Cell Journal (Yakhteh)

198-205

Authors: Zahra Armand , Gholamreza Najafi , Farah Farokhi , Ali Shalizar Jalali

Objective: Cyclosporine (Cs), a cyclic undecapeptide with potent immunosuppressive activity, causes several adverse effects including reproductive toxicity. This study aims to examine the ability of Crataegus monogyna aqueous fruit extract as an antioxidant to protect against Cs-induced reproductive toxicity. Materials and Methods: In this experimental study, 32 adult male Wistar rats were divided into four groups of eight animals each. Rats in two groups received 40 mg/kg/day Cs for 45 days by oral gavage. In addition, one of the two groups received Crataegus monogyna aqueous extract at a dose of 20 mg/kg/day orally four hours after Cs administration. The remaining two groups consisted of a vehicle treated control (Cont) group and a Crataegus monogyna control (Cr) group. Differences between groups were assessed by analysis of variance (ANOVA) using the SPSS software package for Windows. Results: Cs treatment caused a significant decrease in sperm count and viability with an increase in DNA damage and protamine deficiency of the sperm cells. We observed significant decreases in fertilization rate and embryonic development, in addition to an increased rate of embryo arrest in Cs-treated rats. Crataegus monogyna co-administration attenuated all Cs-induced negative changes in the above-mentioned parameters. Conclusion: Supplementation with Crataegus monogyna a queous fruit extract could be useful against reproductive toxicity during Cs treatment in a rat model.

212-217

Authors: Hamid Reza Momeni , Malek Soleimani Mehranjani , Mohammad Ali Shariatzadeh , Mahnaz Haddadi

Objective: Sensory neurons in dorsal root ganglia (DRG) undergo apoptosis after peripheral nerve injury. The aim of this study was to investigate sensory neuron death and the mechanism involved in the death of these neurons in cultured DRG. Materials and Methods: In this experimental study, L5 DRG from adult mouse were dissected and incubated in culture medium for 24, 48, 72 and 96 hours. Freshly dissected and cultured DRG were then fixed and sectioned using a cryostat. Morphological and biochemical features of apoptosis were investigated using fluorescent staining (Propidium iodide and Hoechst 33342) and the terminal Deoxynucleotide transferase dUTP nick end labeling (TUNEL) method respectively. To study the role of caspases, general caspase inhibitor (Z-VAD.fmk, 100 μM) and immunohistochemistry for activated caspase-3 were used. Results: After 24, 48, 72 and 96 hours in culture, sensory neurons not only displayed morphological features of apoptosis but also they appeared TUNEL positive. The application of Z-VAD.fmk inhibited apoptosis in these neurons over the same time period. In addition, intense activated caspase-3 immunoreactivity was found both in the cytoplasm and the nuclei of these neurons after 24 and 48 hours. Conclusion: Results of the present study show caspase-dependent apoptosis in the sensory neurons of cultured DRG from adult mouse.

224-229

Authors: Ali Neshasteh-Riz , Ali Mahmoud Pashazadeh , Seyed Rabie Mahdavi

Objective: To assess relative biological effectiveness (RBE) of 131I radiation relative to 60Co gamma rays in glioblastoma spheroid cells. Materials and Methods: In this experimental study, glioblastoma spheroid cells were exposed to 131I radiation and 60Co gamma rays. Radiation induced DNA damage was evaluated by alkaline comet assay. Samples of spheroid cells were treated by radiation from 131I for four different periods of time to find the dose-response equation. Spheroid cells were also exposed by 200 cGy of 60Co gamma rays as reference radiation to induce DNA damage as endpoint. Results: Resulted RBE of 131I radiation relative to 60Co gamma rays in 100 μm giloblastoma spheroid cells was equal to 1.16. Conclusion: The finding of this study suggests that 131I photons and electrons can be more effective than 60Co gamma rays to produce DNA damage in glioblastoma spheroid cells.

238-243

Authors: Mohammad Reza Bonyadi , Parvin Bastani

Objective: Toxoplasma gondii infection, an intracellular parasite, is often asymptomatic or is caused by different clinical diseases without being detected. Evaluation of IgG, IgA, and IgM in order to diagnose the pending Toxoplasmosis may confront some problems. Several researches has showned that Toxo IgG avidity can be useful in the recent active Toxoplasmosis. In current study, modification and importance of improved Toxoplasma Avidity IgG testing has been evlauated for differentiating Toxoplasma gondii infection in early stage of pregnancy. Materials and Methods: This experimental study included 300 pregnant women with risk of Toxoplasmosis in their initial months of pregnancy. We randomly divided 300 serum samples into A group (n=60) with high avidity and B group (n=40) with borderline avidity. The samples with Toxo IgG levels were classified to four subgroups. IgG avidity was evaluated by enzyme-linked immunosorbent assay (ELISA) method. Results: The mean absorbance of 100 samples in two groups was calculated, and then, two dilutaion curves with plotted absorbance against dilution were drawn for each serum sample. The results of this study showed that in groups with different concentrations of toxo IgG, appropriate dilution of serum is suitable for testing of Avidity. Our findings revealed the subgroups of 1, 2, 3, and 4 with serum dilutios of 1/3 , 1/6, 1/9, and 1/18 respectively, had real and good avidity. Conclusion: One of the issues affectig the results of avidity is high concenteration of Toxo IgG in serum sample. As shown in this study, the best points of dilution for well avidity in both high and borderline avidities are marked with arrows in figures 1-8. This study confirmed that improved methods of measuring Toxo Avidity IgG are very important.

