ISG15, the first ubiquitin-like molecule identified two decades ago, is encoded by interferon stimulated gene 15 ( ISG 15), where its robust expression can be induced by viral infections or interferon treatments. ISG 15 conjugate to other proteins as the ubiquitin and was found to be involved in innate immune response. However, the functions of ISG15 modification remained unclear. We cloned bovine ISG15(bisg15) into a prokaryotic expression vector pET28a( + ) with a His-tag to generate a soluble form of bISG15 fusion protein, and purified with Ni-NTA Sepharose chromatography. The purified protein was concentrated and used to immune Balb/c mice to raise the antiserum, which could specifically recognize bISG15 expressed in eukaryotic cells by Western blot analysis. The concentrated bISG15 protein and its antiserum were then used to establish an in vitro bISG15 modification system. Our studies have demonstrated that cellular proteins could be conjugated to bISG15 with this system.

Objective To evaluate the relationship between angiogenesis and biological character of pancreatic carcinoma (PC). Methods Microvessel density (MVD) in 55 cases of PC were examined by use of immunohistochemical staining,and compared with the pathological features of PC. Results Mean MVD of PC was 54.8?18.6. MVD was positively correlated with tumor cell differentiation (P

Objective To construct a human endostatin adenovirus vector and investigate its inhibitory effect on pancreatic carcinoma in nude mice.Methods Animal model of pancreatic carcinoma bearing nude mice was established by subcutaneous injection of SW1990 cells.All mice were randomized into Ad-hEnd group,Ad-LacZ group and control group with 8 mice in each group.The endostatin gene recombinant adenovirus were intratumorally injected every two days for 4 times.The rate of tumor growth was observed.lmmunohistochemical staining was employed to investigate the expression of vascular endothelial growth factor (VEGF) and micro-vessel density (MVD).TUNEL in situ was used to examine tumor cell apoptosis.Results The tumor formation rate was 100%.4 weeks later,the volumes of the tumors were (921.9±279.7 )mm3,(2804.4±553.5 )mm3 and ( 3040.6±487.6 ) mm3 in Ad-hEnd group,Ad-LacZ group and control group,respectively;the weights of the tumors were (1.19±0.18 ) g,( 2.38±0.42 ) g and ( 2.41±0.47 ) g,respectively;the VEGF positive rates were (36.3±7.1 )%,(81.2±6.6)% and (79.4±6.2)%,respectively;the levels of MVD were 12±4,27±5 and 25±6,respectively;the apoptotic rates were (31.2 ±5.4) %,( 9.4±4.9 ) % and ( 8.5±3.7 ) %,respectively.Compared with Ad-LacZ group and control group,the parameters in Ad-hEnd group were statistically different (P ＜0.01 ).The difference betweon Ad-LacZ group and control group was not statistically different.Conclusions Human endostatin gene mediated by recombinant adenovirus could inhibit tumor growth,angiogenesis and promote cell apoptosis of pancreatic carcinoma and could be used as geue therapy for pancreatic carcinoma.

Objectives To investigate the role of lymphangiogenesis in the perineural micrometastasis of pancreatic adenocarcinoma. Methods The clinical data of 30 pancreatic adenocarcinoma patients who were admitted from Sep. 2005 to Oct. 2006 for extended radical surgery were collected. The samples including pancreatic cancer, adjacent tissue, lower bile duct, pancreatic tail, the structure surrounding the SMA (peripancreatic nerve plexus) and lymph nodes were collected during operation. They were subjected to conventional pathological examination. The lymphatic capillaries weredetected by double immunohistochemical staining and the lymphatic vessel density ( LVD) was measured. Results Intra-pancreatic and/or peripancreatic neural invasion was observed in 25 patients (83. 3% ) , of which 20 were found to have both the peri-pancreatic and intra-pancreatic neural invasion. The other 5 only had the intrapancreatic neural fiber invasion and there was no single patient with peri-pancreatic neural fiber invasion only. Peri-neural invasion was not significantly associated with patients' age, gender, lymph node metastasis, tumor size and the location (P > 0.05) , but was obviously associated with JPS clinical staging ( P < 0. 05 ). The mean intratumoral LVD was (4.2 ±3.4) per field, which was significantly lower than (11.3 ±6.9) per field of adjacent tissue and (10.8 ±4.4)per field of normal pancreatic tissue(P<0.01). The mean intratumoral LVD between adjacent tissue and normal pancreatic tissue was not statistically different. Lymphatic vessel invasion was observed in non-malignant tissues in 18 patients, and there was a distribution correlation between lymphatic vessel invasion and extra-pancreatic neural plexus invasion (P<0.05). Conclusions The incidence of peri-neural invasion was high, peri-neural invasion was associated with JPS clinical staging and lymphatic vessel invasion, which suggested the possibility of the cancer spreading by peritumoral lymphangiogenesis route into the peri-SMA neural plexuses.

