[Mesenchymal stem cells proliferation exposed to hypoxia]

Background and Objective: Some problems such as low viability and apoptosis after injection to the body because of exposure to toxic factors such as hypoxia, thermal stress, oxidative stress and food deprivation are encountered with stem cell application. It is suggested that preconditioning of the cells with cytotoxic factors before injection could enhance their efficiency. This study was done to determine the mesenchymal stem cell proliferation exposed to hypoxia by cobalt chloride

Methods: In this experimental study, Mesenchymal stem cells were isolated from rat bone marrow and cultured at least for four times. The cells were cultured in 96 well plates and treated with different concentration [0, 5, 10, 20, 50, 70, 90, 100, 120, 150 and 200 microM] of cobalt chloride for 6, 12, 24 and 46 hours. Cell proliferation was detected by MTT assay [3-[4,5-Dimethylthiazol-2-Yl]-2,5- Diphenyltetrazolium Bromide]

Results: The cells isolated from bone marrow were propagated easily in culture condition. The cells morphology was not altered after exposure to cobalt chloride. Preconditioning of mesenchymal stem cells with 120 microM for 6 hours, 20 microM for 12 and 24 hours and 5 microM for 48 hours significantly improved cell proliferation after hypoxia in cell culture [P<0.05]

Conclusion: Hypoxia preconditioning increases proliferation of mesenchymal stem cell

Effects of polygonum aviculare herbal extract on proliferation and apoptotic gene expression of MCF-7

One of the most common malignancies in women is breast cancer. Although several treatments for breast cancer are available, application of herbal medicine as a supplementary treatment is a new option to help curing the disease. In this study anticancer effects of Polygonum avicular herbal extract was investigated. Polygonum avicular extract was obtained by methanol. MCF-7 cell line was treated with different concentrations of Polygonum avicular [50, 100, 150, 200, 250, 300,350 400 ng/ micro l] for different time lengths [6, 12, 24, and 48 hrs]. MTT assay and Flow Cytometry were used to evaluate cell proliferation and apoptosis, respectively. RT-PCR was also carried out to evaluate the expression of apoptotic genes. Results showed that Polygonum avicular induced cytotoxicity in MCF- 7 cell line at concentrations higher than 300 ng/ micro l and this was confirmed by the highest rate of cell death as measured by Trypan Blue and MTT assays. RT-PCR results showed up-regulation of P53 and down-regulation of Bcl-2 proteins which implied the ability of Polygonum avicular to induce apoptosis in MCF-7 cells and confirmed its anticancer property. Further studies are required to evaluate effects of the extract on other apoptotic genes

[Effects of different doses of LIF on IVM rate and cumulus expansion in mouse oocytes]

The aim of this research was to study the effects of different doses of LIF on GVBD and MII development rate and cumulus expansion. Immature mice superovulated with HMG and GV oocytes obtained from ovary 48 hours later. The GV oocytes were cultured in TCM199 with 100, 500 and 1000 RI /ml LIF. Cumulus expansions were evaluated with two examiners and numbers of MII oocytes were recorded. For denuding the oocytes hyaloronidase was used. Our results showed that the rate of GVBD and MII development increased in -groups with LIF compared with control group. Rate of MII development with 1000 IU/ml LIF was significantly higher than that of control group [P<0.05]. Cumulus expansion in group with 1000 IU/ml LIF improved significantly compared with control group [p<0.05]. Our results showed that LIF could improve IVM rate in dose dependant. Also cumulus expansion improved in group with LIF and increased oocyte quality

Expression of green fluorescent protein [GFP] using in vitro translation cell free system

