مجله سلول و بافت
سال ششم شماره 3 (پاییز 1394)

Aim: By combining Tet-inducible system and lentivirus vectors, we investigated the induction of Enhanced Green Fluorescent Protein (EGFP) reporter gene in poultry cells.
Material and the EGFP gene was placed under Tet-ON system and its induction was studied in liver cell line LMH. First, we transformed competent bacteria separately with an inducible lentivirus vector carrying EGFP and a second vector carrying Tet transactivator rtTA-M2 and prepared a purified maxi-prep stock of either plasmid DNA. Then, we used DNA-calcium phosphate co-precipitation method to co-transfect LMH cells with both plasmid stocks. In the next step, we added different concentrations of Tet analog doxycycline (DOX) to cell growth medium.
Increase of DOX concentration caused elevation of gene expression Within the first 24 hours after post-transfection, the expression of EGFP for 0, 0.01, 0.1 and 1 µg/ml of DOX was 0.4, 6.2, 10.3 and 16% respectively, and in the next 24 hours the expression changed to 6.4, 26, 28.3 and 29.7% respectively. However, treatment with the overdose of DOX caused the cell death.
Combination of Tet-inducible system originating from bacteria and recombinant lentivirus vectors designed for gene transfer to human cells can jointly promote transgene expression in poultry cells. The concentration of the inducer can directly influence the level of transgene induction.

Aim: In this study the effects of stress on induction of structural chromosomal abnormalities on L929 cell line using micronucleus assay on cytokinesis-blocked binucleated cells were investigated in vitro.
Material and L929 cells were cultured in DMEM containing 10% FBS. The cells were divided into four groups including; control, 2Gy gamma-irradiated cells, and cells treated with doses of 25, 50 and 100 µg/ml hydrocortisone, and cells co-treated with these three doses of hydrocortisone and irradiation. The treated cells were harvested, stained and chromosome abnormalities were scored using micronucleus assay.
Results showed that hydrocortisone did not induce micronuclei as compared to the control group. However, the frequency of micronuclei in cells co-treated with doses of hydrocortisone and irradiation was significantly higher than cells treated only with gamma irradiation (P According to the data of this study, stress hormones are not able to induce any chromosomal abnormalities; however, they are able to increase the cell susceptibility to clastogenic effects of irradiation.

Aim: The effect of chitosan (low molecular weight) as elicitor was evaluated on silymarin production and biochemical factors in hairy root cultures of Silybum marianum.
Material and Different concentrations of chitosan (0, 5, 10, 20 and 30 mg/50 ml of culture media) was added to hairy root cultures of S. marianum (30 days) and sampling were carried out after 12, 24, 48, 72, 96 and 120 hours. The silymarin level, activity of guaiacol peroxidase and ascorbate peroxidase as well as concentration of H2O2 were determined in the samples.
The results indicated that the highest dry weight was in cultures treated with 10 mg /50 ml culture chitosan after 48 and 96 h. Silymarin production was significantly increased from 0.37 in control to 1.19 mg g-1 dry weight in the treated samples, at which the increased level was 1.4-fold higher than the control. Guaiacol peroxidase was activated by chitosan and reaching a pick after 120 h (21.86 ∆OD g-1FW min-1) which was 2.2 fold higher than the control. The ascorbate peroxidase activity kept increasing until 96 h after elicitation (5.14 ∆ODg-1FW).
The results showed that the silymarin production has been promoted by chitosan (low molecular weight). Increased of enzymes activity occurs as a result of free radical elimination and represents a reaction to chitosan as a stress factor.

