فهرست مطالب
Cell Journal (Yakhteh)
Volume:18 Issue: 3, Autumn 2016
- تاریخ انتشار: 1395/06/07
- تعداد عناوین: 20
-
-
Page 289ObjectiveThe organotypic co-culture is a well-known technique to examine cellular interactions and their roles in stem cell proliferation and differentiation. This study aims to evaluate the effects of dermal fibroblasts (DFs) on epidermal differentiation of adipose-derived stem cells (ASCs) using a three-dimensional (3D) organotypic coculture technique.Materials And MethodsIn this experimental research study, rat DFs and ASCs were isolated and cultured separately on electrospun polycaprolactone (PCL) matrices. The PCL matrices seeded by ASCs were superimposed on to the matrices seeded by DFs in order to create a 3D organotypic co-culture. In the control groups, PCL matrices seeded by ASCs were placed on matrices devoid of DFs. After 10 days, we assessed the expressions of keratinocyte-related genes by real-time reverse transcriptase- polymerase chain reaction (RT-PCR) and expression of pan-cytokeratin protein by immunofluorescence in the differentiated keratinocyte-like cells from coculture and control groups. Keratinocyte-like cell morphologies were also observed by scanning electron microscopy (SEM).ResultsThe early, intermediate, and terminal differentiation keratinocyte markers-Cytokeratin14, Filaggrin, and Involucrin significantly expressed in the co-culture groups compared to the control ones (PConclusionThe 3D organotypic co-culture bilayered construct that consisted of DFs and ASCs was an effective technique for epidermal differentiation of ASCs. This co-culture might be useful for epidermal differentiation of stem cells for future applications in skin regeneration.Keywords: Co, Culture Techniques, Fibroblasts, Mesenchymal Stem Cells, Polycaprolactone
-
Page 302ObjectiveNonunion is defined as a minimum of 9 months since injury without any visible progressive signs of healing for 3 months. Recent literature has shown that the application of mesenchymal stromal cells is safe, in vitro and in vivo, for treating long bone nonunion. The present study was performed to investigate the safety of mesenchymal stromal cell (MSC) implantation in combination with platelet lysate (PL) product for treating human long bone nonunion.Materials And MethodsIn this case series clinical trial, orthopedic surgeons visited eighteen patients with long bone nonunion, of whom 7 complied with the eligibility criteria. These patients received mesenchymal stromal cells (20 million cells implanted once into the nonunion site using a fluoroscopic guide) in combination with PL product. For evaluation of the effects of this intervention all the patients were followed up by taking anterior-posterior and lateral X-rays of the affected limb before and 1, 3, 6, and 12 months after the implantation. All side effects (local or systemic, serious or non-serious, related or unrelated) were observed during this time period.ResultsFrom a safety perspective the MSC implantation in combination with PL was very well tolerated during the 12 months of the trial. Four patients were healed; based on the control X- ray evidence, bony union had occurred.ConclusionResults from the present study suggest that the implantation of bone marrow- derived MSCs in combination with PL is safe for the treatment of nonunion. A double blind, controlled clinical trial is required to assess the efficacy of this treatment (Registration Number: NCT01206179).Keywords: Fractures Ununited, Mesenchymal Stromal Cells, Platelet Lysate
-
Page 310ObjectiveThis study examined the in vivo differentiation of mesenchymal stem cells (MSCs) into insulin producing cells (IPCs) on electrospun poly-L-lactide acid (PLLA) scaffolds coated with Matricaria chammomila L. (chamomile) oil.Materials And MethodsIn this interventional, experimental study adipose MSCs (AMSCs) were isolated from 12 adult male New Zealand white rabbits and characterized by flow cytometry. AMSCs were subsequently differentiated into osteogenic and adipogenic lines. Cells were seeded onto either a PLLA scaffold (control) or PLLA scaffold coated with chamomile oil (experimental). A total of 24 scaffolds were inserted into the pancreatic area of each rabbit and placement was confirmed by ultrasound. After 21 days, immunohistochemistry analysis of insulin-producing like cells on protein levels confirmed insulin expression of insulin producing