Cell Journal (Yakhteh)
Year:2013 | Volume:14 | Issue:4 (56)|Papers Count:10

Objective: It has been suggested that the vascular endothelial growth factor (VEGF) gene expression plays an important role in radiation-induced injury to the spinal cord. This study assesses the radio protective effects of N-acetyl-5-methoxytryptamine (melatonin) through its modulation of VEGF expression after localized irradiation of the cervical spinal cord.Materials and Methods: In this experimental study, we divided 192 male rats into four groups: 1. control (n=48); 2. Rats that received an intraperitoneal (IP) injection of melatonin (n=48); 3. Rats that received an IP injection of melatonin 30 minutes prior to cervical spinal cord gamma irradiation [dose: 22 Gy; (n=48)]; and 4. rats that received an IP injection of vehicle prior to spinal cord irradiation (n=48). The changes in VEGF expression were assessed using real-time RT-PCR and enzyme-linked immunosorbent assays. Samples for light microscopy were stained with hematoxylin and eosin (H& E). The differences among the groups were analyzed using the analysis of variance (ANOVA) test followed by Tukey’s multiple comparisons test.Results: Up-regulation of VEGF expression was observed from 8 to 22 weeks after irradiation (p<0.05). Paralysis and other radiation-induced myelopathy manifestations developed within 22 weeks after irradiation. VEGF expression in the melatonin pre-treatment group significantly down-regulated in the 20th and 22nd weeks after irradiation compared to the radiation-only group.Conclusion: The results support the hypothesis that modulation of VEGF expression by melatonin administration may increase the survival rate of irradiated animals.

Objective: Experiments were conducted to find the differences between post-thaw viability and chromosome aberrations in eight-cell mouse embryos at presence of dimethyl sulfoxide (DMSO) and 1, 2-propanediol (PROH) as croprotectants in different storage durations.Materials and Methods: In this case-control study, a total number of 720 mouse embryos from about 250 NMRI mice were vitrified with 30% PROH or DMSO; each diluted with a solution containing 30% ficol plus 0.5 M sucrose. Embryos were exposed to the solutions for 0.5 minute at 25˚C followed by cooling in liquid nitrogen, then after appropriate storage duration, they were rapidly warmed. Besides, there were 100 mouse embryos for each cryoprotectant group (totally 200 embryos) as control. Embryo survival was assessed by in vitro development, and chromosome abnormalities were analyzed by Giemsa staining.Results: The proportion of mitotic abnormalities in PROH/DMSO vitrified embryos was significantly higher than unfrozen control group. This was confirmed also by a reduced viability of the embryos as judged by a culture at the blastocyst stage (p<0.05 in all test groups).Conclusion: It can be deduced that long term cryopreservation may result in chromosomal abnormalities and/or low viability.

Objective: The aim of present study was cloning and expression of phiC31 integrase cDNA in a bacterial expression vector. Thus, an intra-molecular assay vector was applied to show in vitro activity of recombinant protein.Materials and Methods: In this experimental study, phiC31 cDNA was subcloned into a prokaryotic expression vector and transformed into E.coli Bl21 (DE3). Recombinant phiC31 integrase was purified form the bacterial cell lysates and its activity was verified by an in vitro functional assessment.Results: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purifi ed phiC31 integrase confirmed the size of protein (70 kDa). Finally, the functionality of purified phiC31 integrase was verified.Conclusion: The results of this study indicated that the purified integrase has a great potential application for in vitro site-specific integration.

Objective: Electroporation is the most common method used for the transfection of DNA. Although this method has been attributed for various cell using different buffer compositions, the effects of DNA concentration on the transfection efficiency has not yet been studied. Here the correlation between the efficiency of electroporation reaction and increments of DNA concentration has been investigated. Following this investigation, a study was set out to produce a transgenic goat which is capable of secreting recombinant human coagulation factor IX in its milk.Materials and Methods: In this experimental study, a linearized recombinant vector (pBC1) entailing human coagulation factor IX (5.7 kb) cDNA was introduced into goat fetal fibroblast cells (three sub passages) via electroporation in four separate experiments (while other variables were kept constant), beginning with 10 mg DNA per pulse in the first experiment and increments of 10 mg/pulse for the next three reactions. Thereafter, polymerase chain reaction (PCR) -positive cell culture plates were diluted by several factors for further analysis of the transfected wells. Subsequently, positive cells were isolated for fluorescence in situ hybridization. Logistic regression model was used for data analyzing.Significance was defined as p<0.05.Results: The results showed no significant difference among three first concentrations except for group 1 (10 mg/pulse) and group 3 (30 mg/pulse), but there was a significant difference between these groups and the fourth group (p<0.05), as this group (40 mg/pulse) statistically showed the highest rate of transfection. As the fluorescence in situ hybridization (FISH) results indicated the transgene was integrated in a single position in PCR positive cells.Conclusion: Increasing amount of using the vector 40μg/pulse efficiently increased the number of transfected cells. Besides of voltage and buffer strength which had been previously shown to play a critical role in electroporation efficiency, our results showed an increase in DNA concentration could affect an exponential surge in the electroporation efficiency.

