Journal of Pathobiology Reaearch
Volume:14 Issue: 2, 2011

The study of erythropoiesis needs to develop the methods for erythroid progenitor cells (EPCs) culture using stem cell potency to differentiate into variety of hematopoietic cells lineages. In this study, we induced differentiation of cord blood stem cells into erythroid progenitor cells in a semisolid culture media. Cord blood mononuclear cells were isolated and cultured in liquid culture media consist of erythroid differentiation factors (phase I). Non-adherent cells were cultured in semisolid media containing SCF, IL-3, IL-6 and EPO (phase II). After one week, the appeared erythroid colonies were picked up. Characterization of differentiated cells was performed by assessment of CD235a and CD36 using flowcytometry, giemsa cytological staining and gene expression analysis of GATA-1, EKLF, α-globin genes by RT-PCR. Flowcytometry analysis to detect CD235a and CD36 positive cells showed that 94.3% and 95.5% of differentiated cells have erythroid specific cell markers, respectively. Morphology of the cells in giemsa stained slides demonstrated erythroid progenitor cells, ranged from proerythroblast to orthochromatic erythroblast. The RT-PCR results, confirmed the precursor state of erythroid differentiated cells by expression of GATA-1, EKLF, α-globin genes. Cord blood stem cells have high potency to differentiate into erythroid progenitor cells using described method that can be utilized in the experimental studies.

IL18 is a cytokine that plays an important role in the T-cell-helper type 1 (Th1) response and hence, plasmid-encoded IL18 is considered as a potent genetic adjuvant for DNA vaccine studies. In this study, a bicistronic eukaryotic plasmid capable of secreting a more stable mouse IL18 (fused with Fcγ2a fragment) was constructed and expression of this chimer cytokine was also assessed. RNA purified from stimulated mouse spleenocytes and then cDNA corresponding to mouse IL18 (mIL18) and Fcγ2a fragments were constructed by RT-PCR. Sequential subcloning of mIL18 and IgG2aFc fragments first into pSL1180 and then pSecTag2 plasmids resulted in the fusion of mIL18/Fc and addition of immunoglobulin kappa signal sequence (Igk/mIL18/Fc), respectively. Final cloning of Igk/mIL18/Fc sequence downstream of CMV promoter into the NheI/XmaI sites of pIRES2-GFP plasmid and led to the construction of pIRES-Igk/mIL18/Fc plasmid, which was transfected to HEK293T cell line by Turbofec Transfection reagent and expression analysis, was evaluated by ELISA assay. Restriction enzyme analysis of pSL-mIL18، pSL-mIL18/Fc، pSec-mIL18/Fc and pIRES- Igk/mIL18/Fc plasmids with the enzymes that were applied for clonings led to the isolation of fragments with expected size and then plasmid of pIRES- Igk/mIL18/Fc was also confirmed following sequencing reactions. Moreover, expression and secretion of mIL18 to the medium was evidenced in transfected 293T cells, compared to non-transfected controls. pIRES- Igk/mIL18/Fc plasmid possesses the capacity of the cloning and expression of putative antigen gene under the direction of IRES sequence, and also expression of mIL18 as a great secretive genetic adjuvant. This results can be useful to design an efficient DNA vaccine especially for inducing host cellular immune response, moreover, cab be considered a promising for accessing to new generation of DNA vaccine.

Evaluating the effects of p-benzoquinone and hydroquinone on the RUNX2 expression and osteoblastic differentiation of human marrow derived mesenchymal stem cells (MSCs). Bone marrow MSCs obtained by cultivating marrow mononuclear cells, were exposed to 10μM of either p-benzoquinone or hydroquinone. Following chemical treatment, RUNX2 gene expression was assessed by Real-time RT PCR 1, 6, 24 and 48 hours later and osteogenic differentiation was analyzed using alizarin red and alkaline phosphatase staining methods on days 7 and 14 after ostegenic induction. RUNX2 expression was significantly elevated (up to approximately 8 times) due to chemical exposure but the applied chemicals exert no considerable effect on MSCs osteogenic differentiation. According to the literature, despite the necessity of RUNX2 overexpression on the induction of osteogenic differentiation, but it is not sufficient for osteogenesis to occure so increase in RUNX2 expression observed in our study is not the indicator of the induced osteogenic differentiation. Instead, this elevated expression could be the sign of increased activity of the canonical Wnt signaling pathway thereby its involvement in the development of AML due to exposure to benzene and its metabolites. Moreover, this augmented expression of RUNX2 in MSCs can indicate the RUNX2 overexpression in myeloid progenitors as an expected similar effect of exposure to benzene and its metabolites to contribute in myeloid malignancies developed due to benzene exposure.

Curcumin, is the active component in turmeric (Curcuma long). This agent induces apoptosis via inhibiting various signaling pathways. However, its poor aqueous solubility prevents its widespread application. In this study, dendrosomes with water-solubility, nano-sized dimensions and nontoxic properties, was used for curcumin delivery to cells. In the present study, the potential of dendrosomes for use as a drug delivery system was assessed in AGS, HT3, 5637, hBMSC and U87 cell lines. In order to achieve optimal concentration of drug and the most suitable cell line, the effects of different concentrations of free and dendrosomal curcumin was examined on the cell lines. Propidium iodide staining was used for determining apoptosis induction and the expression of Bax gene was investigated by semi-Q RT-PCR. Dendrosomal formulation significantly improved water solubility of highly hydrophobic curcumin in AGS cells. Flow cytometry analysis indicated 48 percent of cells treated with dendrosomal curcumin and 20 percent of cells treated with free curcumin (at the optimal concentration of drug) underwent apoptosis after 18h. Semi-Q RT-PCR results exhibited the increase of expression of Bax pro-apoptotic gene in cells treated with dendrosomal curcumin. Dendrosomal formulation, compared to free curcumin, enhanced curcumin solubility and increased apoptosis induction in treated cells. These data, together with the observation of a 50 % increase of Bax gene expression confirmed the apoptotic effects of dendrosomal formulation of curcumin.

