Imaging and biodistribution of 131I labeled anti-neuropilin-2 monoclonal antibody in lung adenocarcinoma-bearing mice / 中华核医学与分子影像杂志

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.

Effects of acupuncture and moxibustion at different times on the nausea and vomiting induced with chemotherapy: a self cross control study / 中国针灸

<p><b>OBJECTIVE</b>To investigate acupuncture-moxibustion intervention during the different times for chemotherapy-induced nausea and vomiting (CINV) symptoms.</p><p><b>METHODS</b>Eighty patients received cisplatin chemotherapy with nausea and vomiting were assigned into an AB group and a BA group. Self cross control study was carried out. In the AB group, acupuncture and moxibustion were used during the intermission and there were no acupuncture-moxibustion during chemotherapy period in the first chemotherapy cycle; acupuncture and moxibustion were not applied during the intermission and acupuncture-moxibustion intervention were used during chemotherapy in the third cycle of chemotherapy. The intervention times were different correspondingly in the BA group. Acupuncture and moxibustion were not used to in the second cycle of elution period. The vomiting incidence, nausea, vomiting and retching scale (R-INVR), patient satisfaction and compliance during different chemotherapy times after acupuncture and moxibustion intervention were compared.</p><p><b>RESULTS</b>On the first and second days of chemotherapy, the nausea incidences of patients treated with acupuncture and moxibustion during the intermittent period was less than those during chemotherapy period (both <0.05), but continuous 4 days after the third day of chemotherapy, the numbers had no significant difference (all >0.05). The scores of R-INVR were not significantly different between intervention in the chemotherapy period and intermittent period (>0.05), with decreasing trend. The patients were more satisfied with acupuncture and moxibustion in the intermittent period (<0.05).</p><p><b>CONCLUSION</b>Acupuncture and moxibustion in the intermittent period can prevent vomiting induced by the chemotherapy of cisplatin, with satisfaction and compliance. Acupuncture and moxibustion intervention during chemotherapy period have the potential to improve nausea and vomiting.</p>

Inhibitory effect of NRP-1 b1/b2 monoclonal antibody on invasion and migration of gastric cancer cell line BGC-823 and its mechanism / 肿瘤研究与临床

Objective To observe the effect of anti NRP-1 b1/b2 monoclonal antibody (NRP-1mAb) on migration and invasion of gastric cancer cell line BGC-823, and to explore the possible mechanism. Methods NRP-1mAb was prepared in the laboratory, and the purity of antibody was detected by flow cytometry. The different concentrations of NRP-1mAb were added into the culture medium of gastric cancer cell line BGC-823. The migration and invasion of cells after 12 hours was observed by using Transwell method. The phosphorylation of related signal proteins after NRP-1mAb was detected by Western blot analysis. Results When NRP-1mAb prepared by patented technology had the effects on BGC-823 cells after 12 hours, the number of migration and invasion of BGC-823 cells was reduced. The number of cells through the basement membrane in the control group (blank) and the administration group (NRP-1mAb 25, 100, 400 μg / ml) were 167 ± 9, 138 ± 5, 98 ± 5, 36 ± 4, respectively (F = 22.6, P< 0.01); the number of cells through the filtration membrane were 231 ± 40,224 ± 19,176 ± 26,124 ± 34,respectively(F=26.63,P<0.01). There were statistically significant differences between the administered group and the control group at 100 and 400 μg/ml (all P< 0.001). High concentration of NRP-1mAb (100 μg/ml) decreased the phosphorylation level of Akt after 10 minutes' function on gastric cancer cells. However, it was difficult to detect phosphorylated Akt after 30 minutes. Conclusion NRP-1mAb may inhibit the migration and invasion of gastric cancer cell line BGC-823 by decreasing the phosphorylation of Akt, which is positively correlated with the concentration.

