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Objective To prepare 131I-anti-neuropilin-2-monoclonal antibody (131I-anti-NRP-2-mAb),and investigate its biodistribution and imaging in nude mice bearing xenografted lung adenocarcinoma,in order to evaluate its feasibility as an imaging agent targeting to NRP-2 positive tumors.Methods (1)131I-anti-NRP-2-mAb was prepared by Chloramine-T method under the optimum labeling conditions,then the labeling efficiency,radiochemical purity and stability were determined in vitro.(2) The binding fraction and receptor binding affinity of 131I-anti-NRP-2-mAb were measured in A549 human lung cancer cells by cell uptaking and binding experiments.(3) The A549 tumor-bearing mice were randomly divided into 4 groups with direct sampling method and were sacrificed at 6,24,48,and 72 h,respectively,after tail intravenous injection of 0.37 MBq 131I-anti-NRP-2-mAb.The distribution was measured,and the ratios of tumor/muscle (T/M) and tumor/blood (T/B) were calculated.(4) Gamma imaging was performed in 6 mice,including 3 in the competitive inhibition control group (injected with 3.7 MBq 131I-anti-NRP-2-mAb and 100 μg atniNRP-2-mAb),at 6,24,48,and 72 h post-injection to observe the radioactivity in tumor.Two-sample t test was used for data analysis.Results (1) The labeling yield and radiochemical purity of 131I-anti-NRP-2-mAb were (94.69 ± 3.63) ％ and (98.56± 0.48) ％,respectively.The radiochemical purity was more than 85％ after incubating in phosphate-buffered solution at room temperature for 72 h.(2) At 60,120,180 and 240 min post-injection,the binding ratios of 131I-anti-NRP-2-mAb in A549 cells were (3.95±0.18)％,(5.19±0.65) ％,(6.60± 0.36) ％ and (5.58± 0.63) ％,respectively.When excessive anti-NRP-2-mAb were added,the binding ratios were reduced to (0.94±0.31)％,(1.12±0.17)％,(1.24±0.25)％ and (1.04±0.18) ％,respectively,which were significantly lower than those of non-inhibited group (t values:9.89-19.66,all P＜0.05).131I-anti-NRP-2-mAb bound to NRP-2 with high affinity half maximal inhibitory concentration (IC50 =(410.8±1.2) nmol/L).(3) Biodistribution study demonstrated that the T/M and T/B ratios increased with the time extension and were 3.83±0.18 and 1.10±0.20,respectively,at 72 h post-injection.(4) Gamma imaging studies revealed that 131I-anti-NRP-2-mAb could clearly identify A549 tumors 6 h post-injection,especially at 48 h post-injection.Tumors were not observed clearly in competitive inhibition control group.Conclusion 131I-anti-NRP-2-mAb has been successfully prepared,and it could target to NRP-2 specifically.
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Objective To investigate the role and significance of neuropilin-2(NRP2)for regulating the angiogenesis of pancreatic neuroendocrine tumors(PNETs).Methods The NRP2 expression in pancreatic neuroendocrine tumer BON-1 cell line was intevened.The BON-1 cells cultural supernatants in the control group and interference group were used to treat human umbilical vein endothelial cells(HUVEC).CCK-8 was used to detect the cell proliferation,Transwell was used to detected the cell migration and the tubule formation test was used detect the pro-angiogenesis.Results The CCK-8 detection showed that there was no statistically significant difference in the supernatant treated HUVEC proliferations between the interference group and control group medium(P>0.05):the absorbancy in the control group was 0.35±0.04,while which in the interference group was 0.32±0.04.The Transwell test showed that the invasion ability of HUVEC treated with cultural supernatants in the interference group was weakened compared with the control group,the control group was(203±13)/hole,while the interference group was(100±10)/hole(P＜0.01);the tubule formation test showed that HUVEC tubular formation treated by cultural supernatant in the interference group was decreased,the control group was 40±5,while the interference group was 24±3(P＜0.01).Conclusion Interfering NRP2 expression of BON-1 cells can inhibit the vessel formation ability of co-cultured HUVEC,suggesting that NRP2 may have the pro-angiogenesis effect of PNETs,and may be a potential new target for the treatment of PNETs.
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Background and purpose:This study was to investigate the effect of miRNA-486 on the growth of human colorectal cancer cell line SW620 xenograft in nude mice and to explore the possible mechanism of action. Methods:Eighteen mice were randomly divided into three groups, including the experimental group, the negative control group and the blank control group. Each group contained 6 mice. The SW620 cell line was inoculated subcutaneously into nude mice to establish the model of human colorectal cancer xenografts. Peritumoral injection of miRNA-486 overexpres-sion plasmid, or blank vector and PBS were performed every 3 days. The volumes of subcutaneous tumors in each group of inoculated mice were compared. Then mice were sacrificed 3 weeks after infection. Immunohistochemistry and Western blot were used to measure the expression of neuropilin-2 (NRP2).Results:The growth rate of tumors in the experimental group was significantly lower than that in the negative control group and the blank control group. After 21 days, the size of transplanted tumors in the experimental group nude mice was (0.32±0.12) cm3, that in the negative control group was (0.77±0.31) cm3, and that in blank control group was (0.82±0.18) cm3. Tumor mass in the experimental group was sig-nificantly smaller than that in the other two groups (P=0.006<0.05). Tumor mass in the experimental group was (0.40±0.08) g, significantly smaller than that in the negative control group (0.75±0.18) g and in the blank control group (0.79±0.18) g (P=0.008<0.05). Compared with the expression of NRP2 in other groups, the growth of tumor in the experimental group de-clined (P=0.000<0.05).Conclusion:Colorectal cancer cell line SW620 xenografted tumor in nude mice can be suppressed after injection of miR-486, which may decrease the expression of NRP2.