cells (IPSCs). Real-time polymerase chain reaction (PCR) determined the expressions of genes related to pancreatic endocrine development and function.ResultsFourier transform infrared spectroscopy (FTIR) results confirmed the existence of oil on the surface of the PLLA scaffold. The results showed a new peak at 2854 cm-1 for the aliphatic CH2 bond. Pdx1 expression was 0.051 ± 0.007 in the experimental group and 0.009 ± 0.002 in the control group. There was significantly increased insulin expression in the scaffold coated with chamomile oil (0.09 ± 0.001) compared to control group (0.063 ± 0.009, P≤0.05). Both groups expressed Ngn3 and Pdx1 specific markers and pancreatic tissue was observed at 21 days post transplantation.ConclusionThe pancreatic region is an optimal site for differentiation of AMSCs to IPCs. Chamomile oil (as an antioxidant agent) can affect cell adhesion to the scaffold and increase cell differentiation. In addition, the oil may lead to increased blood glucose uptake in pathways in the muscles, liver and fatty tissue of a diabetic animal model by some probable molecular mechanisms.Keywords: Differentiation, Transplantation, Scaffold, Insulin
-
Page 322ObjectiveReceptor activator of nuclear factor-kappa B ligand (RANKL) appears to be an osteoclast-activating factor, bearing an important role in the pathogenesis of multiple myeloma. Some studies demonstrated that U-266 myeloma cell line and primary myeloma cells expressed RANK and RANKL. It had been reported that the expression of myeloid and monocytoid markers was increased by co-culturing myeloma cells with hematopoietic stem cells (HSCs). This study also attempted to show the molecular mechanism of RANK and RANKL on differentiation capability of human cord blood HSC to osteoclast, as well as expression of calcitonin receptor (CTR) on cord blood HSC surface.Materials And MethodsIn this experimental study, CD133 hematopoietic stem cells were isolated from umbilical cord blood and cultured in the presence of macrophage colony stimulating factor (M-CSF) and RANKL. Osteoclast differentiation was characterized by using tartrate-resistant acid phosphatase (TRAP) staining, giemsa staining, immunophenotyping, and reverse transcription-polymerase chain reaction (RT-PCR) assay for specific genes.ResultsHematopoietic stem cells expressed RANK before and after differentiation into osteoclast. Compared to control group, flow cytometric results showed an increased expression of RANK after differentiation. Expression of CTR mRNA showed TRAP reaction was positive in some differentiated cells, including osteoclast cells.ConclusionPresence of RANKL and M-CSF in bone marrow could induce HSCs differentiation into osteoclast.Keywords: Receptor Activator of Nuclear Factor, Kappa B, RANK Ligand, Hematopoietic Stem Cells, Osteoclasts, Calcitonin Receptor
-
Page 332ObjectiveThe label and detection of cells injected into target tissues is an area of focus for researchers. Iron oxide nanoparticles can be used to label cells as they havespecialcharacteristics. The purpose of this study is to examine the effects of iron oxide nanoparticles on human-derived amniotic membrane stem cell (hAMCs) survival and to investigate the magnetic properties of these nanoparticles with increased contrast in magnetic resonance imaging (MRI).Materials And MethodsIn this experimental study, we initially isolated mesenchymal stem cells from amniotic membranes and analyzed them by flow cytometry. In addition, we synthesized superparamagnetic iron oxide nanoparticles (SPIONs) and characterized them by various methods. The SPIONs were incubated with hAMCs at concentrations of 25-800 μg/mL. The cytotoxicity of nanoparticles on hAMCs was measured by the MTT assay. Next, we evaluated the effectiveness of the magnetic nanoparticles as MRI contrast agents. Solutions of SPION were prepared in water at different iron concentrations for relaxivity measurements by a 1.5 Tesla clinical MRI instrument.ResultsThe isolated cells showed an adherent spindle shaped morphology. Polyethylene glycol (PEG)-coated SPIONs exhibited a spherical morphology. The average particle size was 20 nm and magnetic saturation was 60 emu/g. Data analysis showed no significant reduction in the percentage of viable cells (97.86 ± 0.41%) after 72 hours at the 125 μg/ml concentration compared with the control. The relaxometry results of this SPION showed a transverse relaxivity of 6.966 (μg/ml.s)-1ConclusionSPIONs coated with PEG used in this study at suitable concentrations had excellent labeling efficiency and biocompatibility for hAMCs.Keywords: Imaging, Nanoparticles, Stem Cells
