فهرست مطالب

Cell Journal - Volume:20 Issue: 1, 2018
  • Volume:20 Issue: 1, 2018
  • تاریخ انتشار: 1396/10/15
  • تعداد عناوین: 18
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  • Hajar Estiri, Ali Fallah, Masoud Soleimani, Abbas Aliaghaei, Fariba Karimzadeh, Shahnaz Babaei Abraki, Mohammad Hossein Ghahremani Pages 1-9
    Objective
    In this study, we describe an efficient approach for stable knockdown of adenosine kinase (ADK) using lentiviral system, in an astrocytoma cell line and in human Wharton’s jelly mesenchymal stem cells (hWJMSCs). These sources of stem cells besides having multilineage differentiation potential and immunomodulatory activities, are easily available in unlimited numbers, do not raise ethical concerns and are attractive for gene manipulation and cell-based gene therapy.
    Materials And Methods
    In this experimental study, we targeted adenosine kinase mRNA at 3' and performed coding sequences using eight miR-based expressing cassettes of anti-ADK short hairpin RNA (shRNAs). First, these cassettes with scrambled control sequences were cloned into expressing lentiviral pGIPZ vector. Quantitative real time-polymerase chain reaction (qRT-PCR) was used to screen multi-cassettes anti-ADK miR-shRNAs in stably transduced U-251 MG cell line and measuring ADK gene expression at mRNA level. Extracted WJMSCs were characterized using flow cytometry for expressing mesenchymal specific marker (CD44) and lack of expression of hematopoietic lineage marker (CD45-). Then, the lentiviral vector that expressed the most efficient anti-ADK miR-shRNA, was employed to stably transduce WJMSCs.
    Results
    Transfection of anti-ADK miR-shRNAs in HEK293T cells using CaPO4 method showed high efficiency. We successfully transduced U-251 cell line by recombinant lentiviruses and screened eight cassettes of anti-ADK miR- shRNAs in stably transduced U-251 MG cell line by qRT-PCR. RNAi-mediated down-regulation of ADK by lentiviral system indicated up to 95% down-regulation of ADK. Following lentiviral transduction of WJMSCs with anti-ADK miR- shRNA expression cassette, we also implicated, down-regulation of ADK up to 95% by qRT-PCR and confirmed it by western blot analysis at the protein level.
    Conclusion
    Our findings indicate efficient usage of shRNA cassette for ADK knockdown. Engineered WJMSCs with genome editing methods like CRISPR/cas9 or more safe viral systems such as adeno-associated vectors (AAV) might be an attractive source in cell-based gene therapy and may have therapeutic potential for epilepsy.
    Keywords: Adenosine Kinase, Gene Knockdown Techniques, Lentivirus, RNA Interference, Wharton's Jelly
  • Nasrin Khanmohammadi, Hamid Sameni, Moslem Mohammadi, Abbas Pakdel, Majid Mirmohammadkhani, Houman Parsaie, Sam Zarbakhsh Pages 10-18
    Objective
    Although stem cell transplantation has beneficial effects on tissue regeneration, but there are still problems such as high cost and safety issues. Since stem cell therapy is largely dependent on paracrine activity, in this study, utilization of transplantation of bone marrow stromal cells (BMSCs)-secretome instead of the cells, into damaged ovaries was evaluated to overcome the limitations of stem cell transplantation.
    Materials And Methods
    In this experimental study, BMSCs were cultured and 25-fold concentrated conditioned medium (CM) from BMSCs was prepared. Female rats were injected intraperitoneally with cyclophosphamide (CTX) for 14 days. Then, BMSCs and CM were individually transplanted into bilateral ovaries, and the ovaries were excised after four weeks of treatment. The follicle count was performed using hematoxylin and eosin (H&E) staining and the apoptotic cells were counted using TUNEL assay. Ovarian function was evaluated by monitoring the ability of ovulation and the levels of serum estradiol (E2) and follicle-stimulating hormone (FSH).
    Results
    Evaluation of the ovarian function and structure showed that results of secretome transplantation were almost similar to those of BMSCs transplantation and there was no significant differences between them.
