فهرست مطالب

Cell Journal - Volume:14 Issue: 3, 2012
  • Volume:14 Issue: 3, 2012
  • تاریخ انتشار: 1391/09/13
  • تعداد عناوین: 10
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  • Mohammad Hussein Abnosi, Zahra Jafari Yazdi Pages 161-170
    Objective
    Although arsenite is toxic it is currently recommended for the treatment of malignancies. In this study the effects of sub-micromolar concentrations of sodium arsenite on the viability, morphology and mechanism of cell death of rat bone marrow mesenchymal stem cells (BMCs) over 21 days was investigated.
    Materials And Methods
    In this experimental study, BMCs were extracted in Dulbecco’s Modified Eagles Medium (DMEM) containing 15% of fetal bovine serum (FBS) and expanded till the 3rd passage. The cells were treated with 1, 10, 25, 50, 75 and 100 nM of sodium arsenite for 21 days and the viability of the cells estimated using 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyl tetrazolium (MTT) and trypan blue staining. Cells were then treated with the selected dose (25 nM) of sodium arsenite to determine their colony forming ability (CFA) and population doubling number (PDN). Morphology of the cells was studied using florescent dyes, and the integrity of the DNA was investigated using the comet assay and agarose gel electrophoresis. The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and the caspase 3 assay were then applied to understand the mechanism of cell death. Data was analyzed using one way ANOVA, Tukey test.
    Results
    A significant reduction of viability, PDN and CFA was found following treatment of BMCs with 25 nM sodium arsenite (p<0.05). Cytoplasm shrinkage and a significant decrease in the diameter of the nuclei were also seen. Comet assay and agarose gel electrophoresis revealed DNA breakage, while positive TUNEL and activated caspase 3 confirmed the apoptosis.
    Conclusion
    A low concentration of sodium arsenite (25 nM) caused reduction of viability due to induction of apoptosis. Therefore, long term exposure to low dose of this chemical may have unwanted effects on BMCs.
    Keywords: Apoptosis, Cell Viability, Mesenchymal Stem Cell, Rat, Sodium Arsenite
  • Hamid Reza Momeni, Mahsa Jarahzadeh Pages 171-176
    Objective
    The apoptosis of motor neurons is a critical phenomenon in spinal cord injuries. Adult spinal cord slices were used to investigate whether voltage sensitive calcium channels and Na+/Ca2+ exchangers play a role in the apoptosis of motor neurons.
    Materials And Methods
    In this experimental research, the thoracic region of the adult mouse spinal cord was sliced using a tissue chopper and the slices were incubated in a culture medium in the presence or absence of N/L type voltage sensitive calcium channels blocker (loperamide, 100 μM) or Na+/Ca2+ exchangers inhibitor(bepridil, 20 μM) for 6 hours. 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyl tetrazolium (MTT) staining was used to assess slice viability while morphological features of apoptosis in motor neurons were studied using fluorescent staining.
    Results
    After 6 hours in culture, loperamideand bepridil not only increased slice viability, but also prevented motor neuron apoptosis and significantly increased the percentage of viable motor neurons in the ventral horns of the spinal cord.
    Conclusion
    The results of this study suggest that voltage sensitive calcium channels and Na+/Ca2+ exchanger might be involved in the apoptosis of motor neurons in adult spinal cord slices.
    Keywords: Apoptosis, Bepridil, Loperamide, Motor Neuron, Spinal Cord
  • Mehdi Mehdizadeh, Fataneh Dabaghian, Akram Nejhadi, Hassan Fallah, Huseini, Samira Choopani, Nima Shekarriz, Nima Molavi, Arghavan Basirat Ot, Farzaneh Mohammadzadeh Kazorgah, Lirezasamzadeh, Kermani, Sara Soleimani Asl Pages 177-184
    Objective
    The spice Zingiber officinale or ginger possesses antioxidant activity and neuroprotective effects. The effects of this traditional herbal medicine on 3,4-methylenedioxymethamphetamine (MDMA) induced neurotoxicity have not yet been studied. The present study considers the effects of Zingiber officinale on MDMA-induced spatial memory impairment and apoptosis in the hippocampus of male rats.
