[Tyrosine kinase 2 with immunoglobulin-like and epidermal growth factor homology domains RNA interference inhibit the proliferation of human umbilical vein endothelial cells].

OBJECTIVE: The purpose of this study was to investigate the regulatory role of tyrosine kinase 2 with immunoglobulin-like and epidermal growth factor homology domains (Tie2) on apoptosis and proliferation in the endothelial cells. METHODS: RNA interference (RNAi) technique was used to silence Tie2 gene expression by transfecting an expression vector containing short hairpin RNA(shRNA) for Tie2 into human umbilical vein endothelial cells (HUVECs). Real time quantitation reverse transcriptase polymerase chain reaction (QRT-PCR) and Western blot were used to monitor Tie2 mRNA, as well as protein expression. The proliferation of HUVECs was examined by methyl thiazolyl tetrazolium (MTT), and the apoptosis was detected under microscope. HUVECs transfected with pGenesil-hk was negative control, and HUVECs transfected with nothing was empty control. RESULTS: Tie2 mRNA expression was down-regulated 24 h and 48 h after transfection, and Tie2 protein expression was significantly down-regulated at 24 h and 48 h (P< 0.05), especially 48 h after transfection. The apoptosis rate was conspicuously higher in experimental group than in negative control and empty control group after 48 h (P<0.05). The growth monitoring showed that proliferation was also markedly inhibited in experimental group (P<0.05) compared with two control groups. CONCLUSION: Down-regulated expression of Tie2 by RNAi can promotes apoptosis of HUVECs and has an anti-proliferation activity effect on them.

[Intratumor injection of recombinant attenuated salmonella carrying Mycobacterium tuberculosis heat shock protein 70 and herpes simplex virus thymidine kinase genes to suppress murine melanoma growth].

OBJECTIVE: To study the effection of suppression murine melanoma growth by Intratumor injection of recombinant attenuated salmonella carrying heat shock protein 70 and herpes simplex virus thymidine kinase genes. METHODS: Plasmids PCMV-mtHSP70-IRES-TK were electro-transferred into salmonella typhimurium SL7207 to construct recombinant salmonella typhimurium. In vivo, Recombinant bacteria were injected into the mouse melanoma and the antitumor effection was observed. The survival period was recorded and safety analysis for this vaccine in each group. RESULTS: In vivo, the mtHSP70/HSV-tk recombinant bacteria can suppress tumor growth significantly and extend survival. After recombinant Salmonella, 10(9) CFU/mL, was administered as an intratumoral injection, No diarrhea were observed. During therapy, body weight did not change markedly. CONCLUSION: Results of the animal experiment suggests intratumor injection of recombinant attenuated salmonella typhimurium containing mtHSP70 and HSV-tk genes, has targeting ability against B16 tumor cell and could significantly inhibit tumor growth .

Suppression of murine melanoma growth by a vaccine of attenuated Salmonella carrying heat shock protein 70 and Herpes simplex virus-thymidine kinase genes.

Attenuated Salmonella can invade tumor cells and acts as a eukaryotic expression vector for gene propagation. We constructed a bi-gene, eukaryotic co-expression DNA vaccine of Mycobacterium tuberculosis heat shock protein 70 (mtHSP70) and Herpes simplex virus-thymidine kinase (HSV-tk) and used attenuated Salmonella as a vector to treat murine melanoma. In vitro, recombinant Salmonella can carry plasmid stably and can invade into the cytoplasm of B16 tumor cells expressing the protein of the mtHSP70/HSV-tk gene by Western blot assay. In vivo, after the recombinant Salmonella was injected into tumors, the HSV-tk precursor drug ganciclovir (GCV) was administered to start the HSV-tk killing of tumor cells. We found that the mtHSP70/HSV-tk recombinant bacteria can raise CD8+ T lymphocytes in peripheral blood by flow cytometry and in tumor tissues by immunofluorescence detection, increase IFN­Î³ contents in tumor tissue by ELISA and significantly suppress tumor growth.