فهرست مطالب

  • Volume:6 Issue: 1, 2013
  • تاریخ انتشار: 1392/10/08
  • تعداد عناوین: 8
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  • Roghyeh Kordestani, Hamideh Mirshafiee, Seyed Masoud Hosseini, Zohreh Sharifi Page 3
    Background
    The HBV-X (HBX) protein is believed to contribute to the development of HCC. However, the molecular mechanisms involved in HBX-mediated hepatocarcinogenesis remain obscure. In this study, the effect of hepatitis B virus X gene and its protein product HBxAg on expression of p53 gene in Hep G2 cell line was investigated.
    Methods
    Viral DNA extracted from HBV-positive serum and HBX gene region was amplified using polymerase chain reaction (PCR). Then, PCR product was cloned into the pcDNA3 vector. After confirmation of cloning, the recombinant plasmid pcDNA3-X was transfected into HepG2 cell line using lipid-mediated DNA-transfection procedure. SDS-PAGE and western blotting methods were used to identify expression of HBX protein. Relative quantification was used to analyze the p53gene expression using the 2-ΔΔ Ct method.
    Results
    Recombinant plasmid pcDNA3–HBX was confirmed by restriction endonucleases digestion and colony-PCR. The results of SDS-PAGE and western blot assays showed that HBX gene could be expressed in Hep G2 cell line. There was no significant difference between the expression levels of p53 compared with GAPDH gene as housekeeping gene (p<0.05).
    Conclusion
    There was no significant difference in the protein levels between the transfected cells with X gene containing HBX130 and HBX131 double mutations and p53 gene. It is necessary to do more studies on Hepatitis B virus to understand the role of HBX on the development of liver cancer and its function on p53 tumor suppressor protein.
    Keywords: Hepatitis B virus_Hep G2 cell line_p53 gene_X gene
  • Meraj Tabatabaei, Nariman Mosaffa, Shohreh Nikoo, Mahmood Bozorgmehr, Roya Ghods, Somaieh Kazemnejad, Simin Rezania, Bahareh Keshavarzi, Soheila Arefi, Fahimeh Ramezani, Tehrani, Ebrahim Mirzadegan, Amir, Hassan Zarnani Page 10
    Background
    Despite the extensive information available in the literature, cell surface marker signature of human Amniotic Epithelial Cells (hAECs) remains controversial. The aim of the present study was to characterize immuno-phenotypic features, proliferative capacity and immunogenicity of hAECs. We also tested whether expression of some cell surface markers is influenced by the type of trypsin used for tissue digestion.
    Methods
    Single cell suspensions of amniotic membranes from four human placentas were isolated by enzymatic digestion and expression of CD9, CD10, CD29, CD34, CD38, CD44, CD45, CD73, CD105, CD133, HLA-I, HLA-DR, HLA-G, SSEA-4, STRO-1 and OCT-4 was then evaluated by flow cytometry. The differential impact of four trypsin types on the yield and expression of CD105 and HLA-I was also determined. The proliferative capacity of cultured hAECs was assessed and compared in the presence and absence of Epidermal Growth Factor (EGF). To test their immunogenicity, hAECs were injected into Balb/c mice and the reactivity of hyperimmunized sera was examined by immunofluorescence staining.
    Results
    Nearly all purified cells expressed mesenchymal markers, CD9, CD10, CD29, and CD73 and the embryonic marker, SSEA-4. A large proportion of the cells also expressed STRO-1 and OCT-4. The purified cells also expressed HLA-G and HLA-I. A very small proportion of hAECs expressed CD34, CD38, CD44, CD133 and HLA-DR. The type of trypsin used for enzymatic digestion affected both the percentage and expression of HLA-I and CD105. hAECs revealed substantial proliferative capacity only when cultured in the medium supplemented with EGF. These cells were shown to be capable of inducing high amounts of anti-donor antibodies.
    Conclusion
    Here we provided evidence that hAECs are immunogenic cells with high level of HLA-I expression. Furthermore, this work highlighted the impact of isolation procedure on the immunophenotype of hAEC.
    Keywords: Cell proliferation, Epithelial cells, Immunophenotyping, Placenta, Stem cells, Trypsin
  • Abolghasem Esmaeili, Sedigheh Alifarja, Nosrat Nourbakhsh, Ardeshir Talebi Page 21
    Background
    Stem cells from Human Exfoliated Deciduous teeth (SHED) have the capability to differentiate into neural cells. Neurotrophins including Nerve Growth Factor (NGF), Brain-Derived Neurotrophic Factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4) have neurogenesis, neurotrophic, or neuroprotective effects and are expressed in developing teeth. The aim of this study was to measure quantitative changes in mRNA expression levels of neurotrophins in neural-like cells differentiated from dental pulp stem cells.