    Materials And Methods
    In this experimental study, 21 adult male Sprague Dawley rats (200-250 g) were classified into three groups (control, MDMA, and MDMA plus ginger). The groups were intraperitoneally administered 10 mg/kg MDMA, 10 mg/kg MDMA plus 100 mg/kg ginger extract, or 1 cc/kg normal saline as the control solution for one week (n=7 per group). Learning memory was assessed by Morris water maze (MWM) after the last administration. Finally, the brains were removed to study the cell number in the cornu ammonis (CA1) hippocampus by light microscope, Bcl-2 by immunoblotting, and Bax expression by reverse transcription polymerase chain reaction (RT-PCR). Data was analyzed using SPSS 16 software and a one-way ANOVA test.
    Results
    Escape latency and traveled distances decreased significantly in the MDMA plus ginger group relative to the MDMA group (p<0.001). Cell number increased in the MDMA plus ginger group in comparison to the MDMA group. Down-regulation of Bcl-2 and up-regulation of Bax were observed in the MDMA plus ginger group in comparison to the MDMA group (p<0.05).
    Conclusion
    Our findings suggest that ginger consumption may lead to an improvement of MDMA-induced neurotoxicity.
    Keywords: Apoptosis, Ginger, Spatial Memory, MDMA, Hippocampus, Bcl, 2 Family
  • Rokhsareh Meamar, Fereshte Karamali, Seyed Ali Mousavi, Hossein Baharvand, Mohammad Hossein Nasr, Esfahani Pages 185-192
    Objective
    Ecstasy, or 3, 4 (±) methylenedioxymethamphetamine (MDMA), is a potent neurotoxic drug. One of the mechanisms for its toxicity is the secondary release of glutamate. Mouse embryonic stem cells (mESCs) express only one glutamate receptor, the metabotropic glutamate receptor 5 (mGlu5), which is involved in the maintenance and self-renewal of mESCs. This study aims to investigate whether MDMA could influence self-renewal via the mGlu5 receptor in mESCs.
    Materials And Methods
    In this expremental study, we used immunocytochemistry and reverse transcription-polymerase chain reaction (RT-PCR) to determine the presence of the mGlu5 receptor in mESCs. The expression of mGlu5 was evaluated after MDMA was added to mESCs throughout neural precursor cell formation as group 1 and during neural precursor cell differentiation as group 2. The stemness characteristic in treated mESCs by immunofluorescence and flow cytometry was studied. Finally, caspase activity was evaluated by fluorescence staining in the treated group. One-way ANOVA or repeated measure of ANOVA according to the experimental design was used for statistical analyses.
    Results
    In this study mGlu5 expression was shown in mESCs. In terms of neuronal differentiation, MDMA affected mGlu5 expression during neural precursor cell formation (group 1) and not during neural precursor differentiation (group 2). MDMA (450 μM) induced a significant increment in self-renewal properties in mESCs but did not reverse 2-methyl-6(phenylethynyl) pyridine (MPEP, 1 μM), a non-competitive selective mGlu5 antagonist. Fluorescence staining with anti-caspase 3 showed a significant increase in the number of apoptotic cells in the MDMA group.
    Conclusion
    We observed a dual role for MDMA on mESCs: reduced proliferation and maintenance of self-renewal. The lack of decreasing stemness characteristic in presence of MPEP suggests that MDMA mediates its role through a different mechanism that requires further investigation. In conclusion, despite being toxic, MDMA maintains stemness characteristics.
