Cell and Tissue Journal

Abstract Aim: Cancer remains a global health problem, with ovarian cancer ranking fifth among cancers affecting women and the leading cause of cancer-related death in women. There are different types pf ovarian cancer, including Epithelial ovarian cancer, Stromal tumors and Germ cell tumors.  Epithelial ovarian cancer is the most common type that includes several subtypes, including serous carcinoma and mucinous carcinoma. To this end, several factors have been identified to increase your risk of ovarian cancer, including older age, inherited gene changes, family history of ovarian cancer, being overweight or obese, postmenopausal hormone replacement therapy, endometriosis, and never having been pregnant. The current strategies to treat ovarian cancer include surgery, chemotherapy, radiotherapy and targeted therapies, and hormone therapy. Scientists continue to investigate the foundational mechanisms involving cancer development, as well as to find new drugs and metabolites having the capacity to control cancer. Alpha-ketoglutarate (AKG), a critical metabolite in the Krebs cycle involved in cellular energy production and the regulation of gene expression. Recent studies have shown that AKG may have the potential to enhance the efficacy of cancer treatments, by modulating the tumor microenvironment and improving the immune response against cancer cells. This study investigates the effect of AKG on ovarian cancer cells.
Material and methods: SKOV3 cells were obtained from Tehran University and cultured in complete culture medium (RPMI, 10% Fetal bovine serum (FBS), and 1% Penicillin-Streptomycin (Pen/Strep)). To find the proper concentration of AKG, SKOV3 cells were cultures in 96-well plate, and treated with different concentration of AKG (range 20 to 220 µM). After 24 and 48 h, the viability of the cells was determined using MTT assay. Based on the results obtained from viability assay, 200 μM of AKG was selected for the next assessments. To evaluate the effect of AKG on SKOV3 cell proliferation using plotting a growth curve, cells were cultured in the presence (200 μM AKG) and absence of AKG, and counted the number of cells every 24h for one week. To determine the population doubling time (PDT), the cells were cultured in the presence (200 μM AKG) and absence of AKG for 72h, then the cell were collected and the number of living cells was counted using Neubauer Chamber. The PDT was calculated using a related standard method. To assess colony formation potential, the SKOV3 cell were cultured in the presence (200 μM AKG) and absence of AKG. After 7 days, the cells were fixed using 10% formalin solution, then the colonies were stained using crystal violet dye, and the number of colonies were counted using inverted microscope. To investigate the effect of AKG on the migration rate of SKOV3 cells, the cells were cultured in complete medium to reach 85% confluence, then treated with mitomycin (10 μM) for 3h, then Created a scratch in the cell monolayer using a sterile pipette tip. the cells were cultured in the presence (200 μM AKG) and absence of AKG for 3 days. The images of the scratch were taken at regular intervals using a microscope, and the closure of the scratch over time was analyzed. To analyze the cell cycle profile, the SKOV3 cell were cultured in the presence (200 μM AKG) and absence of AKG for 48h. Then, the cells were collected and analyzed using a flow cytometry.
Results: Based on the MTT assay, 200 μM AKG was determined as the proper concentration to investigate other biological behaviors of SKOV3 cells. Colony forming assay showed a decrease in the number and size of colonies in the AKG-treated group (P<0.05). In addition, the cell doubling time increased in the treatment group, indicating slower growth rate (P<0.05). Growth curve analysis confirmed reduced cell growth in treated group. Cell cycle analysis showed a higher percentage of treated cells arrested in S and G1 phases. The scratch assay showed slow cell migration and metastasis in the cells treated with 200 µM AKG.
Conclusion: In conclusion, AKG has an inhibitory effect on the proliferation, viability, migration in SKOV3 ovarian cancer cells, highlighting its potential as an adjuvant treatment with existing therapies. More research is necessary to fully investigate the therapeutic effect of AKG in ovarian cancer.
 

Abstract Aim: This study aimed to access the rapid and improved propagation protocol production of Zamifolia plant using tissue culture.
Material and Methods: For callus formation and regeneration, two independent factorial experiments were carried out in a completely randomized design with 3 replications in the tissue culture laboratory of the National Research Institute of Flowers and Ornamental Plants in 2018. In the first experiment, to induce callogenesis, 2,4-D (0, 1, 2, and 4 mg/l) and BA (0, 1 and 2 mg/l) were used. In the second experiment, or regeneration of explants, NAA (0 and 0.5 mg/l) and BA (0, 0.5, and 1 mg/l) were applied.
Results: The results of the callogenesis experiment showed that a modified basal MS medium with concentration of 2 mg/l BA and 1 mg /l 2,4-D produced the best callus with 85% regeneration. At the regeneration stage of the callus, 0.5 mg/l BA and 0.5 mg NAA produced the highest number of seedlings and regenerated tubers (90%). Subsequently, single tuber seedlings were successfully adapted to 80%-90% in the cocopeat and perlite substrates.
Conclusion: The results were successful and could be possible to achieve the callogenesis and regeneration of Zamifolia plant with the formula presented and recommend as practical a protocol for commercial micropropagation of this valuable ornamental plant.
 