    Conclusion
    BMSCs-secretome is likely responsible for the therapeutic paracrine effect of BMSCs. Stem cell- secretome is expected to overcome the limitations of stem cell transplantation and become the basis of a novel therapy for ovarian damage.
    Keywords: Bone Marrow Stromal Cells, Chemotherapy, Conditioned Medium, Ovary, Transplantation
  • Maryam Mohammadi, Karim Shamsasenjan, Parvin Akbarzadehlaleh Pages 19-24
    Objective
    Angiogenesis, the process of formation of new blood vessels, is essential for development of solid tumors. At first, it was first assumed that angiogenesis is not implicated in the development of acute myeloid leukemia (AML) as a liquid tumor. One of the most important elements in bone marrow microenvironment is mesenchymal stem cells (MSCs). These cells possess an intrinsic tropism for sites of tumor in various types of cancers and have an impact on solid tumors growth by affecting the angiogenic process. But so far, our knowledge is limited about MSCs’ role in liquid tumors angiogenesis. By increasing our knowledge about the role of MSCs on angiogenesis, new therapeutic strategies can be used to improve the status of patients with leukemia.
    Materials And Methods
    In this experimental study, HL-60, K562 and U937 cells were separately co-cultured with bone marrow derived-MSCs and after 8, 16 and 24 hours, alterations in the expression of 10 chemokine genes involved in angiogenesis, were evaluated by quantitative real time-polymerase chain reaction (qRT-PCR). Mono-cultures of leukemia cell lines were used as controls.
    Results
    We observed that in HL-60 and K562 cells co-cultured with MSCs, the expression of CXCL10 and CXCL3 genes are increased, respectively as compared to the control cells. Also, in U937 cells co-cultured with MSCs, the expression of CXCL6 gene was upgraded. Moreover in U937 cells, CCL2 gene expression in the first 16 hours was lower than the control cells, while within 24 hours its expression augmented.
    Conclusion
    Our observations, for the first time, demonstrated that bone marrow (BM)-MSCs are able to alter the expression profile of chemokine genes involved in angiogenesis, in acute myeloid leukemia cell lines. MSCs cause different effects on angiogenesis in different leukemia cell lines; in some cases, MSCs promote angiogenesis, and in others, inhibit it.
    Keywords: Acute Myeloid Leukemia, Angiogenesis, Chemokine, Mesenchymal Stem Cell
  • Ahad Khoshzaban, Peyman Keyhanvar, Elham Delrish, Farhood Najafi, Saeed Heidari, Ikuya Watanabe, Alireza Valanejad, Tahereh Jafarzadeh Kashi Pages 25-30
    Objective
    Alginate, known as a group of anionic polysaccharides extracted from seaweeds, has attracted the attention of researchers because of its biocompatibility and degradability properties. Alginate has shown beneficial effects on wound healing as it has similar function as extracellular matrix. Alginate microcapsules (AM) that are used in tissue engineering as well as Dulbecco’s modified Eagle’s medium (DMEM) contain nutrients required for cell viability. The purpose of this research was introducing AM in medium and nutrient reagent cells and making a comparison with control group cells that have been normally cultured in long term.
    Materials And Methods
    In this experimental study, AM were shaped in distilled water, it was dropped at 5 mL/hours through a flat 25G5/8 sterile needle into a crosslinking bath containing 0.1 M calcium chloride to produce calcium alginate microspheres. Then, the size of microcapsules (300-350 µm) were confirmed by Scanning Electron Microscopy (SEM) images after the filtration for selection of the best size. Next, DMEM was injected into AM. Afterward, adipose- derived mesenchymal stem cells (ADSCs) and Ringer’s serum were added. Then, MTT and DAPI assays were used for cell viability and nucleus staining, respectively. Also, morphology of microcapsules was determined under invert microscopy.
    Results
    Evaluation of the cells performed for spatial media/microcapsules at the volume of 40 µl, showed ADSCs after 1-day cell culture. Also, MTT assay results showed a significant difference in the viability of sustained-release media injected to microcapsules (P
    Conclusion
    According to the results, AM had a positive effect on cell viability in scaffolds and tissue engineering and provide nutrients needed in cell therapy.
