Pub Date : 2019-10-14DOI: 10.22074/cellj.2020.6313
Maryam Fakhimi, Abdolrassul Talei, A. Ghaderi, M. Habibagahi, M. Razmkhah
Objective Mesenchymal stem cells (MSCs) have prominent immunomodulatory roles in the tumor microenvironment. The current study intended to elucidate Treg subsets and their cytokines after exposing naïve T lymphocytes to adipose- derived MSCs (ASCs). Materials and Methods In this experimental study, to obtain ASCs, breast adipose tissues of a breast cancer patient and a normal individual were used. Magnetic cell sorting (MACS) was employed for purifying naïve CD4+T cells from peripheral blood of five healthy donors. Naïve CD4+T cells were then co-cultured with ASCs for five days. The phenotype of regulatory T cells (Tregs) and production of interleukine-10 (IL-10), transforming growth factor beta (TGF-β) and IL-17 were assessed using flow cytometry and ELISPOT assays, respectively. Results CD4+CD25-FOXP3+CD45RA+Tregs were expanded in the presence of cancer ASCs but CD4+CD25+Foxp3+CD45RA+regulatory T cells were up-regulated in the presence of both cancer- and normal-ASCs. This up-regulation was statistically significant in breast cancer-ASCs compared to the cells cultured without ASCs (P=0.002). CD4+CD25+ FOXP3+Helios+, CD4+CD25-FOXP3+Helios+and CD25+FOXP3+CD73+CD39+Tregs were expanded after co-culturing of T cells with both cancer-ASCs and normal-ASCs, while they were statistically significant only in the presence of cancer-ASCs (P<0.05). Production of IL-10, IL-17 and TGF-β by T cells was increased in the presence of either normal- or cancer-ASCs; however, significant effect was only observed in the IL-10 and TGF-β of cancer-ASCs (P<0.05). Conclusion The results further confirm the immunosuppressive impacts of ASCs on T lymphocytes and direct them to specific regulatory phenotypes which may support immune evasion and tumor growth.
{"title":"Helios, CD73 and CD39 Induction in Regulatory T Cells Exposed to Adipose Derived Mesenchymal Stem Cells","authors":"Maryam Fakhimi, Abdolrassul Talei, A. Ghaderi, M. Habibagahi, M. Razmkhah","doi":"10.22074/cellj.2020.6313","DOIUrl":"https://doi.org/10.22074/cellj.2020.6313","url":null,"abstract":"Objective Mesenchymal stem cells (MSCs) have prominent immunomodulatory roles in the tumor microenvironment. The current study intended to elucidate Treg subsets and their cytokines after exposing naïve T lymphocytes to adipose- derived MSCs (ASCs). Materials and Methods In this experimental study, to obtain ASCs, breast adipose tissues of a breast cancer patient and a normal individual were used. Magnetic cell sorting (MACS) was employed for purifying naïve CD4+T cells from peripheral blood of five healthy donors. Naïve CD4+T cells were then co-cultured with ASCs for five days. The phenotype of regulatory T cells (Tregs) and production of interleukine-10 (IL-10), transforming growth factor beta (TGF-β) and IL-17 were assessed using flow cytometry and ELISPOT assays, respectively. Results CD4+CD25-FOXP3+CD45RA+Tregs were expanded in the presence of cancer ASCs but CD4+CD25+Foxp3+CD45RA+regulatory T cells were up-regulated in the presence of both cancer- and normal-ASCs. This up-regulation was statistically significant in breast cancer-ASCs compared to the cells cultured without ASCs (P=0.002). CD4+CD25+ FOXP3+Helios+, CD4+CD25-FOXP3+Helios+and CD25+FOXP3+CD73+CD39+Tregs were expanded after co-culturing of T cells with both cancer-ASCs and normal-ASCs, while they were statistically significant only in the presence of cancer-ASCs (P<0.05). Production of IL-10, IL-17 and TGF-β by T cells was increased in the presence of either normal- or cancer-ASCs; however, significant effect was only observed in the IL-10 and TGF-β of cancer-ASCs (P<0.05). Conclusion The results further confirm the immunosuppressive impacts of ASCs on T lymphocytes and direct them to specific regulatory phenotypes which may support immune evasion and tumor growth.","PeriodicalId":9692,"journal":{"name":"Cell Journal (Yakhteh)","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-10-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"73540548","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-10-14DOI: 10.22074/cellj.2020.6619
H. Azizi, M. Ranjbar, S. Rahaiee, M. Govahi, T. Skutella