Abstract Aim: In this study, the effects of recombinant human growth hormone and Gemcitabine alone and in combination with each other were investigated on the lung fibroblast cell lines.
Material and methods: Human growth hormone at 10-400 ng/ml and Gemcitabine at 1-100 μg/ml concentrations were treated with the human lung fibroblast cells and the analysis were performed with MTT assay, propidium iodide staining and scratch assay.
Results: Studies of the cell cycle and MTT assay revealed the effect of growth hormone on the progression of the cell cycle and exit of G1 phase and the cell proliferation, and the inhibitory effects of the cell cycle and growth process by Gemcitabin compared with the control sample. The results of the scratch test indicated that the growth hormone at its effective concentrations caused increased the cell migration compared to control, while the Gemcitabin reduced the migration of the cell to the control.
Conclusions: Using growth hormone in combination to gemcitabin during chemotherapy treatment, it seems that we can reduce the rate of the neem normal cells death. Since these cells are more sensitive to growth hormone´s effect than cancer cells.
 

Abstract Aim: In the current study, CM anticancer function of the MCF7 cells was investigated in vitro.
Material and Methods: Adipose stem cells were extracted from the cesarean women’s abdominal fat with their written informed consent at the Velayat hospital, Damghan. CM was prepared for fourth passage of hASCs that was cultured in serum-free medium for 72h. MCF7 cells were exposed to CM for 24 and 48 hours. Then cell proliferation rate, survival and apoptotic gene expression were determined using MTT, cell counting (hemocytometer) and RT-PCR.
Results: CM-treated MCF7 for 24 and 48h, showed a significant decrement in cell proliferation and viability as compared to the cells cultured in medium containing serum (control). In addition, caspase3 gene expression of CM-induced cells was increased significantly at 24 and 48h as compared to the control group. While, CM-treated cells showed a significant increment of caspase9 gene expression after 48h induction.
Conclusion: It was concluded that CM induced apoptosis by activation of caspases and reduced proliferation rate and survival of MCF7 cancer cells. Therefore, it seems that conditioned medium can be used as a supplement with other anti-cancer therapies.

Abstract Aim: The aim of this study was study the role of Resveratrol (RSV) on proliferation and colony efficiency of dissociated human embryonic stem cells (hESCs).
Material and methods: In the current study, we used HESC line, RH6. Cells were cultured on the matrigelin supplemented hESC specific medium. Cell proliferation was estimated using cell counting and Brdu incorporation assays. The expression of colony efficiency markers (E-cadherin and β-catenin) was evaluated by western blot technique and β-catenin localization examined by immune-cytochemistry staining.
Results: We have shown that RSV promoted hPSCs proliferation without affecting their colony efficiency.
Conclusion: According to these observations, RSVcan be suggested as a new supplement for hESCs culture.
 
 

Abstract Aim: The aim of this study was to investigate the effects of different treatments of growth regulators on the amount of shooting, rooting, callus production and plant regeneration of hairless catmint (Nepeta nuda), in vitro condition.
Material and Methods: The seeds of this plant were cultured in MS/2, and then the plantlets were sub-cultured. The obtained samples were cultured in different levels of BA and Kinetin, either alone or in combination with IBA. Leaves of explants were used for callus induction with BA and NAA treatments in both medium (light and dark). Also, for regeneration, the same calluses were used with BA and NAA treatments.
Results: The results of this study showed that, in proliferation part, the highest fresh weight (2315.9 mg) and shoot number (15.22 per each bush) of each plantlet were obtained in 1 mgL-1 BA and 0.25 mgL-1 IBA. Moreover, the BA treatment with 2 mgL-1 concentration cause to produce the longest branches (50.5 cm) and the highest number of nodes (53.6 per plantlet) in each plantlet.
The highest length, number and percentage of root were obtained at MS/2 medium and 0.5 mgL-1 IBA. In callus formation part, the highest fresh weight of callus was related to application of 1 mgL-1 BA with 0.5 mgL-1 NAA. In addition, the highest percentage of callus formation was observed in dark medium and no significant differences were observed between the growth regulator treatments. Furthermore, the highest percentage of regeneration of callus related to both growth regulators treatments included 1 mgL-1  BA with 0.2 mgL-1 NAA (83.2 %) and 1 mgL-1  BA with 0.5 mgL-1 NAA (81.66 %) with no significant differences between them.
Conclusion: In total, the most useful growth regulator compound for micro-propagation of hairless catmint, which is endangered extinction species, were 1 mgL-1 BA with 0.5 mgL-1 IBA and NAA.

