Sains Malaysiana 47(9)(2018): 2105–2111

http://dx.doi.org/10.17576/jsm-2018-4709-19

Construction of the pEGFP-N1-p53/MAR Vector and Its Effect on HEP3B Cell Morphology

(Pembinaan Vektor pEGFP-N1-p53/MAR dan Kesannya ke atas Morfologi Sel HEP3B)

PENG SHAN LI¹, XUE QIN LEI^1*, TING SHENG XU¹, PAN LIN WANG¹, ZHEN SONG¹, ZHEN HONG LI¹ & XUE MEI HAN²

¹College of Animal Science and Technology, Henan University of Science and Technology, Luoyang, 471023, China

²Cancer Research, Third Affiliated Hospital of Henan University of Science and Technology, Luoyang, 471003, China

Diserahkan: 28 Januari 2018/Diterima: 9 Mei 2018

ABSTRACT

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.

Keywords: Cell microstructure; cell morphology; gene therapy; p53 gene; matrix attachment region (MAR); non-viral vector

ABSTRAK

Kanser selalu menimbulkan masalah besar yang mengancam kesihatan manusia. Kebelakangan ini, penggunaan terapi gen dalam penyelidikan kanser telah meningkat dengan ketara. Kajian ini bertujuan untuk membina sebuah rekombinan vektor ekspresi eukariot bukan virus, jenis liar, pEGFP-N1-p53/MAR dan mengesahkan mekanisme tindakan dalam sel kanser secara in vitro. Kajian ini memberikan satu strategi baru untuk terapi gen p53 melalui peraturan matriks pelekatan rantau (MAR), berpotensi meletakkan asas yang kukuh untuk penubuhan bioreaktor protein antikanser. Gen p53 diklon daripada darah periferi manusia dan gen MAR telah diamplifikasi daripada tisu hati ayam. Vektor ekspresi rekombinan eukariot pEGFP-N1-p53/MAR telah dibangunkan menggunakan sistem replikasi sendiri E. coli. LipofectamineTM 2000 telah digunakan sebagai ejen transfeksi untuk menghantar plasmid ke dalam titisan sel manusia karsinoma hepar (HEP3B). Kami membahagikan kumpulan seperti berikut: Sel kawalan negatif tanpa transfeksi plasmid, sel kawalan penghantaran transfeksi dengan vektor PEGFP-N1 dan sel uji kaji transfeksi dengan vektor pEGFP-N1-p53/MAR. Sel dalam telaga setiap kawalan sarana dan kumpulan uji kaji telah ditransfeksi dengan 1.6 μg plasmid dan 3 μL liposom. Morfologi sel setiap kumpulan dianalisis menggunakan mikroskop hijau pendarfluor pada 12, 24, 36 dan 48 jam. Kemudian, analisis statistik pada kadar apoptosis antara ketiga-tiga kumpulan telah dijalankan menggunakan perisian SPSS. Ultrastruktur sel telah diperhatikan melalui penghantaran elektron mikroskop selepas transfeksi selama 24 jam. Analisis morfologi menunjukkan bahawa sel kumpulan uji kaji telah dikecut dan dikurangkan saiznya serta sambungan intersel telah hilang. Di samping itu, kadar apoptosis dalam kumpulan uji kaji adalah jauh lebih tinggi daripada kumpulan kawalan dan mikrostruktur sel menunjukkan bahawa jasad heterokromatin dan apoptotik telah ditemui dalam kumpulan uji kaji. Kesimpulannya, berbanding dengan kumpulan kawalan, plasmid pEGFP-N1-p53/MAR berkesan menggalakkan sel apoptosis Hep3B secara in vitro.

Kata kunci: Gen p53; matriks pelekatan rantau (MAR); mikrostruktur sel; morfologi sel; terapi gen; vektor bukan virus

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*Pengarang untuk surat-menyurat; email: xueqinlei@l63.com