Objective We aimed to examine the expression levels of the VASA gene and protein in testis sections of neonate and adult mice as well as testicular cell cultures. Materials and Methods In this experimental study, in order to investigate the expression of this germ cell marker gene in more detail, we analyzed the expression of VASA by immunocytochemistry, immunohistochemistry and fluidigm reverse transcription-polymerase chain reaction (RT-PCR). Results The immunohistochemical assays showed that the VASA protein was exclusively expressed in germ cells in the seminiferous tubules of the neonate and adult testis and not in somatic cells. VASA was not detectable in PLZF positive spermatogonial stem cells (SSCs), was weakly expressed in proliferating spermatogonia, and became abundant in spermatocytes and round spermatozoa. Counting VASA-positive cells in the seminiferous tubules of the neonate and adult testis depicted significant higher expression (P<0.05) of VASA in the adult testis in comparison to its neonate counterpart. SSC colonies were established in vitro after digestion of the testis and characterized by immunocytochemistry for CD90 and stage-specific embryonic antigens 3 (SSEA3). Immunocytochemistry confirmed that in contrast to the not detectable signal in vivo, VASA protein was strongly localized in the cytoplasm of both neonate and adult mouse SSCs under in vitro conditions. The results of Fluidigm RT-PCR revealed a significant higher expression of the germ cell gene VASA in adult SSCs in comparison to neonate SSCs in cell culture (P<0.05). Conclusion The VASA protein is, therefore, an extremely specific marker of testicular germ cell differentiation in vivo and mostly expressed in the adult testis in spermatocytes and round spermatids. The immunohistochemical signal in spermatogonia is very low. So, PLZF positive SSCs are negative for VASA in vivo, while in contrast, once isolated from the testicular niche VASA is also strongly expressed in SSCs under in vitro conditions.
{"title":"Investigation of VASA Gene and Protein Expression in Neonate and Adult Testicular Germ Cells in Mice In Vivo and In Vitro","authors":"H. Azizi, M. Ranjbar, S. Rahaiee, M. Govahi, T. Skutella","doi":"10.22074/cellj.2020.6619","DOIUrl":"https://doi.org/10.22074/cellj.2020.6619","url":null,"abstract":"Objective We aimed to examine the expression levels of the VASA gene and protein in testis sections of neonate and adult mice as well as testicular cell cultures. Materials and Methods In this experimental study, in order to investigate the expression of this germ cell marker gene in more detail, we analyzed the expression of VASA by immunocytochemistry, immunohistochemistry and fluidigm reverse transcription-polymerase chain reaction (RT-PCR). Results The immunohistochemical assays showed that the VASA protein was exclusively expressed in germ cells in the seminiferous tubules of the neonate and adult testis and not in somatic cells. VASA was not detectable in PLZF positive spermatogonial stem cells (SSCs), was weakly expressed in proliferating spermatogonia, and became abundant in spermatocytes and round spermatozoa. Counting VASA-positive cells in the seminiferous tubules of the neonate and adult testis depicted significant higher expression (P<0.05) of VASA in the adult testis in comparison to its neonate counterpart. SSC colonies were established in vitro after digestion of the testis and characterized by immunocytochemistry for CD90 and stage-specific embryonic antigens 3 (SSEA3). Immunocytochemistry confirmed that in contrast to the not detectable signal in vivo, VASA protein was strongly localized in the cytoplasm of both neonate and adult mouse SSCs under in vitro conditions. The results of Fluidigm RT-PCR revealed a significant higher expression of the germ cell gene VASA in adult SSCs in comparison to neonate SSCs in cell culture (P<0.05). Conclusion The VASA protein is, therefore, an extremely specific marker of testicular germ cell differentiation in vivo and mostly expressed in the adult testis in spermatocytes and round spermatids. The immunohistochemical signal in spermatogonia is very low. So, PLZF positive SSCs are negative for VASA in vivo, while in contrast, once isolated from the testicular niche VASA is also strongly expressed in SSCs under in vitro conditions.","PeriodicalId":9692,"journal":{"name":"Cell Journal (Yakhteh)","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-10-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"90724829","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-09-08DOI: 10.22074/cellj.2020.5694