Aim: In the present study we examine the effect of C. mass L. seed extract on learning, memory and some serum parameters in alzheimer induced male mice.
Material and 48 male mice weighing 25-30 gram divided into six groups including 1- Saline-Saline, 2-STZ-Saline, 3- STZ- Extract 25 mg/kg, 4- STZ-Extract 50 mg/kg, 5- STZ-Extract 75 mg/kg, 6- Saline- Effective dosages of Extract (50 mg/kg). For inducing Alzheimer's disease i.c.v. injection of Streptozotocine (3 mg/kg) was used and learning as well as memory was evaluated through passive avoidance test model. All groups received C.mas seed extract or saline intraperitoneally for 3­weeks. Then they entered into learning testes. Finally all animals were killed and blood was taken from right ventricle. Level of glucose, Triglyceride, and total cholestrole in serum were measured. Also during treatment period, weights of the mice were measured at beginning of each week. The data were analyzed using unilateral variance analysis and Tukey test or Analysis of variance with repeated measures.
ICV administration of STZ significantly decreased memory (P0.05). Reduction of mice weight was Significant (P It seems that C. mas seed extract improved memory in disorders neuronal damage such as Alzheimer's due to reduction of risk factors in the blood and adjustment of the glucose and fatty acid metabolism.

Aim: To study the karyotype and chromosomal characteristics of Garra rufa from Semirom River (Karun River drainage) of Isfahan province.
Material and Seven specimens of Garra rufa was collected by a seine net and transferred live to the laboratory. After squashing of gill and head kidney tissues, the tissue was stained by Giemsa. The mitosis was triggered by PHA, followed by other routine procedures of karyology.
From the total 50 mitotic metaphases obtained from 7 individuals, 2n=48 had the highest frequency (52%), consisting of 34 metacentric, 12 submetacentric and 2 acro/telocentric chromosomes. The fundamental number of chromosome arms (NF) was 94. The total lengths of the smallest and largest chromosomes were 6.40 and 1.67 µm, respectively.
The diversity in chromosomal number, chromosomal formulae and the fundamental number of chromosome arms (NF), may indicate the presence of an interspecific polymorphism or the presence of several species in this species which need further investigations, especially from a molecular point of view.

Aim: In the present study the effect of oxidative stress resulted from hydrogen peroxide on some secondary metabolites and changes on intra cellular hydrogen peroxide has been explored.
Material and To induction of callus Periwinkle leaf was sterilized and put on MS medium. Calluses were treated by different concentrations (1, 5 and 10 µM) of hydrogen peroxide for 6 days. Total alkaloid, phenolic components and felavonoids as well as antioxidant potential on the basis of Iron reduction were determined. Data were analyzed using one way ANOVA.
Addition of hydrogen peroxide resulted in significant (P≤0.05) increase of intra cellular hydrogen peroxide compared to the control. Total alkaloids, flavonoids and phenols content showed that hydrogen peroxide caused a significant increase (P Hydrogen peroxide increased intra cellular levels of hydrogen peroxide. Secondary metabolites such as alkaloids and phenolic compounds and total flavonoid were boosted as well. Therefore tensions may be applied to increase them.

Aim: The purpose of this study was to investigate the effect of ultrasound on growth and some physiological parameters in cultures of unicellular alga Dunaliella salina.
Material and Ultrasound waves were applied with a frequency of 40 kHz, and 5W/Cm3 power, at the fourteenth day of subculture in a completely randomized design with 3 replications. Ultrasound exposure times to cells were 0, 2.5, 5 and 10 minutes. The parameters measured were: cell growth, total protein content, photosynthetic pigments, antioxidant potential, membrane lipid peroxidation, amount of phenolic compounds, flavonoids, anthocyanins, soluble sugars, beta-carotene and glycerol.
The results showed, due to increase of ultrasound irradiation time, cell growth and photosynthetic pigments were decreased. In contrast, total protein content, antioxidant potential, membrane lipid peroxidation, phenolic compounds, flavonoids, anthocyanins, beta-carotene and glycerol were increased. Maximum amount of beta-carotene (12.3 mg/l) and glycerol (13.5 mg/l) as the main metabolites were obtained at 10 minutes treatment.
It seems that ultrasound waves increased beta-carotene, and glycerol production of the cell by induction of defensive responses and secondary metabolism.

