Cell Journal (Yakhteh)
Year:2003 | Volume:5 | Issue:19|Papers Count:10

Introduction: Production of Monoclonal antibody against Alkaline phosphatase for application in immunohistochemical and immunocytochemical techniques such as alkaline phosphatase anti-alkaline phosphatase (APAAP) method.   Material and Methods: In this investigation female Balb/c mice were immunized by several injections of alkaline phosphatase and the antibody titer in their sera were measured after each injection. The spleen lymphocytes of immunized mice and Sp2/0 myeloma cells were fused using 50% polyethylen glycol as fusing agent and hybridoma cells were selected by HAT medium. Identification and selection of anti-alkaline phosphatase producing clones were done by performing ELISA test on supernatants of all the resulting clones. Limiting dilution was performed twice on antiboby producing clones for their seperation and the resulted subclones were propagated. Since APAAP complex must be enzymatically active for using in immunohistochemical techniques the adhesion of Ab molecule to enzyme molecule must not affect the enzyme activity. For investigation of this effect, an ELISA technique was planned and the supernatants of selected hybridoma clones were studied by this method. For production of concentrated Ab the hybridoma cells were injected to peritoneal cavity of mice and the ascetic fluids were obtained. Finally the antibodies isotypes were determined.   Results: After 6 fusion experiments 104 hybridoma clones were abtained and two clones (A1G9 and A1G8) which were antibody producing and had the highest absorbance in ELISA test were selected. Using the limiting dilution method finally two monoclonal subclones A1G8F7 and A1G9G3 were selected. ELISA experiments showed that antibodies which were produced by selected hybridoma clones did not react with active site of the enzyme and did not interfer with enzymatic activity. Electrophoresis of ascetic fluids of hybridoma injected mice showed a prominent band in γ position. Isotype determination of monoclonal antibodies showed that both hybridoma clones produce antibody from IgG class with k light chain.   Conclusion: Because monoclonal antibodies which are produced by the obtained hybridoma cell lines are from IgG class and do not affect the enzyme activity, it seem's that they are suitable for APAAP complex formation. Other steps of this study are now being performed until APAAP complex formation and it's application in immunohistochemistry.  

Introduction: The role of adenosine A1 receptors of the amygdala  on entorhinal cortex kindled seizures was investigated.   Material and Methods: Animals were kindled by daily electrical stimutation of entorhinal cortex. In the full kindled animals, N6-cyclohexyladenosine (CHA 10, 50, 100, 500َM), an adenosine A1 receptor agonist and 8-cyclopentyl- 1, 3, dimethylxanthine (CPT, 10, 20 َM), an adenosine A1 receptor antagonist, were microinfused into the amygdala.   Results: Data showed that CHA, only at a concentration of 500َM, decreaed entorhinal cortex and amygdala after discharge duration, stage 5 duration and seizure duration seizure duration and increased patency to stage 4 at 15 min post drug injection. Intraamygdala injection of CPT showed that it only at a concentration of 20 َM, reduced patency to stage 4. Intraamygdala pretreatment of animals by intraamygdala injection of CPT (10 َM), 5 min before CHA (500 َM), resulted in blockage of anticonvulsant effects of CHA.   Conclusion: These results suggest that the amygdala may  have a moderate  role in seizure propagation from the entorhinal cortex and its adenosine A1 receptor activity has anticovulsant effects on entorhinal cortex kindled seizures.        

Introduction: This study was designed to investigate the characters of the human endometrial epithelial cells when cultured on either plastic or ECM (Extracellular Matrix) Gel surface and to study ultrastructural features of cultured cells in comparison to endometrial tissue.    Material and Methods: Human endometrial tissue was obtained from patients who had undergone hysterectomy for uterine myoma or pathology of the oviduct. The tissue was divided into two parts, one part for TEM (Transmission Electron Microscopy) study and the other for culture. Uterine epithelial cells were dispersed with Collagenase type I, and cultured as a nonplarized culture in cell culture dishes. The cells of primary culture were stained with Anticytokeratin 7 to determine nature of the cells. After 5 days, the cells were trypsinized, detached from plastic surface and cultured in a dual chamber system on ECM Gel surface (Polarized culture). Finally the cells that were cultured on plastic and ECM Gel surfaces as well as endometrial tissue were prepared for TEM study.   Results: Immunohistochemical staining indicated the epithelial nature of the cells cultured. Electron micrographs of the cells cultured on plastic showed flat cells with large nucleus and thin cytoplasm with few organells. In the cells, the tight junction that maintains polorized state of cell were not observed. Electron micrograph of the cells cultured on ECM Gel indicated highly polarized columnar cells with basal nucleus. These cells were attached to each other with both tight junction and desmosome. Polarized cells had features in close resembelence to cells of endometrial tissue.   Conclusion: Culturing on ECM Gel causes the endometrial epithelial cell to regain their morphologic features that have been lost during culture on plastic surface.  

Introduction: Embryonic stem (ES) cells are pluripotent cells derived from inner cell mass of blastocyst. These cells can product any cell by spontaneous or directed differentiation. This study was started to produce neuron cells by directed differentiation of  new murine ES cell line in vitro.   Materials and Methods: Murine ES cells (Royan B1 derived from C57BL/6 Strain) were used for in vitro neuron differentiation. After production of embryoid bodies from ES cells, a highly enriched population of neuroepithelial precursor cells were derived from ES cell proliferation in the presence of growth factors (EGF and bFGF). These cells differentiate into both neurons and glia following withdrawal of growth factors and replacement of media. Immunohistochemistry and electrophysiology were used for evaluation of differentiated neurons.   Results: Antibodies raised against microtubule associated proteins (MAP2), glutamic acid decarboxylase (GAD), and tyrosine hydroxylase (TH) antigens revealed neuron cells with GABAergic and dopaminergic characteristics. Also, the neurons produced action potentials and responsed to electrical stimulations.   Conclusion: Embryonic stem cell of Royan B1 can produce functional neuron cells in vitro, by directed differentiation.    

