Author/Authors :
Gharaati Mohammad Reza نويسنده , Baghaban Eslaminejad Mohamadreza نويسنده , Rezaeiani Siamak نويسنده Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehra , Sayadmanesh Ali نويسنده Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehra , Nasrabadi Davood نويسنده Medical Biotechnology Department, Semnan University of Medical Sciences, Semnan, Iran , Shabani Aliakbar نويسنده Medical Biotechnology Department, Semnan University of Medical Sciences, Semnan, Iran
Abstract :
[Background]Parathyroid hormone-related protein (PTHrP) is a multi-functional protein with a sequence “aa 1-34” similar to that of parathyroid hormone (PTH). In spite of growth factor activity, there is no standardized analytical method to determine the biological activity of recombinant PTHrP.[Methods]The current study isolated the cDNA encoding 141 N-terminal amino acid sequence of PTHrP, cloned it in plasmid vector pET32a, and produced recombinant PTHrP in Escherichia coli. The biological activity analysis of the recombinant protein was evaluated using a precise and practical method that measured PTHrP-mediated cell proliferation of a breast cancer cell line MCF-7. Additionally, to investigate the accuracy and precision of MCF-7 method used, chondrogenic differentiation condition of PTHrP treated mesenchymal stem cells (MSCs) was evaluated.[Results]DNA sequencing and protein mass spectrometry analysis confirmed the accuracy of cloned cDNA sequence and expressed the protein. Biological activity analysis showed the increasing effect of recombinant PTHrP on cell proliferation of cancer cell line MCF-7. Alkaline phosphatase and gene expression analysis validated the anti-hypertrophy effects of recombinant PTHrP on chondrogenic differentiation of MSCs.[Conclusions]The current study removed potential challenges and errors in comparability and reproducibility studies focusing on stem cell differentiation and pathogenesis of breast cancer arising from PTHrP biological activity analysis.
