Pathobiology Research
Year:2013 | Volume:16 | Issue:1|Papers Count:10

Objective: Cardiac cell differentiation with the help of miRNAs has recently opened a promising window for the restoration of myocardial infarction. Independent miR-1-2/133a-1 and miR-206/133b clusters are known to be expressed in cardiac and skeletal muscles, respectively. miR-133b differs from miR-133a by only one nucleotide. The sequence similarity of these two miRNAs suggests that they target the same pathways and similar mRNA targets. The present study seeks to determine if miR-133b is expressed during the cardiac cell differentiation and if its expression is in reverse correlation with the SRF and CCND2 (as potential target genes) expression patterns.Methods: Human cardiac progenitor cells were prepared from Royan Stem Cell Bank (RSCB) and differentiated into cardiomyocytes. To initiate differentiation, cells were treated with 5-azacytidine as a demethylation factor. Then, ascorbic acid and TGFB1 were added every other day and twice per week, respectively. Differentiation into cardiomyocytes was confirmed by immunocytochemistry (ICC), flow cytometry and realtime PCR for some of the cardiac marker genes. The expression profiles of hsa-miR-133b and two of its potential target genes were also analyzed during the cardiac differentiation.Results: Three weeks after the first differentiation induction, expression level of hsa-miR- 133b was approximately five times higher than early stage expression (p<0.05). During this process, the expression profile of SRF target gene was inversely correlated with hsamiR- 133b expression.Conclusion: It is known that SRF is critically involved in the cell cycle. Considering increased miR-133b and decreased SRF expression levels during the late stages of heart cell differentiation, here we speculate that elevated expression of miR-133b blocks SRF expression and decreases cardiomyocytes proliferation in order to induce differentiation with direct targeting of SRF. Taken together, our data suggest that miR-133b along with miR-133a may be involved in cardiomyocytes differentiation.

Objective: Development of high producing mammalian cell lines is a major bottleneck in manufacturing of recombinant therapeutic proteins. This study examines the effect of using the matrix attachment region from the human interferon beta gene in combination with promoter activation strategy with E1A 13S protein on human tissue plasminogen activator (t-PA) expression in Chinese hamster ovary (CHO) cells.Methods: The matrix attachment region was cloned in 3' and 5' flanking sides of the t- PA expression cassette in pTPA vector to generate pMTPA. After transfection of the cells with pTPA and pMTPA vectors, stable cell pools were developed and the t-PA expression level determined for each stable cell line. In the next step, E1A 13S expression plasmid was transfected to stable cell pools and t-PA titers were measured after 72 hours.Results: Integration of pTPA and pMTPA vectors in the CHO genome was confirmed by PCR analysis on genomic DNA of stable cell pools. Analysis of the t-PA expression level showed a three-fold enhancement in pMTPA transfected cells compared to pTPAcontaining cells. t-PA expression was further enhanced up to 1771 U/ml by transient expression of E1A 13S in pMTPA stable cell pools.Conclusion: These results have shown that incorporation of matrix attachment region in an expression vector in combination with promoter activation can effectively enhance recombinant protein expression levels in CHO cells.

Objective: The anti-cancer properties of curcumin, a poliphenol extract from the rhizome of curry, has been confirmed by many investigators. However, low levels of uptake, tissue distribution and rapid metabolism has limited its application as an anti-cancer drug. This study is aimed at increasing curcumin's water solubility due to a biodegradable, neutral and non-toxic micellar nano-carrier called dendrosome. This study intends to evaluate the role of dendrosomal-curcumin (DNC) in bladder cancer cell growth.Methods: We performed the MTT assay, flow cytometry and Annexin V-FLUOS (as an apoptosis detection kit) to evaluate cell death. The genetic mechanism of DNC-induced apoptosis was accomplished by a study of the relative expressions of OCT4A, OCT4B1, SOX-2 and Nanog using real-time PCR.Results: DNC-induced cell death complied with a time and dose-dependent paradigm in the 5637 cell line. Cell cycle analysis revealed that the number of cells increased in pre- G1 and gradually decreased in G1 and S phases. This showed the inhibitory property of dendrosomal-curcumin on DNA synthesis. Data from real-time PCR determined that expressions of OCT4A, OCT4B1, SOX-2 and Nanog could be related to 5637 cancer cell growth. Dendrosomal-curcumin significantly suppressed mRNA expression of the above mentioned genes (p<0.01).Conclusion: The data showed that DNC induced apoptosis by suppression of pluripotency genes in 5637 bladder cancer cells, which confirmed the useful characteristic of nano-drug in bladder cancer therapy.

Objective: Antioxidants are essential for sperm motility. Calligonum extract possesses the important antioxidants catechin and quercetin. This study investigates the effects of calligonum extract on sperm parameters and the rate of apoptosis in testes of aging male mice.Methods: We initially performed a dose response test with using three doses of calligonum (10 mg/kg, 30 mg/kg and 50 mg/kg). A total of 25 aging male mice (11-13 months) were divided into the following groups of five mice each: control, sham and three experimental groups. The experimental groups received IP injections of calligonum extract (10 mg/kg, 30 mg/kg, and 50 mg/kg) weekly for up to five weeks. The sham group received IP injections of DMSO. At the end of the injection period, mice were sacrificed and sperm parameters analyzed. To determine apoptosis in testes, we performed TUNEL staining.Results: Our results showed that after calligonum treatment, there were improved sperm parameters in the 30 mg/kg-treated group compared to the other groups (P£0.05).Conclusion: Calligonum extract (30 mg/kg) can improve sperm parameters and decrease apoptosis in the testes of aging male mice. This herbal extract can be employed as an antioxidant component for clinical usage.