    Methods
    Isolated total RNA from SHED, dental pulp and neural-like cells (n=3) were transcribed into cDNA. Then real time PCR was done. Expression levels of mRNA for NGF, BDNF, NT-3, and NT-4 genes were compared in these three cells.
    Results
    In neural like cells, BDNF mRNA increased (372.1113.5) significantly (p<0.01) after differentiation. NGF mRNA increased to more than 266 times the dental pulp level after differentiation. A similar pattern was seen for the expression of NT3 after differentiation. NT4 mRNA enhancement was 1344630.8 and 30.77.9 fold in neural like cells and SHED cells, respectively. Results show alterations with different degrees and direction in neurotrophins mRNA expression levels in these cells.
    Conclusion
    Our results suggest that neurotrophins dental pulp cells, SHED cells and neural like cells derived from SHED cells produce neurotrophic factors. Since the large amounts of neurotrophins are expressed in SHED and neural like cells they may have important role in survival and differentiation of dental pulp stem cells and obtained information may lead to a novel method for tooth regeneration.
    Keywords: Cell differentiation, Neurotrophin, Real, time PCR, Survival
  • Mahboobeh Razmkhah, Mansooreh Jaberipour, Abbas Ghaderi Page 27
    Background
    Mesenchymal Stem Cells (MSCs) are recently introduced as novel immunological gene carriers for treatment of cancer. It is believed that balance between the expression of angiogenic and anti-angiogenic factors, such as SDF-1 and IP-10, may regulate neovascularization within the tumor.
    Methods
    In this study, we compared the expression of important tumor promoting mediators in IP-10-transfected Adipose Derived Stem Cells (ASCs) to those transfected with SDF-1. ASCs were isolated from adipose tissue of a normal subject undergoing cosmetic mamoplasty surgery using collagenase. ASCs were transfected with IP-10 or SDF-1 propagated plasmids by electroporation method and Lipofectamin 2000. Expressions of SDF-1, CXCR4, IP-10, Bcl-2, MMP2, IL-10, IGF-1, and VEGF were detected in transfected ASCs using quantitative Real-Time Polymerase Chain Reaction (qRT-PCR).
    Results
    Results showed that the expressions of SDF-1, CXCR4, Bcl-2, MMP2, IL-10, IGF-1, and VEGF were upregulated in SDF-1-transfected ASCs. In contrast, Bcl-2 and MMP2 transcripts showed 45×103 and 10 fold lower expression in ASCs transfected with IP-10 compared to non-transfected cells.
    Conclusion
    Anti-angiogenic chemokines such as IP-10 may modulate tumor promoting properties of ASCs and would be introduced as novel candidates for tumor immunotherapy; however, further studies are needed to be conducted.
    Keywords: Adipose derived stem cells, IP, 10, SDF, 1, Transfection, Tumor immunotherapy
  • Zahra Amidzadeh, Abbas Behzad Behbahani, Nasrollah Erfani, Sedigheh Sharifzadeh, Reza Ranjbaran, Leili Moezi, Farzaneh Aboualizadeh, Mohammad Ali Okhovat, Parniyan Alavi, Negar Azarpira Page 38
    Background
    Various fixation and permeabilization techniques have been developed for detection of intracellular antigens by flow cytometry; however, there are few studies using flow cytometry to detect the frequency of intracellular nucleic acids, particularly RNA. We tested six different permeabilization methods in order to gain access to a high quality method with minimal damage to intracellular components focusing on 18S rRNA in HeLa cells.
    Methods
    HeLa cells were fixed in 2% paraformaldehyde. A variety of detergents and enzymes including saponin, TritonX-100, Tween-20, NP40, Proteinase K, and streptolysin O were used to optimize a protocol of permeabilization for the flow cytometric enumeration of intracellular 18S rRNA. Treated cells were subjected to standard protocol of flow cytometric in situ hybridization in the presence of FITC-labeled sense and antisense probes to detect 18S ribosomal RNAs. Samples were then analyzed on a FACSCalibur flow cytometer. To evaluate cell morphology, following hybridization the cells were fixed on glass slide, covered with DAPI, and evaluated on a fluorescent microscope with appropriate filter sets.
    Results
    In comparison with other methods, maximum cell frequency in percentage and fluorescent intensity (M1=2.1%, M2=97.9%) were obtained when the cells were treated with 0.2% Tween-20 and incubated for 30 min (p=0.001).
    Conclusion
    Our study indicated that the highest levels of mean fluorescence could be obtained when the cells were treated with Tween-20. However, it should be taken into consideration that for a successful flow cytometric result, other interfering factors such as hybridization conditions should also be optimized.
