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Gene, Cell and Tissue - Volume:4 Issue: 2, Apr 2017

Gene, Cell and Tissue
Volume:4 Issue: 2, Apr 2017

  • تاریخ انتشار: 1396/01/30
  • تعداد عناوین: 9
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  • Tooba Nakhzari Khodakheir, Alireza Pourtalebi-Firoozabadi *, Mohammad Hossein Sangtarash, Abbas Nikravesh Page 1
    Myelin-directed autoimmunity seems to be an important factor in the pathogenesis of multiple sclerosis (MS). Growing productions of both anti-inflammatory and pro-inflammatory cytokines are frequent findings in MS. The IL-10 is an immunosuppressive cytokine in the immune system. In the clinical trial, it is considered to be an anti-inflammatory therapy for inflammatory bowel disease and many other autoimmune diseases such as psoriasis, rheumatoid arthritis, and MS. IL-19 as well as IL-20 belong to the IL-10 family. It appears that polymorphisms, within these genes, affected the level of gene expression. The goal of the present study was investigating the possible associations between IL-19 and IL-20 genes polymorphisms with multiple sclerosis. Therefore, 100 MS patients and 100 healthy controls were recruited in this study. Genotyping for the IL-19 (rs2243158GC, rs2243168GC) and IL-20 1053TG (rs2981572, rs2981573) were executed using Tetra-primer ARMS-PCR. The GC and AT genotypes related to IL-19 polymorphisms, rs2243158 and rs2243168, respectively, were significantly associated with multiple sclerosis (P = 0.004 and P
    Keywords: IL, 19, IL, 20, Multiple Sclerosis, Tetra ARMS, PCR, Genotyping
  • Fatemeh Safari *, Sheyda Jodeiry Zaer Page 2
    Background
    Helicobacter pylori CagA oncoprotein was injected into mammalian host cells via type IV secretion system, where it was tyrosine phosphorylated at the Glu-Pro-Ile-Tyr-Ala (EPIYA) sequence. Tyrosine phosphorylation of CagA was shown to be a prerequisite for the induction of actin cytoskeletal rearrangement in AGS gastric epithelial cells. The needle-like projections, known as the hummingbird phenotype, are thought to be involved in gastric disease. Moreover, Pragmin, a mammalian protein, contains a single EPIYA motif, and tyrosine phosphorylation of Pragmin at EPIYA motif in AGS cells induce cell-morphological changes, which are characterized by elongated cell shape with invasive phenotype that contributes to tumor invasion and metastasis.
    Methods
    In this study, AGS cells were transfected by CagA or/and Pragmin using lipofectamine 2000 reagent, then, cell-morphological changes were investigated using light microscope. Finally, elongated cells were counted and the results were compared.
    Results
    Our results revealed that there is a competition between CagA and Pragmin to interact with CSK via EPIYA motif. We also found that although the cell-morphological changes are particularly dependent on tyrosine phosphorylation at EPIYA motifs in both, changes of cell morphology are different in CagA and Pragmin transfected cells.
    Conclusions
    Our findings suggest that the mechanisms inducing the elongated cell morphology in CagA or Pragmin transfected cells may be different.
    Keywords: Pragmin, Morphological Changes, EPIYA Motif, AGS Cells, CagA Helicobacter pylori
  • Jafar Vatandoost *, Behnaz Dolatabadi Page 3
    Background
    Selecting a host system for the expression of recombinant proteins should be carefully evaluated prior to the initiation of any bio therapeutic development programs. Since different hosts express proteins with various efficiencies and with different posttranslational modifications, changing hosts may impact the expected activity of the protein. The main expression systems have members of the mammalian cell family, however, there are remarkable differences between the species. The most generally used mammalian hosts for the production of recombinant proteins are CHO and HEK293 cells.
    Objectives
    In order to compare the differences between HEK versus CHO cells, the human coagulation factor IX in a transient and stable expression was examined.
    Methods
    After transfection of CHO and HEK cells with an hFIX-expressing mammalian plasmid, pcDNA-hFIX, transient expression was analyzed on the culture supernatant during 72 hours. The stable clones were also recovered after 10 weeks of geneticin selection. The expression and activity of the hFIX was evaluated by performing enzyme-linked immunosorbent assay (ELISA) as well as a coagulation test, respectively. The γ-carboxylation of the hFIX was confirmed by evaluation of the expressed protein, after being precipitated with barium citrate.
    Results
    Although the stable CHO clones secreted hFIX protein 30% higher than HEK cells, the transient HEK cell line proved to be superior in the production of total hFIX protein (%42 increased) and functional hFIX (%29 higher) relative to the CHO cell line. Moreover, specific activity and the fully γ-carboxylated hFIX are almost constant in transient and stable expression, indicating that γ-carboxylation efficiency in both CHO and HEK cell lines is almost equal.
    Conclusions
    In conclusion, transient transfection studies with HEK cells provide a simple and strong method for a high production of recombinant proteins in weeks, a process which is more time consuming in the CHO cell line.
