Comparison of The Melatonin Preconditioning Efficacy between Bone Marrow and Adipose-Derived Mesenchymal Stem Cells

Objective: Mesenchymal stem cells (MSC) from various sources have the potentials to positively affect regenerative medicine. Furthermore, pre-conditioning strategies with different agents could improve the efficacy of cell therapy. This study compares the effects of an anti-inflammatory and antioxidant agent, melatonin, on protection of bone marrow-derived MSCs (BMSCs) and adipose tissue-derived MSCs (ADSCs). Materials and Methods: In this experimental study, rat BMSCs and ADSCs were isolated and expanded. Pre-conditioning was performed with 5 μM melatonin for 24 hours. Cell proliferation and viability were detected by MTT assay. Expression of BAX, BCL2, melatonin receptors and osteocalcin genes were evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR). Also, apoptosis was detected with tunnel assay. Osteogenic differentiation was analyzed using alizarin red staining. Results: No significant increase was found in cell viability between BMSCs and ADSCs after melatonin preconditioning. Following melatonin preconditioning, BAX expression was significantly down-regulated in both ADSCs and BMSCs (P<0.05), with the difference being more significant in ADSCs compared to BMSCs. BCL2 expression was increased significantly in both cell types after preconditioning. Metalothionine 1 and Metalothionine 2 were both upregulated significantly in the two cell types (P<0.05). Melatonin increased osteogenesis capability through increasing osteocalcin expression. However, expression of osteocalcin in BMSCs before and after preconditioning was higher than that in ADSCs. On the other hand, melatonin expression in ADSCs was in higher levels than in BMSCs. Melatonin also improved alizarin red concentration significantly in both BMSCs and ADSCs (P<0.05). Alizarin red staining severity increased significantly in ADSCs after preconditioning compared to BMSCs (P<0.05). Conclusion: Here we have shown that the effects of preconditioning on melatonin expression in ADSCs are higher than those in BMSCs. These findings could be used in adoption of a proper preconditioning protocol based on the sources of MSCs in specific clinical applications, especially in bone regeneration

High-levelexpression of functional recombinant human coagulation factor VII in insect cells

Abstract: Recombinant coagulation factor VII (FVII) is used as a potential therapeutic intervention in hemophilia patients who produce antibodies against the coagulation factors. Mammalian cell lines provide low levels of expression, however, the Spodoptera frugiperda Sf9 cell line and baculovirus expression system are powerful systems for high-level expression of recombinant proteins, but due to the lack of endogenous vitamin K-dependent carboxylase, expression of functional FVII using this system is impossible. In the present study, we report a simple but versatile method to overcome the defect for high-level expression of the functional recombinant coagulation FVII in Sf9 cells. This method involves simultaneous expression of both human γ-carboxylase (hGC) and human FVII genes in the host. It may be possible to express other vitamin K-dependent coagulation factors using this method in the future. Keywords: Baculovirus; γ-carboxylase; Coagulation FVII; Factor VII; Insect cel

In vitro maturation media, cysteamine concentration and glutathione level affect blstocysts development in mouse

Background: Preparation of oocytes is one of the critical factors that determine the developmental competence of embryos produced by in vitro fertilization (IVF). Objective: In this study, the effect of cysteamine, type of media and glutathione (GSH) level on blastocysts development after in vitro maturation of mouse oocytes were investigated. Materials and Methods: Premature female mice were primed with pregnant mare stimulating gonadotrophin (PMSG), and germinal vesicle (GV) stage oocytes were obtained 45 hr later. GV oocytes were cultured in presence of 0, 50, 100, 200 and 500 µm cysteamine in TCM199 and MEME media. After IVM, MII oocytes were in vitro fertilized (IVF) and in vitro cultured (IVC) in order to observe embryo development. A group of In Vivo Ovulated (IVO) oocytes after priming with PMSG and HCG also were included in this study. 5,5-Dithio-bis (2nitrobenzoic acid) DTNB-recycling protocol was used for GSH assay. Results: Rate of IVM and IVF were improved in all oocytes treated with cysteamine in the two medium except 500 µm (81% MII rate in TCM and 64% MII in MEME). Rate of blastocyst in 100 µm cysteamine in TCM1199 and 200 µm in MEME was higher compared to control groups (In TCM 45% and in MEME 35%). In vivo MII and GV oocytes represented the highest and lowest GSH level respectively. Conclusion: Our results revealed that the media and concentration of cysteamine can affects on IVM, IVF and rate of blastocysts development on dose dependant manner

Comparison of The Melatonin Preconditioning Efficacy between Bone Marrow and Adipose-Derived Mesenchymal Stem Cells

