Cell Journal (Yakhteh)

1-8

Authors: Fariba Azimipour, Saeed Zavareh, Taghi Lashkarbolouki

Objective: The unfavorable effects of electromagnetic radiation (EMR) emitted by the cell phone on reproduction health are controversial. Metalloproteinases play a vital role in ovarian follicle development. This study was designed to investigate the effects of exposure to the cell phone on the gelatinolytic activity of in vitro cultured mouse pre-antral follicle. Materials and Methods: In this experimental study, pre-antral follicles were isolated from ovaries of immature mice (n=16) and cultured with or without exposure to the cell phone in talking mode for 60 minutes. The gelatinolytic activity was evaluated through the zymography method, as well as the gene expression of matrix metalloproteinases (MMPs) namely MMP-2 and -9 and tissue inhibitors of metalloproteinases (TIMPs) namely, TIMP-1 and -2 by the real-time polymerase chain reaction (PCR) method. Also, in parallel, the development of pre-antral follicles was assessed. Results: The maturation parameters of the cell phone-exposed pre-antral follicles were significantly lower compared with the control group (P<0.05). The gelatinolytic activity was significantly decreased in the cell phone-exposed preantral follicles compared with the control group (P<0.05). The relative mRNA expression of the MMP-2 gene was significantly (P<0.05) increased in the cell phone-exposed pre-antral follicles whereas the expression rate of the MMP-9 gene was considerably (P<0.05) reduced when compared with the control group. Conversely, the relative expression of the TIMP-1 was markedly (P<0.05) increased in the cell phone-exposed pre-antral follicles while the expression of the TIMP-2 was (P<0.05) significantly diminished in comparison with the control group. Conclusion: Exposure to the cell phone alters the growth and maturation rate of murine ovarian follicle through the changing in the expression of the MMP-2 and -9 genes, as well as the gelatinolytic activity.

17-22

Authors: Maryam Ranjpour, Saima Wajid, Swatantra Kumar Jain

Objective: Liver cancer is the third rank amongst the common malignancies, causing maximum death in the patients diagnosed with cancers. Currently available biomarkers are not enough sensitive for early diagnosis of hepatocellular carcinoma (HCC). This makes difficult management of HCC. With the aim of finding new generation of proteomic-based biomarkers, the represented study was designed to characterize the differentially expressed proteins at different stages of HCC initiation and at progression. This could lead to find potential biomarkers for early detection of HCC. Materials and Methods: In this experimental study, we report induction of HCC by administrating chemical carcinogens in male Wistar rats. Disease progression was monitored by histological evaluation. Serum proteomic analyses such as 2 dimensional (2D)-electrophoresis, MALDI-TOF-MS/MS and Western blot have been used to analyze and characterize the differentially expressed proteins during HCC development. Results: HCC initiation and tumorigenesis were observed at one and four months post carcinogen treatment, respectively. One of the differentially-expressed proteins, namely, cytosolic phospholipase A2 delta was significantly up-regulated at very early stage of HCC development. Its expression continued to increase during cancer progression and hepatotumorigenesis stages. Its elevated expression has been confirmed by Western blot analysis. Consistent to this, analyses of the sera in the clinically confirmed liver cancer patients showed elevated expression of this protein, further validating our experimental results. Conclusion: This study suggests that elevation in the expression of cytosolic phospholipase A2 delta is associated with progression of HCC.

