Cell compatibility is one of the prominent requirements for the fabrication of tissue engineering materials. Silk fibroin (SF) is an excellent material for biomedical applications and shows desirable properties such as good compatibility, minimal tissue reaction, and tailorable degradability. Poly[(R)-3-hydroxybutyrate] (PHB) has a high percentage of crystallinity and a high melting temperature. In this study, PHB and SF were blended together to improve the mechanical properties of the SF fibrous structure and the crystallinity of PHB. Furthermore, the cell-biomaterial interaction on the PHB/SF composite scaffold was expected to be enhanced via the SF. For this purpose, PHB/SF composite scaffolds were prepared through the use of the electrospinning technique, which is a unique method for the production of biomaterials on the nanoscale intended for tissue engineering. PHB/SF scaffolds were prepared with different SF contents and a suitable electrospinning condition was chosen in terms of structure and fiber diameter. The average fiber diameter was 92 ± 2.6 nm at a flow rate of 0.4 mL/h, distance of 20 cm, polymer concentration of PHB/SF-0 of 7%:18% (w/w) or 1:1 (v/v), and voltage of 20 kV. Mechanical and crystalline properties of the PHB/SF scaffold were investigated. Adhesion and proliferation of L929 and HaCaT (mouse fibroblast and immortalized human keratinocyte cell lines) on PHB/SF-0 were examined.
{"title":"Cell-compatible PHB/silk fibroin composite nanofibermat for tissue engineering applications","authors":"Zeynep Karahaliloğlu","doi":"10.3906/BIY-1610-46","DOIUrl":"https://doi.org/10.3906/BIY-1610-46","url":null,"abstract":"Cell compatibility is one of the prominent requirements for the fabrication of tissue engineering materials. Silk fibroin (SF) is an excellent material for biomedical applications and shows desirable properties such as good compatibility, minimal tissue reaction, and tailorable degradability. Poly[(R)-3-hydroxybutyrate] (PHB) has a high percentage of crystallinity and a high melting temperature. In this study, PHB and SF were blended together to improve the mechanical properties of the SF fibrous structure and the crystallinity of PHB. Furthermore, the cell-biomaterial interaction on the PHB/SF composite scaffold was expected to be enhanced via the SF. For this purpose, PHB/SF composite scaffolds were prepared through the use of the electrospinning technique, which is a unique method for the production of biomaterials on the nanoscale intended for tissue engineering. PHB/SF scaffolds were prepared with different SF contents and a suitable electrospinning condition was chosen in terms of structure and fiber diameter. The average fiber diameter was 92 ± 2.6 nm at a flow rate of 0.4 mL/h, distance of 20 cm, polymer concentration of PHB/SF-0 of 7%:18% (w/w) or 1:1 (v/v), and voltage of 20 kV. Mechanical and crystalline properties of the PHB/SF scaffold were investigated. Adhesion and proliferation of L929 and HaCaT (mouse fibroblast and immortalized human keratinocyte cell lines) on PHB/SF-0 were examined.","PeriodicalId":23358,"journal":{"name":"Turkish Journal of Biology","volume":"41 1","pages":"503-513"},"PeriodicalIF":2.2,"publicationDate":"2017-06-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3906/BIY-1610-46","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"48058820","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Characterization of proteinase inhibitor-II gene under OsRGLP2promoter for salt stress in transgenic Nicotiana benthamiana","authors":"S. Rehman, B. Jørgensen, S. Rasmussen, T. Mahmood","doi":"10.3906/BIY-1609-42","DOIUrl":"https://doi.org/10.3906/BIY-1609-42","url":null,"abstract":"* Correspondence: tmahmood@qau.edu.pk","PeriodicalId":23358,"journal":{"name":"Turkish Journal of Biology","volume":"41 1","pages":"494-502"},"PeriodicalIF":2.2,"publicationDate":"2017-06-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3906/BIY-1609-42","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"43279899","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
T. Do, Thao T. T. Nguyen, Thanh Sy Le Nguyen, H. Le
* Correspondence: dttuyen@ibt.ac.vn
*通信：dttuyen@ibt.ac.vn
{"title":"Optimization, purification, and characterization of an extracellular antifungal protein from Serratia marcescens DT3 isolated from soil in Vietnam","authors":"T. Do, Thao T. T. Nguyen, Thanh Sy Le Nguyen, H. Le","doi":"10.3906/BIY-1607-22","DOIUrl":"https://doi.org/10.3906/BIY-1607-22","url":null,"abstract":"* Correspondence: dttuyen@ibt.ac.vn","PeriodicalId":23358,"journal":{"name":"Turkish Journal of Biology","volume":"41 1","pages":"448-457"},"PeriodicalIF":2.2,"publicationDate":"2017-06-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3906/BIY-1607-22","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49186131","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Irfan Ullah, A. Nasir, S. Mehmood, Sohail Ahmed, M. I. Ullah, A. Ullah, A. Aziz, S. I. Raza, K. Shah, Saadullah Khan, M. Hassan, W. Ahmad
