Evaluation of hepatogenic differentiation potential of menstrual blood derived stem cells

Evaluation of hepatogenic differentiation potential of menstrual blood derived stem cells

Abstracts clones selected for further studies. The recombinant plasmid was purified and used for transfection of several E. coli strains including BL...

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Abstracts

clones selected for further studies. The recombinant plasmid was purified and used for transfection of several E. coli strains including BL-21, origami and codon plus. The expressed recombinant protein was purified by Ni-NTA agarose. Finally, the expression level and the IgE reactivity of the produced recombinant proteins were compared with that of natural allergen by the means of SDS PAGE and western blotting. Results: DNA sequencing of the recombinant plasmid confirmed directional insertion of grape LTP. The recombinant protein was expressed under the control of IPTG and purified by metal affinitiy chromatography.SDS PAGE of the expressed protein showed that the recombinant protein was produced in monomeric (14 kDa) and dimmeric (24 kDa) forms. The dimmeric form was predominant in codon plus, whereas origami mainly produced a monomeric protein. Conclutions: In this study, recombinant form of grape LTP was produced in prokaryotic hosts. The imminoreactivity of the purified protein as well as the effect of host species in proper folding of the allergen is under study by biophysical and immunochemical methods. Keywords: LTP, Recombinant protein, Prokaryotic host, SDS-PAGE doi:10.1016/j.clinbiochem.2011.08.1005

Poster – [A-10-781-1] Evaluation of hepatogenic differentiation potential of menstrual blood derived stem cells Sayeh Khanjania, Hale Edalatkhaha, Saeed Talebia, Amirhasan Zarnanib, Manijeh Khanmohammadia, Mahsa Bakhtiari Sania, Somaieh Kazemnejada a Reproductive Biotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran b Nanobiotechnology Research Center, Avicenna Research Institute, ACECR, Tehran, Iran E-mail addresses: [email protected] (S. Khanjani), [email protected] (H. Edalatkhah), [email protected] (S. Talebi), [email protected] (A. Zarnani), [email protected] (M. Khanmohammadi), [email protected] (M. Bakhtiari Sani), [email protected] (S. Kazemnejad) Introduction: Recently, menstrual blood has been identified as an easily accessible and renewable stem cell source. In this study, hepatogenic differentiation capacity of menstrual blood derived stem cells (MenSCs) was investigated. Materials and methods: MenSCs were isolated of menstrual blood samples by discontinuous density gradient centrifugation and plastic adherence. After karyotypic and immunophenotypic analysis, differentiation ability of cultured cells into hepatocyte was evaluated by cytochemical and molecular experiments. Results: Flow cytometric analysis illustrated that MenScs can typically express the surface antigens associated to mesenchymal stem cells such as CD29, CD44, CD73, CD105, and CD10 while lacking CD34, CD38, CD133 and CD45. But great expression of OCT-4 and lack of STRO1 expression distinguished MenSCs from bone marrow derived mesenchymal stem cells (BMSCs). Moreover, MenSCs proliferated at a substantially faster rate than BMSCs as judged by MTT assay (P< 0.05). The result of immunofluorescent staining revealed that well-known media used for hepatogenic differentiation of BMSCs cannot give rise albumin accumulation in treated MenSCs. The cytochemical observations were corroborated with the extent of mRNA expression of albumin and another hepatic specific markers such as cytokeratin-18 (ck-18), cytokeratin-19 (ck-19) and alpha-fetoprotein (AFP) evaluated by real-time PCR analysis. Despite considerable mRNA expression level of ck-18 and ck-19, no gross

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differences in mRNA expression levels of albumin and AFP was obtained between undifferentiated and differentiated MenSCs. Conclusion: MenSCs are unique population cells with higher expansion ability compared with BMSCs. However, more studies are required to achieve robust efficient protocol for hepatogenic differentiation of MenSCs. Keywords: Menstrual blood, Stem cell, Hepatocyte, Differentiation doi:10.1016/j.clinbiochem.2011.08.1006

Poster – [A-10-789-1] Separating and utilizing DNA aptamers in detection of morphine Gholamreza Behrouzia, Mohamad Javad Rasaeeb, Mir Latif Mousavic a No. 40, Golestan 4, Baharestan ndastrial Zone, 5th karaj-Qazvin Highway, Alborz, Iran b 3th Bilding Medical, Jalale Ahmad, Tehran, Iran c Khalij Fars High Way, Shahed University, Iran E-mail addresses: [email protected] (G. Behrouzi), [email protected] (M.J. Rasaee) Introduction and aim: With regard to the characteristics of singlestranded oligonucleotides, including their potential for implemental use in order to detect some key molecules, it was determined to gain experience in obtaining single-stranded DNA (ssDNA) having affinity for binding to morphine molecules. Materials and methods: An oligonucleotide library was prepared, consisting of an 80-nucleotide sequence with fixed length flanked by constant 5′ and 3′ ends, a 19-nucleotide sequence from 3′-end, a 21nucleotide sequence from 5′-end that serve as primer, and a 40nucleotide random sequence in the middle. Four types of nucleic acid and the 40-nucleotide sequence are mathematically equaled with 1015 DNA chains known as oligonucleotide pool. After amplification by PCR, the aptamers were exposed to the matrix-bound morphine molecules. After rinsing with a buffer and eliminating the chains, not bound to morphine molecule, they were separated using washing buffer, having potential to separate DNA chains from the target molecule. This act was repeated 15 times. Lastly, those primers were used in which one fluorescein molecule was placed at its 5′-end and flow cytometry results proved the efficiency of the chain in detection of target molecule (morphine). Conclusion: Morphine molecule was chosen due to its social and commercial importance for governments. For this reason, the separation of this nucleotide segment (oligonucleotide) was designed by applying the experience of other researchers and some segments were eventually obtained, which can be utilized instead of monoclonal antibodies in detecting morphine in rapid kits after detection of sequences. Therefore, we enter to nanobiology and we will achieve applying oligonucleotides in treatment, drug delivery into the target molecule, follow up the effect of the drug on cells, and finally help imaging. Keywords: Aptamer, Morphine, SELEX doi:10.1016/j.clinbiochem.2011.08.1007

Poster – [A-10-801-1] Synthesis and biodistribution studies of 153Sm – DOTA – bevacizumab for new radiopharmaceutical production Kamal Yavaria, Mohammad Ghannadib a Tehran, Iran b Kargar Shomali, Iran