Clinical observation of acupuncture-moxibustion for functional dyspepsia with emotional disorder / 中国针灸

<p><b>OBJECTIVE</b>To compare the clinical effective differences between acupuncture-moxibustion and Chinese herbs for functional dyspepsia with emotional disorder.</p><p><b>METHODS</b>Eighty patients were randomly divided into an observation group and a control group, 40 cases in each one. In the observation group, based on the basic treatment Zusanli (ST 36), Yinlingquan (SP 9), Zhongwan (CV 12), Danzhong (CV 17), Neiguan (PC 6), Taichong (LR 3), Ganshu (BL 18), Geshu (BL 17) and Danshu (BL 19) were selected once every day or every other day. The treatment was given 14 times. In the control group, based on the basic treatment individual therapy of Chinese herbs according to syndrome differentiation was applied,one dose a day. Patients were treated for 4 weeks in the two groups. The scores of self-rating depression scale (SDS), the main symptoms and satisfied degree by self-rating for treatment were observed before and after 4-week treatment in the two groups. Also, the rate and the average time of return visit were compared in 6 months after treatment. Results After treatment, the SDS scores and the main symptoms scores were all improved compared with those before treatment in the two groups (all P< 0. 05). After 4-week treatment, the improvement of the SDS score in the observation group was better than that in the control group (48. 9±8. 5 vs 53. 1±8. 0, P<0. 05). After 1-week treatment and 4-week treatment, the improvements of the main symptoms in the observation group were superior to those in the control group (2. 7 ± 1. 0 vs 3. 3±0. 9, 1. 5±0. 9 vs 2. 3±1. 1, both P<0. 05), and the satisfied degree scores by self-rating were better than those in the control group (3. 0±1. 1 vs 3. 8±1. 3, 2. 0±1. 4 vs 2. 9±1. 5, both P<0. 05). In 6 months after treatment,the return visit rate in the observation group was 42. 5% (17/40), and it was lower than 70. 0% (28/40) in the control group (P<0. 05). The average time of return visit in the observation group was less than that in the control group (1. 0±0.8 vs 1. 9±0. 7, P<0. 05).</p><p><b>CONCLUSION</b>The effect of acupuncture-moxibustion or functional dyspepsia with emotional disorder is better than that of Chinese medicine.</p>

Copracrasia treated with acupuncture and moxibustion: a randomized controlled trial / 中国针灸

<p><b>OBJECTIVE</b>To explore the clinical efficacy and safety of acupuncture and moxibustion for copracrasia.</p><p><b>METHODS</b>By prospective live randomized controlled trial, 40 cases with copracrasia were randomly divided into an acupuncture-moxibustion group and a medication group, 20 cases in each one. In the acupuncture-moxibustion group, acupuncture was applied at Ciliao (BL 32), Changqiang (GV 1) and Tianshu (ST 25) and mild moxibustion was used at Qihai (CV 6). Treatment was given for 12 weeks and 32 times, 3 times a week in the front 8 weeks, 2 times a week in the latter 4 weeks. In the medication group, conventional symptomatic treatment, support therapy, and complications preventing and treating were adopted for 12 weeks. Anal incontinence score (Vaizey incontinence score), effective rate and self-rating score for satisfaction were observed before and after treatment and in the follow-up period.</p><p><b>RESULTS</b>After 12 weeks' treatment in the two groups, Vaizey incontinence' scores were both decreased (both P<0. 05), and after treatment and in the follow-up period the scores in the acupuncture-moxibustion group were lower than those in the corresponding period in the medication group (both P< 0. 05). The effective rate of the acupuncture-moxibustion group was 80. 0% (16/20), which was statistically different from 50. 0% (10/20) in the medication group (P<0. 05). The effective rate in the follow-up period of the acupuncture-moxibustion group was 90. 0% (18/20) and it was not statistically different from 80. 0% (16/20) in the medication group (P>0. 05). The self-rating scores for satisfaction in the acupuncture-moxibustion group were superior to those in the medication group after treatment and in the follow-up period (both P< 0. 05).</p><p><b>CONCLUSION</b>Acupuncture and moxibustion could improve copracrasia and the acupuncture-moxibustion rules and characteristics for the disorder should be paid attention to in the further research.</p>

Biodistribution and imaging of 131I labeled anti-neuropilin-1 monoclonal antibody in malignant gliomas xenografts / 中华核医学与分子影像杂志