Genetic deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS) due to mutations in the GALNS gene results in the pathogenesis of Morquio A syndrome. To date, more than 200 mutations have been reported in GALNS, resulting in variable clinical features. For this reason, bioinformatics analysis of these mutations is important to determine their effect on the structure and functions of the protein and to establish a correlation between genotype and phenotype. In the present study, eight Pakistani consanguineous families with Morquio A syndrome were clinically and genetically evaluated. Linkage analysis followed by sequence analysis of the gene detected four novel (p.Phe216Ser, p.Met38Arg, p.Ala291Ser, p.Glu121Argfs*37) and two reported (p.Pro420Arg, p.Arg386Cys) mutations in the eight families. In silico structural and functional analysis predicted that these mutations disrupt the function of GALNS protein through fluctuating its three-dimensional structure, stability, and binding affinity and produce severe phenotypes. This is the first comprehensive study on molecular analysis of patients with Morquio A syndrome from Pakistan that reports four novel mutations with their structural and functional consequences.
{"title":"Identification and in silico analysis of GALNS mutations causing Morquio A syndrome in eight consanguineous families","authors":"Irfan Ullah, A. Nasir, S. Mehmood, Sohail Ahmed, M. I. Ullah, A. Ullah, A. Aziz, S. I. Raza, K. Shah, Saadullah Khan, M. Hassan, W. Ahmad","doi":"10.3906/BIY-1607-81","DOIUrl":"https://doi.org/10.3906/BIY-1607-81","url":null,"abstract":"Genetic deficiency of N-acetylgalactosamine-6-sulfate sulfatase (GALNS) due to mutations in the GALNS gene results in the pathogenesis of Morquio A syndrome. To date, more than 200 mutations have been reported in GALNS, resulting in variable clinical features. For this reason, bioinformatics analysis of these mutations is important to determine their effect on the structure and functions of the protein and to establish a correlation between genotype and phenotype. In the present study, eight Pakistani consanguineous families with Morquio A syndrome were clinically and genetically evaluated. Linkage analysis followed by sequence analysis of the gene detected four novel (p.Phe216Ser, p.Met38Arg, p.Ala291Ser, p.Glu121Argfs*37) and two reported (p.Pro420Arg, p.Arg386Cys) mutations in the eight families. In silico structural and functional analysis predicted that these mutations disrupt the function of GALNS protein through fluctuating its three-dimensional structure, stability, and binding affinity and produce severe phenotypes. This is the first comprehensive study on molecular analysis of patients with Morquio A syndrome from Pakistan that reports four novel mutations with their structural and functional consequences.","PeriodicalId":23358,"journal":{"name":"Turkish Journal of Biology","volume":"41 1","pages":"458-468"},"PeriodicalIF":2.2,"publicationDate":"2017-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3906/BIY-1607-81","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41464983","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
A. B. Ertan, Halime Kenar, T. Beyzadeoğlu, F. N. Kök, G. Kose
This study reports the generation of a new human muscle tissue equivalent from skeletal muscle-derived stem cells and human umbilical vein endothelial cells (HUVECs). Skeletal muscle stem cells were isolated by the preplate technique and differentiated into neuron-like cells that were positive for neuronal beta-tubulin3 and nestin and negative for the astrocyte marker glial fibrillary acidic protein (GFAP). Coculture of skeletal muscle stem cells with the HUVECs under optimized fetal bovine serum and media conditions resulted in formation of a capillary network among the multinucleated myotubes. The neuron-like cells derived from the human skeletal muscle stem cells were seeded onto vascularized myotubes to obtain the neuromuscular junctions in the coculture. At the end of 24 h of coculture, the neuron-like cells were found to be in association with the myotubes. This model represents a novel complex in vitro human skeletal muscle model containing advanced capillary networks and interacting myotubes and neurons, and it can be used for in vitro drug testing or for skeletal muscle regeneration either through application of cellular therapy or cell-laden tissue-engineered muscle constructs.