    Keywords: Alginate, Cell Culture, Cell Viability, Growth Media, Microcapsule
  • Naeem Erfani Majd, Mohammad Reza Tabandeh, Ali Shahriari, Zahra Soliemani Pages 31-40
    Objective
    Okra (Abelmoschus esculentus) is a tropical vegetable that is rich in carbohydrates, fibers, proteins and natural antioxidants. The aim of the present study was to evaluate the effects of Okra powder on pancreatic islets and its action on the expression of PPAR-γ and PPAR-α genes in pancreas of high-fat diet (HFD) and streptozotocin- induced diabetic rats.
    Materials And Methods
    In this experimental study, diabetes was induced by feeding HFD (60% fat) for 30 days followed by an injection of streptozotocin (STZ, 35 mg/kg). Okra powder (200 mg/kg) was given orally for 30 days after diabetes induction. At the end of the experiment, pancreas tissues were removed and stained by haematoxylin and Eozine and aldehyde fuchsin for determination of the number of β-cells in pancreatic islets. Fasting blood sugar (FBS), Triglycerides (TG), cholesterol, high density lipoprotein (HDL), low density lipoprotein (LDL), and insulin levels were measured in serum. Moreover, PPAR-γ and PPAR-α mRNAs expression were measured in pancreas using real time polymerase chain reaction (PCR) analysis.
    Results
    Okra supplementation significantly decreased the elevated levels of FBS, total cholesterol, and TG and attenuated homeostasis model assessment of basal insulin resistance (HOMA-IR) index in diabetic rats. The expression levels of PPAR-γ and PPAR-α genes that were elevated in diabetic rats, attenuated in okra-treated rats (P
    Conclusion
    Our findings confirmed the potential anti-hyperglycemic and hypolipidemic effects of Okra. These changes were associated with reduced pancreatic tissue damage. Down-regulation of PPARs genes in the pancreas of diabetic rats after treatment with okra, demonstrates that okra may improve glucose homeostasis and β-cells impairment in diabetes through a PPAR-dependent mechanism.
    Keywords: Diabetes, Okra, Pancreas, PPARs, Rat
  • Esameil Rahimi, Amirhossain Ahmadi, Mohammad Ali Boroumand, Bahram Mohammad Soltani, Mehrdad Behmanesh Pages 41-45
    Objective
    ANRIL is an important antisense noncoding RNA gene in the INK4 locus (9p21.3), a hot spot region associated with multiple disorders including coronary artery disease (CAD), type 2 diabetes mellitus (T2DM) and many different types of cancer. It has been shown that its expression is dysregulated in a variety of immune-mediated diseases. CAD is a major problem in T2DM patients and the cause of almost 60% of deaths in these patients worldwide. The aim of the present study was to compare the expression level of ANRIL between T2DM patients with and without CAD.
    Materials And Methods
    In this case-control study, we examined ANRIL expression in peripheral blood mononuclear cell samples by quantitative reverse transcriptionpolymerase chain reaction (RT-qPCR) in 64 T2DM patients with and without CAD (33 CAD and 31 CADpatients respectively, established by coronary angiography).
    Results
    Expression analysis revealed that ANRIL was up regulated (2.34-Fold, P=0.012) in CAD versus CAD diabetic patients. Data from receiver operating characteristic (ROC) curve analysis has shown that ANRIL could act as a potential biomarker for detecting CAD in diabetic patients.
    Conclusion
    The expression level of ANRIL is associated with presence of CAD in diabetic patients and could be considered as a potential peripheral biomarker.
    Keywords: ANRIL, Coronary Artery Disease, Gene Expression, Noncoding RNA, Type 2 Diabetes Mellitus
  • Shahnaz Razavi, Nazem Ghasemi, Mohammad Mardani, Hossein Salehi Pages 46-52
    Objective
    The presence of neurotrophic factors is critical for regeneration of neural lesions. Here, we transplanted combination of neurotrophic factor secreting cells (NTF-SCs) and human adipose derived stem cells (hADSCs) into a lysolecithin model of multiple sclerosis (MS) and determined the myelinization efficiency of these cells.