A. Pourebrahim, I. Goldouzian, A. 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.
目的从法学文献的角度对干细胞技术进行研究。在本分析描述性研究中，我们通过文献检索收集相关资料。我们使用了PubMed, ScienceDirect, Google Scholar，伊朗数据库，如SID，伊朗doc，伊朗法律和伊斯兰资源进行这项研究。结果这些细胞的安全性和对人体的潜在危害存在诸多争议。就像在伊朗一样，有关干细胞的法律并不明确。消除障碍需要起草符合伊朗人民区域和文化信仰的法律。不幸的是，现有的法律跟不上进步的步伐。伊朗司法系统应该根据知识分子法学家和立法者、伊斯兰教和什叶派宗教的观念，聚集熟悉法律的不同政治、科学、医学、社会和思想领域的专家，开展和限制干细胞技术领域的行动。
{"title":"Investigation of Stem cells Technology in The Light of Jurisprudential Documents","authors":"A. Pourebrahim, I. Goldouzian, A. Ramezani","doi":"10.22074/cellj.2020.5694","DOIUrl":"https://doi.org/10.22074/cellj.2020.5694","url":null,"abstract":"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.","PeriodicalId":9692,"journal":{"name":"Cell Journal (Yakhteh)","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"84329439","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-09-08DOI: 10.22074/cellj.2020.6372
Z. Salehi, M. Shams-Ghahfarokhi, M. Razzaghi-Abyaneh
Objective Precise identification of dermatophyte species significantly improves treatment and controls measures of dermatophytosis in human and animals. This study was designed to evaluate molecular tools effectiveness of the gene sequencing and DNA-based fragment polymorphism analysis for accurate identification and differentiation of closely- related dermatophyte species isolated from clinical cases of dermatophytosis and their antifungal susceptibility to the current antifungal agents. Materials and Methods In this experimental study, a total of 95 skin samples were inoculated into mycobiotic agar for two weeks at 28˚C. Morphological characteristics of the isolated dermatophytes were evaluated. DNA was extracted from the fungal culture for amplification of topoisomerase II gene fragments and polymerase chain reaction (PCR) products were digested by Hinf I enzyme. Internal transcribed spacer (ITS) rDNA and TEF-1α regions of the all isolates were amplified using the primers of ITS1/4 and EF-DermF/EF-DermR, respectively. Results Based on the morphological criteria, 24, 24, 24 and 23 isolates were identified as T. rubrum, T. interdigitale, T. tonsurans and E. floccosum, respectively. PCR-restriction fragment length polymorphism (RFLP) results provided identification pattern of the isolates for T. rubrum (19 isolates), T. tonsurans (28 isolates), T. interdigitale (26 isolates) and E. floccosum (22 isolates). Concatenated dataset results were similar in PCR-RFLP, except six T. interdigitale isolates belonging to T. mentagrophytes. Conclusion Our results clearly indicated that conventional morphology and PCR-RFLP were not able to precisely identify all dermatophyte species and differentiation of closely related species like T. interdigitale and T. mentagrophytes, while ITS rDNA and TEF-1α gene sequence analyses provided accurate identification of all isolates at the genus and species level.
