Iranian Biomedical Journal
Volume:13 Issue: 3, Jul 2009

The failure of regeneration after spinal cord injury (SCI) has been attributed to axonal demyelination and neuronal death. Cellular replacement and white matter regeneration are both necessary for SCI repair. In this study, we evaluated the co-transplantation of olfactory ensheathing cells (OEC) and embryonic stem (ES) cell-derived motor neurons (ESMN) on contused SCI. OEC cultured from olfactory nerve rootlets and olfactory bulbs. ESMN was generated by exposing mouse ES cells to retinoic acid and sonic hedgehog. Thirty female rats were used to prepare SCI models in five groups. Control and medium-injected groups was subjected to induce lesion without cell transplantation. OEC or ESMN or both were transplanted into the site of the lesion in other groups. The purity of OEC culture was 95%. Motor neuron progenitor markers (Olig2, Nkx6.1 and Pax6) and motor neuron markers (Isl1, Isl2 and Hb9) were expressed. Histological analysis showed that significantly more (P<0.001) spinal tissue was spared in OEC, ESMN and OEC+ ESMN groups but the OEC+ ESMN group had a significantly greater percentage of spared tissue and myelination than other groups (P< 0.05). The numbers of ESMN in co-transplanted group were significantly higher than ESMN group (P<0.05). A significant (P<0.05) recovery of hindlimb function was observed in rats in the transplanted groups. We found that the co-transplantation of ESMN and OEC into an injured spinal cord has a synergistic effect, promoting neural regeneration, ESMN survival and partial functional recovery.

Cell therapy of many neurodegenerative diseases using bone marrow stromal cells (BMSC) requires the differentiation of BMSC into neuronal subtype. However, the transdifferentiation of BMSC into GABAergic phenotype requires more investigation. In this study, BMSC of adult female rats were pre-induced into neuroblast-like cells using 1 mM β-mercaptoethanol (βME) and 10 mM retinoic acid (RA), followed by 40 mM potassium chloride as inducer. The BMSC were evaluated by fibronectin as well as Oct-4. The percentage of nestin, neurofilaments (NF 68, NF 160, and NF 200) and GABA immuno-reactive cells was used to evaluate the GABAergic differentiation at the pre-induction and induction stages. The statistical analysis was carried out using unpaired student''s t-test and ANOVA with Tukey''s multiple comparison. The BMSC in the fourth passage expressed fibronectin up to 91.24 ± 0.82%. The pre-induced cells after 2 days of RA exposure showed the expression of neuroblastic markers of nestin and NF68 (81.56 ± 2.64% and 82.12 ± 2.65%, respectively). The yield of GABAergic neurons with β-ME for 1 h and RA as pre-inducer for 2 days followed by potassium chloride as inducer (40 mM for 3 days) was 60.64% ± 1.97%. In addition, NF160 and NF200 were detected in the transdifferentiated cells. RT-PCR showed no expression of Oct-4 after the induction and pre-induction stages. GABAergic-like neurons obtained from BMSC can be potentially used in cell transplanting for some neurodegenerative disorders.

Dense granule antigens (GRA antigens) of Toxoplasma gondii induce strong antibody response in humans and are considered as useful diagnostic antigens. Previous studies reported expression of amino terminal GRA8 protein in fusion with large tags such as glutathione-S-transferase. The present study aimed to produce soluble full length immunogenic GRA8 in bacteria. GRA8 complementary DNA (cDNA), encoding amino acids 24 to 258, was amplified from tachyzoites of RH strain and cloned in prokaryotic expression vector pET-28b(+). Expression of recombinant GRA8 (rGRA8) was analyzed by SDS-PAGE. Antigenicity and immunogenicity of the protein were evaluated by Western-blotting. The cloned gene fragment exhibited complete similarity with the published sequence of gra8 gene by sequence analysis. rGRA8 was expressed in Escherichia coli in fusion with a very small tag and the soluble protein was purified by immobilized metal ion affinity chromatography. In immunoblot, serum sample from a rabbit immunized with rGRA8 recognized a single antigen of T. gondii tachyzoite at the expected molecular weight of native GRA8. Sera from acutely-infected pregnant women strongly reacted with rGRA8 in Western-blotting, while sera from chronically-infected or T. gondii-negative women failed to recognize the protein. The full length soluble rGRA8 was successfully produced in E. coli and shown to be a highly immunogenic protein. As a result it could be used in immunological as well as molecular biology experiments.