250-257

Authors: Maryam Shahali , Maryam Kabir-Salmani , Karim Nayernia , Hamid Reza Soleimanpour-Lichaei , Mohammad Vasei , Seyed Javad Mowla , Ehsan Ranaie , Mehdi Shakibaie , Mohammad Hossein Modaresi

Objective: Piwil2, a member of Ago/Piwi gene family containing Piwi and PAZ domains, has been shown to be ectopically expressed in different cancer cells, especially its remarkable expression in cancer stem cells (CSCs), and is also known to be essential for germ line stem cell self-renewal in various organisms. The hypothesis that CSC may hold the key to the central problem of clinical oncology and tumor relapse leads to more anticancer treatment studies. Due to emerging controversies and extreme difficulties in studying of CSC, like the cells using in vivo models, more attempts have expended to establish different in vitro models. However, the progress was slow owing to the problems associated with establishing proper CSC cultures in vitro. To overcome these difficulties, we prompted to establish a novel stable cell line over-expressing Piwil2 to develop a potential proper in vitro CSC model. Materials and Methods: In this experimental study, mouse embryonic fibroblasts (MEFs) were isolated and electroporated with a construct containing Piwil2 cDNA under the control of the cytomegalovirus promoter (CMV). Stable transfectants were selected, and the established MEF-Piwil2 cell line was characterized and designated as CSC-like cells using molecular markers. Functional assays, including proliferation, migration, and invasion assays were performed using characterized CSC like cells in serum-free medium. Additionally, MEF-Piwil2 cell density and viability were measured by direct and indirect methods in normoxic and hypoxic conditions. Results: The results of reverse transcriptase-polymerase chain reaction (RT-PCR), western blot, and immunocytochemistry revealed an overexpression for Piwil2 in the transfected Piwil2 cells both in the RNA and protein levels. Furthermore, analysis of the kinetic and stoichiometric parameters demonstrated that the specific growth rate and the yield of lactate per glucose were significantly higher in the MEF-Piwil2 group compared to the MEF cells (ANOVA, p<0.05). Also, analysis of functional assays including migration and invasion assays demonstrated a significantly higher number of migrated and invaded cells in the MEF-Piwil2 compared to that of the MEF cells (ANOVA, p<0.05). The MEF-Piwil2 cells tolerated hypoxia mimetic conditions (CoCl2) with more than 95% viability. Conclusion: According to the molecular and functional studies, it has been realized that Piwil2 plays a key role(s) in tumor initiation, progression and metastasis. Therefore, Piwil2 can be used not only as a common biomarker for tumor, but also as a target for the development of new anticancer drug. Finally, the main outcome of our study was the establishment of a novel CSC-like in vitro model which is expected to be utilized in understanding the complex roles played by CSC in tumor maintenance, metastasis, therapy resistance or cancer relapse.

266-271

Authors: Hussain Habibi , Saeid Abroun , Abbas Hajifathali , Masoud Soleimani , Saeid Kaviani , Nasim Kalantari , Susan Eslahchi

Objective: Multiple myeloma (MM) is a plasma cell malignancy where plasma cells are increased in the bone marrow (BM) and usually do not enter peripheral blood, but produce harmful factors creating problems in these patients (e.g. malignant plasma cells over activate osteoclasts and inhibit osteoblasts with factors like RANKL and DKK). These factors are a main cause of bone lesion in MM patients. Recently SOST gene which responsible to encodes the sclerostin protein was identify. This protein specifically inhibits Wnt signaling in osteoblasts (inhibition of osteoblast differentiation and proliferation) and decrease bone formation and can also cause bone lesion in MM patients. Materials and Methods: In this experimental study, human myeloma cell lines (U266 b1) were purchased from Pasteur Institute of Iran. Samples consisted of BM aspirates from the iliac crest of MM patients. BM with more than 70% plasma cell were selected for our study (6 patients) and one healthy donor. RNA extraction was done with Qiagen kit. was undertaken on mRNA of samples and cell lines. Also we purchased unrestricted somatic stem cells from Bonyakhte Company to evaluate the effect of soluble factors from myeloma cell lines on osteogenic differentiation medium. Results: Our results showed that SOST is expressed significantly in primary myeloma cells derived from MM patients and myeloma cell lines. In other words, patients with more bone problems, express SOST in their plasma cells at a higher level. In addition, myeloma cells inhibit osteoblast differentiation in progenitor cells from umbilical cord blood stem cell (UCSC) in osteogenic inducing medium. Conclusion: There are many osteoblast maturation inhibitory factors such as DKK, Sfrp and Sclerostin that inhibit maturation of osteoblast in bone. Among osteoblast inhibitory agents (DKK, Sfrp, Sclerostin) sclerostin has the highest specificity and therefore will have less side effect versus non-specific inhibitory agents. Our results also show that based on SOST expression in MM, there is a potential to inhibit sclerostin with antibody or alternative methods and prevent bone lesion in MM patients with the least side effect.