Objective To investigate clinicopathological features、diagnosis and surgical treatment of duodenal stromal tumors. Methods A retrospective clinical analysis was made in 23 cases of duodenal stromal tumors admitted from 1995 to 2004. Results Melena and abdominal pain was the most common presenting symptom. Complete resection was achieved in all cases, including pancreatoduodenectomy in 13 cases, by pylorus-preserving pancreatoduodenectomy in 3 cases, and tumor enucleation in 7 cases. The mean size of the tumor was 6. 6 cm (2 - 21 cm) , among them 10 cm in 6. Pathologically tumors less than 5 cm were all of potential malignancy, whereas those larger than 5 cm were malignant. Follow-up of 2 months to 9 years found all cases alive and healthy except for one who died of liver metastasis one year postoperatively. Conclusion Duodenal stromal tumors were low malignancy tumors. Aggressive surgery is the choice of treatment. Favorable prognosis is expected after complete surgical excision.

AIM: To study the alteration of expression of iNOS mRNA and ecNOS mRNA in peripheral leukocytes of Wistar rats fed with fructose. METHODS: Wistar rats were randomly divided into the control group ( n =10) and the fructose feeding group( n =10). The fructose feeding group drank 12% fructose water for 6 months. The blood glucose, blood insulin, and the expression of iNOS mRNA and ecNOS mRNA in peripheral leukocytes of rats were determined. RESULTS: The levels of blood insulin ( P

OBJECTIVE To optimize the parameters of passive cutaneous anaphylaxis(PCA)in rats immunized by ovalbumin(OVA). METHODS 1-2 month-old Sprague-Dawley rats were immu?nized by ip injection of OVA(0.2,1.0 and 5.0 mg per rat)mixed with complete Freund′s adjuvant once every other day 3 times. Serum was collected on the 12th-16th days after final immunization. Then the rats were intracutaneously injected with sensitized serum and then stimulated by iv injection of the same dose of OVA mixed with Evans blue after a latent period of 0.5,1.5,3,6,12,24,36,48 and 60 h. Finally,the diameters of blue spots in the skin were measured at stimulation. RESULTS Serum total-IgE(T-IgE)and OVA-specific IgE(sIgE)levels increased significantly and reached the peak on the 3rd-7th days and 12th-16th days after final immunization,respectively. There was no correlation between the serum T-IgE level and OVA-sIgE level when the rats were immunized with OVA at OVA 0.2-5.0 mg per rat. The rats experienced PCA after injection of OVA 1.0 and 5.0 mg per rat. Diameters of blue spots in the skin reached the maximum value after rats were sensitized for 0.5-3 h. Moreover,the shape,color and size of blue spots were better 30-60 min after stimulation. CONCLUSION Optimized PCA is as follows:1-2 month-old rats are immunized on the 1st,3rd and 5th days by ip injection of OVA 1.0-5.0 mg. The immunizing serum is collected at 12-16 d after final immunization. The rats are stimulated by OVA and Evans blue after a latent period of 0.5-3 h. Diameters of blue spots in rats′ skin are then measured 30-60 min after stimulation.