Objectives: Controversial findings exist regarding the association between neonate gender and umbilical cord blood lipid levels. This study aims to compare the levels of lipids and lipoprotein B-100 in the umbilical cord blood of male and female newborns and assess the impact of these factors on neonatal anthropometric measurements.Methods: This cross-sectional study was performed on 75 healthy, term (34 male and 41 female) newborns. A total of 5 ml of umbilical cord blood was obtained immediately after delivery and analyzed on the same day to estimate lipid concentrations and apolipoprotein B-100. Additionally, we measured and recorded neonatal anthropometric indicators. The independent sample t-test was used for comparison of mean values in the two groups. The relationship of cord blood lipid profile with anthropometric data was assessed by the Pearson correlation test and multiple linear regression.Results: The cord blood from female newborns had higher levels of low density lipoprotein-cholesterol (LDL-C), high density lipoprotein-cholesterol (HDL-C) and total cholesterol (TC) compared to male newborns, whereas levels of lipoprotein B-100 and triglyceride (TG) were higher in males compared to female newborns. These differences were not statistically significant. In female newborns there was a significant inverse correlation between TG level and head circumference (P=0.038). In males, there was no statistically significant association between lipid levels and anthropometric parameters.Conclusion: Gender did not impact lipid and apolipoprotein B-100 levels in newborns. This study showed a significant inverse correlation between TG level and head circumference in female newborns.

Objective: Various approaches have been offered for resolution of pain resulting from spinal cord injuries. One approach is the use of herbal and natural products. In the present research, as a preliminary study, we investigate the effect of crocin on chronic pain induced by contusion in the rat spinal cord (SCI).Methods: We randomly divided female Wistar rats into five groups. Groups I and II were contused at the L1 level and immediately treated with crocin (50 mg/kg). These groups were sacrificed after 2 hours and 1 week, respectively. The remaining three groups consisted of group III (control group), group IV (treated with crocin and no contusion), and group V (the contused group that underwent no treatment). Groups III-V were sacrificed after one week. The mechanical behavioral test that used Von Frey hairs; the thermal behavioral test that used a hot-plate and the locomotor recovery test with Basso, Beattie and Bresnahn (BBB) scoring were conducted daily to evaluate the extent of injury and recovery of the rats. The calcitonin-gene related peptide (CGRP) was determined in the animals' plasma by the ELISA kit.Results: The results showed a significant increase in plasma CGRP of contused rats that significantly reduced following crocin treatment. The behavioral tests were not changed significantly due to this treatment.Conclusion: The present study shows the beneficial effects of crocin treatment that may occur by decreasing CGRP on chronic pain induced by SCI. This project is continuing using higher dose of crocin for longer time.

Objective: In this study we introduced an RGD-containing peptide of collagen IV origin that possesses potent cell adhesion and proliferation properties. This peptide was immobilized on a nanofibrous polycaprolactone/gelatin scaffold after which we analyzed human bone marrow-derived mesenchymal stem cells (hBMSCs) adhesion and proliferation on this peptide-modified scaffold.Methods: Nanofibrous scaffold was prepared by electrospinning. The peptide was synthesized by solid-phase peptide synthesis and immobilized on electrospun nanofibrous a polycaprolactone/gelatin scaffold by chemical bonding. Native and modified scaffolds were characterized with Scanning Electron Microscope (SEM) and Fourier-Transform Infra-red Spectroscopy (FTIR). Adhesion and proliferation of hBMSCs on native and modified scaffolds were analyzed by the Methylthiazol Tetrazolium (MTT) assay.Results: SEM images showed that electrospun scaffolds had homogenous morphology and were 312±89 nm in diameter. There was no significant difference in scaffold morphology before and after peptide immobilization. FTIR results showed that the peptide was successfully immobilized on the scaffold. Based on MTT assay, cell adhesion studies indicated that peptide immobilization improved cell adhesion on RGD-modified scaffolds at all corresponding time points (p<0.05). RGD immobilization led to increased cell proliferation potential of the scaffold compared with tissue culture plate and native scaffold (P<0.05).Conclusion: This novel peptide and modified nanofibrous scaffold, having improved cell adhesion and proliferation properties, can be used for tissue engineering and regenerative medicine by using hBMSCs.

Objective: In recent decades, b-glucans have been used as important complementary and alternative medicines for numerous immunocompromised individuals and those with advanced cancer. The most active form of b-glucans is b (1, 3) D-glucan and its most common source is cell wall of Candida albicans. Recently it has been introduced as a nano particle design to be used as a carrier for drug delivery. The current study researches a rapid method for the extraction of b (1, 3) D-glucans.Methods: The present study was conducted at Tarbiat Modares Medical University in 2012. Candida solubilized b-glucans were obtained by oxidation of the cell wall with sodium hypochlorite and sodium hydroxide. The particle part could be solubilized by treatment with dimethylsulfoxide (DMSO) and zymolyase digestion to extract b (1, 3)Dglucan. The soluble fractions were lyophilized. We performed the Callose test to verify the presence of b (1, 3) D-glucans. Solubilized fractions were dissolved in D2O and 1HNMR spectra were measured.Results: The soluble b (1, 3) D-glucan fraction which was derived from 1 g of dried Candida albicans germ tube weighed 190 mg. b (1, 3) D-glucan was verified by the Callose test and 1H-NMR test compared with Curdlan (standard). 1H-NMR spectra verified the existence of b (1, 3) D-glucan in the final product.Conclusion: In the present study, extraction of b (1, 3) D-glucan by oxidation of the cell wall using sodium hypochlorite yielded more pure b (1, 3) D-glucans in comparison with other extraction methods. Thus it might represent a rapid method of extraction.