Cytogentic analysis of human dermal fibroblasts (HDFs) in early and late passages using both karyotyping and comet assay techniques

Human dermal fibroblasts (HDFs) are a potential source of somatic cells for genetic manipulation and tissue engineering. Confirmation of cytogenetic stability of these cells is an essential step for cell nuclear transfer and generation of a suitable and functional induced pluripotent stem cells line. HDF cells were isolated and cultured from human foreskin samples. Cytogenetic stability of these cells was evaluated in early (3-4) and late (10-15) passages using karyotype test and alkaline comet assay techniques. HDF cells in early and late passages showed normal karyotype but by comet assay abnormality and DNA damages in late passages of HDFs were observed. Also, the parameters of alkaline comet assay in early passages of HDFs compared with late passages and positive control groups more significantly were different (p < 0.05). These findings indicate that single-strand breaks or DNA damage after many passages may have occurred in HDF cells. Our results demonstrate that only early passages of HDF cells maintain cytogenetic stability and are good candidates for gene reprogramming. In conclusion, karyotype testing alone can not be used for detection of all signs of cytogenetic abnormality and DNA damages of cells. So, for precise evaluation of DNA damage and cytogenetic instability of fibroblast cells comet assay and karyotype techniques could complement each other

Effects of lentiviral vectors on DNA damage of human dermal fibroblasts (HDFs)

In present study we evaluated the DNA damages and cytogenetic stability of transducted and non-transducted human dermal fibroblasts (HDFs) by enhanced green fluorescent protein (eGFP) lentiviral vector using karyotyping, comet assay, and molecular techniques. HDFs were isolated from human foreskin samples and eGFP-expressing lentiviral vector were transfected into HEK-293T cells to produce lentiviruses. Then, HDFs at passage 2 were transducted with concentrated eGFP lentivirus and transducted HDFs were detected by fluorescent microscope. The expression levels of cell cycle genes include two subunits of anaphase promoting complex (APC) in transducted and nontransducted HDFs were measured by quantitative real-time PCR and finally, karyotype test and comet assay was performed to evaluate the DNA damages and cytogenetic stability in both groups. The results of karyotype analysis were not showed any abnormalities in karyotype of transducted HDFs by eGFP in compared to normal cells. The mean values of alkaline comet assay parameters on non-transducted (normal cells), eGFP-transducted group and positive control (H2O2 treatment) were calculated by CaspLab software. The comparison of mean difference of comet assay parameters include tail length, comet length, tail moment, and Olive tail moment by T test between eGFP-transducted HDFs and other groups (positive control and non-transducted HDFs) were statistically significant (p≤0.05). The alkaline comet assay on HDFs in eGFP-transducted group was showed small tail and indicated slight genetic damage compared with non-transducted group. Furthermore, the analysis of real-time PCR on expression of APC2 and APC7 genes in non-transducted HDFs compared with eGFP-transducted HDFs were not significant (p≤0.05). These findings indicated that integration of lentiviral vectors in first passage of transducted HDFs could not disturb the DNA structure and create chromosome instability. So in genetic engineering and gene transformation these vectors in first passages are useful

A study of cytogenetic stability of induced pluripotent stem cells using karyotyping and comet assay techniques

Background & Aims: Induced pluripotent stem cells (iPSCs) have the capability to undergo unlimited selfrenewal and differentiation into all cell types in the body. These cells are artificially derived from a nonpluripotent cell, typically human dermal fibroblasts (HDFs). The study of cytogenetic stability of these cells, in order to use iPS cells and apply studies in therapeutic applications, is essential. Methods: In the present experimental study, HDFs were isolated and cultured from human foreskin samples. The cytogenetic stability of these cells was evaluated in early passages (1-3) of HDFs using karyotype test and alkaline comet assay technique. The HDF cells treatment with hydrogen peroxide (H2O2) was used as a positive control for alkaline comet assay. The iPS cells with low passage (4-7) derived from reprogrammed HDFs were cultured on mouse embryonic fibroblast (MEF) feeder layer and cytogenetic stability of these cells were evaluated in early passages using karyotype test and alkaline comet assay technique. Results: The iPS cells in early passages (4-7) had normal karyotype (46, XY) and DNA damage and comet were not observed in these cells. In addition, HDF cells showed normal karyotype in early passages (1-3), but using comet assay, abnormality and DNA damages were observed in positive control (HDFs treated with H2O2). The comparison of alkaline comet assay parameters of iPS and HDF cells with positive control group showed statistically significant differences (P < 0.05). Conclusion: Since the comet assay is a sensitive technique for finding DNA damage, it is best if cytogenetic stability of these cells were evaluated before performing functional experiments on iPS cells. Therefore, for the precise evaluation of DNA damage and cytogenetic stability of iPS cells, the two techniques could complement each other. © 2015, Kerman University of Medical Sciences. All rights reserved