Objective: Mesenchymal stem cells (MSC) from various sources have the potentials to positively affect regenerative medicine. Furthermore, pre-conditioning strategies with different agents could improve the efficacy of cell therapy. This study compares the effects of an anti-inflammatory and antioxidant agent, melatonin, on protection of bone marrow-derived MSCs (BMSCs) and adipose tissue-derived MSCs (ADSCs). Materials and Methods: In this experimental study, rat BMSCs and ADSCs were isolated and expanded. Pre-conditioning was performed with 5 μM melatonin for 24 hours. Cell proliferation and viability were detected by MTT assay. Expression of BAX, BCL2, melatonin receptors and osteocalcin genes were evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR). Also, apoptosis was detected with tunnel assay. Osteogenic differentiation was analyzed using alizarin red staining. Results: No significant increase was found in cell viability between BMSCs and ADSCs after melatonin preconditioning. Following melatonin preconditioning, BAX expression was significantly down-regulated in both ADSCs and BMSCs (P<0.05), with the difference being more significant in ADSCs compared to BMSCs. BCL2 expression was increased significantly in both cell types after preconditioning. Metalothionine 1 and Metalothionine 2 were both upregulated significantly in the two cell types (P<0.05). Melatonin increased osteogenesis capability through increasing osteocalcin expression. However, expression of osteocalcin in BMSCs before and after preconditioning was higher than that in ADSCs. On the other hand, melatonin expression in ADSCs was in higher levels than in BMSCs. Melatonin also improved alizarin red concentration significantly in both BMSCs and ADSCs (P<0.05). Alizarin red staining severity increased significantly in ADSCs after preconditioning compared to BMSCs (P<0.05). Conclusion: Here we have shown that the effects of preconditioning on melatonin expression in ADSCs are higher than those in BMSCs. These findings could be used in adoption of a proper preconditioning protocol based on the sources of MSCs in specific clinical applications, especially in bone regeneration

Effects of Different Doses of Hyaloronan on Human Sperm Motility, Vitality and Morphology

Important aspect of sperm function such as motility and capacitation appear to be mediated at least partially though hyaloronic acid (HA). Present study investigated effects of different doses of HA on sperm motility and vitality in human. Sperm was obtained from 20 male from IVF clinic in Imam Khomeini Hospital. Sperm motility and vitality in human semen was analyzed according to WHO criteria before and 4 hours after treatment with different doses of HA (0.750, 1000 and 1250 &amp;micro;g/ml). The results showed that in 1000 &amp;micro;g/ml the percent of stage 3 and 4 increased compare to control group. Percent of stage 1 and 2 decreased in group with 1000 &amp;micro;g/ml HA, there was an increase in the percentage of stage 3 and 4 and decrease in percentage of stage 1 and 2 compare to control. In the group treated with 1250 &amp;micro;g/ml stage 1 and 2 increased while stage 3 and 4 decreased. Vitality in all groups decreased except of the group treated with 1000 &amp;micro;g/ml HA. The group with 1250 &amp;micro;g/ml showed significantly decrease in vitality compare to fresh group (P &amp;lt; 0.05). The present study showed that the effects of HA on sperm motility and vitality is dose dependant and 1000 &amp;micro;g/ml HA had the effective role on sperm parameters

The Expression of Heme Oxygenase-1 in Human-Derived Cancer Cell Lines

Background: Heme oxygenase-1 (HO-1) is a cytoprotective and antiapoptotic enzyme, which has been involved in maintaining cellular homeostasis, and plays an important protective role by modulating oxidative injury. Up-regulation of (HO-1) has contributed to tumorogenicity of some cancers. In this study we investigated the expression pattern of the HO-1, in five different human-derived cancer cell lines with high incidence in Iran. Methods: Total cell RNA were extracted from HepG2 (hepato carcinoma), A549 (lung adenocarcinoma), MCF-7 (breast cancer), K562 (myeloid leukemia) and LS174T (colon cancer) cell lines. Human embryonic kidney (HEK293) cell line was used as a control. cDNAs were synthesized and expression of HO-1 was examined using RT-PCR. Results: The expression of HO-1 was not detected in the control cell line (HEK293), but it was observed to express following ultraviolet (UV) exposure indicating that HO-1 is not constantly expressed. The examined cancer cell lines constitutively expressed different variety of HO-1 on mRNA level. Strong expression of HO-1 was observed in HepG2, MCF-7 and A549 cells. A moderate expression of HO-1 was observed in K562 cells, and LS174T cells showed no expression of HO-1. Conclusion: Heme oxygenase-1 could be considered as a new marker in the diagnosis of some cancers, especially hepatomacarcinoma. Our results also suggest that up-regulation of HO-1 may contribute to tumorogenicity of some cancers. Therefore, the combination of gene-silencing effect of HO-1 and chemo-therapy might be considered as a new modality for the treatment of cancers in which the expression HO-1 is up-regulated