-
Page 340ObjectiveThe out-of-field/non-target effect is one of the most important phenomena of ionizing radiation that leads to molecular and cellular damage to distant non-irradiated tissues. The most important concern about this phenomenon is carcinogenesis many years after radiation treatment. In vivo mechanisms and consequences of this phenomenon are not known completely. Therefore, this study aimed to evaluate the oxidative damages to out-of-field lung tissues 24 and 72 hours after pelvic irradiation in rats.Materials And MethodsIn this experimental- interventional study, Sprague Dawleymale rats (n=49) were divided into seven groups (n=7/each group), including two groups of pelvisexposed rats (out-of-field groups), two groups of whole body- exposed rats (scatter groups), two groups of lung-exposed rats (direct irradiation groups), and one control sham group. Outof- field groups were irradiated at a 2×2 cm area in the pelvis region with 3 Gy using 1.25 MeV cobalt-60 gamma-ray source, and subsequently, malondialdehyde (MDA) and glutathione (GSH) levels as well as superoxide dismutase (SOD) activity in out-of-field lung tissues were measured. Results were compared to direct irradiation, control and scatter groups at 24 and 72 hours after exposure. Data were analyzed using Mann-Whitney U test.ResultsSOD activity decreased in out-of-field lung tissue 24 and 72 hours after irradiation as compared with the controls and scatter groups. GSH level decreased 24 hours after exposure and increased 72 hours after exposure in the out-of-field groups as compared with the scatter groups. MDA level in out-of-field groups only increased 24 hours after irradiation.ConclusionPelvis irradiation induced oxidative damage in distant lung tissue that led to a dramatic decrease in SOD activity. This oxidative stress was remarkable, but it was less durable as compared to direct irradiation.Keywords: Bystander Effect, Out of Field, Radiation, Lung, Oxidative Stress
-
Page 346ObjectiveRadiation effects induced in non-irradiated cells are termed radiation-induced bystander effects (RIBE). The present study intends to examine the RIBE response of QU-DB bystander cells to first, second and third radiation fractions and compare their cumulative outcome with an equal, single acute dose.Materials And MethodsThis experimental study irradiated three groups of target cells for one, two and three times with 60Co gamma rays. One hour after irradiation, we transferred their culture media to non-irradiated (bystander) cells. We used the cytokinesis block micronucleus assay to evaluate RIBE response in the bystander cells. The numbers of micronuclei generated in bystander cells were determined.ResultsRIBE response to single acute doses increased up to 4 Gy, then decreased, and finally at the 8 Gy dose disappeared. The second and third fractions induced RIBE in bystander cells, except when RIBE reached to the maximum level at the first fraction. We split the 4 Gy acute dose into two fractions, which decreased the RIBE response. However, fractionation of 6 Gy (into two fractions of 3 Gy or three fractions of 2 Gy) had no effect on RIBE response. When we split the 8 Gy acute dose into two fractions we observed RIBE, which had disappeared following the single 8 Gy dose.ConclusionThe impact of dose fractionation on RIBE induced in QU-DB cells depended on the RIBE dose-response relationship. Where RIBE increased proportionally with the dose, fractionation reduced the RIBE response. In contrast, at high doses where RIBE decreased proportionally with the dose, fractionation either did not change RIBE (at 6 Gy) or increased it (at 8 Gy).Keywords: Bystander Effects, Dose Fractionation, Micronucleus Assay, Radiation
-
