Inhibitory Effect of Eukaryotic Expression Vector Bearing TFPI-2 Gene on SHI-1 Cell Growth.
10.19746/j.cnki.issn.1009-2137.2019.06.017
- Author:
Jun-Jun LI
1
;
Pei LIAO
1
;
Feng WEN
1
;
Ze-Yu LUO
1
;
Yi-Xiong CAO
2
Author Information
1. Department of Hematology, The First Affiliated Hospital of University of South China, Hengyang 421001,Hunan Province, China.
2. Department of Hematology, The First Affiliated Hospital of University of South China, Hengyang 421001,Hunan Province, China E-mail：caoyixiongcyx@163.com.
- Publication Type:Journal Article
- MeSH:
Eukaryota;
Genetic Vectors;
Glycoproteins;
metabolism;
Green Fluorescent Proteins;
Humans;
Transfection
- From:
Journal of Experimental Hematology
2019;27(6):1812-1819
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE:To construct a eukaryotic expression vector of human tissue factor pathway inhibitor-2 (TFPI-2) and to investigate the effect of TFPI-2 gene on the growth of acute monocytic leukemia cell line (SHI-1).
METHODS:The cDNA of TFPI-2 was obtained by genetic chemical synthesis, the TFPI-2 gene and the linear vector fragment were ligated and inserted into the multiple cloning site of PEGFP-N1 vector, and the eukaryotic expression vector PEGFP-N1-TFPI-2 was transfected SHI-1 cells, then the obtained SHI-1 cells was observed by fluorescence microscopy; MTT assay was used to detect the effect of TFPI-2 gene on the relative growth rate of SHI-1 cells at the different time-point; RT-PCR was used to detect TFPI-2 mRNA expression levels in the cells of each group before and after TFPI-2 transfection; TFPI-2 protein expression was detected by Western blot. The cells which successfully transfected with PEGFP-N1-TFPI-2 vector were named as SHI-1-TFPI-2 (experimental group), and the cells transfected with the empty vector pEGFP-N1 and the untransfected cells were named as SHI-1-V and SHI-1-P and used as the control group.
RESULTS:The human TFPI-2 gene eukaryotic expression vector PEGFP-N1-TFPI-2 was successfully constructed, then the transfected into SHI-1 cells, observed by fluorescence microscopy 24 hours later, as a result, the PEGFP-N1-TFPI-2 was successfully transferred into SHI-1 cells, and the number of fluorescent cells increased after 48 h and 72 h. RT-PCR showed that the gray scale ratio of TFPI-2 gene to β- actin in the experimental group was higher than that in the control group. The gray scale ratio was 0.51±0.04 in SHI-1-V group, 0.52±0.03 in SHI-1-P group, 0.87±0.08 in SHI-1-TFPI-2 group, and the difference between SHI-1-TFPI-2 and SHI-1-V, SHI-1-P group was statistically significant (P＜0.05).
CONCLUSION:The expression of TFPI-2 gene in PEGFP-N1-TFPI-2 can inhibit the growth of SHI-1 cells, which provides a research direction for gene therapy of leukemia in the future.
