Cell Journal (Yakhteh)
Year:2007 | Volume:9 | Issue:1 (33)|Papers Count:12

Introduction: Monitoring the engraftment of donor cell after allogeneic blood stem cell transplantation is important for the early diagnosis of graft failure or relapse of disease. So Analysis of donor chimerism has become a routine method for documentation of engraftment after allogeneic stem cell transplantation. In humans, the amelogenin gene is present on both the X and the Y chromosomes. However, there are size differences in this gene between these chromosomes, which have been utilized for sexing in forensic casework and prenatal identification. The aim of the present study was to evaluate the application of the amelogenin gene for assessment of chimerism in PBL and/or BM samples of patients who received sex-mismatched BMT.Material and Methods: PCR techniques using a set of amelogenin gene primer were preformed on whole WBC and/or BMA samples, and in some cases on isolated T-cell and granulocytes subsets of recipient patients. We have investigated 30 allogeneic stem cell transplantation suffering from different types of leukemia (n=18) or non-malignant hematologic disorders (n=12) by close molecular monitoring during 1-12 months after transplantation.Results: The PCR amplification of this gene resulted in 106bp and 112bp amplicons from the X- and Y-chromosome respectively. The ratio of X/Y fragments was reported as the mixed chimerism. The sensitivity of the test was as low as 1-2%.Conclusion: The advantage of this approach is the use of a single set of primer to amplify both X and Y chromosomes. The PCR-based assay was extremely sensitive and did not need donor and recipient samples for the assessment of chimerism. The test can be used routinely, alone or in conjunction with STR-PCR, to analyze BM and PBL of post allogenic sex- mismatched BMT to detect early engraftment or rejection and for observing kinetics of engraftment.

Introduction: The purpose of this study was to evaluate the effect of different doses of retinoic acid on resumption of meiosis, in vitro maturation of oocytes and resulting embryo development in mice.Materials and Methods: Germinal vesicle of NMRI female mouse (6-8 weeks old) oocytes were collected from ovaries and cultured in maturation medium MEM-a contained: 100 mlIU/ml rFSH+7.5 IU/ml hCG+5% FCS (Control group). All-trans retinoic acid (t-RA) at concentrations of 0.25, 0.5, 1, 2mM and ethanol alone (Sham group) 0.2% (v/v), were added to oocytes maturation medium. After 24 hours the matured oocytes were inseminated with spermatozoa in T6 medium and cultured for 5 days.Results: The difference between our experimental and control groups in resumption of meiosis increased by t-RA dose from 0.25mM (p<0.01) to 0.5mM (p<0.001), 1mM (p<0.001) and 2mM (p<0.0001), but higher doses like 5mM and 10mM reduced it. The rate of oocytes developing to the Metaphase II stage of maturation was significantly increased with t-RA doses of 1mM (p<0.01) and 2mM (p<0.0001). The rate of embryos reaching to 2-cell after 24 hours was significantly increased with 1 (p<0.05) and 2mM t-RA (p<0.01). The rate of embryo developing to blastocyst stage was significantly increased in medium containing 2mM retinoic acid (p<0.01).Conclusion: All- trance retinoic acid enhanced mouse oocytes maturation in vitro and improved embryo development in a dose dependent manner.

Introduction: There are the evidences to suggest that bone marrow stromal cells (BMSCs) not only differentiate into mesodermal cells, but also adopt the fate of endodermal and ectodermal cell types. BMSCs can be a valuable cell source as an autograph for clinical application involving regeneration of the central nervous system. Bone marrow stromal cells can be expanded rapidly in vitro and have the potential with appropriate treatment, can be differentiated into neuronal, glial-like cell types. The study of induction of deprenyl on trans differentiation of BMSCs into neuron and glial-like cells in vitro.Materials and Methods: In the study, bone marrow was extracted from the femur and tibia of adult rat, and then bone marrow stromal cells with 5 passages were proliferated and cultured. Bone marrow stromal cells were differentiated using deprenyl 10-8M to neuronal-and glial-like cells and were assessed with immunocytochemical method. Before and after neuronal induction with deprenyl, were assessed by anti neurofilament 68 (KD), 200 (KD), GFAP and Oligo antibodies.Results: Differentiation of the cells that induced by deprenyl were positive reaction for neuronal markers. For determination of the percentage of the neuron and glial-like cells, were counted by immunocyochemical method using anti NF 200 and anti GFAP antibodies. Results showed that 82.71% neuronal-like cells and 25.99% astrocyte-like cells of the cells were positive reaction. Conclusion: Hence we concluded that bone marrow stromal cells can be differentiated into neuronal-and glial-like cells with deprenyl 10-8M as inducer in vitro.