    Keywords: 18S rRNA, Flow cytometry, HeLa cells
  • Kambiz Gilany, Roudabeh Sadat Moazeni Pouraci, Mohammad Reza Sadeghi Page 47
    Background
    The seminal plasma is an excellent source for noninvasive detection of spermatogenesis. The seminal plasma of normospermic and azoospermic men has been analyzed for detection of spermatogenesis.
    Methods
    Optical spectroscopy (Attenuated Total Reflectance-Infrared spectroscopy (ATR-IR) and Fourier Transform infrared spectroscopy (FT-IR) has been used to analyze the seminal plasma and the metabolome of seminal plasma for detection of spermatogenesis.
    Results
    The seminal plasma of normospermic and azoospermic men has been analyzed by ATR-IR. The results show that there is a pattern variation in the azoospermic men compared to normospermic men. However, the seminal plasma is too complex to show significant pattern variation. Therefore, the metabolome which is a subcomponent of the seminal plasma was analyzed. The seminal plasma metabolome of normospermic and azoospermic men has been analyzed by FT-IR. A significant pattern change was observed. The data combined with chemometrics analysis showed that significant changes are observed at metabolome level.
    Conclusion
    We suggest that FT-IR has the potential as a diagnostic tool instead of testicular biopsy.
    Keywords: Azoospermia, Fourier transform infrared spectroscopy, Seminal plasma, Metabolome
  • Sadegh Alijani, Shaban Alizadeh, Ahmad Kazemi, Zahra Kashani Khatib, Masoud Soleimani, Mohamadreza Rezvani, Neda Minayi, Farshid Karami, Behnoosh Tayebi Page 53
    Background
    The major hemoglobin in the fetus is hemoglobin F (𝛼2𝛾2), whereas in adult humans, hemoglobin A (𝛼2𝛽2) is predominately expressed. Several studies have indicated that expression of the HbF subunit 𝛾-globin might be regulated post-transcriptionally. This could be done by small non-coding RNAs called microRNAs which target mRNAs in a sequence-specific manner and lead to translational repression or mRNA decay. The aim of this study is to evaluate the effect of miR-26b up-regulation on 𝛾-globin gene expression in K-562 cell line.
    Methods
    These cells were grown in RPMI 1640 and pre miR-26b and were transfected within K-562 cell line using lentiviral vector. After RNA extraction and cDNA synthesis in selected days, miRNA up-regulation was confirmed by miRNA real time PCR and then 𝛾and 𝛽chain and GATA-1 expression were investigated by RT and QRT-PCR.
    Results
    The viability of cells before transfection was 90%. Three and 7 days after transfection, through the use of relative Q-PCR, the 𝛾 chain expression increased 3.7, 6.8 and 3.8 folds and GATA-1 expression increased 2.1, 6.0 and 8.0 in comparison with untransfected cells.
    Conclusion
    The data suggest that miR-26b can be involved in the increase of 𝛾-globin gene expression in K-562 cell line. We suggest that miR-26b may be a significant therapeutic target for increasing HbF levels in patients with sickle cell disease and 𝛽-thalassemia.
    Keywords: K, 562, MicroRNAs, MiR, 26b
  • Mojtaba Kafi, Seyed Fakhroddin Mesbah, Najmeh Davoodian, Ali Kadivar Page 57
    Background
    The following study was carried out to determine the ultrastructural features of the oocyte of the ovulatory-sized follicles in relation to concentrations of steroids and IGF-I in the follicular fluid and serum in the dromedary camel.
    Methods
    Camel follicles with a clear and healthy appearance were categorized into three classes: follicles 10 to 13.9, 14-17.9 and 18-30 mm diameter. The Follicular Fluid (FF) and serum samples were assayed for estradiol-17β, progesterone and IGF-I. Recovered Cumulus-Oocyte Complexes (COCs) were prepared for transmission electron microscopy.
    Results
    The mean (±SD) FF concentrations of progesterone and IGF-I was significantly (p<0.05) higher in follicles 18 to 30 mm diameter compared to other groups of follicles. There was no difference in the mean (±SD) serum estradiol-17β, progesterone and IGF-I concentrations between camels with different ovulatory-sized follicles (p>0.05). Oocytes from follicles 18 to 30 mm diameter (group 3) showed more advanced signs of maturation including the disappearance of the nuclear envelope, increased number of microvilli in erect position, the increase in number and size of vesicles and more even distribution of the mitochondria throughout the ooplasm.
    Conclusion
    The final stages of oocyte maturation in dromedary camel is associated with increasing progesterone and IGF-I concentrations and constant high estradiol concentration in the follicular fluid which are paralleled with well-defined ultrastructural changes in oocytes.
    Keywords: Camel, IGF, I, Oocyte, Steroids, Ultrastructure