Objective: This study defines the relationship between salivary beta-2 microglobulin (b2-M) and intensity of uremia in male patients diagnosed with chronic renal failure (CRF).Materials and Methods: In total of 42 males were enrolled in a case-control study. There were 21 cases of CRF and 21 control cases. We collected 10cc of saliva plus 5 cc of blood from all patients to determine b2-M, blood urea nitrogen (BUN) and creatinine (Cr) levels.Results: There was a correlation between the level of serum BUN and salivary urea in controls and patients, which was statistically significant for controls (p=0.028).The correlation between serum and salivary Cr was 0.195 in controls (p=0.398) and 0.598 in patients (p=0.006), which was statistically significant in patients. The correlation between serum and saliva was 0.133 (p=0.566) in controls and 0.078 (p=0.737) in patients, which was not statistically significant. The correlation between serum BUN and b2-M was 0.168 (p=0.469) in the control group and 0.629 (p=0.002) in patients, which was statistically significant in patients. The correlation between serum Cr and b2-M was 0.110 (p=0.635) in the control group and 0.678 (p=0.001) in patients, which was statistically significant in patients. The correlation between serum BUN and salivary b2-M was 0.093 (p=0.0690) in controls and 0.152 (p=0.152) in patients, which was not statistically significant. The correlation between serum Cr and salivary b2-M was 0.072 (p=0.070) in the control group and 0.286 (p=0.209) in patients, which was not statistically significant in either group.Conclusion: The results of the study indicated that salivary b2-M cannot be used as a non-invasive indicator to detect the severity of renal failure.

Objective: Human basic fibroblast growth factor (bFGF) plays an important role in cellular proliferation, embryonic development, and angiogenesis as well as in several signaling pathways of various cell types. bFGF is an essential growth factor for the maintenance of undifferentiated human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs).Materials and Methods: In this experimental study, we present a straightforward method to produce biologically active recombinant human bFGF protein in E. coli that has long-term storage ability.Results: This procedure provides a rapid, cost effective purification of a soluble human bFGF protein that is biologically active and functional as measured in hESCs and hiPSCs in vitro and in vivo.Conclusion: The results show no significant difference in function between our in-house produced and commercialized bFGF.

Objective: Ionizing radiation interacts with biological systems to induce excessive fluxes of free radicals that attack various cellular components. Melatonin has been shown to be a direct free radical scavenger and indirect antioxidant via its stimulatory actions on the antioxidant system. The aim of this study was to evaluate the antioxidant role of melatonin against radiation-induced oxidative injury to the rat liver after whole body irradiation.Materials and Methods: In this experimental study, thirty-two rats were divided into four groups. Group 1 was the control group, group 2 only received melatonin (30 mg/kg on the first day and 30 mg/kg on the following days), group 3 only received whole body gamma irradiation of 10 Gy, and group 4 received 30 mg/kg melatonin 30 minutes prior to radiation plus whole body irradiation of 10 Gy plus 30 mg/kg melatonin daily through intraperitoneal (IP) injection for three days after irradiation. Three days after irradiation, all rats were sacrificed and their livers were excised to measure the biochemical parameters malondialdehyde (MDA) and glutathione (GSH). Each data point represents mean ±standard error on the mean (SEM) of at least eight animals per group. A one-way analysis of variance (ANOVA) was performed to compare different groups, followed by Tukey’s multiple comparison tests (p<0.05).Results: The results demonstrated that whole body irradiation induced liver tissue damage by increasing MDA levels and decreasing GSH levels. Hepatic MDA levels in irradiated rats that were treated with melatonin (30 mg/kg) were significantly decreased, while GSH levels were significantly increased, when compared to either of the control groups or the melatonin only group.Conclusion: The data suggest that administration of melatonin before and after irradiation may reduce liver damage caused by gamma irradiation.

