To express recombinant arresten in Escherichia coli (E. Coli) and investigate its biological activity, prokaryotic expression vector of human arresten gene was constructed by gene engineering. Human arresten gene was amplified from recombinant plasmid pGEMArr by polymerase chain reaction (PCR), and inserted into prokaryotic expression vector pRSET containing T7 promoter. Restriction analysis and DNA sequencing verified that the arresten gene was correctly cloned into the expression vector. The recombinant plasmid pRSETAt was subsequently transformed intoE. coli BL21 (DE3), and the target gene was expressed under induction of IPTG. SDS-PAGE analysis revealed that the recombinant protein with a molecular weight of 29 kD (1 kD=0.992 1 ku) amounted to 29% of the total bacterial proteins. After purification and renaturation, the recombinant protein could significantly suppress the proliferation of human umbilical vein endothelial cells (HUVECs). These results suggested that the expression of a biologically active form of human arresten in the pRSET expression system laid a foundation for further study on the mechanistic insight into arresten action on angiogenesis and the development of powerful anti-cancer drugs.

In order to investigate the effect of tumor necrosis factor-α (TNFα) on resistin expression in 3T3-L1 adipocytes, and further explore its mechanisms, the differentiated 3T3-L1 adipocytes were incubated with 0, 1, 10, 100 ng/mL TNFα respectively for 24 h, and then the expression of resistin was determined. The differentiated 3T3-L1 adipocytes were incubated with 100 ng/mL TNFα for 3, 6, 24 h respectively, and then the expression of resistin mRNA was analyzed. 3T3-L1 adipocytes were induced to differentiate into mature adipocytes. The cells were randomly divided into 4 groups for culture. In the control group, no drugs were added. Cells of TNFα group were treated with 100 ng/mL TNFα. In Ro-31-8220 group, 5 μmol/L protein kinase C inhibitor Ro-31-8220 was added. With TNFα+Ro-31-8220 group, 100 ng/mL TNFα were added 1 h after the addition of 5 μmol/L Ro-31-8220. All adipocytes were cultured for 24 h. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were employed to detect the expression of resistin gene. Our results showed that resistin protein and mRNA in 3T3-L1 adipocytes were inhibited by TNFα at different concentrations (P<0.01), and the inhibitory effect increased with the concentration (P<0.01). At the same concentrations, the inhibitory effect increased with time (P<0.01). Ro-31-8220 could inhibit its expression and the inhibitive effect remained unchanged with addition of TNFα(P>0.05). It was concluded that TNFα could inhibit the expression of resistin in 3T3-L1 adipocytes. The mechanism may be that the expression of resistin is partly controlled by protein kinase C signal conduction pathway.

To explore the mechanism of epilepsy induced by IL-1β and IL-6, the changes of glutamic acid (Glu) and GABA immunoreaction in the cerebral cortex and hippocampus of rats with seizure induced by IL-1β or IL-6 were studied. Rats were randomly divided into 3 groups: control group (i. c. v. injection of IL-6). 120 min after the icv injection of reagents of IL-1β or IL-6, behavioral changes were observed and Glu and GABA in the cerebral cortex and hipocampus were examined by means of immunohistochemistry. Our results showed that no seizure developed in the control group, while moderate seizure was observed in IL-1β group and IL-6 group. Compared with the controls, the immunoreaction of Glu was significantly increased, while GABA was obviously decreased in IL-1β group and IL-6 group after 120 min. Our study suggested that the IL-1β and IL-6 might promote and induce epilepsy by increasing Glu and decreasing GABA in the cerebral cortex and hippocampus.