Introduction: The present study was undertaken to produce alkaline phosphatase anti- alkaline phosphatase (APAAP) complex and to use it in one of the most practical methods of determining the site of antigen in tissue or in cell, i.e. APAAP technique, and its comparison with similar foreign products. Materials and Methods: In this basic and applied study the secreted antibodies of the two hybridomas A1 G9 G3 and A1 G8 F7 produced in tarbiat Modarres university were concentrated and purified and their affinities were determined. Then the complexes  of monoclonal antibody against alkaline phosphatase and alkaline phosphatase enzyme (APAAP complex) were prepared in their optimal concentration and used in immunocytochemistry and immunohistochemistry staining. The results were compared with similar foreign sample (DAKO).Results: Both cell colonies had saved the potency of producing anti-alkaline phosphatase monoclonal antibody with high affinity. The produced APAAP complex was very effective and its sensitivity and specificity were comparable to the similar foreign kit (i.e. DAKO).Conclusion: The high affinity of the monoclonal antibodies of the two hybridomas and also the stability of the resulted complex from mixing monoclonal antibodies with the enzyme make it possible to use these two cell colonies to produce trading amounts of APPAP. The produced APAAP complex could be used in immunocytochemistry and immunohistochemistry staining.

Introduction: An investigation of bone, cartilage and fat differentiation potentials of canine bone marrow-derived mesenchymal stem cells. Materials and methods: Bone marrow aspirates were obtained from 10 canine’s iliac crest under general anesthesia. Each sample was separately loaded on lymphodex and centrifuged at 1200 rpm for ten minutes to obtain mononuclear cell fraction. 5×104 cell/cm2 of mononuclear cells was cultured in 150 cm2-flask in a DMEM medium containing 15% fetal bovine serum. Fibroblastic cells were then purified and expanded through several subcultures. Passaged-3 cells were differentiated into osteogenic, chondrogenic and adipogenic lineages for a period of 3 weeks. The differentiated cells were evaluated by histochemistry and RT-PCR analysis.Results: Passaged-3 fibroblastic cells cultured in osteogenic medium were stained red with Alizarin red method, indicating that the matrix was mineralized. The cell also produced a large amount of bone specific markers including collagen I and osteopontine. In the adipogenic cultures, lipid droplets were stained red by oil red method. Furthermore, RT-PCR analysis indicated that these cells produced a large amount of adiopocyte specific genes including lipoprotein lipase and PPARG2. Sections from chondrogenic cultures turned into purple upon safranin O staining that was indicative of significant amount of glycosaminoglycans in the cultures. In addition, RT-PCR analysis revealed that the mRNA of Decorin and collagen II as cartilage specific genes were produced in these cells but aggreacan was not expressed. Conclusion: Canine bone marrow fibroblastic cells could easily be differentiated into bone and fat but in chondrogenic differentiation, while glycoseaminoglycan-rich matrix containing collagen II and Decorin was produced, the agreacan gene was not expressed.

Introduction: Promoter methylation has an inhibitory effect on gene expression. Thus we investigated methylation patterns of p15 gene in Esophageal Squamous Cell Carcinoma (ESCC), one of the most frequent and fetal cancers in the world. Materials and Methods: p15 promoter methylation status of 30 ESCC and 10 normal specimens was investigated using methylation specific PCR. DNA extracted from the specimens was digested by restriction enzyme. The digested DNA was modified by sodium bisulfite. Using methylated and unmethylated specific primers, methylation status was determined based on the presence of the methylated and unmethylated specific PCR products. Also DNA extracted from normal blood and CpG methylase (SssI)–treated genomic DNA were used as the negative and positive controls respectively.Results: Our results showed that all normal specimens were unmethyalted and only 5 out of 30 tumor samples (16.6%) were methylated. Statistical analysis (Fisher’s exact tests) showed no significant correlation between p15 gene promoter methylation and ESCC cancer.Conclusion: According to the results of the study, p15 gene does not play a significant role in aberrant methylation process in ESCC, other mechanisms may involve in p15 inactivation during ESCC development. However, due to limited number of specimens, we recommend further investigation using a larger sample.

Introduction: Duchene/ Becker (DMD/BMD) muscular dystrophy is the most frequent neuromuscular disease in children which is inherited as an X-Linked recessive trait. The disease is caused by partial deletion in dystrophin gene. We developed a rapid and robust method for direct identification of female carriers of deletions and duplications in the dystrophin gene, in order to prevent the affected newborn males to be born.Materials and Methods: Qantitative real-time PCR assay for deletion/duplication within two major hot spots in dystrophin gene (exon 6 and 47) was developed and an X-linked gene was considered as a normalizer (i.e., a fragment of human coagulation factor IX gene).The quantitative Real-time PCR test was conducted using comparative threshold method (DDCT).Results: The results showed 2-DDCT of 1.09±0.21 for normal individuals and 0.51±0.1 for carrier samples. Carrier status was accurately attributed in 100% of cases.Conclusion: This method is simple, rapid, reliable and cost-effective. It may be applied for direct determination of deletion/duplication in potential DMD/BMD carriers and may easily be adapted for other genetic conditions involving deletions and duplications.

Differentiation of stem cells from a pluripotent to a committed state involves global changes in genome expression patterns, the best known as “epigenetic modifications”. Epigenetic mechanisms, including histone modifications, DNA methylation and non-coding RNA-mediated regulatory events are considered as the essential factors to control the heritable cellular memory of gene expression during development. With complete genome sequence in hand, understanding the epigenetic control of genome is the next step towards comprehending how the same DNA sequence gives rise to different cells and lineages, the phenomenon referred to as cell fate specification of stem cells. Here we discuss the epigenetic mechanisms which serve as the master regulators of cellular destiny through development.