Objective To synthesize 131I labeled anti-neuropilin-1 monoclonal antibody A6 (131IA6) and evaluate its biodistribution and imaging in malignant glioma xenografts.Methods (1) A6 was labeled with 131I by Iodogen method under the optimum labeling conditions,then the labeling efficiency,radiochemical purity and stability were measured in vitro.(2) In vitro bioactivity,cellular uptake and receptor affinity of 131I-A6 with U87MG cells were measured.(3) The nude mice bearing human U87MG cells were randomly divided into 5 groups with 5 in each group.The nude mice were sacrificed by cervical dislocation and dissected at 24,48,72,96,and 120 h,respectively,after intravenous injection of 1.2 MBq 131I-A6.The biodistribution of the agent was measured as ％ID/g,and the ratios of tumor/blood (T/B) and tumor/muscle (T/M) were calculated.(4) SPECT/CT imaging was performed in 6 mice including 3 in the competitive inhibition control group at 24,48,72,96,and 120 h post injection.Two-sample t test was used for data analysis.Results (1) The labeling yield of 131I-A6 was (95.46±3.34)％,and the radiochemical purity was more than 95％.At 96 h of incubation in PBS,the radiochemical purity was more than 85％.(2)131I-A6 had rapid accumulation in U87MG cells and reached the peak of (15.80±1.30)％ at 1 h.When the probe was incubated with large excesses of non-radioactive A6,the uptake level of 131I-A6 in U87MG cells was significantly inhibited (t=2.862,P＜0.05).Kd of 131I-A6 binding to NRP-1 was (1.67±0.14) nmol/L in U87MG cells.(3) Biodistribution study showed that the uptake in blood,liver and tumor was (8.00±1.42),(7.68±1.56) and (6.00±1.24) ％ID/g at 24 h,respectively.The uptake in muscle,brain and bone was lower.The T/B and T/M were 0.78±0.10 and 3.20±0.30 at 24 h,and they reached the highest level of 1.87±0.50 and 7.13±0.24 at 120 h.(4) The SPECT imaging showed that the tumors could be visualized at 24 h and delineated more clearly at 120 h post injection of 131I-A6.Conclusions 131I-A6 can be easily synthesized by Iodogen method with high radiochemical purity.The specific tumor uptake of 131I-A6,which correlates with NRP-1 expression in gliomas,suggests that it may be a new promising tumor targeting radiotracer.

Effect of Helicobacter pylori on the function of peripheral blood monocyte-derived dendritic cells in gastric cancer patients / 中国肿瘤临床

Objective: This study aimed to compare and analyze the functional differences between peripheral blood mono-cyte-derived dendritic cells (DCs) of Helicobacter pylori-positive and H. pylori-negative patients with gastric cancer. Methods:H. py-lori infection was detected in 84 patients with gastric cancer in our hospital from January 2011 to October 2012 by the 14C-urea breath test. DCs were generated from monocytes isolated by an adherent method from the two groups of patients and cultured in the presence of rhIL-4, rhGM-CSF, and rhTNF-α. Furthermore, the expression of surface marker molecules was determined by fluorescence-activat-ed cell sorting analysis. The cytotoxicity of DCs pulsed T cells against gastric carcinoma cell was assessed by the lactate dehydroge-nase-releasing assay. The secretion of IL-12 and IFN-γin the supernatant was determined by enzyme-linked immunosorbent assay. Re-sults:No difference was observed in the morphological change of the maturation process. The mean expression of CD1a, CD80, CD83, CD86, and HLA-DR molecules in DCs of H. pylori-infected patients was higher than that in DCs of H. pylori-negative group, and the differences were statistically significant except for CD1a and HLA-DR. The cytotoxicity activities, IL-12 release, and IFN-γrelease in the H. pylori-positive group were significantly higher than those in the H. pylori-negative group (P<0.05). Conclusion:H. pylori infec-tion has no effect on the morphological change of the maturation process of monocyte-derived DCs. These data clearly demonstrate that monocyte-derived DCs of H. pylori-infected patients with gastric cancer can induce stronger maturation and activation than those of H. pylori-negative patients.

Influence of bear bile on rat hepatocarcinoma induced by diethylnitrosamine / 药学学报

To investigate the influence of bear bile on rat hepatocarcinoma induced by diethylnitrosamine (DEN), a total of 40 rats were randomly divided into 4 groups: normal control group, model group, and two bear bile treatment groups. The rat liver cancer model was induced by breeding with water containing 100 mg x L(-1) DEN for 14 weeks. The rats of the bear bile groups received bear bile powder (200 or 400 mg x kg(-1)) orally 5 times per week for 18 weeks. The general condition and the body weight of rats were examined every day. After 18 weeks the activities of serum alanine transaminase (ALT), aspartate transaminase (AST) and total bilirubin (TBIL) were detected. Meanwhile, the pathological changes of liver tissues were observed after H&E staining. The expression of proliferative cell nuclear antigen (PCNA) and a-smooth muscle actin (alpha-SMA) in liver tissue were detected by immunohistochemical method. After 4 weeks the body weights of rats in normal group were significantly more than that in other groups (P < 0.05); and that in the two bile groups was significantly more than that in the model group. Compared with normal group, the level of serum glutamic-pyruvic transaminase and total bilirubin increased significantly in other groups; compared with model group, these two indexes decreased significantly in two bile groups. Hepatocellular carcinoma occurred in all rats except for normal group; there were classic cirrhosis and cancer in model group while there were mild cirrhosis and high differentiation in two bile groups. There were almost no expressions of PCNA and alpha-SMA in normal group while there were high expressions in model group; the two bile groups had some expressions but were inferior to the model group, and alpha-SMA reduced markedly. It indicated that bear bile restrained the development of liver cancer during DEN inducing rat hepatocarcinoma, which may be related to its depressing hepatic stellate cell activation and relieving hepatic lesion and cirrhosis.