Aim: In this research morphological, cell culture, immunochemical and inductive properties of rat’s claw dermal cells were investigated.
Material and First, the toes and fingers of rat were amputated at the point of the last joint and the amputated parts were processed for histological studies. Moreover, the dermal tissue located beneath the claw’s plate was dissected and cultured in vitro. Cultured cells at passage two or three were transferred on the slide and treated with α-smooth muscle actin (ASMA) antibody. Finally the cultured cells were stained with DiI and implanted into the vibrissae semi-follicles and ears.
The rat’s claw dermal cells in terms of morphological, cell culture and expression of ASMA demonstrated similarity with dermal papilla cells of hair follicles. These cells showed a bipolar spindle-shaped morphology having an extracellular matrix rich in glycosaminoglycans. Majority of the cells expressed ASMA in cytoplasm with a higher intensity in lamelopodia. However, with respect to hair follicular dermal papilla cells, the claw dermal cells were not able to induce an ectopic epidermis to form the skin appendage.
The inductive capability which has been seen in hair follicle’s dermal papilla cells can not be generalized to dermal cells present in other skin appendages, including claw. Based on the results presented here, the inductive factors exist in hair follicle dermal papilla cells is not expressed in the claw dermal cells.

Aim: In this project, production of haploid parthenogenetic embryonic stem cells has been studied in presence of the ERK and TGF-β signaling pathway inhibitors.
Material and First the parthenogenetic blastocyst- produced with chemical activation of oocytes- was cultured in R2i culture condition (containing PD0325901 and SB431542 which inhibit ERK and TGF-β signaling pathways respectively). Then parthenogenetic embryonic stem cell lines was subsequently expanded and preserved in the same culture medium. Using immunofluorescent staining and qRT-PCR, expression of the pluripotent markers were evaluated. Moreover, differentiation potential of these cells was assessed by spontaneous differentiation and teratoma formation assay. Degree of haploid in this cell lines was determined by flowcytometry methods.
In R2i culture condition, about 50% of parthenogenetic embryos produce cell lines, out of which 10-13% are haploid. Established parthenogenetic stem cell lines demonstrate pluripotent stemness characteristics including morphology, passagability as well as expression of pluripotent specific genes in mRNA and protein levels. In addition, these cells were able to differentiate spontaneously to embryonic germ layers and form teratoma.
Inhibition of ERK and TGF-β signaling pathways can provide an appropriate condition for producing haploid stem cells from parthenogenetic embryos.

Aim: In this study the efficiency of lentiviral gene transfer and cytomegalovirus promoter mediated overexpression has been investigated. Furthermore, the effect of small molecule-mediated inhibition of epigenetic pathways on gene overexpression has been studied.
Mouse ES cells were transduced with different doses of lentiviruses harboring Green Fluorescent Protein (GFP) and the percentage of GFP expressing cells was quantified with flowcytometery. The persistence of GFP expression was assessed after 8 days of transduction. Using embryonic stem (ES) cells transduced with a lentiviral vector harboring pancreatic and duodenal 1 (Pdx1) gene, we studied the influence of 5-azacytidin (5-AZA), DZNep, and BIX01294 small molecules on the gene expression system.
Lentiviral mediated gene transfer to ES cells with 10 and 20 multiplicities of infection (MOI) resulted in more than 90 percent transgenesis. However, the expression of transgene showed a dramatic decrease during 8 days of post-transduction. Treatment with 5-AZA leads to a 2.5 folds increase in the transgene expression in a permissive culture medium. DZNep also elevated the transgene expression up to 26.5 and 5.9 folds in pluripotency and permissive media respectively. The expression of endogenous Pdx1 gene also increased following DZNep treated.
Lentiviral transduction with MOIs more than 10, is an efficient method for transgenesis of mouse ES cells. However, co-application of this method along with the CMV promoter leads to inactivation of the gene expression in long term. DZNep treatment results in reactivation of transgene expression but also can influence the expression of endogenous genes.