Cell Journal (Yakhteh)
Year:2006 | Volume:8 | Issue:3 (31)|Papers Count:13

Introduction: The aim of this study was to differentiate human mesenchymal stem cells (hMSCs) into cartilage in a micromass culture system and study of their structure by light and electron microscopy.Material and Methods: Human bone marrow cells obtained from volunteer patients were plated in 75-cm2 flasks and their MSCs were expanded through several sub-cultures. The passage 4 cells were used to establish micromass culture system for chondrogenic differentiation. For this purpose, 200,000 fibroblastic cells were placed in centrifuge tubes and pelleted at 250 g for 5 minutes. About 0.5 ml chondrogenic induction medium was then added to the pellet and the culture incubated in 5% CO2 at 37°C for 21 days. Then, some pellets were utilized to evaluate chondrogenic differentiation by either RT-PCR analysis of some cartilage marker molecules or specific staining for detecting cartilage matrix, and other pellets were used for light and electron microscopic study of differentiated tissue.Results: Primary culture of the bone marrow cells were initially composed of the spindle- and round shaped cells, from which the spindle cells remained and expanded through several passages. At the end of differentiation period, RT-PCR analysis showed high production of collagen II and X and aggrecan mRNA inside the differentiated cells, and toluidine blue staining indicated intermediate accumulation of the metachromatic matrix among the induced cells. In general, light micrograph indicated a rather cellular state of the differentiated tissue in which the peripheral part had more metachromatic matrix than central zone. More detailed study of the sections revealed that induced aggregates of the cells were composed externally of very thin layer of elongated cells reminiscent of perichondrium and internally a mass of oval cells comprising the main part of the pellet. Ultra-thin sections showed that the cells in perichondrium-like layer were very similar to fibroblastic cells and those located centrally had a set of well-developed organelles, characteristic of highly active cells. Some fat cells were seen in central zone. Conclusion: Cartilage tissue differentiated from MSCs in micromass culture system seemed to be structurally very similar to developing cartilage not to adult mature cartilage.

Introduction: Heavy metals are important occupational and environmental pollutants that cause damage to various organs. Although there is no effective therapy for such a poisoning, metallothionein has been shown to play a key role in the detoxification of cadmium (Cd). Evidence in the literature suggests that superoxide dismutase, glutathione peroxidase, and catalase constitute important defense mechanisms against oxygen toxicity in the cells. The aim of this study was to investigate the effect of cadmium chloride and Pb-acetate on antioxidant enzymes in the human skin fibroblast cells (HF2FF.( Material and Methods: The human skin fibroblast (HF2FF) cells were incubated in serum-free medium containing 20 µM CdCl2 for 18 hr three times a week. The same exposure to an equimolar dose of Pb-acetate was performed. After each exposure and after three times exposure the cells were collected and cell viability, the contents of superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-Px), GSH and malondialdehyde (MDA) were measured. Results: Cd caused cytotoxicity and inhibition of glutathione peroxidase (GSH-Px) and SOD activity, as well as depletion of the reduced form of glutathione (GSH) in the cell. The level of lipid peroxidation (LP) was increased, but catalase activity was not significantly altered. These defects were increased with repeated exposures. The same exposure to an equimolar dose of Pb-acetate evoked only inhibition of GSH-Px and SOD. The values of GSH, catalase and LP activity remained unchangedConclusion: The inhibition of GSH-Px and SOD may be considered as an important biomarker of the toxic effect of metals.