Page 353ObjectiveMicro-RNAs (miRNAs) are a class of posttranscriptional regulators that play crucial roles in various biological processes. Emerging evidence suggests a direct link between miRNAs and development of several diseases including type 2 diabetes (T2D). In this study, we aimed to investigate the effect of predicted miRNA and target genes on insulin resistance.Materials And MethodsThis experimental study was conducted on the C2C12 cell line. Using bioinformatics tools miRNA-135 and two respective target genes-insulin receptor (Insr) and vesicle associated membrane protein 2 (Vamp2)- were selected as potential factors involved in insulin resistance process. Levels of glucose uptake miRNA expression and respective gene targets were determined after cell transfaction by miR-135.ResultsIt was determined that Insr gene expression was significantly down-regulated in miR-135 transfected C2C12 cell line (P≤0.05). Interestingly; these transfected cells have shown a significant difference in glucose uptake incomparision the positive control cells, while it was similar to the insulin resistant cell line (P≤0.05). In contrast, no significant alteration of Vamp2 gene expression was observed.ConclusionOur data indicated no change on the Vamp2 expression level after miRNA transfection, while expression level of Insr was reduced and miR-135 expression was contrarily increased leading to poor stimulation of glucose uptake through insulin, and development of insulin resistance phenotype in C2C12 cell line.Keywords: Insulin Resistance, MiR, 135, Insulin Receptor, C2C12
-
Page 362ObjectiveThis study aimed to evaluate the effects of royal jelly (RJ) on serum biochemical alterations and oxidative stress status in liver and pancreas of streptozotocin (STZ)- induced diabetic rats.Materials And MethodsIn this experimental study, thirty two male Wistar rats were divided into the following four groups (n=8/group): i. Control (C), ii. Diabetic (D), iii. Royal jelly (R), and iv. Royal jelly-treated diabetic (D/R) groups. Diabetes was induced by single intraperitoneal (IP) injection of STZ (60 mg/kg). The RJ [100 mg/kg body weight (BW)] was administered orally for 42 days. Blood samples were used to determine serum levels of insulin, high density lipoprotein cholesterol (HDL-c), total protein (TP), albumin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and fasting blood glucose (FBG). Also, the antioxidant status was evaluated by determining the levels of malondialdehyde (MDA), catalase (CAT) and ferric reducing antioxidant power (FRAP) in liver and pancreas. Data were analyzed by one-way analysis of variance (ANOVA) with PResultsSTZ-induced diabetic rats showed a significant elevation in the serum levels of AST, ALT, ALP and FBG, whereas there was a significant decrease in serum levels of insulin, albumin, HDL-c and TP (PConclusionRJ improves oxidative damage induced by STZ in the liver and pancreas of rats; therefore, it can be considered as an effective and alternative treatment for diabetes.Keywords: Diabetes Mellitus, Liver, Oxidative Stress, Pancreas, Royal Jelly
-
Page 371ObjectiveMicroRNAs (miRNA) are a class of non-coding RNAs which play key roles in post-transcriptional gene regulation. Previous studies indicate that miRNAs are dysregulated in patients with multiple sclerosis (MS). Th17 and regulatory T (Treg) cells are two subsets of CD4 T-cells which have critical functions in the onset and progression of MS. The current study seeks to distinguish fluctuations in expression of CD4 T-cell derived miR-223 during the relapsing-remitting (RR) phase of MS (RR-MS), as well as the expressions of Th17 and Treg cell markers.Materials And MethodsThis experimental study used real-time quantitative polymerase chain reaction (qRT-PCR) to evaluate CD4 T cell derived miR-223 expression patterns in patients that experienced either of the RR-MS phases (n=40) compared to healthy controls (n=12), along with RNA markers for Th17 and Treg cells. We conducted flow cytometry analyses of forkhead box P3 (FOXP3) and RAR-related orphan receptor γt (RORγt) in CD4 T-cells. Putative and validated targets of miR-223 were investigated in the miRWalk and miRTarBase databases, respectively.ResultsmiR-223 significantly upregulated in CD4 T-cells during the relapsing phase of RR-MS compared to the remitting phase (P=0.000) and healthy individuals (P=0.036). Expression of RORγt, a master transcription factor of Th17, upregulated in the relapsing phase, whereas FOXP3 upregulated in the remitting phase. Additionally, potential targets of miR-223, STAT1, FORKHEAD BOX O (FOXO1) and FOXO3 were predicted by in silico studies.ConclusionmiR-223 may have a potential role in MS progression. Therefore, suppression of miR-223 can be proposed as an appropriate approach to control progression of the relapsing phase of MS.Keywords: CD4+ T, cell, MicroRNAs, MiR, 223, Multiple Sclerosis, Th17