{"title":"An in vitro human skeletal muscle model: coculture of myotubes, neuron-like cells, and the capillary network","authors":"A. B. Ertan, Halime Kenar, T. Beyzadeoğlu, F. N. Kök, G. Kose","doi":"10.3906/BIY-1611-22","DOIUrl":"https://doi.org/10.3906/BIY-1611-22","url":null,"abstract":"This study reports the generation of a new human muscle tissue equivalent from skeletal muscle-derived stem cells and human umbilical vein endothelial cells (HUVECs). Skeletal muscle stem cells were isolated by the preplate technique and differentiated into neuron-like cells that were positive for neuronal beta-tubulin3 and nestin and negative for the astrocyte marker glial fibrillary acidic protein (GFAP). Coculture of skeletal muscle stem cells with the HUVECs under optimized fetal bovine serum and media conditions resulted in formation of a capillary network among the multinucleated myotubes. The neuron-like cells derived from the human skeletal muscle stem cells were seeded onto vascularized myotubes to obtain the neuromuscular junctions in the coculture. At the end of 24 h of coculture, the neuron-like cells were found to be in association with the myotubes. This model represents a novel complex in vitro human skeletal muscle model containing advanced capillary networks and interacting myotubes and neurons, and it can be used for in vitro drug testing or for skeletal muscle regeneration either through application of cellular therapy or cell-laden tissue-engineered muscle constructs.","PeriodicalId":23358,"journal":{"name":"Turkish Journal of Biology","volume":"41 1","pages":"514-525"},"PeriodicalIF":2.2,"publicationDate":"2017-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3906/BIY-1611-22","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"44022803","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The collaboration between the Human Genome Project and the Sanger Institute, spurred on by their contemporary- Celera Genomics, in the race to sequence the human genome, opened the door to a generation of research concerned with the study of the nucleic manual. Following this monumental achievement, the genomic era was born, heralding the development of gene therapy, personalized medicine, pharmacogenomics, and CRISPR/Cas9 gene-editing systems. These 'omic' disciplines offer a wide range of applications in the treatment of disease and the betterment of the quality of human life. Their notability, however, manifests in the unique mix of sociological and scientific discourse that ensues. Current genomic-era milestones present a unique opportunity to view scientific advance in a thoughtful light, lacing together discoveries, history, and ethical questions, while encouraging the public to consider the varied facets of this scientific evolution with the same curiosity and enthusiasm as the trailblazers of the Human Genome Project. Consider it an invitation to reflect on the fruits of the genomic era.