30-39

Authors: Ommolbanin Asadpour, Fatemeh Rahbarizadeh

Objective: The purpose of this study was to develop multivalent antibody constructs via grafting anti-HER2 antibodies, including Herceptin and oligoclonal-variable domain of heavy chain antibodies (VHHs), onto liposome membranes to enhance antibody activity and compare their effect on phospholipase C (PLC) signaling pathway with control. Materials and Methods: In this experimental study, SKBR3 and BT-474 cell lines as HER2 positive and MCF10A cell line as normal cell were screened with anti-HER2 antibodies, including constructs of multivalent liposomal antibody developed with Herceptin and anti-HER2 oligoclonal-VHHs. To confirm the accuracy of the study, immunofluorescent assay, migration assay and immuno-liposome binding ability to HER2 were evaluated. Finally, the antibodies effect on PLCγ1 protein level was measured by an immunoassay method (ELISA). Results: In the present study, by using multivalent form of antibodies, we were able to significantly inhibit the PLCγ1 protein level. Interestingly, the results of migration assay, used for study the motility of different types of cell, shows correspondingly decreased number of immigrated cells in SKBR3 and BT-474 cell lines. Since MCF10A cells show no overexpression of HER2, as expected, the result did not show any change in PLCγ1 level. Moreover, immunofluorescent assay has confirmed high expression of HER2 in SKBR3 and BT-474 cell lines and low HER2 expression on MCF10A cell line. High binding of immuno-liposome to SKBR3 and BT-474 cells and low binding to MCF10A confirmed that in this study anti-HER2 antibodies have conserved binding ability to HER2 even after conjugation with liposome. Conclusion: PLCγ1 protein levels did indeed decrease after treatment with immuno-liposome form of compounds in both two tested cell lines, verifying the inhibition ability of them. Moreover, an elevated antibody activity is associated with liposomes conjugation suggesting that immuno-liposome may be a potential target for enhancing the antibody activity.

55-59

Authors: Nazanin Karimi, Homa Mohseni Kouchesfahani, Mohammad Hossein Nasr-Esfahani, Marziyeh Tavalaee, Abdolhossein Shahverdi, Hamid Choobineh

Objective: The aim of this blind randomised clinical trial study was to assess the clinical efficiency of combined density gradient centrifugation/Zeta (DGC/Zeta) sperm selection procedure compared to conventional DGC in infertile men candidates for intracytoplasmic sperm injection (ICSI). The literature shows that DGC/Zeta is more effective compared to DGC alone in selection of sperms with normal chromatin and improves the clinical outcome of the ICSI procedure. Therefore, this study re-evaluates the efficiency of DGC/Zeta in improving the clinical outcomes of ICSI in an independent clinical setting. Materials and Methods: In this randomized, single-blind, clinical trial, a total of 240 couples with male factor infertility and at least one abnormal sperm parameter were informed regarding the study and 220 participated. Based on inclusion and exclusion criteria, 103 and 102 couples were randomly allocated into the DGC/Zeta and DGC groups, respectively. ICSI outcomes were followed and compared between the two groups. Results: Although there was no significant difference in fertilization rate (P=0.67) between the DGC/Zeta and DGC groups, mean percentage of good embryo quality (P=0.04), good blastocysts quality (P=0.049), expanded blastocysts (P=0.007), chemical pregnancies (P=0.005) and clinical pregnancies (P=0.007) were significantly higher in the DGC/ Zeta group compared to DGC. In addition, implantation rate was insignificantly higher in DGC/Zeta compared to DGC (P=0.17). Conclusion: This is the second independent study showing combined DGC/Zeta procedure improves ICSI outcomes, especially the pregnancy rate, compared to the classical DGC procedure and this is likely related to the improved quality of sperm selected by the DGC/Zeta procedure (Registration number: IRCT20180628040270N1).