-
Page 381ObjectiveVascular endothelial growth factor (VEGF) and VEGF receptors (VEGFRs) play important roles in angiogenesis of different developmental mechanisms such as wound healing, embryogenesis and diseases, including different types of cancer. VEGFR2 is associated with cell proliferation, migration, and vascular permeability of endothelial cells. Blocking VEGF and its receptors is suggested as a therapeutic approach to prevent tumor growth. In this study, we aim to block VEGF signaling via small interfering RNA (siRNA) inhibition of VEGFR2.Materials And MethodsIn this experimental study, we used the RNA interference (RNAi) mechanism to suppress expression of the VEGFR2 gene. We conducted the 3-(4,5 dimethylthiazol- 2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay, real-time polymerase chain reaction (PCR), Western blot, and flow cytometry analyses of VEGFR2 expression.ResultsReal-time PCR and Western blot results showed that VEGFR2 expression significantly downregulated. This suppression was followed by inhibition of cell proliferation, reduction of viability, and induction of apoptosis in the cancer cells.ConclusionThese findings suggest that VEGFR2 has a role in cell proliferation and tumor growth. Accordingly, it is suggested that VEGFR2 can be a therapeutic target for controlling tumor growth and proliferation.Keywords: VEGFR2, Downregulation, siRNA, Apoptosis
-
Page 389ObjectivePulp regeneration within the root canal of necrotic teeth is considered an ideal treatment to allow for continued root development and recover teeth vitality. This study aims to evaluate the inductive effect of platelet-rich plasma (PRP) on expression of angiogenesis factors and pulpal revascularization of immature necrotic teeth.Materials And MethodsIn this experimental animal study, we randomly divided 28 immature premolars from two mixed breed dogs into four groups, two experimental, negative and a positive control. Premolars in negative control group were left intact to develop normally. In the positive control and experimental groups, we removed the pulps and induced pulp necrosis, after which the chambers were sealed. Then, we applied the revascularization protocol in the experimental teeth located in the right quadrant. Two months later, the same protocol was applied to the left quadrant. The root canals were disinfected by irrigation with sodium hypochlorite (NaOCl) solution and application a triple antibiotic past. Following the induction of a blood clot (BC) inside the canal space, the coronal portion of the canals was assigned to either of two experimental groups: group 1 [BC㴓 mineral trioxide aggregate (MTA)], group 2 (BC). Access cavities were sealed with a Glass Ionomer. The jaws that held the teeth were processed for histologic analysis of newly formed tissue and immunohistochemical evaluation according to vascular endothelial growth factor (VEGF) and factor VIII expressions in the canals.ResultsHistological analysis demonstrated no significant difference in the formation of new vital tissue inside the root canals between groups1 (42.8%) and 2 (43.5%, P 0.05). Based on immunohistochemical evaluation, micro-vessel density (MVD) of the granulation tissues in both groups were similar and were higher compared with the normal pulp. We observed strongly positive expressions of VEGF and factor VIII in the stromal and endothelial cells, with severe intensity after one month. Both factors showed downregulation at three months postoperative.ConclusionPRP could not increase the formation of new vital tissue. The immunohistochemical results showed that VEGF and factor VIII played a pivotal role in the formation of new vessels inside the root canals of immature, non-vital teeth.Keywords: Platelet, Rich Plasma, Revascularization, Vascular Endothelial Growth Factor
-