{"title":"Aftermath of the Human Genome Project: an era of struggle and discovery","authors":"Zainab Hamdoun, Hashimul Ehsan","doi":"10.3906/BIY-1609-77","DOIUrl":"https://doi.org/10.3906/BIY-1609-77","url":null,"abstract":"The collaboration between the Human Genome Project and the Sanger Institute, spurred on by their contemporary- Celera Genomics, in the race to sequence the human genome, opened the door to a generation of research concerned with the study of the nucleic manual. Following this monumental achievement, the genomic era was born, heralding the development of gene therapy, personalized medicine, pharmacogenomics, and CRISPR/Cas9 gene-editing systems. These 'omic' disciplines offer a wide range of applications in the treatment of disease and the betterment of the quality of human life. Their notability, however, manifests in the unique mix of sociological and scientific discourse that ensues. Current genomic-era milestones present a unique opportunity to view scientific advance in a thoughtful light, lacing together discoveries, history, and ethical questions, while encouraging the public to consider the varied facets of this scientific evolution with the same curiosity and enthusiasm as the trailblazers of the Human Genome Project. Consider it an invitation to reflect on the fruits of the genomic era.","PeriodicalId":23358,"journal":{"name":"Turkish Journal of Biology","volume":"41 1","pages":"403-418"},"PeriodicalIF":2.2,"publicationDate":"2017-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3906/BIY-1609-77","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42445458","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
This study aims to explore the role and the possible underlying molecular mechanism of SNF5 in the pathogenesis of rheumatoid arthritis (RA). MH7A cells were respectively transfected with pc-SNF5 (pcDNA3.1 containing the Brg1 coding sequence), short hairpin RNA against SNF5 (shSNF5), and their negative controls (pcDNA3.1 and shNC). The alterations of SNF5 expression were assessed by qRT-PCR and western blot analysis. MTT assay, flow cytometry, and western blot analysis were performed to evaluate proliferation, apoptosis, and expression levels of p16 and JNK pathway associated proteins, respectively. Finally, the effect of SNF5 was verified in fibroblast-like synoviocytes (FLSs) obtained from a rat model with adjuvant-induced arthritis. Results showed that the expression of SNF5 was increased in the pc-SNF5 group (P < 0.05) while it was decreased in the shSNF5 group (P < 0.05). Afterwards, cell viability after transfection was reduced by SNF5 overexpression (P < 0.05, P < 0.01, or P < 0.001), whereas it was enhanced by SNF5 knockdown (P < 0.05 or P < 0.001). In terms of apoptosis, SNF5 overexpression promoted cell apoptosis (P < 0.01). The western blot analysis showed that the phosphorylated levels of proteins involved in the JNK pathway were downregulated by SNF5 overexpression while they were upregulated by SNF5 knockdown (P < 0.05, P < 0.01, or P < 0.001). However, the effect of SNF5 on the expression of p16 was the opposite. Finally, the effect of SNF5 was validated in murine FLSs. In conclusion, SNF5 suppresses proliferation and induces apoptosis of fibroblast-like cells through overexpression of p16 and suppression of the JNK pathway.
{"title":"Role of SNF5 in rheumatoid arthritis by upregulation ofp16 and inactivation of JNK pathway","authors":"Shupeng Wu, Jing Wang, Fang Li","doi":"10.3906/BIY-1610-40","DOIUrl":"https://doi.org/10.3906/BIY-1610-40","url":null,"abstract":"This study aims to explore the role and the possible underlying molecular mechanism of SNF5 in the pathogenesis of rheumatoid arthritis (RA). MH7A cells were respectively transfected with pc-SNF5 (pcDNA3.1 containing the Brg1 coding sequence), short hairpin RNA against SNF5 (shSNF5), and their negative controls (pcDNA3.1 and shNC). The alterations of SNF5 expression were assessed by qRT-PCR and western blot analysis. MTT assay, flow cytometry, and western blot analysis were performed to evaluate proliferation, apoptosis, and expression levels of p16 and JNK pathway associated proteins, respectively. Finally, the effect of SNF5 was verified in fibroblast-like synoviocytes (FLSs) obtained from a rat model with adjuvant-induced