In this study, two conserved genes (M1 and NP) of influenza virus were expressed in a bicistronic vector in order to develop a universal gene based vaccine. Plasmids M1-pIRES2-EGFP, pIRES2-NP were constructed by cloning the PCR products of M1 and NP genes which were amplified from the A/Peurto Rico/8/34 (H1N1) influenza virus strain into the plasmid expression vector pIRES2-EGFP, respectively. For construction of M1-pIRES2-NP bicistronic plasmid, M1 gene was extracted from M1-pIRES-EGFP plasmid and sub-cloned into pIRES2-NP construct. Finally, simultaneous expression of both genes was assessed by transient transfection of bicistronic plasmid into BHK-21 cell lines and subsequent immunofluorescence staining. The results of enzymatic double digestions on the constructed plasmids and sequencing demonstrated the success of cloning processes of above mentioned genes. Correct expression of these genes was confirmed by M1-pIRES2-NP plasmid expression in BHK-21 cell lines confirmed by immunofluoresence microscopy. Simultaneous expression of influenza M1 and NP genes from a bicistronic plasmid containing “IRES” sequence is achievable.

The objective of this study is to develop and assess targeted PAMAM-PEG nanocarrier with anti-TAG72 nanobody for t-Bid gene coding construct delivery into the human colonic adenocarcinoma cells. Nanobody (Nb) coding sequence was subcloned into pSJ expression vector for large-scale production and then Nb was purified by Ni++ affinity chromatography. SDS-PAGE and western blot analysis were performed to verify purifiction. PAMAM was reacted with PEG at the ratio 1:2 (mol/mol) and anti-TAG72 Nb at the ratio 1:1 (mol/mol). Surface charge and size of resulting nanoparticles were evaluated by Malvern zeta sizer and Nanosight. Efficiency of constructed gene carrier for t-Bid, a killer gene, delivery into colonic adenocarcinoma cells in in vitro was assessed using real time PCR and cell counting assays. Production of nanoparticles with the average size of 162±92 nm and +4.57±0.52 zeta potential was confirmed by nanosight and Malvern zeta sizer in order. Gel retardation assay result verified efficiency of carrier for pDNA comlexation. Real time PCR results confirmed the target gene overexpression in the cancerous cell lines. The results of this research confirms the efficiency of PAMAM dendrimers for gene transferring, positive effect of PEGylation and targeting of nanoparticles by anti-TAG72 nanobody.

Recent studies have shown that the use of prolonged and intermittent normobaric hyperoxia (95%) decrease the infarct volume of stroke. The aim of current study was to study the effect of an oxygen radicals-scavenger applied during intermittent normobaric hyperoxia on infarct volume in the rat brain and neurologic deficit. Male Wistar rats (250-350 g) were divided to two main groups. These groups were respectively subjected to room air (21% O2; RA) or normobaric hyperoxia (95% O2; HO) 4 hours per day for 6 days. Each main group was divided to three subgroups which received nothing (RA and HO), normal saline (HO-S and RA-S) or Dimethyltiourea (HO-MT and RA-MT. Afterward, all rats were subjected to 60 min middle cerebral artery occlusion (MCAO). After 24 h, neurologic deficit scores and infarct volume were assessed. The medians of neurologic deficit scores in RA, RA-MT, RA-S, HO-MT, HO-S, and HO were 2, 2, 2, 2, 0, 0, respectively. The infarct volume in RA, RA-MT, RA-S, and HO-MT versus HO-S, and HO were increased. Above results showed that neurologic deficit score and infarct volume were restored by MT significantly. The effective neuroprotection induced by intermittent normobaric hyperoxia seems to be mediated at least partially by oxygen radicals.

Evaluation of the effects of Toxoplasma gondii infection on spermatogenesis in male rats. The RH strain of T. gondii tachyzoites were injected interaperitoneally in an infected group of 35 rats, while 21 rats were used as controls. Each ten days from 10- 70 days of post-infection (PI), 5 rats from infected group and 3 rats from control group were scarified. The percentage of body weight to testis weight ratio (BTR) as well as sperm parameters and fructose levels in seminal vesicles and coagulating glands (SVCG) were investigated. An IgG ELISA kit was designed for serologic diagnosis of infection in the rats. All rats injected with T. gondii tachyzoites were infected from 10-70 PI. Sperm motility from 10-70 PI, sperm viability from 10-60 PI and sperm concentration from 20-60 PI were significantly decreased in the infected group (P<0.05); sperm abnormality was significantly increased in the infected group on days 30, 40 and 50 PI (P < 0.05). BTR in the infected group was not significantly changed compared to control group (P>0.05). Fructose level in SVCG in the infected group was significantly decreased on days 10-50 PI (P < 0.05) compared to control. According to the results, toxoplasmosis can cause impermanent impairment on the spermatogenesis in the male rats.