Page 397ObjectiveMethylmalonic acidura (MMA) is a rare autosomal recessive inborn error of metabolism. In this study we present a novel nucleotide change in the mutase (MUT) gene of two unrelated Iranian pedigrees and introduce the methods used for its functional analysis.Materials And MethodsTwo probands with definite diagnosis of MMA and a common novel variant in the MUT were included in a descriptive study. Bioinformatic prediction of the splicing variant was done with different prediction servers. Reverse transcription- polymerase chain reaction (RT-PCR) was done for splicing analysis and the products were analyzed by sequencing.ResultsThe included index patients showed elevated levels of propionylcarnitine (C3). Urine organic acid analysis confirmed the diagnosis of MMA, and screening for mutations in the MUT revealed a novel C to G variation at the 3´ splice acceptor site in intron 12. In silico analysis suggested the change as a mutation in a conserved sequence. The splicing analysis showed that the C to G nucleotide change at position -3 in the acceptor splice site can lead to retention of the intron 12 sequence.ConclusionThis is the first report of a mutation at the position -3 in the MUT intron 12 (c.2125-3C>G). The results suggest that the identified variation can be associated with the typical clinical manifestations of MMA.Keywords: Amino Acid Metabolism, Inborn Errors, Methylmalonyl, CoA Mutase, 3´ Splice Acceptor Site
-
Page 405ObjectivePompe disease is a rare neuromuscular genetic disorder and is classified into two forms of early and late-onset. Over the past two decades, mitochondrial abnormalities have been recognized as an important contributor to an array of neuromuscular diseases. We therefore aimed to compare mitochondrial copy number and mitochondrial displacement-loop sequence variation in infantile and adult Pompe patients.Materials And MethodsIn this retrospective study, the mitochondrial D-loop sequence was analyzed by polymerase chain reaction (PCR) and direct sequencing to detect possible variation in 28 Pompe patients (17 infants and 11 adults). Results were compared with 100 healthy controls and sequences of all individuals were compared with the Cambridge reference sequence. Real-time PCR was used to quantify mitochondrial DNA copy number.ResultsAmong 59 variants identified, 37(62.71%) were present in the infant group, 14(23.333%) in the adult group and 8(13.333%) in both groups. Mitochondrial copy number in infant patients was lower than adults (PConclusionThe 317-318 ins CCC was detected as a new mitochondrial variant in Pompe patients.Keywords: Pompe, Mitochondrial DNA, D, Loop, Copy Number
-
Page 416ObjectiveFollowing traumatic brain injury, disruption of blood-brain-barrier and consequent brain edema are critical events which might lead to increasing intracranial pressure (ICP), and nerve damage. The current study assessed the effects of aqueous date fruit extract (ADFE) on the aforementioned parameters.Materials And MethodsIn this experimental study, diffused traumatic brain injury (TBI) was generated in adult male rats using Marmarous method. Experimental groups include two pre-treatment (oral ADFE, 4 and 8 mL/kg for 14 days), vehicle (distilled water, for 1 days) and sham groups. Brain edema and neuronal injury were measured 72 hours after TBI. Veterinary coma scale (VCS) and ICP were determined at -1, 4, 24, 48 and 72 hours after TBI. Differences among multiple groups were assessed using ANOVA. Turkeys test was employed for the ANOVA post-hoc analysis. The criterion of statistical significance was sign at PResultsBrain water content in ADFE-treated groups was decreased in comparison with the TBI뷨扲 group. VCS at 24, 48 and 72 hours after TBI showed a significant increase in ADFE groups in comparison with the TBI뷨扲 group. ICP at 24, 48 and 72 hours after TBI, was decreased in ADFE groups, compared to the TBI뷨扲. Brain edema, ICP and neuronal injury were also decreased in ADFE group, but VCS was increased following on TBI.ConclusionADFE pre-treatment demonstrated an efficient method for preventing traumatic brain deterioration and improving pathological parameters after TBI.Keywords: Brain Injury, Brain Edema, Intracranial Pressure
-
Page 425ObjectiveElectroporation can be a highly efficient method for introducing the foreign genetic materials into the targeted cells for transient and/or permanent genetic modification. Considering the application of this technique as a very efficient method for drug, oligonucleotide, antibody and plasmid delivery for clinical applications and production of transgenic animals, the present study aimed to optimize the transfection efficiency of sheep testicular cells including spermatogonial stem cells (SSCs) via electroporation.Materials And MethodsThis study is an experimental research conducted in Biotechnology Research Center (Avicenna Research Institute, Tehran, Iran) from September 2013 to March 2014. Following isolation and propagation of one-month lamb testicular cells (SSCs