Tarbiat Modares University
Pathobiology Research
2538-3000
2538-5887
14
2
2011
6
1
Differentiation of cord blood stem cells into erythroid progenitor cells in semisolid culture media containing SCF, IL-3, IL-6 and EPO
1
12
FA
Amir
Atashi
Department of Hematology and Blood Banking, Faculty of Medical Sciences, Tarbiat Modares University
N
Saeid
Kaviani
Department of Hematology and Blood Banking, Faculty of Medical Sciences, Tarbiat Modares University
Y
Masoud
Soleimani
N
Mehrdad
Norouzinia
Department of Medical Genetic, Faculty of Medical Sciences, Tarbiat Modares University
N
Yousef
Mortazavi
Associated Professor, Department of Pathology, Faculty of Medicine, Zanjan University of Medical Sciences
N
Maryam
Hafizi
Department of Stem Cell Biology, Stem Cell Technology Research center
N
Objectives: 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.
Materials and Methods: 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.
Results: 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.
Conclusions: Cord blood stem cells have high potency to differentiate into erythroid progenitor cells using described method that can be utilized in the experimental studies.
Erythroid Progenitor Cells,Cord Blood Stem Cells,Eerythropoiesis
http://mjms.modares.ac.ir/article-30-4184-en.html
http://mjms.modares.ac.ir/article-30-4184-en.pdf
Tarbiat Modares University
Pathobiology Research
2538-3000
2538-5887
14
2
2011
6
1
Designing and construction of bicistronic plasmid pIRES-Igk/mIL18/Fc: potential implications for vaccine investigations
13
23
FA
Mohammah Hassan
Pouriayevali
Department of Hepatitis and AIDS, Pasteur Institute of Iran, Tehran
N
Arash Reza
Memarnejadian
Asisstant Professor, Department of Hepatitis and AIDS, Pasteur Institute of Iran
N
Seyad Mehdi
Sadat
Ph.D., Department of Hepatitis and AIDS, Pasteur Institute of Iran,
N
Mahdi
Zavva
Department of Hepatitis and AIDS, Pasteur Institute of Iran
N
Seyad Davar
Siadat
Associated Professor, Department of Hepatitis and AIDS, Pasteur Institute of Iran
N
Christine
Hartoonian
Ph.D, Department of Virology, Pasteur Institute of Iran
N
Mohammad Reza
Aghasadeghi
Asisstant Professor, Department of Hepatitis and AIDS, Pasteur Institute of Iran
Y
Objective: 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.
Materials and Methods: 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.
Results: 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.
Conclusion: 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.
IRES,IRES,Bicistronic Plasmid,Genetic Adjuvant,IL-18
http://mjms.modares.ac.ir/article-30-175-en.html
http://mjms.modares.ac.ir/article-30-175-en.pdf
Tarbiat Modares University
Pathobiology Research
2538-3000
2538-5887
14
2
2011
6
1
The effects of p-benzoquinone and hydroquinone on the RUNX2 gene expression and osteoblastic differentiation of human marrow derived mesenchymal stem cells
25
35
FA
Fatemeh
Zolghadr
Ph.D. Student, Department of Genetics, Faculty of Biological Sciences, Tarbiat Modares University
N
Majid
Sadeghizadeh
Professor, Department of Genetics, Faculty of Biological Sciences, Tarbiat Modares University
Y
Naser
Amirizadeh
Associated Professor, Research Center of Iranian Blood Transfusion Organization
N
Mehrdad
Behmanesh
Associated Professor, Department of Genetics, Faculty of Biological Sciences, Tarbiat Modares University
N
Saman
Hosseinkhani
Associated Professor, Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University
N
Maryam
Amani
Research Center of Iranian Blood Transfusion Organization
N
Objective: Evaluating the effects of p-benzoquinone and hydroquinone on the RUNX2 expression and osteoblastic differentiation of human marrow derived mesenchymal stem cells (MSCs).
Materials and Methods: 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.
Results: 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.
Conclusion: 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.
Human Marrow Derived Mesenchymal Stem Cells (MSC),Runx2 Gene,Osteogenic Differentiation,Hydroquinone (HQ),ρ-Benzoquinone (BQ)
http://mjms.modares.ac.ir/article-30-11357-en.html
http://mjms.modares.ac.ir/article-30-11357-en.pdf
Tarbiat Modares University
Pathobiology Research
2538-3000
2538-5887
14
2
2011
6
1
Investigation of free and dendrosomal curcumin effects on apoptosis induction in stem cells and cancer cell lines
37
49
FA
Najmeh
Ranji
MSc,department of microbiology,faculty of basic sciences,Rasht branch, Islamic azad university,Rasht,Iran
N
Majid
Sadeghizadeh
Professor, Department of Genetics, Faculty of Biological Sciences, Tarbiat Modares University
Y
Abbas
padeganeh
MSc, department of genetics,faculty of biology sciences,tarbiat modares university
N
Objective: 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.
Materials & Methods: 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.
Results: 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.