    Keywords: HEK Cells, CHO Cells, Transient, Stable Expression, Coagulation Factor IX
  • Marzie Bahadori, Fatemeh Amiri, Marjan Movahed, Amaneh Mohammadi Roushandeh, Mehryar Habibi Roudkenar * Page 4
    Background
    Hemolytic anemia is associated with intravascular heme release and oxidative stresses that lead to endothelial dysfunctions. Hemopexin (HPX) is a plasma heme-binding β1-glycoprotein, which plays a pivotal role in heme transfer to hepatocytes and iron recycling. Recently, HPX administration in hemolytic patients attenuated hemolysis-driven oxidative damages and endothelial disorders that led to a rise in the strategy of HPX therapy. Human HPX production using recombinant DNA technology could provide a real alternative to plasma-derived HPX. The purpose of this study was to generate a stable Chinese hamster ovary (CHO) cell line expressing human rHPX.
    Methods
    Total RNA was extracted from HepG2 cells, and HPX gene was amplified by Real Time-Polymerase Chain Reaction (RT-PCR). Then, the HPX gene was cloned in pcDNA3.1 () shuttle vector. The recombinant pcDNA3.1-HPX was transformed to E. coli TOP10 strain. Gene cloning was verified by colony PCR, restriction digestion, and DNA sequencing. The CHO cells were chemically transfected with pcDNA3.1-HPX, and screening was performed by G418 sulfate effective concentration to develop stable single cell clones. The rHPX expression was verified by RT-PCR and Western blot.
    Results
    Cloning confirmation analyses showed that HPX gene was successfully cloned in pcDNA3.1 () vector. Screening of transfected cells with G418 sulfate enriched the population of single cell clones expressing human rHPX. The RT-PCR and Western blot analyses confirmed rHPX expression in CHO cell line both at transcriptional and translational levels.
    Conclusions
    Human rHPX protein was stably expressed in CHO cells. This study was a pioneering work for the future production of therapeutic rHPX.
    Keywords: Hemopexin, Hemolysis, Hemolytic Anemia, CHO Cells, Gene Expression
  • Atefeh Tahervand, Behnaz Parnian, Mehryar Habibi Roudkenar, Amaneh Mohammadi Roushandeh* Page 5
    Background
    Digoxin, which is used in heart failure patients, contributes to inhibition of cell proliferation and induction of apoptosis. Therefore, digoxin could have a therapeutic potential in helping determine which individuals may suffer from cancer. This study was conducted to determine whether digoxin declines HeLa cells proliferation and viability or induces senescence.
    Methods
    HeLa cell line was cultured with different concentrations of digoxin in RPMI-1640 medium for 6, 12, 24, and 48 hours. Cell proliferation and viability were assessed with MTT and trypan blue, respectively. To detect cell morphology and senescence, hematoxilin and gimsa staining were used. One way ANOVA was used for data analysis using SPSS Version 20 software.
    Results
    Cell proliferation and viability decreased in all concentrations of digoxinin compared to controls. Colony formation was inhibited significantly after digoxin treatment (P
    Conclusions
    Our findings clearly revealed that dixogin inhibited HeLa cell growth, but it was not time and dose dependent. The apoptosis and senescence, which were detected in the present study, were based on non-advance methods. Therefore, for reliable results, it is necessary to prove the anticancer properties of digoxin through advanced methods. Although the antitumoral effects of digoxin are still being investigated, more studies should be conducted to clarify the related mechanism in cellular, biochemical, and molecular aspects.
    Keywords: Digoxin, HeLa Cell Line, Cancer, Apoptosis, Senescence
  • Mehrnaz Narooie-Nejad *, Somayeh Nezam-Abadi, Morad-Ali Ranaei, Mahdie Bagherzade-Yazdi, Mojgan Mokhtari Page 6
    Background
    Recurrent miscarriage (RM) is defined as the presence of two or more spontaneous abortions. It has been estimated that 1% to 3% of couples experience RM. Furthermore, RM is considered as a multifactorial disease and genetic disorders are one of the suggested issues. In this study, 30 couples with RM were evaluated for their karyotypes, for the first time in Sistan and Baluchestan province.
    Methods
    In this study, 60 individuals (30 couples) with a history of recurrent miscarriage were recruited for cytogenetic evaluation through karyotyping of peripheral blood. All the participants in this study took part in immunologic, hematologic, and anatomic evaluations and no abnormality was found.
    Results
    Among all male and female subjects of this study, only 2 females (6.7%) showed chromosomal aberration: mos45, X(3)/46XX(47) and 46, XX, and inv (8)(p12q21). Both of these females had no children with a record of 3 miscarriages. These females and their partners had no history of miscarriage in their family.
    Conclusions
    The number of chromosomal abnormalities found in this Study (6.7%) was less than other studies conducted in Iran, including the city of Mashad, Yazd, Tehran, and Ahvaz. This might be because of the low number of participants taking part in this investigation. Therefore, it is a necessity to perform furhter evaluations in this regard.
    Keywords: Recurrent Miscarriage, Chromosomal Disorders, Karyotype
  • Ramin Saravani *, Hamid Reza Galavi Page 7
  • Maryam Moossavi, Mostafa Ibrahimi, Milad Mohammadoo-Khorasani * Page 9
  • Sayed Alireza Mirsane *, Shima Shafagh, Nasrin Oraei Page 10