and somatic testicular cells including; Sertoli, Leydig, and myoid cells), the effect of different electroporation parameters including total voltages (280, 320, and 350 V), burst durations (10, 8, and 5 milliseconds), burst modes (single or double) and addition of dimethyl sulfoxide (DMSO) were evaluated on transfection efficiency, viability rate and mean fluorescent intensity (MFI) of sheep testicular cells.ResultsThe most transfection efficiency was obtained in 320 V/8 milliseconds/single burst group in transduction medium with and without DMSO. There was a significantly inverse correlation between transfection efficiency with application of both following parameters: addition of DMSO and double burst. After transfection, the highest and lowest viability rates of testicular cells were demonstrated in 320 V/8 milliseconds with transduction medium without DMSO and 350 V/5 milliseconds in medium containing DMSO. Addition of DMSO to transduction medium in all groups significantly decreased the viability rate. The comparison of gene expression indicated that Sertoli and SSCs had the most fluorescence intensity in 320 V/double burst/DMSO positive. However, myoid and Leydig cells showed the maximum expression in 320 V/single burst and/or 350 V/double burst/ DMSO positive.ConclusionWe optimized the electroporation method for transfection of sheep testicular cells and recommended the application of 320 V/8 milliseconds/single pulse/DMSO negative for transduction of plasmid vector into these cells. Among testicular cells, the most external gene expression was demonstrated in SSC population.Keywords: Electroporation, Testicular Cells, Transfection, Sheep, Spermatogonial Stem Cells
-
Page 438ObjectiveGlobozoospermia is a rare type of teratozoospermia with incidence of 0.1% among infertile individuals. Phospholipase C zeta (PLCζ) and postacrosomal sheath WW domain binding protein (PAWP) are the main candidates in sperm taking responsibility for oocyte activation during fertilization. Therefore, we aimed to evaluate the expression of these two genes at RNA and protein levels in globozoospermic individuals and compare the results with fertile individuals.Materials And MethodsIn this experimental study, semen samples of 21 infertile men with globozoospermia and 25 fertile men were collected. Expression of PLCζ and PAWP at RNA and protein levels were assessed and compared between two groups by quantitative real time polymerase chain reaction (qPCR) and Western blot, respectively.ResultsExpression of both PLCζ and PAWP were significantly reduced at RNA and protein levels in infertile men with globozoospermia compared to fertile men.ConclusionThis is the first study that simultaneously assessing the respective factors in a large population of globozoospermia, suggested that intra-cytoplasmic sperm injection (ICSI) along with artificial oocyte activation may rescue failed fertilization in routine ICSI.Keywords: Globozoospermia, PLCζ PAWP
-
Page 446ObjectiveEpididymo-orchitis (EO) potentially results in reduced fertility in up to 60% of affected patients. The anti-inflammatory effects of Korean red ginseng (KRG) and its ability to act as an immunoenhancer in parallel with the beneficial effects of this ancient herbal medicine on the reproductive systems of animals and humans led us to evaluate its protective effects against acute EO.Materials And MethodsThis animal experimental study was conducted in the Department of Anatomical Sciences, Faculty of Medicine, Zanjan University of Medical Sciences (ZUMS), Zanjan, Iran during 2013-2015. We divided 50 Wistar rats into five following groups (n=10 per group): i. Control-intact animals, ii. Vehicle-phosphate buffered saline (PBS) injection into the vas deferens, iii. KRG-an intraperitoneal (IP) injection of KRG, iv. EO-an injection of uropathogenic Escherichia coli (UPEC) strain M39 into the vas deferens, and v. EO/ KRG-injections of both UPEC strain M39 and KRG. The treatment lasted seven days. We then evaluated sperm parameters, number of germ cell layers, Johnsons criteria, germ cell apoptosis, body weight and relative sex organs weight.ResultsAcute EO increased the relative weight of prostate and seminal vesicles (P0.05). It also reduced sperm quality such as total motility, sperm concentration (P≤0.01), and the percentage of normal sperm (P≤0.001). Moreover, acute EO decreased Millers (P≤0.05) and Johnsens scores and increased apoptotic indexes of spermatogenic cells (P≤0.001). KRG treatment decreased prostate weight gain (P≤0.05) and improved the percentage of sperm with normal morphology, total motility (P≤0.01), and progressive motility (P 0.05). The apoptotic indexes of spermatogenic cells reduced (P≤0.001), whereas both Johnsens (P≤0.01) and Millers criteria increased in the KRG-treated EO testis (P≤0.05).ConclusionConsequently, KRG ameliorated the devastating effects of EO on the sperm retrieved from either epididymis or testicle in rats.Keywords: Ginseng, Uropathogenic Escherichia coli, Sperm, Rat, Testis