Introduction: Colorectal Carcinoma is a main health problem in many countries and the third common cancer in Iran. This malignancy at present is the most curable carcinoma of gastrointestinal tract. Variation in the expression of the proteins produced by P53, P21, P16, E-cadherin, and β-catenin genes have been noted in this malignancy and may be important in the prognosis and therapeutic response rate. The aim of this study was to compare the frequency and pattern of expression of these proteins in tumoral and nontumoral colonic mucosa. The correlation with prognostic factors including tumor stage, grade, and vascular and perineural invasion was also determined. Material and Methods: The paraffin blocks from tumoral and nontumoral parts of the colon obtained from 58 patients with colorectal adenocarcinoma were studied along with 50 colectomic cases in individuals without malignancy. Cylindrical tissue fragments were obtained from appropriate parts of donor blocks by using a 2.5 mm punch biopsy instrument. Each 30 samples were manually arrayed in one tissue array block. Expression of above genes was investigated after sectioning the blocks and immunohistochemical staining of slides. Results: The expression of P53 in tumor cells was significantly more common than in colonic nontumor cells and colon of individuals without tumor (p<0.001); expression of this protein in tumoral tissues was directly related to vascular invasion (p=0.017). The expression frequency of P21 and P16 in tumor cells was less than nontumoral tissues of patients with cancer and patients without cancer (p<0.001). These two gene products showed no correlation with prognostic factors. The expression frequency of membranous E-cadherin and β-catenin in tumor cells was not different from controls, while the membranous expression of E-cadherin was inversely related to cell differentiation (p=0.023) and vascular invasion (p=0.025). In addition, the membranous expression of β-catenin was inversely related to vascular invasion (p=0.049). Cytoplasmic and nuclear expression of β-catenin in tumor cells were significantly higher than their expression in the controls (p<0.001). Cytoplasmic expression of this marker was inversely related to disease stage (p=0.013), while its nuclear expression was inversely related to cell differentiation (p=0.012).Conclusion: According to our data, it seems that we are able to predict aggressive capacity of the colorectal tumor by determining the frequency and pattern of expression of P53, E-cadherin and β-catenin proteins. These studies can be done simply on formalin-fixed small biopsy samples before surgery to provide valuable information for surgeons, gastroenterologists, and oncologists to choose the best therapeutic approach and predict the therapeutic response. Manual tissue array method is believed to be an economical technique for similar research projects.

Introduction: Epineural suture and autologous graft are two routine techniques in peripheral nerve surgery. However, their efficiency can be highly limited depending on the type of lesion and the gap between two nerve stumps and because of deficient proper nerve donors. So much interest has been focused on the development of alternative instruments for bridging the nerve gaps. In the present study, we have used charged polyvinelidene fluoride (PVDF) tube filled with nerve growth factor (NGF) and collagen gel as a substitute for nerve autograft and compared the results with other current surgical techniques. We studied the changes of spinal motoneurons to evaluate the effect of repairing techniques.Material and Methods: In this study, 30 male Wistar rats weighing 200-250 g were divided randomly in five groups: axotomy, epineural suture, autograft, nerve guidance channel, and sham operation. In all experimental groups, the left sciatic nerve was transected at mid-thigh level. The nerve was not repaired in axotomy group. In epineural suture group, it was sutured end-to-end. In autograft group, a 10 mm piece of nerve was rotated 180° and sutured again in the nerve gap. Finally, in nerve guidance channel group, a piece of PVDF tube containing NGF7s (100 ng/ml) and collagen gel (1.28 mg/ml) was replaced in the gap. After one week, one month, and two months, L4-6 segments of spinal cord were removed and 5 µm paraffin sections were prepared for bax immunohistochemical study. In all groups contralateral spinal cord was used as the control. The proportion of Bax-positive apoptotic motoneurons was studied in all groups to evaluate the efficiency of different repairing techniques.Results: Mean percentage of Bax-positive neurons to the total number of motor neurons in left side was analyzed. One way ANOVA showed significant difference after two months. LSD post hoc test showed that mean percentage of Bax-positive neurons in axotomized group was significantly higher compared to other surgical groups (p<0.05). The number of apoptotic neurons after one week, one month and two months in each type of surgical approach showed no significant difference between one week and one month and between one month and two months. Comparison of motoneuron population in left side (experimental) with right side (control) showed no significant differences after one week, but significant differences were seen (p<0.01) after one month and two months.In sham group, no Bax-positive neuron was found after one week, one month, and two months.Conclusion: A PVDF tube filled with NGF and collagen gel can be used as a proper substitute for autografts and protect motoneurons following peripheral nerve injury.