Comparing the expression levels of mRNA for MMP-7 in gastric mucosa of patients with H. pylori infection and uninfected patients

Background and purpose: The expression of growth factors, proteolytic enzymes, fibrogenic factors, and cytokines are altered in Helicobacter pylori (H. pylori) infected gastric mucosa. Matrix metalloproteinases (MMP) are a family of zinc-dependent homologous enzymes digesting most of the components of the extracellular matrix and basement membrane and are involved in remodeling and functioning of the biological processes. The purpose of this study was to compare gene expression of matrix metalloproteinase-7 (MMP-7) in patients with H. pylori-infected and uninfected individuals with gastrointestinal diseases. Materials and methods: This study was conducted in 50 H. pylori-negative patients and 50 H. pylori-positive patients being admitted to Shahrekord Hajar Hospital due to gastrointestinal diseases in 2014. The participants’ demographic information was collected and sampling was done. First DNA was extracted, and then PCR was performed to check for the presence of 16sRNA and UreC. The RNA from each sample was also extracted and cDNA was prepared. Afterwards, the expression of MMP-7 was measured by real time-PCR using specific primers and probes. Results: MMP-7 mRNA expression was significantly higher in biopsies of H. pylori-infected patients compared to that in H. pylori-uninfected patients (P<0.0001). Conclusion: Increased expression of MMP-7 can be effective in inflammatory response and development of the disease. It could be used as a key marker for early diagnosis of gastrointestinal diseases and gastric cancer. © 2016, Mazandaran University of Medical Sciences. Engineering. All rights reserved

Association between H. pylori BabA virulence factor with clinical outcome and ABO blood groups

Helicobacter pylori infection is a prevalence infection 50% of the human population. The main H. pylori adhesin is the BabA, which was the first identified factor. The aim of this study was to determine the relationship between the ABO blood groups and various gastrointestinal diseases 140 patients, were included in this study. Gastric biopsies were taken for recognition of H.pylori by RUT and PCR. Blood samples were tested for ABO blood group. In the present study 140 H.pylori positive samples examined for the presence or absence of babA gene by PCR. From 140 patients, 35% were positive for babA gene and 65% were negative for this gene. Positivity rate of H. pylori babA infection was 42.4 % in blood group O, 18.8 % in blood group A, 100% in blood group B and 44.8 % in blood group AB. The frequencies of ABO blood group among endoscopic findings are predominant for Gastritis for group A. In our study, There was statistically significant difference in babA (+) and babA (–) were compared in endoscopy finding (P<0.001) and there was statistically significant difference in positivity rate of H.pylori infection among ABO blood groups (p< 0.001). The higher incidence of Gastritis and peptic ulcer was in patients with blood group A and AB and there was statistically significant between these symptoms (p= 0.02). Our results showed that the prevalence of babA genotype is associated with gastritis and gastric ulcer and there is a relation with ABO blood group

Prevalence of cagA and babA2 genes in Helicobacter Pylori strains Isolated from Iranian gastrointestinal disorder patients and their gas-tritis classification

Helicobacter pylori is a spiral gram negative flagellate bacteria and localize in the stomach. H.p infection is a worldwide health problem and identified as an important cause of gastritis and gastric cancer and its ability to develop such disorders is related to its virulence factors and environment. cagA is the most important Hp virulence factor that directly penetrate into gastric epithelial cells by bacterial secretion system (T4SS) from pathogenicity island (PAI) and disrupts cell homeostasis. Adherence factors are significant for bacterial colonization and suitable function of other virulence factor. Blood group antigen binding adhesion (babA) is an outer membrane protein (OMP) that binds to ABO blood group antigen and can stimulate inflammatory response in gastric cells. Our main target was to determine the roles and prevalence of cagA and babA2 virulence factor in gastrointestinal disorders in Iranian patients. Existences of These factors were determined by PCR in 218 patients with gastrointestinal disorders. Semi-quantitative methods of scoring according to the Updated Sydney classification system were used for detection of H.pylori density, neutrophil and monocyte cells infiltration. A high prevalence of cagA positive (81.4%) and babA2 positive (35%) were found. The most combined genotype (cagA&babA2) prevalence was found in gastritis & ulcer (100%) (P < 0.001). High prevalence of cagA positive observed in active inflammation phase 76.9% and high prevalence of babA2 positive was in active phase 61.1% of H.pylori gastritis (P=0.001) . Results of this study showed information about the high prevalence of cagA genes in H.pylori infected patients and their rolls in active gastrointestinal disorder