66-70

Authors: Afsaneh Goudarzi, Amir Amiri-Yekta

Objective: Acyl-CoA synthetase short-chain family member 2 (ACSS2) activity provides a major source of acetyl-CoA to drive histone acetylation. This study aimed to unravel the ACSS2 expression during mouse spermatogenesis, where a dynamic and stage-specific genome-wide histone hyperacetylation occurs before histone eviction. Materials and Methods: In this experimental study, ACSS2 expression levels during spermatogenesis were verified by Immunodetection. Testis paraffin-embedded sections were used for IHC staining with anti-H4 pan ac and anti-ACSS2. Co-detection of ACSS2 and H4K5ac was performed on testis tubular sections by immunofluorescence. Proteins extracts from fractionated male germ cells were subjected to western-blotting and immunoblot was probed with anti- ACSS2 and anti-actin. Results: The resulting data showed that the commitment of progenitor cells into meiotic divisions leads to a robust accumulation of ACSS2 in the cell nucleus, especially in pachytene spermatocytes (P). However, ACSS2 protein drastically declines during post-meiotic stages, when a genome-wide histone hyperacetylation is known to occur. Conclusion: The results of this study are in agreement with the idea that the major function of ACSS2 is to recycle acetate generated after histone deacetylation to regenerate acetyl-CoA which is required to maintain the steady state of histone acetylation. Thus, it is suggested that in spermatogenic cells, nuclear activity of ACSS2 maintains the acetate recycling until histone hyperacetylation, but disappears before the acetylation-dependent histone degradation.

75-84

Authors: Amir Bajouri, Zahra Orouji, Ehsan Taghiabadi, Abdoreza Nazari, Atefeh Shahbazi, Nasrin Fallah, Parvaneh Mohammadi, Mohammad Rezvani, Zahra Jouyandeh, Fatemeh Vaezirad, Zahra Khalajasadi, Mahshid Ghasemi, Aslan Fanni, Sara Haji Hosseinali, Ahad Alizadeh, Hossein Baharvand, Saeed Shafieyan, Nasser Aghdami

Objective: Recently, the promising potential of fibroblast transplantation has become a novel modality for skin rejuvenation. We investigated the long-term safety and efficacy of autologous fibroblast transplantation for participants with mild to severe facial contour deformities. Materials and Methods: In this open-label, single-arm phase IIa clinical trial, a total of 57 participants with wrinkles (n=37, 132 treatment sites) or acne scars (n=20, 36 treatment sites) who had an evaluator’s assessment score of at least 2 out 7 (based on a standard photo-guide scoring) received 3 injections of autologous cultured fibroblasts administered at 4-6 week intervals. Efficacy evaluations were performed at 2, 6, 12, and 24 months after the final injection based on evaluator and patient’s assessment scores. Results: Our study showed a mean improvement of 2 scores in the wrinkle and acne scar treatment sites. At sixth months after transplantation, 90.1% of the wrinkle sites and 86.1% of the acne scar sites showed at least a one grade improvement on evaluator assessments. We also observed at least a 2-grade improvement in 56.1% of the wrinkle sites and 63.9% of the acne scar sites. A total of 70.5% of wrinkle sites and 72.2% of acne scar sites were scored as good or excellent on patient assessments. The efficacy outcomes remained stable up to 24-month. We did not observe any serious adverse events during the study. Conclusion: These results have shown that autologous fibroblast transplantation could be a promising remodeling modality with long-term corrective ability and minimal adverse events (Registration Number: NCT01115634).

92-95

Authors: Mahtab Fattahi, Nahid Eskandari, Fattah Sotoodehnejadnematalahi, Vahid Shaygannejad, Mohammad Kazemi