Evaluierung der kombinierten Therapie bestehend aus transarterieller Chemoembolisation (TACE) und lokaler Immunotherapie des hepatozellulären Karzinoms (HCC) am Kleintiermodell. Bei männlichen ACI-Ratten (n=30) erfolgte nach subkapsulärer Implantation eines soliden Morris Hepatoms (2 mm³) in den linken Leberlappen (Tag 0) die magnettomographische Tumorvolumenbestimmung (V1: Tag 13). Nach retrograder Katheterisierung der Arteria gastroduodenalis wurden die Tiere entsprechend den folgenden Therapieschemata behandelt (Tag 14): (A) TACE (0,1 mg Mitomycin C+0,1 ml Lipiodol)+(TNF (7,5μg)+IL-2 (5×10³IU) (n=10); (B) TACE+OK-432 (0,05 mg) + IL-2 (5×10³ IU) (n=10); (C) TACE allein (Kontrollgruppe,n=10). Zu Beurteilung des Therapieerfolges der unterschiedlichen Behandlungsprotokolle wurde das Tumorvolumen (V2) erneut magnettomographisch ermittelt (Tag 28). Das durchschnittliche Tumorvolumenverhältnis (V2/V1) betrug 7, 30, 6, 53 und 9, 14 in Gruppe A, B und C, Im Vergleich zur Kontrollgruppe zeigten die Therapiegruppen A (P<0,05) und B (P<0,01) ein statistisch signifikant geringeres Tumorvolumenverhältnis (V2/V1). Das Wachstum des induzierten Morris Hepatom wurde unter der kombinierten Therapie von TACE und lokaler Immunotherapie im Tiermodell signifikant, im Vergleich zur Kontrollgruppe, gehemmt.

Evaluation der Effektivität der Kombinationsbehandlung von TACE und LITT im Kleintiermodell. Lebermetastase wurde nach subkapsulärer Injektion von den Tumorzellen (CC531) in den WAG Ratte (n=30) implantiert (Tag 0). An dem 13. Tag danach wurde die Tumorvolumina (V1) magnettomographisch bestimmt. Im Anschluß erfolgte nach Laparatomie die folgende Therapieprotokolle: (A) TACE allein (0,1 mg Mitomycin C+0,1 ml Lipiodol+5,0 mg DSM) (n=10); (B) LITT allein (n=10): (C) TACE+LITT (n=10). Zur Effektivitätsbeurteilung der unterschiedlichen Therapieprotokolle erfolgte eine erneute magnetto-mographische Bestimmung der Tumorvolumina (V2). Das durchschnittliche Tumorvolumen der Gruppen A, B und C vor und nach interventioneller Therapie betrug 0,1100 cm³ und 0,5988 cm³, 0,1108 cm³ und 0,6763 cm³ sowie 0,1110 cm³ und 0,3503 cm³. Das errechnete durchschnittliche Verhältnis (V2/V1) betrug 5,42, 6, 14 sowie 3, 14. Im Vergleich zu Kontrollegruppen A und B konnte für Therapiegruppe C eine signifikant (P<0,01) geringere Tumorvolumenzunahme im Beobachtungszeitraum ermittelt werden. Im Vergleich zu den Kontrollgruppen konnte das Wachstum der induzierten lebertumoren unter Behandlung mittels TACE+LITT im Tiermodell statistisch signifikant gehemmt werden.

The specific anti-tumor immune response induced by mouse bone marrow dendritic cells (DCs) transfected with recombinant adenovirus carrying mutant k-ras genes was investigated. DCs were generated from mouse bone marrow in the presence of rmGM-CSF (3.3 ng/mL) and rmIL-4 (1.3 ng/mL) and detected by FACS, and then transfected with the recombinant adenovirus encoding mutant k-ras gene. The efficacy of transfection and T cell stimulating activity of DCs were detected. CTL activity of the mice vaccinated with DCs was observed. The results showed that DCs had dendritic veiled morphology. BmDCs highly expressed B7-1 (80%), B7-2 (77%), MHC II (70%), CD11c (65%), CD40 (70%) and CD54 (96%) with FACS, and no significant difference in the expression was observed before and after the transfection (P>0.05). The DCs transfected by mutant k-ras gene could significantly stimulate lymphocytes proliferation as compared with those transfected by Ad-c or non-modified DCs (P<0.05). DC vaccine transfected by mutant k-ras gene could induce CTL activity against Lewis lung cancer, but not against B16. The specific cytotoxicity against Lewis lung cancer in Ad-k-ras/12-transduced DC group was significantly higher than those in the control, vector and non-transfected DCs groups (P<0.05). It was concluded that special antitumor response could be induced by DCs transfected with recombinant adenovirus carrying mutant k-ras genes.