    Materials And Methods
    In this experimental study, 50 adult rats were randomly divided into five groups: control, lysolecithin, vehicle, hADSCs transplantation and NTF-SCs/ hADSCs co-transplantation group. Focal demyelization was induced by lysolecithin injection into the spinal cord. In order to assess motor functions, all rats were scored weekly with a standard experimental autoimmune encephalomyelitis scoring scale before and after cell transplantation. Four weeks after cell transplantation, the extent of demyelination and remyelination were examined with Luxol Fast Blue (LFB) staining. Also, immunofluorescence method was used for evaluation of oligodendrocyte differentiation markers including; myelin basic protein (MBP) and Olig2 in the lesion area.
    Results
    Histological study show somewhat remyelinzation in cell transplantation groups related to others. In addition, the immunofluorescence results indicated that the MBP and Olig2 positive labeled cells were significantly higher in co-cell transplantation group than hADSCs group (P
    Conclusion
    Our results indicated that the remyelinization process in co-cell transplantation group was better than other groups. Thus, NTF-SCs/ hADSCs transplantation can be proper candidate for cell based therapy in neurodegenerative diseases, such as MS.
    Keywords: Adult Stem Cells, Lysophosphatidylcholines, Multiple Sclerosis, Myelinization, Nerve Growth Factors
  • Tae Woo Kim, Myung Chul Lee, Hyun Cheol Bae, Hyuk Soo Han Pages 53-60
    Objective
    Coculture of chondrocytes and mesenchymal stem cells (MSCs) has been developed as a strategy to overcome the dedifferentiation of chondrocytes during in vitro expansion in autologous chondrocyte transplantation. Synovium-derived stem cells (SDSCs) can be a promising cell source for coculture due to their superior chondrogenic potential compared to other MSCs and easy accessibility without donor site morbidity. However, studies on coculture of chondrocytes and SDSCs are very limited. The aim of this study was to investigate whether direct coculture of human chondrocytes and SDSCs could enhance chondrogenesis compared to monoculture of each cell.
    Materials And Methods
    In this experimental study, passage 2 chondrocytes and SDSCs were directly cocultured using different ratios of chondrocytes to SDSCs (3:1, 1:1, or 1:3). glycosaminoglycan (GAG) synthetic activity was assessed using GAG assays and Safranin-O staining. Expression of chondrogenesis-related genes (collagen types I, II, X, Aggrecan, and Sox-9) were analyzed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry staining.
    Results
    GAG/DNA ratios in 1:1 and 1:3 coculture groups were significantly increased compared to those in the chondrocyte and SDSC monoculture groups. Type II collagen and SOX-9 were significantly upregulated in the 1:1 coculture group compared to those in the chondrocyte and SDSC monoculture groups. On the other hand, osteogenic marker (type I collagen) and hypertrophic marker (type X collagen) were significantly downregulated in the coculture groups compared to those in the SDSC monoculture group.
    Conclusion
    Direct coculture of human chondrocytes and SDSCs significantly enhanced chondrogenic potential, especially at a ratio of 1:1, compared to chondrocyte or SDSC monocultures.
    Keywords: Chondrocyte, Chondrogenesis, Coculture, Mesenchymal Stem Cells, Synovium
  • Sharif Moradi, Thomas Braun, Hossein Baharvand Pages 61-72
    Objective
    Embryonic stem cells (ESCs) are regulated by a gene regulatory circuitry composed of transcription factors, signaling pathways, metabolic mediators, and non-coding RNAs (ncRNAs). MicroRNAs (miRNAs) are short ncRNAs which play crucial roles in ESCs. Here, we explored the impact of miR-302b-3p on ESC self-renewal in the absence of leukemia inhibitory factor (LIF).