arthritis. Results showed that the expression of SNF5 was increased in the pc-SNF5 group (P < 0.05) while it was decreased in the shSNF5 group (P < 0.05). Afterwards, cell viability after transfection was reduced by SNF5 overexpression (P < 0.05, P < 0.01, or P < 0.001), whereas it was enhanced by SNF5 knockdown (P < 0.05 or P < 0.001). In terms of apoptosis, SNF5 overexpression promoted cell apoptosis (P < 0.01). The western blot analysis showed that the phosphorylated levels of proteins involved in the JNK pathway were downregulated by SNF5 overexpression while they were upregulated by SNF5 knockdown (P < 0.05, P < 0.01, or P < 0.001). However, the effect of SNF5 on the expression of p16 was the opposite. Finally, the effect of SNF5 was validated in murine FLSs. In conclusion, SNF5 suppresses proliferation and induces apoptosis of fibroblast-like cells through overexpression of p16 and suppression of the JNK pathway.","PeriodicalId":23358,"journal":{"name":"Turkish Journal of Biology","volume":"41 1","pages":"535-542"},"PeriodicalIF":2.2,"publicationDate":"2017-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3906/BIY-1610-40","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45882882","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Li-jing Chen, Li Zhang, W. Qi, M. Irfan, Jing-wei Lin, Hui Ma, Zhi-Fu Guo, Ming Zhong, Tianlai Li
Cold environmental conditions influence the growth and development of plants, causing crop reduction or even plant death. Under stress conditions, cold-inducible promoters regulate cold-related gene expression as a molecular switch. Recent studies have shown that the chloroplast-expressed GPAT gene plays an important role in determining cold sensitivity. However, the mechanism of the transcriptional regulation of GPAT is ambiguous. The 5'-flanking region of GPAT with length of 1494 bp was successfully obtained by chromosome walking from Lilium pensylvanicum. The cis-elements of GPAT promoters were predicted and analyzed by a plant cis-acting regulatory DNA element database. There exist core promoter regions including TATA-box and CAAT-box and transcription regulation regions, which involve some regulatory elements such as I-box, W-box, MYB, MYC, and DREB. Full-length and four 5'-deletion fragments linked with GUS vectors were constructed and transformed into Nicotiana tabacum by Agrobacterium-mediated transformation. Transient transformation and histochemical staining of leaves indicated that the activity of the GPAT promoter was strong and induced by low-temperature stress. The deletion of a -294 bp region suggested that the DRE-motif was functionally an essential element for cold induction, and the deletion of -1494 bp and -1194 bp regions suggested that negative regulation exists in the promoter. Our results show that the GPAT gene promoter is a key regulator under cold stress and we think that this study will have significant impact on lily molecular breeding and improving the resistance of plants.
{"title":"Characterization of the promoter region of the glycerol-3-phosphate-O-acyltransferase gene in Lilium pensylvanicum","authors":"Li-jing Chen, Li Zhang, W. Qi, M. Irfan, Jing-wei Lin, Hui Ma, Zhi-Fu Guo, Ming Zhong, Tianlai Li","doi":"10.3906/biy-1611-56","DOIUrl":"https://doi.org/10.3906/biy-1611-56","url":null,"abstract":"Cold environmental conditions influence the growth and development of plants, causing crop reduction or even plant death. Under stress conditions, cold-inducible promoters regulate cold-related gene expression as a molecular switch. Recent studies have shown that the chloroplast-expressed GPAT gene plays an important role in determining cold sensitivity. However, the mechanism of the transcriptional regulation of GPAT is ambiguous. The 5'-flanking region of GPAT with length of 1494 bp was successfully obtained by chromosome walking from Lilium pensylvanicum. The cis-elements of GPAT promoters were predicted and analyzed by a plant cis-acting regulatory DNA element database. There exist core promoter regions including TATA-box and CAAT-box and transcription regulation regions, which involve some regulatory elements such as I-box, W-box, MYB, MYC, and DREB. Full-length and four 5'-deletion fragments linked with GUS vectors were constructed and transformed into Nicotiana tabacum by