Conclusion: 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.
Apoptosis,Dendrosome,Curcumin,Nanocurcumin,AGS Cells
http://mjms.modares.ac.ir/article-30-7881-en.html
http://mjms.modares.ac.ir/article-30-7881-en.pdf
Tarbiat Modares University
Pathobiology Research
2538-3000
2538-5887
14
2
2011
6
1
Construction of a bicistronic eukaryotic vector expressing M1 and NP genes of influenza virus
51
62
FA
Mohammad
Shenagari
Ph.D. Student, Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University
N
Farzaneh
Sabahi
Associated Professor, Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University
Y
Masoumah
Tavasoti Kheiri
Assistant Professor, Department of Influenza, Pasteur Institute of Iran
N
Kambiz
Forghan Parast
Associated Professor, Department of Bacteriology, Faculty of Medicine, Gilan University of Medical Sciences, Iran
N
Abbas
Jamali
Post-Doctoral fellow, Department of Influenza, Pasteur Institute of Iran, Iran
N
Hamidreza
Hashemi
Ph.D. Student, Department of Virology, School of Public Health, Tehran University of Medical Sciences, Iran
N
Shadi
Khodamoradi
M.Sc. Student, Department of Influenza, Pasteur Institute of Iran, Iran
N
Objective: 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.
Materials and Methods: 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.
Results: 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.
Conclusion: Simultaneous expression of influenza M1 and NP genes from a bicistronic plasmid containing “IRES” sequence is achievable.
Influenza Virus,bicistronic vector,Simultaneous Expression,M1,NP
http://mjms.modares.ac.ir/article-30-8566-en.html
http://mjms.modares.ac.ir/article-30-8566-en.pdf
Tarbiat Modares University
Pathobiology Research
2538-3000
2538-5887
14
2
2011
6
1
Construction of PEG-PAMAM dendrimer-based TAG72-targeting nanocarrier for t-Bid gene delivery to colorectal tumor cells
63
74
FA
Fatemeh
Safarian
M.Sc. Student, Department of Medical Biotechnology, Faculty of Medical Science, Tarbiat Modares University, Iran
N
Fatemeh
Rahbarizadeh
Assistant Professor, Department of Medical Biotechnology, Faculty of Medical Science, Tarbiat Modares University, Iran
Y
Said
Amanpour
Assistant Professor, Cancer Research Center, Institute of Cancer, Tehran University of Medical Sciences, Iran
N
Zahra
Sharifzadeh
Ph.D. Student, National cell bank of Iran, Pasteur Institute, Iran
N
Objective: 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.
Materials and Methods: 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.
Results: 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.
Conclusion: 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.
Anti-TAG72 Nanobody,Targeted Gene Therapy,Human Colonic Adenocarcinoma,PAMAM-PEG Nanocarrier
http://mjms.modares.ac.ir/article-30-3676-en.html
http://mjms.modares.ac.ir/article-30-3676-en.pdf
Tarbiat Modares University
Pathobiology Research
2538-3000
2538-5887
14
2
2011
6
1
The role of oxygen radicals-scavenger in neuroprotection-induced by normobaric hyperoxia on infarct volume in the rat brain
75
82
FA
Mahdiye
Asheghabadi
M.Sc., Department of Physiology, Faculty of Biological Sciences, Beheshti University, Iran
N
Mohammad Reza
Bigdeli
Asisstant Professor, Department of Physiology, Faculty of Biological Sciences, Beheshti University, Iran
Y
Objective: 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.
Materials and Methods: 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.
Results: 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.
Conclusion: The effective neuroprotection induced by intermittent normobaric hyperoxia seems to be mediated at least partially by oxygen radicals.
Normobaric Hyperoxia,stroke,Neuroprotection,Neurologic Deficit,Free Radical Oxygen
http://mjms.modares.ac.ir/article-30-7630-en.html
http://mjms.modares.ac.ir/article-30-7630-en.pdf
Tarbiat Modares University
Pathobiology Research
2538-3000
2538-5887
14
2
2011
6
1
Effect of Toxoplasma gondii infection on spermatogenesis of male rats
83
91
FA
Amir
Abdoli
M.Sc. Student, Department of Parasitology, Faculty of Medical Sciences, Tarbiat Modares University, Iran
N
Abdolhossein
Dalimi
Professor, Department of Parasitology, Faculty of Medical Sciences, Tarbiat Modares University, Iran
Y
Mansoureh
Movahedin
Professor, Department of Anatomy, Faculty of Medical Sciences, Tarbiat Modares University, Iran
N
Objective: Evaluation of the effects of Toxoplasma gondii infection on spermatogenesis in male rats.
Materials and Methods: 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.
Results: 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.
Conclusion: According to the results, toxoplasmosis can cause impermanent impairment on the spermatogenesis in the male rats.
Toxoplasma gondii,spermatogenesis,Male Rat
http://mjms.modares.ac.ir/article-30-5535-en.html
http://mjms.modares.ac.ir/article-30-5535-en.pdf