{"title":"Internal Transcribed Spacer rDNA and TEF-1α Gene Sequencing of Pathogenic Dermatophyte Species and Differentiation of Closely Related Species Using PCR-RFLP of The Topoisomerase II","authors":"Z. Salehi, M. Shams-Ghahfarokhi, M. Razzaghi-Abyaneh","doi":"10.22074/cellj.2020.6372","DOIUrl":"https://doi.org/10.22074/cellj.2020.6372","url":null,"abstract":"Objective Precise identification of dermatophyte species significantly improves treatment and controls measures of dermatophytosis in human and animals. This study was designed to evaluate molecular tools effectiveness of the gene sequencing and DNA-based fragment polymorphism analysis for accurate identification and differentiation of closely- related dermatophyte species isolated from clinical cases of dermatophytosis and their antifungal susceptibility to the current antifungal agents. Materials and Methods In this experimental study, a total of 95 skin samples were inoculated into mycobiotic agar for two weeks at 28˚C. Morphological characteristics of the isolated dermatophytes were evaluated. DNA was extracted from the fungal culture for amplification of topoisomerase II gene fragments and polymerase chain reaction (PCR) products were digested by Hinf I enzyme. Internal transcribed spacer (ITS) rDNA and TEF-1α regions of the all isolates were amplified using the primers of ITS1/4 and EF-DermF/EF-DermR, respectively. Results Based on the morphological criteria, 24, 24, 24 and 23 isolates were identified as T. rubrum, T. interdigitale, T. tonsurans and E. floccosum, respectively. PCR-restriction fragment length polymorphism (RFLP) results provided identification pattern of the isolates for T. rubrum (19 isolates), T. tonsurans (28 isolates), T. interdigitale (26 isolates) and E. floccosum (22 isolates). Concatenated dataset results were similar in PCR-RFLP, except six T. interdigitale isolates belonging to T. mentagrophytes. Conclusion Our results clearly indicated that conventional morphology and PCR-RFLP were not able to precisely identify all dermatophyte species and differentiation of closely related species like T. interdigitale and T. mentagrophytes, while ITS rDNA and TEF-1α gene sequence analyses provided accurate identification of all isolates at the genus and species level.","PeriodicalId":9692,"journal":{"name":"Cell Journal (Yakhteh)","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"73994837","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-09-08DOI: 10.22074/cellj.2020.6704
O. Asadpour, F. 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.
{"title":"Phospholipase-Cγ1 Signaling Protein Down-Regulation by Oligoclonal-VHHs based Immuno-Liposome: A Potent Metastasis Deterrent in HER2 Positive Breast Cancer Cells","authors":"O. Asadpour, F. Rahbarizadeh","doi":"10.22074/cellj.2020.6704","DOIUrl":"https://doi.org/10.22074/cellj.2020.6704","url":null,"abstract":"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.","PeriodicalId":9692,"journal":{"name":"Cell Journal (Yakhteh)","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"85153287","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-09-08DOI: 10.22074/cellj.2020.6532
H. Azizi, M. Koruji, T. Skutella
Objective Spermatogonial stem cells (SSCs), as unipotent stem cells, are responsible for the production of sperm throughout the male’s life. Zinc finger and BTB domain containing 16 (ZBTB16/PLZF) genes provide various functions in the cell development, signaling pathway, growth regulatory and differentiation. Here, we aimed to investigate expression of the PLZF germ cell gene marker in testis, SSCs, pluripotent embryonic stem cells (ES cells) and ES-like cells of mouse testis. Materials and Methods In this experimental study, we examined the expression of the PLZF germ cell marker in the testis section and testicular cell culture of neonate and adult mice by immunohistochemistry (IMH), immunocytochemistry (ICC) and Fluidigm Real-Time polymerase chain reaction (PCR). Results IMH data indicated that the PLZF protein was localized in the neonate testis cells of the tubules center as well as the basal compartment of adult testis seminiferous tubules. Counting PLZF IMH-positive cells in the sections of seminiferous tubules of adult and neonate testis revealed significant expression of positive cells in adult testis compared to the neonate (P<0.05). Under in vitro conditions, isolated SSC colonies were strongly ICC-positive for the PLZF germ cell marker, while ES cells and ES-like cells were negative for PLZF. Fluidigm Real-Time-PCR analysis demonstrated a significant expression of the PLZF germ cell gene in the neonate and adult SSCs, compared to ES cells and ES-like cells (P<0.05). Conclusion These results indicate that PLZF is a specific transcription factor of testicular germ cell proliferation, but it is down- regulated in pluripotent germ cells. This can be supportive for the analysis of germ cells development both in vitro and in vivo.