Objective To investigate the clinical features, diagnosis and management of pancreatic lesions in patients with von Hippel-Lindau (VHL) disease, and improve the current understanding on pancreatic lesions in VHL disease.Methods The clinical data of three VHL disease patients with pancreatic lesions were analyzed retrospectively, including clinical features, laboratory findings, imaging, pathological features, operation method and follow-up.Results Two patients had a family history of hemangioblastoma in central neural system, and 1 patient had retinal multiple angioma and angioma in central neural system, who were diagnosed as VHL.One patient with pancreatic portal hypertension had splenectomy and biopsy of left renal tumor.During the surgery, pancreatic cystic lesions were observed and the transparent cysts were diffusively distributed on the surface.The other 2 patients were diagnosed as pancreatic multiple cysts and non functional pNETs, and pancreatic multiple cysts, respectively, based on the imaging.All the three patients had stable disease status, and followed up by outpatient visits.Conclusions VHL disease can manifest as simple pancreatic cyst, pancreatic serous cystadenoma and pNETs.The optimal individualized treatment should be determined by Multidisciplinary team (MDT) according to the general condition of patient.

Objective:To evaluate the effect of setup errors from daily cone-beam computed tomography (CBCT) on the accumulated dose under different image-guidance (IG) strategies, aiming to investigate the appropriate IG strategies during radiotherapy for the spine metastases.Methods:A total of 720 CBCT scans of 36 vertebral lesions were obtained. All 36 lesions were divided into the simultaneous boosting (PTV 40 Gy/20f, GTV 60 Gy/20f, n=20) and conventional radiotherapy groups (PTV 40 Gy/20f, n=16). The actual fractionated plan was recalculated simulatively after transferring the isocenter of the initial plan according to the interfraction setup error. Under no daily image-guidance (no-DIG) strategies including twice imaging guidance weekly (TIG), initial 5 days then weekly imaging guidance (5D+ WIG), WIG and no imaging guidance (NIG), the dose deviation was calculated between the delivered dose accumulated by each actual fractionated plan and the dose distribution under DIG. The tolerance of dose deviation for the target was within ±5% and the D max of the spinal cord was limited below 45 Gy. Results:Under different image-guidance strategies of TIG, 5D+ WIG, WIG and NIG, the median dose deviation was approximately ±1% for the CTV D 95% and D max of spinal cord. However, the median dose deviation was beyond -5% for the PTV D 95% when conventional radiotherapy was given. The median dose deviation was approximately 10% for the D max of spinal cord and the proportion of cases whose maximum irradiated dose of spinal cord was more than 4500 cGy was ≥70%. Also, the median dose deviation was beyond -5% for the GTV D 95% and PTV D 95% when simultaneous boosting was delivered. Conclusions:Because the dose deviation of CTV and spinal cord is within the tolerance limit, the image-guidance strategies could be chosen according to the clinical practice when conventional radiotherapy is delivered. However, the dose deviation of spinal cord, GTV and PTV exceeds the tolerance limit under no-DIG strategies when simultaneous boosting is delivered. Hence, it is necessary to perform daily IGRT for the spine metastases.

Objective:To investigate the promotive effect of 4,5,6,7-tetrabromobenzotriazole (TBB) on the apoptosis of the human cervical cancer HeLa cells,and to clarify its effect mechanism in related signaling pathways.Methods:The human cervical cancer HeLa cells at logarithmic growth phase were divided into control group(without TBB) and experiment group(with TBB).MTT assay was used to detect the survival rate of the HeLa cells;the morphology of HeLa cells was observed under inverted microscope;Annexin Ⅴ-FITC/PI double staining and flow cytometry(FCM) were used to determine the apoptotic rates of the HeLa cells;the expression levels of apoptosis-related proteins p-Akt,Akt,Bcl-2,Bax,cleaved-caspase-3,and Pro-caspase-3 were detected by Western blotting method.Results:The MTT results showed that the survival rates of the HeLa cells in experiment group were significantly decreased(P<0.01) in a dose-dependent manner compared with control group.The apoptotic morphology of the HeLa cells in experiment group were found as cell shrinkage and karyopyknosis under inverted microscope.The Annexin Ⅴ-FITC/PI double staining and FCM results showed that the apoptotic rates of the HeLa cells in experiment group (3,6,12 and 24 h) were higher than that in control group (P<0.05 or P<0.01).The Western blotting results showed that compared with control group,the expression levels of the anti-apoptotic proteins p-Akt and Bcl-2 in HeLa cells in experiment group were decreased obviously,whereas the expression levels of the pro-apoptotic proteins Bax and cleaved-caspase-3 were increased and the expression levels of Pro-caspase-3 were decreased.Conclusion:TBB may promote the apoptosis of human cervical cancer HeLa cells by inhibiting the Akt signaling pathway.