    Keywords: Embryonic Stem Cells, Ecstasy, MDMA, Self Renewal, mGlu Receptor
  • Mahboobeh Ashrafi, Seyedeh Zahra Bathaie, Saeid Abroun Pages 193-202
    Objective
    N-nitroso-N-methylurea (NMU) induces breast cancer in rodents, particularly in rats. This model of breast cancer is very similar to human breast cancer. As a continuation of our recent work, we investigated the expressions of cyclin D1 and p21 in NMU-induced breast cancer of Wistar Albino rats.
    Materials And Methods
    In this experimental study, mammary carcinoma was induced in female Wistar Albino rats by a new protocol which included the intraperitoneal injection of NMU (50 mg/kg) at 50, 65, and 80 days of the animal’s age. The animals were weighed weekly and palpated in order to record the numbers, location, and size of tumors. Subsequently tumor incidence (TI), latency period (LP), and tumor multiplicity (TM) were reported. About four weeks after the tumor size reached 1.5 cm3, rats were sacrificed. Cyclin D1 and p21 expressions in tumors and normal mammary glands from normal rats were measured by reverse-transcription polymerase chain reaction (RT- PCR) and Western blot analysis. Statistical analysis of the data was performed using SPSS software version 16.0.
    Results
    The efficiency of tumor induction was 65%, LP was 150 days, and a TM of 1.43 ± 0.53 per rat was noted. RT-PCR and Western blot data indicated significant (p<0.05) induction of both cyclin D1 and p21 expressions in rat mammary tumors compared with normal tissue from the control group.
    Conclusion
    These results indicate an efficient mammary tumor induction protocol for this type of rat, which is accompanied by an increase in cyclin D1 and p21 expressions.
    Keywords: Breast Cancer, N, Nitroso, N, Methylurea (NMU), Cyclin D1, p21 Expression
  • Zakieh Asgari, Fatemeh Ghasemian, Mina Ramezani, Mohammad Hadi Bahadori Pages 203-208
    Objective
    Melatonin is a scavenger agent that has been used to promote in vitro embryo development. This study was designed to show the effects of melatonin on the quality and quantity rate of preimplantation mouse embryo development and pregnancy.
    Materials And Methods
    In this experimental study, super ovulated, mated mice were killed by cervical dislocation to collect two-cell zygotes from the oviduct of pregnant 1 day NMRI mice. Zygotes were cultured to the hatching blastocyst stage and the numbers of embryos at different stages were recorded under an inverted microscope. The cleavage rates of two-cell zygotes were assayed until the blastocyst and hatching blastocyst stage in drops of T6 medium that contained either melatonin (1, 10, and 100×106, 10 and 100×109 M) or no melatonin. The cell numbers of blastocysts were determined by differential staining, implantation outcomes were studied, and development and pregnancy rate were compared by the Chi-square (development) and Fisher’s exact (pregnancy rate) tests.
    Results
    The addition of 10 and 100 nM melatonin to the embryo culture media promoted the development of the two-cell stage embryos to blastocyst and hatching blastocysts (p<0.01) and caused a significant increase in total cell number (TCN), trophoectoderm (TE), and inner cell mass (ICM) of the blastocysts (p<0.01). A difference was observed in the percentage of transferred embryos that were successfully implanted between the control and treatment groups (p<0.05).
    Conclusion
    The data indicate that 10 and 100 nM of melatonin positively impact mouse embryo cleavage rates, blastocyst TCN, and their implantation. Therefore, melatonin at low concentrations promotes an embryonic culture system in mice.
    Keywords: Development, Implantation, Melatonin, Differential Staining, Cleavage
  • Shaghayegh Anvari, Shahin Najar Peerayeh, Mehrdad Behmanesh, Mina Boustanshenas Pages 209-214
    Objective
    Vibrio cholerae (V. cholerae) causes a potentially lethal disease named cholera. The cholera enterotoxin (CT) is a major virulence factor of V. cholerae. In addition to CT, V. cholerae produces other putative toxins, such as the zonula occludens toxin (Zot) and accessory cholera enterotoxin (Ace). The ace gene is the third gene of the V. cholerae virulence cassette. The Ace toxin alters ion transport, causes fluid accumulation in ligated rabbit ileal loops, and is a cause of mild diarrhea. The aim of this study is the cloning and overexpression of the ace gene into Escherichia coli (E. coli) and determination of some characteristics of the recombinant Ace protein.