-
Page 458ObjectiveAlcohol consumption is habitually accompanied by the use of other psychoactive substances, mostly tobacco. Nicotine and alcohol affect male accessory reproductive glands function. Most studies have been done on pathologic features of prostate, but there has been no systematic study on the seminal vesicle. Therefore, the aim of current study was to investigate the distribution of androgen receptor (AR) and estrogen receptors beta (ER-β) immune reactivities following long-term treatment of alcohol, nicotine or a combination of both substances.Materials And MethodsIn this experimental study, a total of 40 adult Wistar rats, nine weeks of age, were used. Animals were randomly divided into four groups, including: i. Control group receiving normal saline 0.09%, ii. Ethanol group receiving ethanol 20% (2 ml/kg, via gavage), iii. Nicotine group receiving nicotine (0.1 mg/kg, subcutaneous injection), and iv. Ethanol-nicotine group receiving simultaneous ethanol 20% (2 ml/kg) and nicotine (0.1 mg/kg) treatment. All treatment lasted for eight weeks. Prior to intracardiacperfusion, blood sample was collected from left ventricle. The seminal vesicles were isolated and processed for paraffin blocking. The sample tissues were then studied for distribution of AR and ER-β immunereactivities using immunohistochemical (IHC) staining method. One way analysis of variance (ANOVA) and Tukeys test were performed for data analysis. A value of PResultsOur results revealed that the lowest mean number of positive cells belonged to the animals of ethanol-nicotine group that was followed by the ethanol, nicotine, and control groups, respectively. However, there was no significant difference regarding serum testosterone level among experimental groups.ConclusionIt was concluded that combination of both ethanol and nicotine may be a crucial factor in the expression levels of AR and ER-β.Keywords: Alcohol, Nicotine, Androgen Receptor, Estrogen Receptor, Seminal Vesicle
-
Page 464ObjectiveChlorpyrifos (CPF) is a neurotoxic organophosphorus (OP) insecticide. Its mechanism of action includes oxidative stress, excitotoxicity, and inhibition of the acetylcholinesterase enzyme (AChE). The aim of the present study is to investigate CPF toxicity in mature and immature cerebellar granule neurons (CGNs), as well as its effect on glutamate induced excitotoxicity.Materials And MethodsThis study was an in vitro experimental study performed on mice cultured CGNs. Immature and mature neurons were exposed to different concentrationsof CPF (1-1000 μM) and glutamate (10-600 μM) for 48 hours after which we used the MTT assay to measure cytotoxicity. Immature neurons had exposure to CPF for 5 days in order to evaluate the cytotoxic effect on developing neurons. Mature neurons received sub-lethal concentrations of CPF (10, 100 μM) combined with different concentrations of glutamate. AChE activity and reactive oxygen species (ROS) generation were assessed after treatments.ResultsImmature CGNs had increased sensitivity to CPF toxicity compared to mature neurons. We observed significantly greater ROS production in immature compared to mature neurons, however AChE activity was more inhibited in mature neurons. Although CPF toxicity was not well correlated with AChE inhibition, it correlated well with ROS production. Glutamate toxicity was potentiated by sub-lethal concentration of CPF, however glutamate induced ROS production was not affected. The results suggested that CPF potentiated glutamate toxicity by mechanisms other than oxidative stress.ConclusionCPF toxicity differed in mature and immature neurons. Potentiated glutamate toxicity by CPF implied that CPF exposure might be a risk factor for neurodegenerative disease.Keywords: Chlorpyrifos, Neurotoxicity, Glutamate Toxicity, CGNs, Oxidative Stress