One of the major concerns about recombinant protein production is its possible toxicity for the organism. Purification of the recombinant protein is another challenge in this respect. Recently In Vitro translation cell free system that provides a coupled transcription-translation reaction for protein synthesis to overcome the above mentioned problems has been emerged. The aim of this study was expression of GFP as a marker for gene expression and protein in In Vitro translation system. pIVEX2.3-GFP plasmid was cloned to E. coli and the plasmid DNA extracted. In Vitro translation was performed with RTS 100 E. coli Hy kit according to manufacture's instructions. Expression of recombinant fusion protein, His- GFP, was determined by SDS-PAGE, ELISA and western blot analysis. Expected size of recombinant protein was detected in SDS-PAGE and further confirmed by western blot analysis and ELISA. Results showed that In Vitro translation is suitable for expression of recombinant protein and fusion of the recombinant protein with His-tag facilitates the purification

Presence of antioxidant in vitro maturation medium and its effects on glutathione level, spindle area and rate of in vitro fertilization

Effect of different doses of cysteamine on rate of in vitro maturation [IVM], in vitro fertilization [IVF] and glutathione [GSH] level was studied. Metaphase II [MII] spindle area was analyzed for quantification of shape and size of oocytes. Female mice were primed with 5 IU of pregnant mare's stimulating gonadotrophin. Germinal vesicle [GV] oocytes were retrieved 48 hrs later. IVM medium was supplemented with 0, 50, 100, 200 and 500 mM of cysteamine. For IVM and IVF assessment in each group, 150 GV oocytes were used. Experiments also included a group of ovulated oocytes [matured in vivo] after priming with pregnant mare's stimulating gonadotrophin and human chorionic gonadotropin. GSH level was measured by 5, 5-Dithio-bis [2nitrobenzoic acid] DTNB-GR recycling protocol in GV and MII oocytes. For IVF, MII oocytes were inseminated with mature mouse sperm and rate of two-cell embryo was measured. For immunocytochemistry of microtubule and chromosomes, MII oocytes were fixed by methanol and immunostained with alpha-and beta-microtubule antibody and Hoechst. The spindle area was then analyzed. A dose-dependent improvement was observed in IVM and IVF rate. MII development and two-cell embryo formation were increased significantly in group which received 200 micro m cysteamine compare to the control group. GSH level was increased in presence of cysteamine in group which received 200 micro m cysteamine. Spindle area was increased in all groups in vitro except for the group which received 500 micro m cysteamine. The difference between spindle area in 200 micro m cysteamine and in vivo group was not significant [P>0.05]. Administration of cysteamine improves IVM and IVF rate in a dose-dependant manner. Also cysteamine induces glutathione synthesis in MII oocyte and improves microtubule when administered at a dose of 200 micro m. Therefore, addition of cysteamine as an antioxidant can improve IVM and IVF rate by increasing of oocytes quality

Isolation, cloning, expression and purification of recombinant RhD antigen from cord blood

Rh [Rhesus] is a highly complex blood group system in man deeply rooted in transfusion medicine. Isolation of RhD from cord blood, cloning and expression of recombinant RhD antigen in bacterial expression system was the aim of this study. Total RNAs were extracted from cord blood [O[+]]. The quality of RNA was determined by electrophoresis. In order to obtain coding sequence of RhD antigen cDNA was synthesized and Rh D gene was amplified by RT-PCR. The isolated RhD gene was cloned to pUCIS vector and transformed to DH5alpha. The confirmed construct was sub cloned into expression vector, pBADgIII/A, and expressed in Top 10 E.coli. The expressed protein was characterized by SDS-PAGE and western blot analysis. Antigenicity of the expressed protein was assessed by ELISA using commercially available human anti-RhD polyclonal antibody with peroxidase conjugated goat anti-human IgG, IgM, IgA as secondary antibody. RhD gene was successfully cloned and expressed. The expected size of recombinant RhD protein was detected in SDS-PAGE, and confirmed by dot and western blot analysis. RhD antibody reacted with recombinant RhD antigen as well as with RhD polypeptide extracted from RBCs membrane. The recombinant RhD may be helpful to further investigate the molecular basis of RhD protein and could be applicable for production anti- D antibody in an animal model