Objective: Endothelial progenitor cells (EPCs) have a potential application for cell therapy, however, their biological nature is not well-understood. EPCs also possess some stemness features, such as their clonogenicity and differentiation capacity. The main aim of this study was to evaluate the expression of certain transcription factors regulating self-renewal property of stem cells.Materials and Methods: In this experimental study, peripheral blood mononuclear cells were isolated from fresh human blood of several volunteers and were cultured in fibronectin-coated plates. EPCs were identified based on their morphology and growth characteristic. Then, the expression of some markers implicated in self-renewal capacity was assessed in the isolated cells using reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry.Results: Expression of the cell surface markers, CD31 and CD34, was determined by RT-PCR and immunocytochemistry. Furthermore, these cells had the ability for Di-AC-LDL incorporation as well as attachment to lectin I. EPCs did not express the main stem cell markers, like OCT4-A, Nanog, and Sox2; nevertheless, they expressed the weaker pluripotent markers, including OCT4B and OCT4-B1 spliced variants, such as Nucleostemin and ZFX. Furthermore, the expression of Nucleostemin and ZFX genes revealed a decreasing pattern from days 4th to 11th.Conclusion: The main regulators of stem cell self-renewal genes, including OCT4-A, Nanog, and Sox2 are not expressed in EPCs. Forced expression of these genes can elevate the stemness property and clinical application of EPCs.

Objective: The aim of the present study was to evaluate three thawing rates on the post thaw motility, viability and chromatin structure of buffalo semen frozen in 0.5-ml straws.Materials and Methods: In this experimental study semen was collected with artificial vagina (42˚C) from four buffalo bulls.Split pooled ejaculates (n=4) were extended at 37˚C with a Bioxcell® extender. Semen was cooled to 4˚C within 2 hours, equilibrated at 4˚C for 4 hours, then filled in 0.5 ml French straws, and frozen in programmable cell freezer before plunging into liquid nitrogen.Straws were thawed at water bath temperatures of 37, 50 or 70˚C for 30, 15 and 6 seconds, respectively. Semen was incubated at 37˚C for 2 hours and evaluated for post thaw motility, viability, acrosomal and DNA integrity of spermatozoa.Analysis of variance (ANOVA) was used for comparisons of means. When the ANOVA test showed statistical differences, the mean of the treatments were compared using Duncan’s multiple range tests.Results: The initial post thaw motility (0 hour) averaged 62.7 ± 7.2%, 73.1 ± 9.77%, and 74.9 ± 8.58% for the three thaw rates, respectively. Kinematic parameters such as average path velocity, linearity and beat/cross frequency in the thaw rate of 70˚C for 6 seconds were superior to other rates studied (p<0.05). After 2 hours of incubation, proportions of progressive motility and Kinematic parameters decreased in all groups (p>0.05). A positive correlation was detected between sperm motility and thawing rate after two hours incubation times. The percentage of viable spermatozoa and spermatozoa with an intact acrosome and plasma membrane integrity were not different between the groups of samples thawed at different temperatures (p>0.05). The percentage of spermatozoa with chromatin dispersion forthe thaw rate of 70˚C for 6 seconds was significantly higher than for the to other rates studied (p<0.05). In contrast with motility and viability, the DNA integrity of post thaw spermatozoa remained unaffected during 2 hours incubation.Conclusion: The post thaw motility and kinematic parameters of buffalo spermatozoa were significantly improved immediately after thawing by increasing the thawing rate from 37˚C in 30 seconds to70˚C in 6 seconds. However, this relative advantage had disappeared after incubation in a water bath at 37˚C for two hours. A thaw rate of 70˚C for 6 seconds was associated with higher chromatin dispersion than the other thaw rates studied. Sperm thawing over at 50 degrees could be safely used to improve motility recovery after sperm cryopreservation in buffalo bulls.

Medicinal plants are presumed to be natural sources of antioxidants that protect organisms from oxidative stresses. The present investigation aims to study the anti-oxidative stress activity of the Stachys lavandulifolia (S. lavandulifolia) plant. This trial was conducted on 26 healthy human subjects. The study was done in a before after fashion. The included subjects were asked to consume the prepared infusion from 3 g aerial parts of S. lavandulifolia on a daily basis. Doses were administered in every morning and evening for 14 days. At the beginning and the end of the study, blood samples were acquired to determine the level of cellular lipid peroxidation and the total content of serum antioxidants. Biomarkers analyzed from samples obtained before start of treatment and 14 days post treatment, were subjected to paired t test analysis. Total blood antioxidants increased and reached from 2.3 ± 0.84 mmol/ml to 3.3 ±0.54 mmol/ml. The lipid peroxidation reduced and reached from 8.38 ± 1.78 to 11.6 ± 2.64 nmol/ml. The results suggest that S. lavandulifolia possesses marked anti-oxidative stress activity and it can be useful as a supplement in the management of diseases related to oxidative stress (Registration Number: IRCT2013012210003N2).