Expression and activities analysis of a fusion protein CREKA/tTF / 中国生化药物杂志

Purpose To prepare a novel fusion protein of CREKA and tTF as a universal carrier targeting to cancer,and to analyze its activities.Methods CREKA and tTF gene were acquired by PCR,and inserted into plasmid pET22b(+)to construct recombinant plasmid CREKA/tTF/pET22b(+),and the fusion gene was expressed in E.coli BL21.The fusion protein Wag purified through Nickel-affinity chromatography column.After purifying,the fusion protein was refold by subsequent dialysis.The activities of the fusion proteins were measured by coagulation timing and quantitative fluorescence test in vitro.Results The recombinant plasmid CREKA/tTF/pET22b(+)with correct sequence was obtained.The fusion protein was highly expressed in E.coli BL21.The coagulation of the fusion protein Was determined by the coagulation test.And the capability of the fusion protein effectively binding to clotted plasma proteins is identified in quantitative fluorescence test.Conclusion The recombinant plasmid CREKA/tTF/pET22b(+)with correct sequence was built.The fusion protein CREKA/tTF with both TF and CREKA activity was successfully obtained.

Experimental study of (RGD)_3-tTF fusion proteins binding specifically to tumor vasculature in the colonic carcinoma nude mice model / 中国癌症杂志

Background and purpose: Tumor vasculature is increasingly recognized as a target for cancer therapy. In recent years, a fusion protein consisting of the extra cellular domain of tissue factor (truncated tissue factor, tTF) was fused to the antibody selectively binding to tumor vasculature. Antibody-truncated tissue factor(Ab-tTF) fusion protein specifically induced thrombotic occlusion of tumor vessels resulting in tumor growth retardation or regression in some types of solid tumors. However, there were still some disadvantages in the above approach. We constructed and expressed that the (RGD)_3-tTF fusion protein with peptides arginine-glycine-aspartic acid (GRGDSP, abbr. RGD)as the carrier of tTF to explore whether it bad the capability of targeting to tumor vasculature in the colonic carcinoma model. Methods: The (RGD)_3-tTF fusion gene consisting of the tTF was fused to three series-wound peptides RGD. The (RGD)_3-tTF construct was expressed in Escherichia coil BL21(DE_3). The fusion protein was purified through Nickel affinity chromatography column. The activity of inducing blood coagulation was detected by clotting assay and coagulation factor X (FX) activation assay. The specific binding to integrins α_vβ_3 was analyzed by indirect enzyme linked immunosorbent assay (ELISA). All these were compared with the fusion protein RGD-tTE Colonic nude mice models were randomly divided into 3 groups (1 nude mice per group).Tumors were stained by the (RGD)_3-tTE RGD-tTF fusion protein and tTF which were labeled with Fluorescein Isothiocyanate(FITC). The location of the (RGD)_3-tTF fusion protein in the colonic carcinoma bearing nude mice tissue was analyzed by immunofluorescence assay. Results: The (RGD)_3-tTF fusion protein retained tissue factor thrombogenic activities. With increasing concentration, the clotting time was shortened correspondingly. Under the conditions of Ca~(2+), the clotting time was 9.96±0.56 min when the concentration was 6 μmol/L(P<0.01). The (RGD)_3-tTF fusion protein could activise F X above 6 μmol/L concentration, which was similar to RGD-tTF fusion (F=0.147, P>0.05). The ability of the (RGD)_3-tTF fusion protein binding specifically to integrins α_vβ_3 was stronger than that of the RGD-tTF fusion protein in the same concentration (F=164.81, P<0.01), which was apparently indicated by the A_(405nm) 1.25 and 0.95 when the concentration was 0.24 μmol/L. Immunofluorescence assay showed that the (RGD)_3-tTF fusion protein was assembling in the tumor vasculature of the colonic carcinoma bearing nude mice. Conclusion: The (RGD)_3-tTF fusion protein which retained tissue factor thrombogenic activities could bind specifically and efficiently to tumor vasculature in the colonic carcinoma bearing mice through binding to the tumor marker integrins α_vβ_3. It might be a promising foundation for further studies on the colon cancer molecular targeted therapy with tTF as an effective factor.