    Materials And Methods
    In this experimental study, the ace gene was amplified from V. cholerae strain 62013, then cloned in a pET28a expression vector and transformed into an E. coli (DH5 α) host strain. Subsequently, the recombinant vector was retransformed into E. coli BL21 for expression, induced by isopropythio-β-D-galctoside (IPTG) at a different concentration, and examined by SDS-PAGE and Western blot. A rabbit ileal loop experiment was conducted. Antibacterial activity of the Ace protein was assessed for E. coli, Stapylococcus aureus (S. aureus), and Pseudomonas aeruginosa (P. aeruginosa).
    Results
    The recombinant Ace protein with a molecular weight of 18 kDa (dimeric form) was expressed in E. coli BL21. The Ace protein showed poor staining with Coomassie blue stain, but stained efficiently with silver stain. Western blot analysis showed that the recombinant Ace protein reacted with rabbit anti-V. cholerae polyclonal antibody. The Ace protein had antibacterial activity at a concentration of ≥200 μg/ml and caused significant fluid accumulation in the ligated rabbit ileal loop test.
    Conclusion
    This study described an E. coli cloning and expression system (E. coli BL21- pET-28a-ace) for the Ace protein of V. cholerae. We confirmed the antibacterial properties and enterotoxin activity of the resultant recombinant Ace protein.
    Keywords: Vibrio cholerae, Accessory Cholerae Enterotoxin, pET28a, E. coli
  • Catsper, Sperm Cation, Male Fertility, Contraception, Antibody Pages 215-224
    Objective
    CatSper is a voltage-sensitive calcium channel that is specifically expressed in the testis and it has a significant role in sperm performance. CatSper (1-4) ion channel subunit genes, causes sperm cell hyperactivation and male fertility. In this study, we have explored targeting of the extracellular loop as an approach for the generation of antibodies with the potential ability to block the ion channel and applicable method to the next generation of non-hormonal contraceptive.
    Materials And Methods
    In this experimental study, a small extracellular fragment of CatSper1 channel was cloned in pET-32a and pEGFP-N1 plasmids. Then, subsequent methods were performed to evaluate production of antibody: 1) pEGFP-N1/CatSper was used as a DNA vaccine to immunize Balb/c mice, 2) The purified protein of pET-32a/CatSper was used as an antigen in an enzyme-linked immunosorbent assay (ELISA) and western- blot, and 3) The serum of Balb/-c mice was used as an antibody in ELISA and western-blot. The statistical analysis was performed using the Mann Whitney test.
    Results
    The results showed that vaccination of the experimental group with DNA vaccine caused to produce antibody with (p< 0.05) unlike the control group. This antibody extracted from Balb/c serum could recognize the antigen, and it may be used potentially as a male contraception to prevent sperm motility.
    Conclusion
    CatSpers are the promising targets to develop male contraceptive because they are designed highly specific for sperm; although, no antagonists of these channels have been reported in the literature to date. As results showed, this antibody can be used in male for blocking CatSper channel and it has the potential ability to use as a contraceptive.
    Keywords: Mahboobeh Nazari, Manouchehr Mirshahi, Seyed, Javad Mowla, Taravat Bamdad, Sina Sarikhani
  • Bahar Movaghar, Saeedeh Askarian Pages 225-230
    Objective
    The appropriate interaction between a blastocyst and the endometrium is essential for successful implantation. Numerous factors, including hormone receptors (progesterone receptor), cytokines [leukemia inhibitory factors (LIF)], and adherence molecules such as E-cadherin are involved in the cross-talk that occurs between the embryo and endometrium. Studies show that a lack of these genes impact endometrial receptivity. In this study, we compare the expression levels of E-cadherin, LIF, and progesterone receptor (PgR) genes in blastocysts that have been obtained from superovulated mice to those obtained from natural cycles.