While articular chondrocytes are among those appropriate candidates for cartilage regeneration, the cell dedifferentiation during monolayer culture has limited their application. Several investigations have indicated the usefulness of alginate, but the topic of proliferation and differentiation of chondrocytes in alginate culture has still remained controversial. Rat articular chondrocytes were released by enzymatic digestion, plated at 5 × 104 cells/cm2 and culture-expanded. Passaged-5 cells were then cultivated in alginate as 2-mm beads for a period of two months during which the expansion rate and the level of cell differentiation were determined by [3-(A, 5- dimethylthiazolyl- 2-yl)-1, 5-diphenyl tetrazolium bromide] assay and real-time PCR analysis respectively and compared with those of chondrocytes in monolayer culture. Average population doubling time in alginate cultures (10.04 ± 0.9 days) tended to be significantly (P<0.05) higher than that in monolayer cultures (2.94 ± 0.3 days). The period of alginate culture could be subdivided into expansion phase (Days 0-40); during which proliferation appeared to be high and differentiation phase (Days 40-60) during which the expression of cartilage-specific genes including collagen II and aggrecan tended to be upregulated. During both the proliferation and differentiation phases, the expression of collagen I was low. At chondrocytes monolayer cultures, the proliferating cells appeared to have a very low expression level of cartilage-specific genes and a high expression level of collagen I gene during the entire culture period (P<0.05). It seems that alginate provides conditions in which rat articular chondrocytes are able to undergo proliferation and differentiation in certain time point of cultivation period.

Patients with diabetes mellitus type II suffer from hyperglycemia because they are not able to use the insulin that they produce, often due to inadequate function of insulin receptors. There are some evidences that this deficiency is inherited in a dominant autosomal manner and leads to the malfunction of the pancreatic beta cells resulting in insulin excretion disorders. In this study, we sought to identify mutations in the insulin receptor (INSR) gene, which can cause insulin resistance in type II diabetic patients. DNA was extracted from peripheral blood cells of the patients (n = 128) diagnosed with type II diabetes. All 22 exons of the INSR gene of the patients were analyzed for mutations running PCR, conformation-sensitive gel electrophoresis and DNA sequencing, consecutively. Approximately 26% of the patients had genetic mutations; however, most of them were not reported. These mutations include exon 2 (His171Asn, Ile172Ser, Cys196Ser and Ser210Arg), exon 3 (Gly227Asp and Gly232Ser), exon 8 (Thr543Ser), exon 9 (a heterozygote was observed with no change in phenylalanine at position 669), exon 13 (two heterozygotes: Arg890Pro with Asn865 remaining unchanged), exon 14 (Ala906Gly and Pro918Trp with Arg902 unchanged), exon 17 (Val1086Glu) and exon 19 (His1157Gln with Thr1172 unchanged). The lack of similar mutation records in literature and genetic data banks may suggest a geographic pattern for these INSR gene variants in our population.

Extensive full-thickness burns require replacement of both epidermis and dermis. In designing skin replacements, the goal has been to re-create this model and make a product which has both essential components. In the present study, we developed procedures for establishing confluent, stratified layers of cultured human keratinocytes on the surface of modified collagen-chitosan scaffold that contains fibroblasts. The culture methods for propagation of keratinocytes and fibroblasts isolated from human neonatal foreskin were developed. The growth and proliferation of normal human keratinocytes were evaluated in serum-free (keratinocyte growth medium) and our modified medium. Characterization of human keratinocytes was determined by using pan-keratin and anti-involucrin monoclonal antibodies. For fabrication of relevant biodegradable and biocompatible collagen-chitosan porous scaffold with improved biostability, modified method of freeze-gelation was used. In generating organotypic co-cultures, epidermal keratinocytes were plated onto the upper surface of scaffold containing embedded fibroblasts. The results showed that the growth of isolated human skin fibroblasts and keratinocytes in our modified medium was more than that in the serum-free medium. The different evaluations of collagen-chitosan scaffold showed that it is relevant to growth of cells (fibroblast and keratinocyte) and has a good flexibility in manipulation of the living skin equivalents. These findings indicate that the integration of collagen-chitosan scaffold with co-cultured keratinocyte and fibroblast in vitro provides a potential source of living skin for grafting in vivo.

The herpes simplex virus (HSV) UL41 gene product, virion host shutoff (Vhs) protein, mediates the rapid degradation of both viral and cellular mRNA. This ability suggests that Vhs protein can be used as a suicide gene in cancer gene therapy applications. The recent reports have shown that the degradation of cellular mRNA during herpes simplex infection is selective. RNA containing AU-rich elements (ARE) in their 3’ untranslated ends are the targets for the Vhs protein. RNA that are not subject to Vhs protein-dependent degradation are up-regulated during HSV infection. ARE are frequently found in mRNA that encode proto-oncogenes, nuclear transcription factors, and cytokines. In many human cancers, the AU-rich stretch of proto-oncogenes and regulatory genes has impaired. To investigate whether Vhs protein might be useful for inhibition of tumor cell proliferation, a eukaryotic expression vector containing Vhs protein gene was constructed. Cell degradation and RNA content of HeLa and MRC-5 tumor cells after transfection with the constructed vector were studied. The results showed a strong inhibitory activity in proliferation of transfected tumor cells and a sharp decrease in their RNA content. These data suggest that Vhs protein can be considered as a candidate for suicide cancer gene therapy.