{"title":"Comparison of PLZF Gene Expression between Pluripotent Stem Cells and Testicular Germ Cells","authors":"H. Azizi, M. Koruji, T. Skutella","doi":"10.22074/cellj.2020.6532","DOIUrl":"https://doi.org/10.22074/cellj.2020.6532","url":null,"abstract":"Objective Spermatogonial stem cells (SSCs), as unipotent stem cells, are responsible for the production of sperm throughout the male’s life. Zinc finger and BTB domain containing 16 (ZBTB16/PLZF) genes provide various functions in the cell development, signaling pathway, growth regulatory and differentiation. Here, we aimed to investigate expression of the PLZF germ cell gene marker in testis, SSCs, pluripotent embryonic stem cells (ES cells) and ES-like cells of mouse testis. Materials and Methods In this experimental study, we examined the expression of the PLZF germ cell marker in the testis section and testicular cell culture of neonate and adult mice by immunohistochemistry (IMH), immunocytochemistry (ICC) and Fluidigm Real-Time polymerase chain reaction (PCR). Results IMH data indicated that the PLZF protein was localized in the neonate testis cells of the tubules center as well as the basal compartment of adult testis seminiferous tubules. Counting PLZF IMH-positive cells in the sections of seminiferous tubules of adult and neonate testis revealed significant expression of positive cells in adult testis compared to the neonate (P<0.05). Under in vitro conditions, isolated SSC colonies were strongly ICC-positive for the PLZF germ cell marker, while ES cells and ES-like cells were negative for PLZF. Fluidigm Real-Time-PCR analysis demonstrated a significant expression of the PLZF germ cell gene in the neonate and adult SSCs, compared to ES cells and ES-like cells (P<0.05). Conclusion These results indicate that PLZF is a specific transcription factor of testicular germ cell proliferation, but it is down- regulated in pluripotent germ cells. This can be supportive for the analysis of germ cells development both in vitro and in vivo.","PeriodicalId":9692,"journal":{"name":"Cell Journal (Yakhteh)","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"83695320","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-09-08DOI: 10.22074/cellj.2020.6527
Maryam Ranjpour, S. Wajid, S. 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 A2delta 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 A2delta is associated with progression of HCC.
{"title":"Elevated Expression of Cytosolic Phospholipase A2Delta Is Associated with Lipid Metabolism Dysregulation during Hepatocellular Carcinoma Progression","authors":"Maryam Ranjpour, S. Wajid, S. Jain","doi":"10.22074/cellj.2020.6527","DOIUrl":"https://doi.org/10.22074/cellj.2020.6527","url":null,"abstract":"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 A2delta 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 A2delta is associated with progression of HCC.","PeriodicalId":9692,"journal":{"name":"Cell Journal (Yakhteh)","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"88806209","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-09-08DOI: 10.22074/cellj.2020.6604
Evren Gumus
Objective Decellularized tissue scaffolds provide an extracellular matrix to control stem cells differentiation toward specific lineages. The application of mesenchymal stem cells for artificial ovary production may enhance ex vivo functions of the ovary. On the other hand, the scaffold needs interaction and integration with cells. Thus, the development of ovarian engineered constructs (OVECs) requires the use of efficient methods for seeding of the cells into the ovarian and other types of scaffolds. The main goal of the present study was to develop an optimized culture system for efficient seeding of peritoneum mesenchymal stem cells (PMSCs) into human decellularized ovarian scaffold. Materials and Methods In this experimental study, three methods were used for cellular seeding including rotational (spinner flask) and static (conventional and injection) seeding cultures. OVECs were evaluated with Hematoxylin and Eosin staining and viability analyses for the seeded PMSCs. Then, immunohistochemistry analysis was performed using the best method of cellular seeding for primordial germ cell-like cells, mesenchymal stem cells and proliferation markers. Stereology analysis was also performed for the number of penetrated cells into the OVECs. Results Our results showed that rotational seeding increases the permeability of PMSCs into the scaffold and survival rate of the seeded PMSCs, comparing to the other methods. On the other hand, rotationally seeded PMSCs had a more favorable capability of proliferation with Ki67 expression and differentiation to ovarian specific cells with expression of primordial germ cell line markers without mesenchymal stem cells markers production. Furthermore, stereology showed a more favorable distribution of PMSCs along the outer surfaces of the OVEC with further distribution at the central part of the scaffold. The average total cell values were determined 2142187 cells/mm3 on each OVEC. Conclusion The rotational seeding method is a more favorable approach to cell seeding into ovarian decellularized tissue than static seeding.