Preparation and activity analysis of hu3D3/core-streptavidin fusion protein as a universal carrier targeting to lung cancer / 中华微生物学和免疫学杂志

Objective To prepare a novel fusion protein of hu3D3 and core-streptavidin(hu3D3/cSA) as a universal carrier targeting to lung cancer,and analyze its activities. MethodscSA gene was acquired by PCR,and inserted into plasmid hu3D3/pET-22b(+)to construct a recombinant plasmid hu3D3/cSA/pET-22b(+)．The fusion gene was expressed in E. Coli BL21(DE3).The fusion protein was purified through nickel-affinity chromatography column and renatured using TEA buffer. The tetrameric hu3D3/cSA complex was analyzed by SDS-PAGE and Western blot.The hu3D3/cSA protein was labeled with FITC,then its antigen-binding activity was analyzed using fluorescence microscopy,and the functional affinity of hu3D3/cSA and hu3D3 were analyzed and compared by flow cytometry. The biotin-binding activity of hu3D3/cSA was tested bv EUSA and Western blot. Results The recombinant plasmid hu3D3/cSA/pET-22b(+)with correct sequence was obtained. The fusion protein was found after expression in E. Coli BL21 (DE3)mainly in the form of inclusion body. After being purified and refolded,tetrameric complex was formed. The purified tetrameric hu3D3/cSA complex retained both antigen-binding activity of hu3D3 moiety and biotin-binding activity of cSA moiety:furthermore,the avidity of the hu3D3/cSA to its target antigen increased by about 14 times as compared with that of monomeric hu3D3. Conclusion The fusion protein hu3D3/cSA with both antigen- and biotin-binding activity is SHCCessfully prepared,and the avidity of hu3D3 moiety to 3D3 antigen is enhanced. Consequently,hu3D3/cSA could be a universal carrier targeting to lung cancer, and could be an alternative,convenient method to realize target therapy to lung cancer by the combination of multiple biotinylated anti-tumor drugs or agents.

Expression and activity analysis of catalytic domain of PTP1B / 生物工程学报

The amino acid sequence (1-301aa) coding the human PTP1B catalytic domain (PTP1Bc) was obtained from the GenBank. The PTP1Bc gene was constructed by overlapping PCR, then was inserted into vector pET-22b(+) and expressed efficiently in E. coli BL21(DE3) under optimum condition after IPTG induction. The proteins were expressed mainly as inclusion bodies with the yield of more than 30% of total bacterial proteins. The expressed products were purified through Ni(2+)-affinity chromatographic column. After purification, the purity of the proteins was more than 95%. Western blotting analysis suggested that the purified proteins could specially combine with anti-PTP1B antibody. Enzyme activity assay showed that the protein has phosphatase activities.

Expression and characterization of fusion protein RGD/tTF for targeting therapy of cancer / 中国免疫学杂志

Objective:To express and purify a new fusion protein(RGD/tTF) for targeting therapy of cancer, and analyze its activities.Methods:The fused gene RGD/tTF was constructed by PCR, and then was inserted into vector pET22 b(+),expressed in E.coli BL21(DE3).The fusion protein was purified through Nickel-affinity chromatography column. The tTF activity of the fusion protein was analyzed by clotting assay and FⅩ activation assay. The specific binding of RGD/tTF to ?_v?_3 was analyzed by indirect ELISA.Results:The recombinant plasmid pET22 b(+)/RGD/tTF with correct sequence was obtained. The fusion protein was expressed with high yield in E.coli BL21(DE3). The purified fusion protein could activiate FⅩ and cause blood coagulation, and bind to ?_v?_3 specifically.Conclusion:The recombinant plasmid pET22 b(+)/RGD/tTF was constructed.The fusion protein retained TF activity and binding specificity to ?_v?_3, lays the foundation for studying the function of inducing thrombosis in tumor vasculature in vivo.