Inhibitory Effect of Eukaryotic Expression Vector Bearing TFPI-2 Gene on SHI-1 Cell Growth / 中国实验血液学杂志

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.

Inhibitory Effect of Serum Containing Fuzheng Jiedu Decoction on the Leukemia K562/A02 Multi-drug Resistance Cells and Its Mechanism / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To study the inhibitory effect of serum containing Fuzheng Jiedu decoction on leukemia multi-drug-resistance K562/A02 cells and its possible mechanism.</p><p><b>METHODS</b>The MTT method was used to detect the inhibitory rate of K562/AO2 cells treated with serum containing Fuzheng Jiedu decoction; the flow cytometry was used to detect the inhibitory effect of serum containing medicin on growth of K562/AO2 cells and P-gp expression; the Q-PCR was used to assay the BCL-2 mRNA expression; the Western blot was used to detect the BCL-2 protein expression.</p><p><b>RESULTS</b>MTT cytotoxic test showed serum containing Fuzheng Jiedu decoction could inhibit K562/A02 cell growth, and the inhibitory rate increased with the increase of drug concentration; the flow cytometry showed that the serum containing Fuzheng Jiedu decoction could promote K562/A02 cell apoptosis in a concentration-dependent manner. qPCR and Western blot showed that serum containing Fuzheng Jiedu decoction could down-regulate the protein expression of BCL-2. Fuzheng Jiedu decoction could reduce the protein expression of P-gp on the K562/A02 cell membrane.</p><p><b>CONCLUSION</b>serum containing Fuzheng Jiedu decoction can promote K562/A02 cell apoptosis, its mechanism of inducing apoptosis may be related with the inhibition of BCL-2 and P-gp protein expression.</p>