{"title":"Optimizing The Cell Seeding Protocol to Human Decellularized Ovarian Scaffold: Application of Dynamic System for Bio-Engineering","authors":"Evren Gumus","doi":"10.22074/cellj.2020.6604","DOIUrl":"https://doi.org/10.22074/cellj.2020.6604","url":null,"abstract":"Objective Decellularized tissue scaffolds provide an extracellular matrix to control stem cells differentiation toward specific lineages. The application of mesenchymal stem cells for artificial ovary production may enhance ex vivo functions of the ovary. On the other hand, the scaffold needs interaction and integration with cells. Thus, the development of ovarian engineered constructs (OVECs) requires the use of efficient methods for seeding of the cells into the ovarian and other types of scaffolds. The main goal of the present study was to develop an optimized culture system for efficient seeding of peritoneum mesenchymal stem cells (PMSCs) into human decellularized ovarian scaffold. Materials and Methods In this experimental study, three methods were used for cellular seeding including rotational (spinner flask) and static (conventional and injection) seeding cultures. OVECs were evaluated with Hematoxylin and Eosin staining and viability analyses for the seeded PMSCs. Then, immunohistochemistry analysis was performed using the best method of cellular seeding for primordial germ cell-like cells, mesenchymal stem cells and proliferation markers. Stereology analysis was also performed for the number of penetrated cells into the OVECs. Results Our results showed that rotational seeding increases the permeability of PMSCs into the scaffold and survival rate of the seeded PMSCs, comparing to the other methods. On the other hand, rotationally seeded PMSCs had a more favorable capability of proliferation with Ki67 expression and differentiation to ovarian specific cells with expression of primordial germ cell line markers without mesenchymal stem cells markers production. Furthermore, stereology showed a more favorable distribution of PMSCs along the outer surfaces of the OVEC with further distribution at the central part of the scaffold. The average total cell values were determined 2142187 cells/mm3 on each OVEC. Conclusion The rotational seeding method is a more favorable approach to cell seeding into ovarian decellularized tissue than static seeding.","PeriodicalId":9692,"journal":{"name":"Cell Journal (Yakhteh)","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"82482495","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-09-08DOI: 10.22074/cellj.2020.6306
