsAg)-positive and 18 negative individuals, was recruited. Based on the previous research and the Online Mendelian Inheritance in Man database (OMIM, https://www.omim.org/ ), targeted promoter methylation sequencing was employed to investigate 28 paternally imprinted genes associated with various diseases.

Results: Bioinformatic analyses revealed 42 differentially methylated sites across 29 CpG islands within 19 genes and four differentially methylated CpG islands within four genes. At the gene level, an increase in methylation of DNMT1 and a decrease in methylation of CUL7, PRKAG2, and TP53 were observed. DNA methylation haplotype analysis identified 51 differentially methylated haplotypes within 36 CpG islands across 22 genes.

Conclusions: This is the first study to explore the effects of HBV infection on sperm DNA methylation and the potential underlying mechanisms of intergenerational influence of paternal HBV infection.

Background: Circulating microRNAs have been implicated in a diverse array of biological and pathological phenomena. Their potential utility as noninvasive biomarkers for screening and diagnosing various diseases has been proposed.

Objective: This study aimed to explore the potential role of the miRNAs miR-122 and miR-486 as molecular biomarkers in the pathogenesis of hepatitis C virus (HCV) infection. Thus, miR-122 and miR-486 were detected in the serum of HCV patients and healthy controls. Moreover, the potential correlations of miR-122 and miR-486 with viral complications, such as physical activity, pain, muscle fatigue, and HCV infection, were identified.

Methods: A total of 150 subjects aged 30 to 66 years were included in this study. The patients were classified as patients with chronic hepatitis C virus (CHC) (n = 110) or healthy controls (n = 40). Real-time polymerase chain reaction (PCR) analyses were performed to determine miR-122 and miR-486 expression. Physical activity (PA), pain score, HCV genotyping, viral overload, aspartate transaminase (AST), alanine transaminase (ALT), lactic acid dehydrogenase (LDH), creatine kinase (CK), and antioxidant status were also estimated by using prevalidated questionnaires, PCR, and spectrophotometric analyses.

Results: Compared with those in normal controls, significant increases in the serum levels of miR-122 and miR-486 were reported in patients with CHC. In physically active CHC patients, there was a significant correlation between the expression of miRNAs and increased alanine transaminase (ALT), aspartate transaminase (AST), fibrosis scores, and inflammation activity, but no association was reported for hepatitis C virus (HCV) RNA or viral load. Additionally, significant decreases in LDH, CK, GSSG, and pain scores and increases in TAC, GSH, and the GSH/GSSG ratio were reported. Moreover, the expression of miR-122 and miR-486 was positively correlated with changes in body mass index (BMI) and liver fibrosis stage, as well as negatively correlated with sex, PA, TAC, GSH, GSSG, and the GSH/GSSG ratio.

Conclusion: MiR-122 and miR-486 expression levels were strongly correlated with physical activity, pain perception, and muscle fatigue biomarkers in HCV-infected patients. These miRNA levels were associated with elevated AST, ALT, fibrosis scores, LDH, CK, and antioxidant status, thus suggesting their potential as biomarkers for disease severity and oxidative stress. However, no correlation was observed with viral load or HCV-RNA expression, thus implying that these miRNAs may impact disease progression and symptoms through host factors, rather than directly affecting viral replication. In summary, the results demonstrated that molecular studies of miR-22 and miR-468 and their associations with PA, pain, adiposity, sex differences, and muscle fatigue, as well as routine biomarkers, could be

Background: Transfection is an important analytical method for studying gene expression in the cellular environment. There are some barriers to efficient DNA transfection in host cells, including circumventing the plasma membrane, escaping endosomal compartmentalization, autophagy, immune sensing pathways, and translocating the nuclear envelope. Therefore, it would be very useful to introduce an optimum transfection approach to achieve a high transfection efficiency in the Vero cell line. The aim of this study was to compare various transfection techniques and introduce a highly efficient method for gene delivery in Vero cells.

Methods: In the current study, three transfection methods were used, including chemical transfection, electroporation, and lentiviral vector transduction, to obtain the optimum transfection conditions in the Vero cell line. Vero cells were cultured and transfected with chemical transfection reagents, electroporation, or HIV-1-based lentivectors under different experimental conditions. Transfection efficiency was assessed using flow cytometry and fluorescence microscopy to detect GFP-positive cells.

Results: Among the tested methods, TurboFect™ chemical transfection exhibited the highest efficiency. Optimal transfection conditions were achieved using 1 µg DNA and 4 µL TurboFect™ in 6 × 10⁴ Vero cells.

Conclusion: TurboFect™, a cationic polymer transfection reagent, demonstrated superior transfection efficiency in Vero cells compared with electroporation and lentivirus particles, and is the optimal choice for chemical transfection in the Vero cell line.