Agrobacterium-mediated transformation. Transient transformation and histochemical staining of leaves indicated that the activity of the GPAT promoter was strong and induced by low-temperature stress. The deletion of a -294 bp region suggested that the DRE-motif was functionally an essential element for cold induction, and the deletion of -1494 bp and -1194 bp regions suggested that negative regulation exists in the promoter. Our results show that the GPAT gene promoter is a key regulator under cold stress and we think that this study will have significant impact on lily molecular breeding and improving the resistance of plants.","PeriodicalId":23358,"journal":{"name":"Turkish Journal of Biology","volume":"41 1","pages":"552-562"},"PeriodicalIF":2.2,"publicationDate":"2017-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3906/biy-1611-56","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46959158","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
M. Ren, Qiwei Yang, Yuanyuan Song, Ao Wang, Qingyu Wang, Xiaonan Wang, Shuhong Hao, Zhitao Wang, Zhenwu Du, Guizhen Zhang, Jincheng Wang
The selection of stably expressed reference genes is crucial for quantitative real-time polymerase chain reaction (qRT-PCR) assay when assessing osteosarcoma treated with cyclophosphamide (CTX) and Ginkgo biloba L. extract (EGB). We aimed to identify the reference genes that are stably expressed in human osteosarcoma cell lines after the above treatments. We used qRT-PCR assay to determine the expression levels of 18S, ACTB, ALAS1, GAPDH, TBP, HPRT1, RPL29, HMBS, PPIA, PUM1, GUSB, and B2M in the cell lines MG-63, Saos-2, and U2OS, which were treated with CTX or EGB or both. We analyzed the expression stability of these genes using geNorm, NormFinder, and BestKeeper. The optimal reference gene combinations were 18S + PPIA + HPRT1 in the total group, HMBS + ALAS1 + 18S in the EGB-treated group, PPIA + RPL29 in the EGB + CTX-treated group, PPIA + HPRT1 in the MG-63 group, 18S + PPIA in the Saos-2 group, and HPRT1 + ALAS1 + GAPDH in the U2OS group. We conclude that no single reference gene was most stably expressed in all three treated cell lines. Our findings provide a suitable approach through which qRT-PCR can be applied to investigate the pharmacological effects and the molecular mechanisms of CTX and EGB on human osteosarcoma.
{"title":"Reference gene expression in human osteosarcoma cell lines treated by EGB and CTX","authors":"M. Ren, Qiwei Yang, Yuanyuan Song, Ao Wang, Qingyu Wang, Xiaonan Wang, Shuhong Hao, Zhitao Wang, Zhenwu Du, Guizhen Zhang, Jincheng Wang","doi":"10.3906/BIY-1607-15","DOIUrl":"https://doi.org/10.3906/BIY-1607-15","url":null,"abstract":"The selection of stably expressed reference genes is crucial for quantitative real-time polymerase chain reaction (qRT-PCR) assay when assessing osteosarcoma treated with cyclophosphamide (CTX) and Ginkgo biloba L. extract (EGB). We aimed to identify the reference genes that are stably expressed in human osteosarcoma cell lines after the above treatments. We used qRT-PCR assay to determine the expression levels of 18S, ACTB, ALAS1, GAPDH, TBP, HPRT1, RPL29, HMBS, PPIA, PUM1, GUSB, and B2M in the cell lines MG-63, Saos-2, and U2OS, which were treated with CTX or EGB or both. We analyzed the expression stability of these genes using geNorm, NormFinder, and BestKeeper. The optimal reference gene combinations were 18S + PPIA + HPRT1 in the total group, HMBS + ALAS1 + 18S in the EGB-treated group, PPIA + RPL29 in the EGB + CTX-treated group, PPIA + HPRT1 in the MG-63 group, 18S + PPIA in the Saos-2 group, and HPRT1 + ALAS1 + GAPDH in the U2OS group. We conclude that no single reference gene was most stably expressed in all three treated cell lines. Our findings provide a suitable approach through which qRT-PCR can be applied to investigate the pharmacological effects and the molecular mechanisms of CTX and EGB on human osteosarcoma.","PeriodicalId":23358,"journal":{"name":"Turkish Journal of Biology","volume":"41 1","pages":"439-447"},"PeriodicalIF":2.2,"publicationDate":"2017-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.3906/BIY-1607-15","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"47775608","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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