effects of melilotus officinalis extract on expression of daxx, nfkb and vegf genes in the streptozotocin-induced rat model of sporadic alzheimer's disease

Background: Possible mechanisms of Alzheimer Disease [AD] such as inflammation and oxidative stresses in the brain led us to investigate potential AD therapeutics of Melilotus officinalis, an herbal extract, with possible role as an anti-inflammatory and anti-oxidant agent. Among different genes which had important role in Sporadic AD [SAD], three genes including DAXX, NFkB and VEGF have shown significant statistical diversity in the brains of Alzheimer patients

Methods: These genes were chosen to be investigated for neuroprotective effects of the extract by comparing the expression level in the hippocampus of Sporadic AD [SAD] rat model using quantitative polymerase chain reaction [qPCR] in the treated and untreated groups. In addition, therapeutic effects at the behavioral, learning and memory level by Morris Water Maze [MWM] test were investigated

Results: The results represented significant decreased expression in Daxx, Nfkb and Vegf genes in the SAD rat's model treated with the herbal extract compared to the Streptozotocin-induced [STZ-induced] rats. Furthermore, no significant changes were seen in swimming distance and time for finding the hidden platform in the herbal-treated compared to the STZ-induced group. In memory level, no significant changes were observed among treated and untreated groups

Conclusion: It seems that the herbal extract may have significant effect on Alzheimer-related gene expression changes but not on clinical levels

Optimizing a lipocomplex-based gene transfer method into heLa cell line

One of the most significant steps in gene expression studies is transferring genes into cell cultures. Despite there are different methods for gene delivery such as viral and non-viral producers, some cationic lipid reagents have recently developed to transfect into mammalian cell lines. The main aim of this study was optimizing and improving lipocomplex based transient transfection procedures into HeLa cell line which is being used widely as a typical cell in biological studies. This study was an experimental research. In this work, pCMV: Beta -Gal DNA plasmid was used as a reporter DNA for determining the rate of gene transfection into HeLa cells. To accomplish the highest gene delivery into HeLa cells, optimizing experiments were carried out in different volumes of FuGENE-HD, Lipofectamine[TM] 2000 and X-tremeGENE. Also, we investigated tranasfection efficiency in presence of various cell densities of HeLa cells. Then, transfection efficiency and cell toxicity were measured by beta gal staining and trypan blue methods, respectively. Using FuGENE-HD in volume of 4 Micro l along with 10[5] HeLa cells, transfection efficiency was higher [43.66 +/- 1.52%] in comparison with the cationic lipids lipofectamine[TM] 2000 and X-tremeGENE. In addition, the rate of cell toxicity in presence of FuGENE-HD was less than 5%. In summary, the cationic lipid FuGENE-HD indicates a suitable potential to transfer DNA into HeLa cells and it can be an efficient reagent for gene delivery for HeLa cells in vitro. Moreover, it is worth designing and optimizing gene transfer experiments for other cell lines with FuGENE-HD due to its low toxicity and high efficiency

[Evaluation of knocking down of the RNAi mediated gamma globin repressor into K562 cells, for gene therapy of beta-thalassemia]

Background and Aim: Beta-thalassemia is a genetic disorder manifested by the presence of anemia in adult patients. One approach to treatment of beta-thalassemia is induction of the fetal gamma-globin gene. One of the gamma-globin repressors is a complex called DRED [Direct repeat erythroid-definitive]. DRED is composed of TR2 and TR4 DNA binding subunits. The aim of this study was to set up the RNAi system to increase the expression of the gamma-globin gene

Materials and Methods: In this study, lipofectamin[Trade Mark] 2000 was used to transfect siRNA molecules and pSV-beta-Galactosidase vector was used as a reporter to monitor transfection efficiency. Real-time PCR method was used to measure TR4 knockdown and gamma-globin expression levels

Results: Our findings showed that K562 cells were transfected by 40% using Lipofectamine[Trade Mark] 2000. Also, the level of TR4 expression decreased by 44% after using TR4siRNA, even though the expression of gamma-globin gene was induced by 1.18 times

Conclusion: Despite a 44% knockdown of TR4, no increase in gamma-globin mRNA was observed. Two factors may count for this observation; first, TR4 knockdown may have been limited by our transfection efficiency. Second, simultaneous knockdown of TR2 and TR4 may lead to increased gamma-globin levels. In conclusion, although knocking down of TR4 expression occurred by using RNAi system, this can not be a solitary efficient way to induce the gamma-globin expression

[Assessing the levels of cAMP response element binding protein 1 [CREB1] after siRNA mediated knockdown in K562 cells]

CREB1 is an important downstream protein for many signaling pathways. By designing efficient siRNAs against CREB1, it may be possible to assess the role of molecules involved in signaling pathways in different cell types. In this research the efficiency of CREB1 knockdown by two different siRNAs in K562 cells has been studied. siRNAs have been designed according to the criteria suggested by Reynolds et al. K562 cells were transfected by siRNA using Lipofectamine 2000. The efficiency of CREB knockdown has been assessed by quantitative relative Real-time PCR. Our results have shown that only one of the siRNAs has a high level of inhibitory effect on CREB1 gene expression. The expression of CREB1 by this siRNA was knocked-down by 87% in K562 cells. In this research, although two siRNAs were designed according to the Reynolds et al. criteria, only one showed an inhibitory effect. Reasons other than the aforementioned criteria may be involved in effectiveness of siRNAs