ABSTRACT
OBJECTIVE: Specific expression of therapeutic genes in cancer therapy has been per used for many years. One of the innovative strategies that have recently been introduced is employing miRNA response elements (MREs) of microRNAs (whose expression are reduced or inhibited in cancerous cells) into the 3´UTR of the therapeutic genes for their specific expression. Accordingly, MREs of anti-metastatic miRNA family have been used in 3´UTR of the metastasis suppressor gene in the corresponding cells to evaluate the level of metastatic behavior. MATERIALS AND METHODS: In this experimental study, 3´UTR of the ZEB1 gene with 592 bp length, encompassing multiple MREs of miR-141, miR-429, miR-200b and miR-200c, was employed to replace BRMS1 3´UTR. The obtained vector was then assessed in the context of MCF-10A, MDA-MB231 and MCF-7 cells. RESULTS: It was shown that the employed MREs are able to up-regulate BRMS expression in the metastatic MDAMB231 cells (almost 3.5-fold increase), while it was significantly reduced within tumorigenic/non-metastatic MCF-7 cells. Specific expression of BRMS1 in metastatic cells led to a significant reduction in their migratory and invasive characteristics (about 65% and 55%, respectively). Two-tailed student's t test was utilized for statistical analysis. CONCLUSION: It was demonstrated that a chimeric vector containing BRMS1 which is regulated by miR-200 family response element may represent a promising therapeutic tool. This is due to the capability of the chimeric vector for cell type-specific expression of anti-metastatic genes with lowest side-effects. It consequently prohibits the invasive characteristics of metastatic cells.
ABSTRACT
BACKGROUND: The accurate and early diagnosis of tuberculosis is important for its effective management. During the last decade, several molecular methods for detection of Tuberculosis (TB) have been developed. Since RNA especially mRNA has a generally much shorter half-life than DNA, its detection may be useful for the assessment of viability of bacteria. This research is a Nucleic Acid Sequence Based Amplification-Enzyme Linked Immunosorbent Assay (NASBA-ELISA) which was designed and developed for rapid detection of viable Mycobacterium tuberculosis (M. tuberculosis). METHODS: Oligonucleotide primers targeting tuf gene encoding viability marker EF-Tu mRNAs were selected and used for the amplification of mycobacterial RNA by the isothermal NASBA Digoxigenin (DIG) labeling process and incorporated with DIG-UTP, reverse transcriptase and T7 RNA polymerase. RESULTS: Using the NASBA-ELISA system, as little as 17.5 pg of RNA of M. tuberculosis was detected within 4 hr and no interference was encountered in the amplification and detection of viable M. tuberculosis in the presence of non-target RNA or DNA. Results obtained from the clinical specimens showed 97 and 75% of sensitivity and specificity, respectively. CONCLUSION: The NASBA-ELISA system offers several advantages in terms of sensitivity, rapidity and simplicity for detection of M. tuberculosis. Furthermore, due to its simplicity and high sensitivity feature, it could be used in limited access laboratories in a cost-effective manner.
ABSTRACT
OBJECTIVE(S): Ex vivo expansion of hematopoitic stem cells is an alternative way to increase umbilical cord blood (UCB)-CD34+ cells for bone marrow transplantation. For this purpose demineralized bone matrix (DBM) and mineralized bone allograft (MBA) as two scaffolds based on bone matrix and stem cell niche, were simultaneously used to enhance the effect of human mesenchymal progenitor cells (MPCs) - unrestricted somatic stem cells (USSCs) - as a feeder layer. MATERIALS AND METHODS: USSCs were isolated and characterized by morphological and immunological analysis then seeded on both scaffolds as a feeder layer. UCB-CD34(+) were isolated by MACS method and were co-culture expanded by USSC in 3D and 2D environments. After 3 weeks expansion, cells were counted and were assessed by karyotype, flow cytometry, clonogenic activity, and long-term culture-initiating cells (LTC-IC). RESULTS: Co-culture expansion in DBM and MBA was 29.22-fold and 27.77-fold, no significant differences in colony and LTC-IC were obtained. Maximum number of colonies belonged to the day 14 with the 73% CFU-GM (Colony Forming Unit- Granulocyte/Macrophage) in contrast to the day 0 which was BFU-E/CFU-E (Burst/Colony Forming Unit-Erythroid). Flow cytometry indicated that the percentage of CD34+ marker was decreased in USSC co-culture and the highest percentage was observed in simple 2D culture. CONCLUSION: Because of acid extraction in the DBM production process, mineral materials were removed and the protein background that was more flexible was presented. Therefore these results suggest that USSC-DBM can be a suitable ex vivo mimicry niche by intensifying of surface/volume ratio and supporting the stem cell differentiation and expansion.