A. 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.
目的乙酰辅酶a合成酶短链家族成员2 (ACSS2)活性是驱动组蛋白乙酰化的主要乙酰辅酶a来源。本研究旨在揭示小鼠精子发生过程中ACSS2的表达，其中动态和阶段特异性的全基因组组蛋白超乙酰化发生在组蛋白排出之前。材料与方法本实验研究采用免疫检测方法验证ACSS2在精子发生过程中的表达水平。睾丸石蜡包埋切片进行抗h4 pan ac和抗acss2免疫组化染色。采用免疫荧光法在睾丸小管切片上同时检测ACSS2和H4K5ac。对分离的男性生殖细胞进行western-blotting和免疫印迹，检测抗ACSS2和抗肌动蛋白。结果表明，祖细胞参与减数分裂导致细胞核中ACSS2的大量积累，特别是在粗线精母细胞(P)中。然而，在减数分裂后阶段，当全基因组组蛋白超乙酰化发生时，ACSS2蛋白急剧下降。结论本研究结果与ACSS2的主要功能是回收组蛋白去乙酰化后产生的乙酸，再生维持组蛋白乙酰化稳定状态所需的乙酰辅酶a的观点一致。因此，我们认为在生精细胞中，ACSS2的核活性维持了乙酸循环直到组蛋白超乙酰化，但在乙酰化依赖性组蛋白降解之前消失。
{"title":"Regulated Acyl-CoA Synthetase Short-Chain Family Member 2 Accumulation during Spermatogenesis","authors":"A. Goudarzi, Amir Amiri-Yekta","doi":"10.22074/cellj.2020.6306","DOIUrl":"https://doi.org/10.22074/cellj.2020.6306","url":null,"abstract":"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.","PeriodicalId":9692,"journal":{"name":"Cell Journal (Yakhteh)","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"75563124","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2019-09-08DOI: 10.22074/cellj.2020.6290
M. Zangi, Mohammad Bagher Bagherieh Najjar, M. Golalipour, Mahnaz ghdasi
Objective: DNA methylation systems are essential for proper embryo development. Methylation defects lead to developmental abnormalities. Furthermore, changes in telomerase gene expression can affect stability of chromosomes and produces abnormal growth. Therefore, defects in both methylation and telomerase gene expression can lead to developmental abnormalities. We hypothesized that mutation in the methylation systems may induce developmental abnormalities through changing telomerase gene expression. Materials and Methods: In this experimental study, we used Arabidopsis thaliana (At) as a developmental model. DNA was extracted from seedlings leaves. The grown plants were screened using polymerase chain reaction (PCR) reactions. Total RNA was isolated from the mature leaves, stems and flowers of wild type and met1 mutants. For gene expression analysis, cDNA was synthesized and then quantitative reverse transcription PCR (qRT-PCR) was performed. Results: Telomerase gene expression level in homozygous met1 mutant plants showed ~14 fold increase compared to normal plants. Furthermore, TERT expression in met1 heterozygous was~ 2 fold higher than the wild type plants. Conclusion: Our results suggested that TERT is a methyltransferase-regulated gene which may be involved in developmental abnormities causing by mutation in met1 methyltransferase system.
{"title":"met1 DNA Methyltransferase Controls TERT Gene Expression: A New Insight to The Role of Telomerase in Development","authors":"M. Zangi, Mohammad Bagher Bagherieh Najjar, M. Golalipour, Mahnaz ghdasi","doi":"10.22074/cellj.2020.6290","DOIUrl":"https://doi.org/10.22074/cellj.2020.6290","url":null,"abstract":"Objective: DNA methylation systems are essential for proper embryo development. Methylation defects lead to developmental abnormalities. Furthermore, changes in telomerase gene expression can affect stability of chromosomes and produces abnormal growth. Therefore, defects in both methylation and telomerase gene expression can lead to developmental abnormalities. We hypothesized that mutation in the methylation systems may induce developmental abnormalities through changing telomerase gene expression. Materials and Methods: In this experimental study, we used Arabidopsis thaliana (At) as a developmental model. DNA was extracted from seedlings leaves. The grown plants were screened using polymerase chain reaction (PCR) reactions. Total RNA was isolated from the mature leaves, stems and flowers of wild type and met1 mutants. For gene expression analysis, cDNA was synthesized and then quantitative reverse transcription PCR (qRT-PCR) was performed. Results: Telomerase gene expression level in homozygous met1 mutant plants showed ~14 fold increase compared to normal plants. Furthermore, TERT expression in met1 heterozygous was~ 2 fold higher than the wild type plants. Conclusion: Our results suggested that TERT is a methyltransferase-regulated gene which may be involved in developmental abnormities causing by mutation in met1 methyltransferase system.","PeriodicalId":9692,"journal":{"name":"Cell Journal (Yakhteh)","volume":null,"pages":null},"PeriodicalIF":0.0,"publicationDate":"2019-09-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"75720790","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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