Objective: Multiple sclerosis (MS) is an inflammatory disease resulting in demyelination of the central nervous system (CNS). T helper 17 (Th17) subset protects the human body against pathogens and induces neuroinflammation, which leads to neurodegeneration. MicroRNAs (miRNAs) are a specific class of small (~22 nt) non-coding RNAs that act as post-transcriptional regulators. The expression of the miR-326 is highly associated with the pathogenesis of MS disease in patients through the promotion of Th17 development. Recently, studies showed that disease-modifying therapies (DMTs) could balance the dysregulation of miRNAs in the immune cells of patients with relapsing-remitting MS (RRMS). Interferon-beta (IFN-β) has emerged as one of the most common drugs for the treatment of RR-MS patients. The purpose of this study was to evaluate the expression of the miR-326 in RRMS patients who were responders and nonresponders to IFN-β treatment. Materials and Methods: In this cross-sectional study, a total of 70 patients (35 responders and 35 non-responders) were enrolled. We analyzed the expression of the miR-326 in peripheral blood mononuclear cells (PBMCs) of RRMS patients at least one year after the initiation of IFN-β therapy. Real-time polymerase chain reaction (RT-PCR) was applied to measure the expression of the miR-326. Results: The results showed no substantial change in the expression of the miR-326 between responders and nonresponders concerning the treatment with IFN-β. Although the expression of the miR-326 was slightly reduced in IFN-β-responders compared with IFN-β-non-responders; however, the reduction of the miR-326 was not statistically significant. Conclusion: Overall, since IFN-β doesn’t normalize abnormal expression of miR-326, this might suggest that IFN-β affects Th17 development through epigenetic mechanisms other than miR-326 regulation.

106-114

Authors: Florian Strube, Manfred Infanger, Markus Wehland, Xenia Delvinioti, Alexander Romswinkel, Carlo Dietz, Armin Krau

Objective: Weightlessness simulation due to the simulated microgravity has been shown to considerably affect behavior of tumor cells. It is aim of this study to evaluate characteristics of human breast cancer cells in this scaffoldfree 3D culture model. Materials and Methods: In this experimental study, the cells were exposed to simulated microgravity in a randompositioning machine (RPM) for five days. Morphology was observed under phase-contrast and confocal microscopy. Cytofilament staining was performed and changes in expression level of cytofilament genes, proliferation/differentiation genes, oncogenes and tumor suppressor genes were detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR), followed by western blot confirmation. Results: After five days, distinct spheroid formation was observed. Rearrangement of the cytoskeleton into spherical shape was visible. VIM gene expression was significantly up-regulated for adherent cells and spheroids (3.3x and 3.6x respectively, P<0.05 each). RHOA also showed significant gene up-regulation for adherent cells and spheroids (3.2x and 3.9x respectively, P<0.05 each). BRCA showed significant gene up-regulation in adherent cells and spheroids (2.1x and 4.1x respectively, P<0.05 each). ERBB2 showed significant gene up-regulation (2.4x, P<0.05) in the spheroids, but not in the adherent cells. RAB27A showed no significant alteration in gene expression. MAPK) showed significant gene up-regulation in adherent cells and spheroids (3.2x, 3.0x, P<0.05 each). VEGF gene expression was down-regulated under simulated microgravity, without significance. Alterations of gene expressions could be confirmed on protein level for vimentin and MAPK1. Protein production was not increased for BRCA1, human epidermal growth factor receptor 2 (HER2) and VEGF. Contradictory changes were determined for RHOA and its related protein. Conclusion: Microgravity provides an easy-to handle, scaffold-free 3D-culture model for human breast cancer cells. There were considerable changes in morphology, cytoskeleton shape and gene expressions. Identification of the underlying mechanisms could provide new therapeutic options.

121-127

Authors: Ahmad Pourebrahim,Iraj Goldouzian,Ahmad Ramezani

Objective: The aim of this study is investigation of Stem cells Technology in The Light of Jurisprudential Documents. Materials and Methods: In this analytical-descriptive research, we collected the relevant data through a literature search. We have used PubMed, ScienceDirect, Google Scholar, Iranian databases like SID, Iran doc, Iranian law and also Islamic resources for this study. Results: There are so many controversies about safety of these cells and possible dangers for human body. As in Iran, laws of stem cells are not clear. Elimination of barriers requires drafting laws compatible with regional and cultural beliefs of Iranian people. Unfortunately, available laws could not keep up with the advances. Conclusion: Iran juridical system should conduct and restrict actions in the area of stem cells technology by gathering experts of different political, science, medicine, social and mindful who are familiar with law, according to notions of intellectual jurists and legislators, Islam and Shia religious.