RESUMO
The E1A gene was obtained by PCR with QBI-293A cell genome DNA as template. After enzyme digestion, the E1A gene was ligated to transfer vector pAdTrack-CMV. The positive clone pAdTrack-CMV-E1A were lineared by PmeI and co-transformed with pAdEasy-1 in BJ5183 E. coli. The recombinant adenovirus vector pAdEasy-1-pAdTrack-CMV-E1A were digested by PacI and transfected into QBI-293A cells with liposomes. The oncolytic recombinant adenovirus Ad-E1A was obtained after 7 days. The results showed that this oncolytic adenovirus Ad-E1A can replicate in ECV304 cells and inhibit growth of ECV304 cell. In addition, it also decreased the secretion of VEGF and expression of NF-kappaB of ECV304 cells, indicating that Ad-E1A have potential of inhibition of tumor metastasis.
Assuntos
Humanos , Adenoviridae , Genética , Fisiologia , Proteínas E1A de Adenovirus , Genética , Proliferação de Células , Células Cultivadas , Células Endoteliais , Biologia Celular , Metabolismo , Terapia Viral Oncolítica , Vírus Oncolíticos , Genética , Fisiologia , Regiões Promotoras Genéticas , Veias Umbilicais , Biologia Celular , MetabolismoRESUMO
Study effect and mechanisms of growth-suppression of hepatocelluar carcinoma (HCC) in nude mice. The construction of the pAdeasy-1-pTrack-CMV-hIL-24 recombined adenovirus vector (Ad-hIL-24) was completed and lineared with PacI. Ad-hIL-24 were transfected into QBI-293 cells and obtained. 16 nude mice of the subcutaneous tumor models were established with SMMC-7721 HCC and were randomly divided into NS, 5-Fu, Ad and Ad-hIL-24 groups. Then 100 microL NS, Ad (10(7) pfu) and Ad-hIL-24 (10(7) pfu) for each one were given respectively QOD, and 5-Fu (20 microg/kg) were injected Q.D., for 5 times, with intratumor injections. After 15 d, 16 mice were sacrificed and subcutaneous tumors were taken out. The volumes (before administration, 1 week and 2weeks after administration) were measured and the weights of tumor were weighed and ratios of tumor-suppression were calculated. The morphological changes of apoptotic tumor cells were observed under microscope. Caspase3, P53 and P27, CD34 and VEGF were tested in immunohistochemistry. In tumor subcutaneous model, compared with NS group, the ratios of tumor-suppression of Ad-hIL-24 group and 5-Fu group were 68.52% (P < 0.01) and 65.64 (P < 0.01), respectively. Caspase3 protein in Ad-hIL-24 group was higher than other 3 groups significantly (P < 0.01). The expression of P27 also differed from NS group (P < 0.01). CD34 and VEGF protein in Ad-hIL-24 group can inhibit neovascularization obviously (P < 0.001), compared with NS and Ad groups. Ad-hIL-24 inhibits the growth of SMMC-7721 HCC on nude mice's. The mechanisms of tumor-suppression may be multi-pathways such as the induction of caspase3 pathway, P27 activities and the antiangiogenic mechanism.
Assuntos
Animais , Humanos , Camundongos , Adenoviridae , Genética , Carcinoma Hepatocelular , Genética , Metabolismo , Patologia , Linhagem Celular Tumoral , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Vetores Genéticos , Genética , Imuno-Histoquímica , Interleucinas , Genética , Neoplasias Hepáticas , Genética , Metabolismo , Patologia , Camundongos Nus , Plasmídeos , GenéticaRESUMO
The hIL24 cDNA sequence was cloned into prokaryotic high expressive vector pET-21a(+) and recombinant hIL24 was expressed in E. coli with IPTG induction. The purified recombinant hIL24 exhibits following functions in HeLa cell: inhibiting cell growth, inducing apoptosis, inducing PMBC to secrete IL-6, TNF-alpha, IFN-r and inhibiting blood vessel formation. Our preliminary results suggest that the apoptosis induced by rhIL24 is through down-regulating expression of anti-apoptosis factor Bcl-2 and activation of mitochondria apoptosis pathway.