Introduction: Deficiency in mu opioid receptor signaling pathway is partially responsible for morphine tolerance. This deficiency may be due to uncoupling of opioid receptors and related G proteins, which in turn, it may be caused by their structural changes such as phosphorylation or by decreasing their abundance. In this study, we tried to investigate the effect of chronic administration of morphine on Gai/o and Gb protein gene expression in rat lumbar spinal cord. Material and Methods: Daily injections of morphine 20 mg/kg for 4 days were used to introduce morphine tolerance in NMRI male rats. In 5th day, the animals were decapitated and their lumbar spinal cord were extracted and stored in-70ْc. After RNA extraction using RNX+ solution, cDNA was synthesized using Oligo-dT primer and M-MuLV reverse transcriptase.Parts of cDNA related to b-actin, Gai/o and Gb protein were amplified by PCR (semi-quantitative PCR) using specific primers. Electro phoresis of the samples was performed on 1.5% agarose gel and the ratio of band density of Gai/o and Gb to b-actin were calculated and compared with control group. Results: Chronic morphine administration did not change Gai/o gene expression but increased Gb gene expression (P< 0.05). Conclusion: It is concluded that parts of morphine tolerance is due to the over expression of Gb gene.These results are supported with several reports concerning adenylyl cyclase activation by Gb proteins 

Introduction: To evaluate the effect of electroactivation on fertilization and embryo development up to 8-cell stage, after intracytoplasmic injection of severely amorphous sperm. Material and Methods: Oocytes (168) from 13 patients with severe- teratospermia undergoing ICSI were divided into control (83) and electro activation (85) groups. In the latter group, oocytes were electrically activated with 1.2 kV/cm for 50 َs using the electrofusion system, model CF-150B (CF-150 Biological laboratory service, H-1165 A.U. 31 Hungary iCF-150) post ICSI. Results: The results show that there is a significant difference in fertilization rate post ICSI between the control (42%) and electroactivation (71%) groups (P=0.04). No significant difference was observed for embryo developmental rates between the two groups during the 66 hr post ICSI (P>0.150). However the mean percentage of 8-cells, with respect to total number of injected oocytes, were significantly different between control (15%) and electroactivation group (50%) (P=0.009).Conclusion: Electroactivation seems to improve fertilization rate and to a certain extent embryo developmental rate 30 minutes post ICSI in severe-teratospermic patients.

Introduction: The aim of this study was to determine a proper cell line for adherance of Helicobacter pylori and a suitable method for attachment of this bacteria to cell lines in vitro. Material and Methods: A total of 22 H.pylori strains,isolated from the antral biopsies of 49 patients with dyspepsia,gastritis,gastric ulcer,duodenal ulcer, etc., were evaluated by ELISA(UPR)to determine the diversity of attachment to 7 mamalian cell lines. Results: H.pylori can attach to all 7 cell lines,there are no significant differences between 22 H.pylori strains in cell attachment.The attachment pattern of H.pylori to the cells showed a significant reduction respectly from HepII, HeLa, SW742, AGS, HT29/219, HT29 to Caco-2. Maximum attachment was seen to HepII, HeLa and SW742 cells, and among these HepII was the best cell for this purpose. The concentration of H.pylori and cell suspension, condition, and temperature can alter the attachment rate. Best bacterial concentration was equal to 1 Mc farland, and for cell suspension it was 5x104 cells/ml. An incubation period of 90 minutes in 37°C resulted in maximum attachment. Conclusion: Our study suggests using the introduced method for the study of H.pylori adhesion, attachment, inhibition of attachment and detachment assays and that HepII cell could serve as the best in-vitro model in this regard.

Introduction: Both initiation and maintenance of spermatogenesis are hormonally regulated by FSH and testosterone. In this study, the effects of FSH and testosterone on the in vitro maturation of fresh and frozen- thawed round spermatids were determind. Material and Methods:  Cell suspension was isolated from testis of NMRI male mice (8-10 weaks old) and divided into two groups .The first part was used to culture freshly and the second part quickly cryopreserved using 18% raffinose (w/v) and 3% skim milk (w/v) in distilled water. Each part of cell suspension was divided into two groups: the fresh specimen was cultured in medium containing DMEM and 10% FBS and medium supplemented with FSH (50 IU/L) and testosterone (1Çmol/l). The frozen -thawed specimen cultured on DMEM & 10% FBS and DMEM & 10% FBS was supplemented with rFSH & testosterone. The number of round, elongating and elongated spermatids, before and during culture were counted up daily for 96 h using light microscope. Survival rates of all kinds of spermatids were evaluated using trypan blue test and the results of every group were compared statistically by a repeated measure ANOVA. Results: The results of this study showed that in fresh specimen cultured in medium suppelemented with FSH and testosterone, on the first 24h, the numbers of round spermatid cells were reduced but elongating and elongated spermatid cells increased. Between 24 to 96h, all kinds of spermatid cells were reduced .In frozen-thawed group cultured in medium supplemented with FSH & testosterone, although there was a severe reduction in the number of round spermatids, the number of elongating and elongated cells did not change during the first day of culture. Viability rates of all kinds of spermatid cells reduced during 96h culture in the both fresh and frozen-thawed specimens. Conclusion: Results of this study confirm that round spermatid cells can progress into elongating and elongated cells only over the first 24h culture period in fresh and frozen-thawed specimen cultured in medium supplemented with FSH and testosterone.