Introduction: Toxoplasma gondii is a protozoan parasite which is globally prevalent in human and animals. Toxoplasma infection is commonly asymptomatic, but can cause serious medical problems in immunocompromised individuals and in fetus. Recombinant antigens of the parasite may be helpful in diagnosing the infection more precisely. The goal of this study was to construct and evaluate the functionality of a prokaryotic expression plasmid pGEX-P35, harboring P35 surface antigen gene of T. gondii and to perform preliminary studies on its ability to detect T. gondii specific antibodies.Material and Methods: A 450 bp fragment of the P35 gene was amplified and inserted into pGEM-T plasmid, sequenced, cut, and then inserted into pGEX-4T-1 plasmid to produce the recombinant plasmid pGEX-P35. In order to confirm that the plasmid construct was capable of expressing P35 in bacterial cells, it was transformed into BL21 strain of E. coli and expressed. The resultant recombinant protein was purified and subjected to SDS-PAGE and Western-blot analysis.Results: A 450 band of PCR product was visualized on 1% agarose gel. Comparison of resultant DNA sequence with GenBank databases showed 100% identity with AF01275. Restriction enzyme analysis confirmed subcloning and correction of orientation. SDS-PAGE analysis showed a 42 kDa band of purified expressed protein. Western-blot analysis using mouse antibody against RH strain of T. gondii showed that the recombinant P35 antigen could be recognized by specific antibodies.Conclusion: Purified and specific recombinant antigens obtained by molecular biology techniques are attractive alternatives for detection of serum antibodies. We amplified and cloned a fragment from P35 gene of Toxoplasma gondii encoding P35 tachyzoite-specific surface antigen. Sequenced fragment was accepted by GenBank with an accession number of DQ092625.  This confirms previous results from other countries estimating just 1% divergence at the level of DNA sequence between lineages isolated from different geographical areas. As the recombinant P35 (rP35) antigen is recognized by specific antibodies, it is suggested to evaluate the (rP35) for diagnosis of clinical Toxoplasma gondii infections.

Introduction: At present, most recombinant proteins are produced in prokaryotes especially E coli. Yeasts and CHO also are used as eukaryotic hosts. Leishmania tarentolae, a parasite of lizards, a member of Trypanosomatidae family is one of the new systems for expression of heterologous proteins. In this system, some of the parasitic protozoa features are used in expression of mammalian proteins.Material and Methods: For evaluation of the protozoa for expression of human complex proteins, we cloned cDNA of tPA gene containing native human signal sequence. We used vectors containing 3´ and 5´ sequences of Leishmania 18s rRNA for integration of the vectors in 18s rRNA gene and severe transcription.Results: RT-PCR test showed production of specific mRNA of tPA gene in the recombinant cells. Southern blot analysis confirmed the cloning of t-PA in the genome of the Leishmania.Conclusion: This study showed native human signal sequence mediate transport and secretion of the protein. Hence, L. tarentolae is the first useful biotechnologically protozoan and tPA is the most complex protein expressed in it.

Introduction: In the present study, we hypothesized that a novel approach to promote vascularization would be to create injectable three dimensional (3-D) scaffolds within growth factor that enhance the sustained release of growth factor and induce the angiogenesis.Material and Methods: We demonstrate that a 3-D scaffold can be formed by mixing of peptide-amphiphile (PA) aqueous solution with hepatocyte growth factor (HGF) solution. PA was synthesized by standard solid phase chemistry that ends with the alkylation of the NH2 terminus of the peptide. The sequence of arginine-glycineaspartic acid (RGD) was included in peptide design as well. A 3-D network of nanofibers was formed by mixing HGF suspensions with dilute aqueous solution of PA.Results: Scanning electron microscopy (SEM) examination revealed the formation of fibrous assemblies with an extremely high aspect ratio and high surface areas with mean diameter of less than 200 nm. In vitro HGF release profile of 3-D nanofibers was investigated while angiogenesis induced by the released HGF was being assessed. In vivo potential ability of PA nanofibers to induce angiogenesis was assessed through subcutaneous injection of PA solution, HGF solution, and PA in combination with HGF solutions. Injection of PA with HGF induced significant angiogenesis around the injected site, in marked contrast to HGF injection alone and PA injection alone.Conclusion: The combination of HGF-induced angiogenesis is a promising procedure to improve tissue regeneration.

Cord blood is a rich source of hematopoietic stem cells and could be potentially used for transplantation instead of conventional sources of stem cells (bone marrow or peripheral blood). Cord blood cells are successfully used in pediatric and adult patients, but their major limitation is the low number of hematopoietic stem cells for patients of large body size. There are several possible solutions for this problem, including use of third party donor, use of multi-unit cord blood, and finally ex-vivo expansion of cord blood hematopoietic stem cells to accelerate engraftment of transplanted cells. In this article we will discuss exvivo expansion from bench and clinical points of view.