Abstract Aim: The aim of the present study was to evaluate the effects of different concentrations of Chir99021 on ovine fetal marrow-derived mesenchymal stem cells (BM-MSCs) expansion in culture.  Material and Methods: BM-MSCs were isolated from ovine fetal and cultured. Passaged-3 cells were examined for their differentiation potential into osteocytes and adipocytes. In the present study, BM-MSCs from ovine fetal were plated in the presence of 0, 0.5, 1, 1.5, 3 and 5 μM of Chir9902. During the cultivation period, the cultures were statistically compared in terms of induces of cell growth including the number of colonies, population doubling number (PDN), doubling time (DT) and the number of viable cells. Furthermore, expression of the beta-catenin was evaluated. The culture without Chir99021 was taken as the control group.  Results: Our findings indicated that, addition of 0.5 and 1 µM of Chir99021 to medium significantly improved overall proliferation compared to that of control group as well as 5µM of Chir99021(p < 0.05). Furthermore, Five days after treatment with small molecule showed that the treatments with 0.5 and 1 µM Chir99021 formed higher Colony Forming Unit-Fibroblast (CFU-F) compared to control and 5 µM of Chir99021. The expression of beta-catenin was significantly higher in culture supplemented with 1 μM of Chir99021 compared to that in groups containing 0.5 and 1.5 μM (p < 0.05). Conclusion: In conclusion, using Chir99021 at concentration of 1 μM could enhance in vitro proliferation of ovine fetal BM-MSC; however administration of high concentration of this small molecule had toxic effects on these cells proliferation.

Abstract Aim: Appendicitis is one of the most common abdominal inflammatory diseases. The function of appendix is not clearly defined. In this study to understand the immunological function of the appendix, we investigated the function and phenotypic pattern of lymphocyte in appendix of patients with normal and inflamed appendix tissue.
Material and Methods: Appendix tissue and appendiceal mononuclear cells obtained from 81 patients (mean age; 23±10.5), who was clinicallysuspected of having appendicitis were collected. Based on histopathological examination, twenty-five patients had normal appendix while 40 and 16 were diagnosed with suppurative and gangrene appendicitis, respectively. The phenotypic characteristics of lymphocyte subsets in appendix was analyzed using three color-flow cytometry. In addition, the proliferative response of tissue mononuclear cells was assayed by MTT method.
Results: A significant difference (p < 0. 01) was observed  in the percentage of CD19/HLA-DR, HLA-DR and CD19 cellsfrom patients suffering from suppurative appendicitis in comparison with the patients having normal appendix or gangrene one.  There was also a significant difference (p < 0.01) in the proliferative responses of the appendiceal mononuclear cells to PHA and LPS from pathients with suppurative appendicitis when compare with other groups.
Conclusion: The phenotypic and function of lymphocytes are different between normal and inflamed appendix tissue. These results showed that the appendix tissue with special lymphocyte profile may be effective in preventing or reducing intestinal infections.
 

Abstract Aim: During recent years many studies have suggested some developmental roles for embryonic cerebrospinal fluid. Here we examined the effect of embryonic cerebrospinal fluid on proliferation and self-renewal of embryonic ventricular zone derived neurosphere.
Material and Methods: Cortex from 15.5 day old embryonic Wistar rat dissected out and enzymaticaly dissociated to form single cell suspension. Cell suspension seeded in DMEM/F12 medium supplemented with N2 and mitogenes (10ng.ml^-1 EGF and 20ng.ml^-1 bFGF). The CSF-treated culture from different embryonic ages (E16, E18, and E20) in 10/100 ratio (v/v) was considered as experimental groups. Neurospheres number was counted for proliferation assay and cell viability evaluated with MTT assay. The data were analyzed with One-way ANOVA with turkey’s post hoc test.
Results: Embryonic cerebrospinal fluid (e-CSF) enhanced neurosphere number. Also, embryonic cerebrospinal fluid (e-CSF) enhanced cell viability and growth.
Conclusion: Embryonic cerebrospinal fluid (e-CSF) enhanced proliferation and viability of neural progenitor cells in age dependent manner. Moreover, this fluid play important role in establishment of neuroprogenitor cells self-renewal.