    Materials And Methods
    In this experimental study, ESCs were cultured in the presence of 15% fetal bovine serum (FBS) and induced to differentiate by LIF removal. miR-302b-3p overexpression was performed by transient transfection of mature miRNA mimics. Cell cycle profiling was done using propidium iodide (PI) staining followed by flow cytometry. miRNA expression was quantified using a miR-302b-3p-specific TaqMan assay. Data were analyzed using t test, and a P
    Results
    We observed that miR-302b-3p promoted the viability of both wild-type and LIF-withdrawn ESCs. It also increased ESC clonogenicity and alkaline phosphatase (AP) activity. The defective cell cycling of LIF-deprived ESCs was completely rescued by miR-302b-3p delivery. Moreover, miR-302b-3p inhibited the increased cell death rate induced by LIF removal.
    Conclusion
    miR-302b-3p, as a pluripotency-associated miRNA, promotes diverse features of ESC self-renewal in the absence of extrinsic LIF signals.
    Keywords: Differentiation, Embryonic Stem Cells, MicroRNA, miR-302, Self-Renewal
  • Fahimeh Mollaahmadi, Ashraf Moini, Reza Salmanyazdi, Mehrdad Behmanesh Pages 73-77
    Objective
    Infertility is a common human disorder which is defined as the failure to conceive for a period of 12 months without contraception. Many studies have shown that the outcome of fertility could be affected by DNA damage. We attempted to examine the association of two SNPs (rs1127354 and rs7270101) in ITPA, a gene encoding a key factor in the repair system, with susceptibility to infertility.
    Materials And Methods
    This was a case-control study of individuals with established infertility. Blood samples were obtained from 164 infertile patients and 180 ethnically matched fertile controls. Total genomic DNA were extracted from whole blood using the standard salting out method, and stored at -20˚C. Genotyping were based on mismatch polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method in which PCR products were digested with the XmnI restriction enzyme and run on a 12% polyacrylamide gel.
    Results
    All genotype frequencies in the control group were in Hardy-Weinberg equilibrium. A significant association (in allelic, recessive and dominant genotypic models) was observed between infertile patients and healthy controls based on rs1127354 (P=0.0001), however, no significant association was detected for rs7270101. Also, gender stratification and analysis of different genotype models did not lead to a significant association for this single- nucleotide polymorphis (SNP).
    Conclusion
    ITPA is likely to be a genetic determinant for decreased fertility. To the best of our knowledge, this is the first report demonstrating this association, however, given the small sample size and other limitations, genotyping of this SNP is recommended to be carried out in different populations with more samples.
    Keywords: Infertility, ITPA, Genotyping, Single Nucleotide Polymorphism
  • Hossein Eslami, Ali Eslami, Raha Favaedi, Ummolbanin Asadpour, Shabnam Zari Moradi, Poopak Eftekhari-Yazdi, Tahereh Madani, Maryam Shahhoseini, Anahita Mohseni Meybodi Pages 78-83
    Objective
    The diminished ovarian reserve (DOR) is a condition characterized by a reduction in the number and/or quality of oocytes. This primary infertility disorder is usually accompanied with an increase in the follicle-stimulating hormone (FSH) levels and regular menses. Although there are many factors contributing to the DOR situation, it is likely that many of idiopathic cases have genetic/epigenetic bases. The association between the FMR1 premutation (50-200 CGG repeats) and the premature ovarian failure (POF) suggests that epigenetic disorders of FMR1 can act as a risk factor for the DOR as well. The aim of this study was to analyze the mRNA expression and epigenetic alteration (histone acetylation/methylation) of the FMR1 gene in blood and granulosa cells of 20 infertile women.
    Materials And Methods
    In this case-control study, we analyzed the mRNA expression and epigenetic altration of the FMR1 gene in blood and granulosa cells of 20 infertile women. These women were referred to the Royan Institute, having been clinically diagnosed as DOR patients. Our control group consisted of 20 women with normal antral follicle numbers and serum FSH level. All these women had normal karyotype and no history of genetic disorders. The number of CGG triplet repeats in the exon 1 of the FMR1 gene was analyzed in all samples.
