Construction of the pEGFP-N1-p53/MAR vector and its effect on HEP3B cell morphology

Cancer always presents a big problem that endangers human health. In recent years, the use of gene therapy in cancer research has significantly increased. This study aimed to construct a non-viral, wild-type, recombinant eukaryotic expression vector, pEGFP-N1-p53/MAR and verify its mechanism of acti...

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Main Authors:	Li, Peng Shan, Lei, Xue Qin, Xu, Ting Sheng, Wang, Pan Lin, Song, Zhen, Li, Zhen Hong, Han, Xue Mei
Format:	Article
Language:	English
Published:	Penerbit Universiti Kebangsaan Malaysia 2018
Online Access:	http://journalarticle.ukm.my/12401/ http://journalarticle.ukm.my/12401/ http://journalarticle.ukm.my/12401/1/19%20Peng%20Shan%20Li.pdf

id	ukm-12401
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spelling	ukm-124012018-12-07T23:35:28Z http://journalarticle.ukm.my/12401/ Construction of the pEGFP-N1-p53/MAR vector and its effect on HEP3B cell morphology Li, Peng Shan Lei, Xue Qin Xu, Ting Sheng Wang, Pan Lin Song, Zhen Li, Zhen Hong Han, Xue Mei Cancer always presents a big problem that endangers human health. In recent years, the use of gene therapy in cancer research has significantly increased. This study aimed to construct a non-viral, wild-type, recombinant eukaryotic expression vector, pEGFP-N1-p53/MAR and verify its mechanism of action in cancer cells in vitro. This investigation provides a novel strategy for p53 gene therapy via regulation of the matrix attachment region (MAR), potentially laying a foundation for the establishment of an anticancer protein bioreactor. The p53 gene was cloned from human peripheral blood and the MAR gene was amplified from chicken liver tissue. The recombinant eukaryotic expression vector pEGFP-N1-p53/MAR was constructed using an E. coli self-replication system. LipofectamineTM 2000 was used as the transfection agent to deliver the plasmid into the human hepatic carcinoma (HEP3B) cell line. We divided the groups as follows: negative control cells without plasmid transfection, vehicle control cells transfected with the PEGFP-N1 vector, and experimental cells transfected with the pEGFP-N1-p53/MAR vector. Cells in each well of the vehicle control and experimental groups were transfected with 1.6 μg of plasmid and 3 μL of liposome. The cellular morphology of each group was analysed using green fluorescence microscopy at 12, 24, 36 and 48 h. Then, statistical analysis of the apoptosis rates among the three groups was performed using SPSS. The ultrastructures of the cells were observed via transmission electron microscopy after transfection for 24 h. Morphological analysis showed that the cells of the experimental group were shrunken and reduced in size and their intercellular connections had disappeared. Additionally, the apoptosis rate in the experimental group was significantly higher than that in the control groups and the cellular microstructure showed that heterochromatin and apoptotic bodies were found in the experimental group. In conclusion, compared with the control groups, the pEGFP-N1-p53/MAR plasmid can effectively promote Hep3B cell apoptosis in vitro. Penerbit Universiti Kebangsaan Malaysia 2018-09 Article PeerReviewed application/pdf en http://journalarticle.ukm.my/12401/1/19%20Peng%20Shan%20Li.pdf Li, Peng Shan and Lei, Xue Qin and Xu, Ting Sheng and Wang, Pan Lin and Song, Zhen and Li, Zhen Hong and Han, Xue Mei (2018) Construction of the pEGFP-N1-p53/MAR vector and its effect on HEP3B cell morphology. Sains Malaysiana, 47 (9). pp. 2105-2111. ISSN 0126-6039 http://www.ukm.my/jsm/english_journals/vol47num9_2018/contentsVol47num9_2018.html
repository_type	Digital Repository
institution_category	Local University
institution	Universiti Kebangasaan Malaysia
building	UKM Institutional Repository
collection	Online Access
language	English
description	Cancer always presents a big problem that endangers human health. In recent years, the use of gene therapy in cancer research has significantly increased. This study aimed to construct a non-viral, wild-type, recombinant eukaryotic expression vector, pEGFP-N1-p53/MAR and verify its mechanism of action in cancer cells in vitro. This investigation provides a novel strategy for p53 gene therapy via regulation of the matrix attachment region (MAR), potentially laying a foundation for the establishment of an anticancer protein bioreactor. The p53 gene was cloned from human peripheral blood and the MAR gene was amplified from chicken liver tissue. The recombinant eukaryotic expression vector pEGFP-N1-p53/MAR was constructed using an E. coli self-replication system. LipofectamineTM 2000 was used as the transfection agent to deliver the plasmid into the human hepatic carcinoma (HEP3B) cell line. We divided the groups as follows: negative control cells without plasmid transfection, vehicle control cells transfected with the PEGFP-N1 vector, and experimental cells transfected with the pEGFP-N1-p53/MAR vector. Cells in each well of the vehicle control and experimental groups were transfected with 1.6 μg of plasmid and 3 μL of liposome. The cellular morphology of each group was analysed using green fluorescence microscopy at 12, 24, 36 and 48 h. Then, statistical analysis of the apoptosis rates among the three groups was performed using SPSS. The ultrastructures of the cells were observed via transmission electron microscopy after transfection for 24 h. Morphological analysis showed that the cells of the experimental group were shrunken and reduced in size and their intercellular connections had disappeared. Additionally, the apoptosis rate in the experimental group was significantly higher than that in the control groups and the cellular microstructure showed that heterochromatin and apoptotic bodies were found in the experimental group. In conclusion, compared with the control groups, the pEGFP-N1-p53/MAR plasmid can effectively promote Hep3B cell apoptosis in vitro.
format	Article
author	Li, Peng Shan Lei, Xue Qin Xu, Ting Sheng Wang, Pan Lin Song, Zhen Li, Zhen Hong Han, Xue Mei
spellingShingle	Li, Peng Shan Lei, Xue Qin Xu, Ting Sheng Wang, Pan Lin Song, Zhen Li, Zhen Hong Han, Xue Mei Construction of the pEGFP-N1-p53/MAR vector and its effect on HEP3B cell morphology
author_facet	Li, Peng Shan Lei, Xue Qin Xu, Ting Sheng Wang, Pan Lin Song, Zhen Li, Zhen Hong Han, Xue Mei
author_sort	Li, Peng Shan
title	Construction of the pEGFP-N1-p53/MAR vector and its effect on HEP3B cell morphology
title_short	Construction of the pEGFP-N1-p53/MAR vector and its effect on HEP3B cell morphology
title_full	Construction of the pEGFP-N1-p53/MAR vector and its effect on HEP3B cell morphology
title_fullStr	Construction of the pEGFP-N1-p53/MAR vector and its effect on HEP3B cell morphology
title_full_unstemmed	Construction of the pEGFP-N1-p53/MAR vector and its effect on HEP3B cell morphology
title_sort	construction of the pegfp-n1-p53/mar vector and its effect on hep3b cell morphology
publisher	Penerbit Universiti Kebangsaan Malaysia
publishDate	2018
url	http://journalarticle.ukm.my/12401/ http://journalarticle.ukm.my/12401/ http://journalarticle.ukm.my/12401/1/19%20Peng%20Shan%20Li.pdf
first_indexed	2023-09-18T20:02:31Z
last_indexed	2023-09-18T20:02:31Z
_version_	1777406943592185856

Construction of the pEGFP-N1-p53/MAR vector and its effect on HEP3B cell morphology

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