ABSTRACT
Objective:To explore the inhibitory effect of Tum-5 gene on the tumor growth and its influence in the angiogenesis of hepatocellular carcinoma (HCC),and to illustrate its mechanism involved in antitumorigenesis.Methods:The human HepG2 cells were selected in in vitro experiment and treated with different multiplicity of infection (MOI) (0,1,5,10,25 and 50) of pLXSN and pLXSN-Tum-5 virus particles for 72 h.The proliferation rates of cells in various groups were detected by MTT method.In vivo,the H22 tumor-bearing mouse models were established.The mice were divided into saline group,pLXSN group and pLXSN-Tum-5 group (n=5).The mice in saline group were intratumorally injected with normal saline,and the mice in pLXSN group and pLXSN-Tum-5 group were intratumorally injected with virus particles (MOI =5),5 times every other day.The volumes and weights of transplanted tumor and the weights of mice in various groups were measured.The CD31 expressions in transplanted tumor tissue were detected by immunohistochemical method and the microvessel density (MVD) was calculated.Results:Compared with pLXSN group,the proliferation rates of cells in pLXSN-Tum-5 group after infected with different MOI of virus particles were not significantly different (P＞ 0.05).The volumes and weights of transplanted tumor of the mice in pLXSN-Tum-5 group were significantly smaller than thosein pLXSN group and saline group after intratumoral injection (P＜ 0.05 or P＜ 0.01).The immunohistochemical results showed that there was irregular angiogenesis in each group.Compared with saline group and pLXSN group,the mean value of MVD of the transplanted tumor tissue of the mice in pLXSN-Tum-5 group was significantly decreased (P ＜ 0.05).Conclusion:Tum-5 can exert its antitumor activity by inhibiting the formation of neovascularization in HCC.
ABSTRACT
Objective To obtain the Tat-GFP fusion proteins with penetrating activity and labeled with green fluorescence protein (GFP), and to explore the cell membrane penetrating activity of Tat-GFP in MCF-7 cells. Methods The plasmid pET-24a-Tat-GFP was transformed into Escherichia coli BL21 cells. Different concentrations (0.5 and 1.0 mmol · L-1 ) of isopropyl-β-D-thiogalactopyranoside (IPTG ) and cell culture temperatures (22℃ and 37℃)were used to optimize the protein expression.The Tat-GFP proteins in supernatant were purified using Ni-IDA resins. Western blotting analysis was used to identify the Tat-GFP protein, and confocal laser scanning microscope (CLSM ) was used to examine the cell penetration of Tat-GFP protein. Results There was no significant difference in the Tat-GFP protein production induced by 0.5 and 1.0 mmol·L-1 IPTG;however,the low temperature (22℃)-induced BL21 cells expressed more Tat-GFP proteins than that at 37℃ induction.The Western blotting analysis results showed that GFP antibody could specifically recognize the proteins in PVDF membranes in dose-dependent manner;the CLSM results indicated the distribution of green fluorescence in cytoplasm and nucleus of MCF-7 cells.Conclusion The Tat-GFP protein highly expresses in the supenatant of Escherichia coli i BL2 1 cells at low temperature;the obtained Tat-GFP protein with green fluorescence preserves the cell penetrating activity.