    Materials And Methods
    In this experimental study, for the experimental group, a total of 17 virgin female NMRI mice (6- 8 weeks old) were injected with 7.5 IU pregnant mare serum gonadotropin (PMSG). Their blastocysts (approximately n= 120) were flushed out after 3.5 days, following administration of human chorionic gonadotropin (hCG). The control group consisted of blastocysts from 62 female mice that were mated with male mice. The natural cycle blastocysts were flushed out from the female mice uteri 3.5 days after mating. The expression levels of E-cadherin, LIF, t PgR genes were examined by quantitative real-time reverse-transcriptase polymerase chain reaction (RT-PCR). Data were analyzed by the student’s t-test (one sample t-test).
    Results
    Expression levels of all studied genes were significantly lower in the hormone-treated group compared to the natural cycle blastocysts (p<0.05).
    Conclusion
    Although ovarian stimulation is utilized to obtain more oocytes in ART cycles, it seems that this could disadvantageous to implantation because of the decrease in expression levels of certain genes. Because of the important roles of E-cadherin, LIF, and progesterone receptor in the implantation process, we have shown lower expression levels of these genes in mouse blastocysts obtained from ovarian-stimulated mice than those derived from the natural cycle. The results observed in this study have shown the possibility of an unfavorable effect on implantation and pregnancy rate.
    Keywords: Blastocyst, Ovarian Stimulation, Progesterone Receptor, Leukemia Inhibitory Factor, E, Cadherin
  • Mansooreh Soleimani, Majid Katebi, Akram Alizadeh, Farzaneh Mohammadzadeh, Mehdi Mehdizadeh Pages 231-236
    Objective
    Ecstasy, also known as 3, 4-methylenedioxymethamphetamine (MDMA), is a psychoactive recreational hallucinogenic substance and a major worldwide recreational drug. There are neurotoxic effects observed in laboratory animals and humans following MDMA use. MDMA causes apoptosis in neurons of the central nervous system (CNS). Withdrawal signs are attenuated by treatment with the adenosine receptor (A2A receptor). This study reports the effects of glutamyl cysteine synthetase (GCS), as an A2A receptor agonist, and succinylcholine (SCH), as an A2A receptor antagonist, on Sprague Dawley rats, both in the presence and absence of MDMA.
    Materials And Methods
    In this experimental study, we used seven groups of Sprague Dawley rats (200-250 g each). Each group was treated with daily intraperitoneal (IP) injections for a period of one week, as follows: i. MDMA (10 mg/kg); ii. GCS (0.3 mg/kg); iii. SCH (0.3 mg/kg); iv. GCS + SCH (0.3 mg/kg each); v. MDMA (10 mg/kg) + GCS (0.3 mg/kg); vi. MDMA (10 mg/kg) + SCH (0.3 mg/kg); and vi. normal saline (1 cc/kg) as the sham group. Bax (apoptotic protein) and Bcl-2 (anti-apoptotic protein) expressions were evaluated by striatum using RT-PCR and Western blot analysis.
    Results
    There was a significant increase in Bax protein expression in the MDMA+SCH group and a significant decrease in Bcl-2 protein expression in the MDMA+SCH group (p<0.05).
    Conclusion
    A2A receptors have a role in the apoptotic effects of MDMA via the Bax and Bcl-2 pathways. An agonist of this receptor (GCS) decreases the cytotoxcity of MDMA, while the antagonist of this receptor (SCH) increases its cytotoxcity.
    Keywords: Ecstasy or MDMA, Neurotoxicity, Adenosine Receptor, Agonist of A2A Receptor, Antagonist of A2A Receptor