    Results
    Results clearly demonstrated significantly higher expression of the FMR1 gene in blood and granulosa cells of the DOR patients with the FMR1 premutation compared to the control group. In addition, epigenetic marks of histone 3 lysine 9 acetylation (H3K9ac) and di-metylation (H3K9me2) showed significantly higher incorporations in the regulatory regions of the FMR1 gene, including the promoter and the exon 1, whereas tri-metylation (H3K9me3) mark showed no significant difference between two groups.
    Conclusion
    Our data demonstrates, for the first time, the dynamicity of gene expression and histone modification pattern in regulation of FMR1 gene, and implies the key role played by epigenetics in the development of the ovarian function.
    Keywords: Epigenetic, FMR1 Gene, Histone Modification
  • Oranous Bashti, Mehrdad Noruzinia, Masoud Garshasbi, Morteza Abtahi Pages 84-89
    Objective
    Endometriosis is a prevalent gynecologic disease affecting 10% of women in reproductive age. Endometriosis is diagnosed by laparoscopy that was followed by histologic confirmation. Early diagnosis will lead to a more effective treatment with much less morbidity. As miR-31 and miR-145 are shown to be directly or indirectly correlated to biological processes involved in endometriosis, the aim of this study was to examine the association of miR-31 and miR-145 expression in plasma with the presence of endometriosis.
    Materials And Methods
    In this case control study, the plasma samples of 55 patients with endometriosis and 23 women without endometriosis were collected, extracted and analyzed by real time quantitative polymerase chain reaction (qPCR) for the expression of miR-145 and miR-31.
    Results
    Our findings showed that miR-31 expression levels in stage 3 or 4 and stage 1 or 2 were significantly down- regulated (less than 0.01-fold, P
    Conclusion
    Different cellular biological processes, such as differentiation, proliferation, mitochondrial function, reactive oxygen species (ROS) production, invasion and decidualization, are deregulated in endometriosis. miR-31 and miR-145 are microRNAs (miRNAs) with potential roles, as shown in pathologies like cancers. We found that miR- 31 was under-expressed in patients with endometriosis, while miR-145 was over-expressed in stage 1 or 2, indicating that they were relatively down-regulated in the more severe forms. Our findings suggested that these two miRNAs may be considered as potential biomarkers with probable implications in early diagnosis and even follow-up of patients with endometriosis.
    Keywords: Biomarker, Endometriosis, miR-145, miR-31, miRNA
  • Parvin Dorfeshan, Marefat Ghaffari Novin, Mohammad Salehi, Reza Masteri Farahani, Fatemeh Fadaei Fathabadi, Ronak Sehatti Pages 90-97
    Objective
    In vitro maturation technique (IVM) is shown to have an effect on full maturation of immature oocytes and the subsequent embryo development. Embryonic genome activation (EGA) is considered as a crucial and the first process after fertilization. EGA failure leads to embryo arrest and possible implantation failure. This study aimed to determine the role of IVM in EGA-related genes expression in human embryo originated from immature oocytes and recovered from women receiving gonadotrophin treatment for assisted reproduction.
    Materials And Methods
    In this experimental study, germinal vesicle (GV) oocytes were cultured in vitro. After intracytoplasmic sperm injection of the oocytes, fertilization, cleavage and embryo quality score were assessed in vitro and in vivo. After 3-4 days, a single blastomere was biopsied from the embryos and then frozen. Afterwards, the expression of EGA-related genes in embryos was assayed using quantitative reverse transcriptase-polymerase chain reaction (PCR).
    Results
    The in vitro study showed reduced quality of embryos. No significant difference was found between embryo quality scores for the two groups (P=0.754). The in vitro group exhibited a relatively reduced expression of the EGA- related genes, when compared to the in vivo group (all of them showed P=0.0001).
    Conclusion
    Although displaying the normal morphology, the IVM process appeared to have a negative influence on developmental gene expression levels of human preimplanted embryos. Based on our results, the embryo normal morphology cannot be considered as an ideal scale for the successful growth of embryo at implantation and downstream processes.