The Lcn2-engineered HEK-293 cells show senescence under stressful condition

Objective(s): Lipocalin2 (Lcn2) gene is highly expressed in response to various types of cellular stresses. The precise role of Lcn2 has not been fully understood yet. However, it plays a key role in controlling vital cellular processes such as proliferation, apoptosis and metabolism. Recently it was shown that Lcn2 decreases senescence and increases proliferation of mesenchymal stem cells (MSC) with finite life span under either normal or oxidative stress conditions. However, Lcn2 effects on immortal cell line with infinite proliferation are not defined completely.  Materials and Material and Methods: HEK-293 cells were transfected with recombinant pcDNA3.1 containing Lcn2 fragment (pcDNA3.1-Lcn2). Expression of lipocalin2 in transfected cells was evaluated by RT-PCR, real time RT-PCR, and ELISA. Different cell groups were treated with H2O2 and WST-1 assay was performed to determine their proliferation rate. Senescence was studied by β-galactosidase and gimsa staining methods as well as evaluation of the expression of senescence-related genes by real time RT-PCR. Results: Lcn2 increased cell proliferation under normal culture condition, while the proliferation slightly decreased under oxidative stress.  This decrease was further found to be attributed to senescence. Conclusion: Our findings indicated that under harmful conditions, Lcn2 gene is responsible for the regulation of cell survival through senescence

HESA-A Exerts Its Cytoprotective Effects through Scavenging of Free Radicals: An in Vitro Study

Background: Natural medicines have been recently considered more reasonable for human use most notably due to their safety and tolerance. HESA-A is a marine-originated herbal medicine with a variety of healing effects. However, its exact biological mechanism is not clear. The present study aimed at the evaluation of the HESA-A antioxidant effect.Methods: Chinese hamster ovary (CHO) and human embryonic kidney (HEK293T) cells were treated with different concentrations of HESA-A and H2O2 followed by cell proliferation assays. The antioxidant effect of the HESA-A preparations was evaluated by an antioxidant assay kit.Results: The viability of CHO and HEK293T cells were about 89% following their incubation with 100 and 200 ng/ml HESA-A, respectively for 1.5 hrs. However, when the cells were incubated with concentrations of 300 ng/ml or more, the cell viability significantly decreased to 48% compare to the control cells. The cytotoxic effects of H2O2 were observed after 2 hrs of incubation of the HEK293T or CHO cells with 10 mM or 16 mM H2O2, respectively, while in the presence of HESA-A the cytotoxicity was significantly decreased. Antioxidant assay revealed that HESA-A scavenges free radicals. Conclusion: The findings indicate that HESA-A had cytoprotective effects in vitro, and that such an effect might be due to antioxidant properties

High Frequency Electromagnetic Field Induces Lipocalin 2 Expression in

Objective(s)Neutrophil gelatinase-associated lipocalin (NGAL/Lcn2), comprise a group of small extracellular proteins with a common β-sheet-dominated 3-dimensional structure. In the past, it was assumed that the predominant role of lipocalin was acting as transport proteins. Recently it has been found that oxidative stress induces Lcn2 expression. It has been also proved that electromagnetic field (EMF) produces reactive oxygen species (ROS) in different tissues. Expression of Lcn2 following exposure to electromagnetic field has been investigated in this study. Materials and MethodsBalb/c mice (8 weeks old) were exposed to 3 mT, 50 HZ EMF for 2 months, 4 hr/day. Afterwards, the mice were sacrificed by cervical dislocation and livers were removed. The liver specimens were stained with Haematoxylin- Eosin (H&E) and analyzed under an optical microscope. Total RNA was extracted from liver and reverse transcription was performed by SuperScript III reverse transcriptase with 1 µg of total RNA. Assessment of Lcn2 expression was performed by semiquantitative and real time- PCR.ResultsThe light microscopic studies revealed that the number of lymphocyte cells was increased compared to control and dilation of sinosoids was observed in the liver. Lcn2 was up-regulated in the mice exposed to EMF both in mRNA and protein levels.ConclusionTo the extent of our knowledge, this is the first report dealing with up-regulation of Lcn2 in liver after exposure to EMF. The up-regulation might be a compensatory response that involves cell defense pathways and protective effects against ROS. However, further and complementary studies are required in this regards