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Objective To obtain a chimera composed of annexin Bl (AnxBl) and melittin (MLT) and to investigate its inhibitory effect on phosphatidylserine liposome activity and SMMC7721 and HepG2 cell proliferation. Methods A fusion gene AnxBl-MLT was constructed by overlap extension gene splicing and then was inserted into plasmid pGEX-5T. The recombinant plasmid was transformed into E. coli strain K802 and induced by IPTG at low temperature. The expression condition was optimized and GST affinity chromatography column was used for purification. Calcium-dependent phospholipid binding assay was used to determine whether the chimera kept the activity of AnxBl. CCK8 analysis was employed to investigate the effect of AnxBl-MLT on SMMC7721 and HepG2 cell proliferation. Results After being inserted into the expression plasmid, AnxBl-MLT protein expressed in K802 cells, with a high level recombinant protein induced by 0. 2 mmol/ L IPTG at 22-24»C for 4 h. The protein AnxBl-MLT was purified by using GST affinity chromatography column (a band at 63 000) and the purification of the final purified protein was >95%. AnxBl-MLT was able to bind to phosphatidylserine liposome in a calcium-dependent manner. CCK8 analysis indicated that AnxBl-MLT inhibited SMMC7721 and HepG2 cell proliferation at a dose-dependent manner. Conclusion We have successfully constructed a chimera AnxBl-MLT, which retains the calcium-dependent phospholipid binding activity of AnxB1, and can inhibit the proliferation of hepatic cancer cell lines SMMC7721 and HepG2.
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Background and purpose: One of the main mechanism of chemotherapy is inducing tuomr apoptosis. Molecular imaging can allow noninvasively and dynamically monitor tumor apoptosis in vivo, and help to drug screening and therapeutic evaluation. The purpose of this study was to evaluate the feasibility of 18F-SFB-Annexin B1 in detecting apoptosis at an early phase after chemotheraphy. Methods:Annexin B1 was labeled with 18F using SFB as a chelating agent. Tissue distribution of 18F-SFB-Annexin B1 was studied in healthy mice by the dissection method. W256 tumor-bearing rats were injected with 18F-SFB-Annexin B1 intravenously at 24 h after the treatment of cyclophosphamide (CTX 200 mg/kg) or saline. Then imaging was acquired at 1, 2, 3, and 4 h postinjection on a PET/CT, and the tumor-to-muscle ratio of SUVmax (T/M) and the AI from TUNEL testing were compared. Results: 18F-SFB-Annexin B1 had a radiochemical pruity (RCP)>95%. Biodistribution of this probe showed a predominant uptake in the kidney, then was liver, spleen, and myocardium, rapid clearance from blood and urinary was observed. The radios of T/M were 4.38±0.56, 6.75±1.16, 6.44±1.12, 4.81±0.17, respectively at 1, 2, 3, 4 h post injection of the chemotherapy group, much higher than that of the saline group (2.35±0.14, 2.99±0.55, 3.04±0.41, 2.33±0.47, respectively). The differences between the two groups were significant (F=23.790, 16.913, 14.046, 77.517, respectively, all P<0.05). TUNEL staining revealed that chemotherapy treatment significantly increased the percentage of apoptosis cells with an AI of (21.00±0.04)%in the chemotherapy group, higher than that in the saline group (8.58±0.01)%, the difference was significant (F=21.539, P<0.05). The radios of T/M were significantly correlated with the values of AI (r=0.91, P<0.05). Conclusion: 18F-SFB-Annexin B1 can be used to apoptosis imaging and early therapeutic evaluation in vivo because it can reflect apoptosis at an early stage after chemotheraphy.
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Objective To evaluate 18F-N- succinimidyl -4-fluorobenzoate (SFB)-Annexin B1 in detectingin vitro andin vivo apoptosis. Methods Anti-Fas antibody was used to induce apoptosis in Jurkat cells. Apoptosis in Jurkat cells was confirmed by flow cytometer (FCM). Unilateral renal ischemia/reperfusion injury was induced by transient (45 min) ligation of the renal artery in the rabbit. The rabbit was then administrated with 18F-SFB-Annexin B1 intravenously 24 h later and then imaged by PET/CT at 10,30,60,90,120 and 240 min postinjection. Apoptosis in kidney was confirmed by terminal deoxynucleotidyl transferase mediated dUTP biotin nick end labeling (TUNEL) assay and HE staining. Results The apoptosis rate induced by anti-Fas antibody was 25.98%(120 min) while that in the control group was only 1.81%. The uptake of 18F-SFB-Annexin B1in apoptosis group was greater than that in the control group. PET/CT images at 240 min showed higher uptake in the ligated kidney than the non-ligated kidney. TUNEL assay and HE staining confirmed great amount apoptotic cells in the ligated kidney. Conclusion 18 F-SFB-Annexin B1may be potentially useful in detecting apoptosis both in vitro and in vivo.
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Objective:To prepare monoclonal antibody against Annexin B for further studying the physiological function of Annexin B1.Methods: Annexin B1 was expressed and purified in E.coli and its antigenicity was confirmed.The product was used to immunize BALB/c mice by intrasplenic embedding.Spleen cells of BALB/c mice were obtained and fused with myeloma cell line SP2/0 in a HAT medium containing 50% PEG 4000 and were then subjected to incubation in 37℃,5% CO_(2)incubator for several days.ELISA was used to assay the type and titer of the McAbs in the supernatant.Results: A hybridoma cell line 2B10H5,which can steadily secrete specific McAbs,was obtained.Conclusion: The McAbs of Annexin B1 have been successfully prepared,which pave a way for further investigation of Annexin B1.