    Keywords: Embryonic Development, Intracytoplasmic Sperm Injection, In Vitro Maturation, Ovarian Stimulation
  • Mohammad Hadi Sekhavati, Sayed Morteza Hosseini, Mojtaba Tahmoorespur, Kamran Ghaedi, Farnoosh Jafarpour, Mehdi Hajian, Kyanoosh Dormiani, Mohammad Hossein Nasr Esfahani Pages 98-107
    Objective
    The Streptomyces phage phiC31 integrase offers a sequence-specific method of transgenesis with a robust long-term gene expression. PhiC31 has been successfully developed in a variety of tissues and organs for purpose of in vivo gene therapy. The objective of the present experiment was to evaluate PhiC31-based site-specific transgenesis system for production of transgenic bovine embryos by somatic cell nuclear transfer and intracytoplasmic sperm injection.
    Materials And Methods
    In this experimental study, the application of phiC31 integrase system was evaluated for generating transgenic bovine embryos by somatic cell nuclear transfer (SCNT) and sperm mediated gene transfer (SMGT) approaches.
    Results
    PhiC31 integrase mRNA and protein was produced in vitro and their functionality was confirmed. Seven phiC31 recognizable bovine pseudo attachment sites of phage (attP) sites were considered for evaluation of site specific recombination. The accuracy of these sites was validated in phic31 targeted bovine fibroblasts using polymerase chain reaction (PCR) and sequencing. The efficiency and site-specificity of phiC31 integrase system was also confirmed in generated transgenic bovine embryo which successfully obtained using SCNT and SMGT technique.
    Conclusion
    The results showed that both SMGT and SCNT-derived embryos were enhanced green fluorescent protein (EGFP) positive and phiC31 integrase could recombine the reporter gene in a site specific manner. These results demonstrate that attP site can be used as a proper location to conduct site directed transgenesis in both mammalian cells and embryos in phiC31 integrase system when even combinaed to SCNT and intracytoplasmic sperm injection (ICSI) method.
    Keywords: Intracytoplasmic Sperm Injection, Somatic Cell Nuclear Transfer, Transgenesis
  • Maryam Ezzati, Leila Roshangar, Jafar Soleimani, Nahid Karimian Pages 108-112
    Objective
    Infertility is a worldwide health problem which affects approximately 15% of sexually active couples. One of the factors influencing the fertility is melatonin. Also, protection of oocytes and embryos from oxidative stress inducing chemicals in the culture medium is important. The aim of the present study was to investigate if melatonin could regulate hyaluronan synthase-2 (HAS2 ) and Progesterone receptor (PGR ) expressions in the cumulus cells of mice oocytes and provide an in vitro fertilization (IVF) approach.
    Materials And Methods
    In this experimental study, for this purpose, 30 adult female mice and 15 adult male mice were used. The female mice were superovulated using 10 U of pregnant mare serum gonadotropin (PMSG) and 24 hours later, 10 U of human chorionic gonadotropin (hCG) were injected. Next, cumulus oocyte complexes (COCs) were collected from the oviducts of the female mice by using a matrix-flushing method. The cumulus cells were cultured with melatonin 10 µM for 6 hours and for real-time reverse transcription-polymerase chain reaction (RT-PCR) was used for evaluation of HAS2 and PGR expression levels. The fertilization rate was evaluated through IVF. All the data were analyzed using a t test.
    Results
    The results of this study showed that HAS2 and PGR expressions in the cumulus cells of the mice receiving melatonin increased in comparison to the control groups. Also, IVF results revealed an enhancement in fertilization rate in the experimental groups compared to the control groups.
    Conclusion
    To improve the oocyte quality and provide new approaches for infertility treatment, administration of melatonin as an antioxidant, showed promising results. Thus, it is concluded that fertility outcomes can be improved by melatonin it enhances PGR .
    Keywords: Hyaluronan Synthase-2, Melatonin, Mouse Oocyte, Progesterone Receptor
  • Mahsa Rezaeepoor, Mazdak Ganjalikhani-Hakemi, Shima Shapoori, Nahid Eskandari, Mohammadreza Sharifi, Masoud Etemadifar, Marjan Mansourian Pages 113-119
    Objective
    Semaphorin-3A (SEMA3A) and its receptors are found on some immune cells and act as suppressors of immune cells over-activation. Considering the role of SEMA3A and its down-regulation in some autoimmune diseases, as well as our bioinformatics predictions, we assumed that miR-145-5p might affect SEMA3A expression. So, we aimed to determine the effect of miR-145-5p on SEMA3A gene expression level.
    Materials And Methods
    In this experimental study, we evaluated the effect of miR-145-5p transfection on SEMA3A expression in peripheral blood mononuclear cells (PBMCs) using ELISA and quantitative real-time polymerase chain reaction (PCR) methods.
    Results
    Our results showed that miR-145-5p is able to decrease SEMA3A expression at both protein and mRNA levels. These data confirmed our previous bioinformatic prediction about the inhibitory effect of miR-145-5p on SEMA3A expression.
    Conclusion
    These results enlightened us about an unknown aspect of SEMA3A role in some autoimmune disorders like multiple sclerosis (MS) and rheumatoid arthritis (RA) and also proposed SEMA3A as a potential therapeutic approach.
    Keywords: microRNAs, miR-145-5p, Semaphorin-3A
  • Morteza Yaftian, Fatemeh Yari, Mehran Ghasemzadeh, Vahid Fallah Azad, Mansoureh Haghighi Pages 120-126
    Objective
    The in vitro treatment of tumor cells with platelet (Plt) causes inhibition of tumor cell growth, although mechanism of this effect is not clear yet. Induction of apoptosis has been proposed as a mechanism of Plt effects on tumor cells. The purpose of this study was to clarify the role of Plts and Plt-derived components in the induction of apoptosis in the blood mononuclear cells of patients with leukemia.
    Materials And Methods
    In this experimental study, peripheral blood mononuclear cells (PBMCs) were isolated from whole blood of five patients with childhood B-precursor acute lymphoblastic leukemia (pre-B ALL) and encountered with Plts, Plt-derived microparticles (Plt-MPs) as well as purified soluble CD40L (sCD40L). After 48 hours of co-culture, the anti-cancer activity of the aforementioned factors was surveyed using examination of apoptosis markers of the cells including active caspase-3 and CD95 using ELISA and flow cytometer techniques, respectively. Additionally, staining of the cells with 7-Aminoactinomycin D (7-AAD) was evaluated by flow cytometer technique. Trypan blue exclusion test and WST-1 method were also used to compare the death/survival status of the cells.
    Results
    Levels of CD95 and caspase-3 were significantly increased in the all treated groups (P
    Conclusion
    This study can show the ability of Plts, Plt-MPs and sCD40L for the induction of apoptosis in PBMCs of pre-B-ALL patients. Further studies are necessary to elucidate the different effects of platelets on cancer cells in vitro and in vivo.
    Keywords: Acute Lymphoblastic Leukemia, Apoptosis, CD40 Ligand, Microparticles, Platelet
  • Farah Talebi, Farideh Ghanbari Mardasi, Javad Mohammadi Asl, Saeed Tizno, Marziyeh Najafvand Zadeh Pages 127-131
    Autosomal recessive non-syndromic hearing loss (ARNSHL) is defined as a genetically heterogeneous disorder. The aim of the present study was to screen for pathogenic variants in an Iranian pedigree with ARNSHL. Next-generation targeted sequencing of 127 deafness genes in the proband detected two novel variants, a homozygous missense variant in PTPRQ (c.2599 T>C, p.Ser867Pro and a heterozygous missense variant in MYO1A (c.2804 T>C, p.Ile935Thr), both of which were absent in unaffected sibs and two hundred unaffected controls. Our results suggest that the homozygous PTPRQ variant maybe the pathogenic variant for ARNSHL due to the recessive nature of the disorder. Nevertheless, the heterozygous MYO1A may also be involved in this disorder due to the multigenic pattern of ARNSHL. Our data extend the mutation spectrum of PTPRQ and MYO1A, and have important implications for genetic counseling in unaffected sibs of this family. In addition, PTPRQ and MYO1A pathogenic variants have not to date been reported in the Iranian population.
    Keywords: Hearing Loss, MYO1A, Novel Variant, PTPRQ