Expression and purification of CDNF and preparation of its polyclonal antibodies / 中国免疫学杂志

Objective: To obtain purified and functional CDNF-his recombinant protein and prepare its polyclonal antibodies.Methods:Preparation of recombinant CDNF-his was carried out in HEK 293 T cells with pVR1012-CDNF-his successfully constructed transfected into them.Then,the recombinant protein was purified by Ni-NTA immunoaffinity chromatography.The purity was analyzed by SDS-PAGE and the protein′s identity was tested by Western blot.MTT was used to verify the biological function of the protein purified.New Zealand white rabbits were immunized with purified CDNF-his protein for preparation of polyclonal antibodies.Results:pVR1012-CDNF-his expressed successfully in HEK 293 T cells.The purity of protein was up to more than 90%after purification.MTT showed that CDNF-his was able to protect PC 12 cells from damage by 6-OHDA.The polyclonal antibody was detected at the end of animal immunizing process.Conclusion: A method to express and purify protein using HEK 293T cell and following Ni-NTA immunoaffinity chromatography has been built.CDNF-his with biological activity is obtained based that.Finally, polyclonal antibodies of CDNF were generated successfully.

Increased expression of MUC-1 has close relation with patient survivor in high-grade salivary gland mucoepidermoid carcinoma.

PURPOSE: High-grade mucoepidermoid carcinoma (MEC) is an infrequent malignant neoplasm originates most commonly in the salivary glands with poor prognosis. Thus far, there is little knowledge on MUC-1 expression in high-grade MEC or how it may relate to patient survival. The aim of this study was to determine the extent of MUC-1 expression in high-grade MEC and correlate expression with clinic outcome. METHODS: Surgical specimens from 62 cases of primary high-grade MEC and 10 cases of normal salivary gland tissue were examined by immunohistochemistry. The relationship between MUC-1 expression and the clinicopathological data and patient survival was analyzed. RESULTS: The disease-free survival rates were 54.6% (3 years) and 37.7% (5 years) for high-grade MEC patients. Patient who with primary location at submandibular gland have the worst prognosis (P = 0.042) in comparing with other sites. High expression of MUC-1 in high-grade MEC was significantly correlated with lymphatic/regional metastasis (P = 0.003) and clinic stage (P = 0.02), and high expression of MUC-1 (>75%) was confirmed to be significant independent prognostic factors in high-grade MEC patients [HR 2.929 (95% CI 1.246-6.881); P < 0.0137]. CONCLUSIONS: The findings suggest that high levels of MUC-1 expression may be a potential marker for worse outcome in high-grade MEC and could be used as a new molecule target to improve outcomes for these patients in the future.

Toll-like receptor-9 agonists increase cyclin D1 expression partly through activation of activator protein-1 in human oral squamous cell carcinoma cells.

Increasing evidence suggests that malignant transformation can result from chronic infection, and Toll-like receptors (TLRs) may play an important role in this process. We have previously reported that the increased expression of TLR-9 is associated with tumor cell proliferation in oral cancer. However, the mechanisms involved have not been elucidated. The aim of this study was to investigate whether CpG-oligodeoxynucleotides (CpG-ODN), a special TLR-9 agonist, is able to exert the proliferation-promoting effect in human oral squamous cell carcinoma (OSCC), and to explore the possible underlying molecular mechanism. Flow cytometry, MTT, and colony formation assay were used to evaluate cell proliferation and cell cycle distribution. The mRNA and protein levels were analyzed by quantitative RT-PCR and Western blot assay. Luciferase reporter gene, EMSA, and ChIP assays were used to detect the activity of activator protein-1 (AP-1) and nuclear factor-κB (NF-κB) in HB cells. Results showed that CpG-ODN could stimulate proliferation of HB cells in a dose- and time-dependent manner with a promoted G(1) /S cell cycle progression. Increased cyclin D1 expression was detected in the nuclear region after CpG-ODN treatment. Moreover, CpG-ODN promoted nuclear translocation and activation of AP-1, which appeared to be required for TLR-9-mediated cyclin D1 expression and subsequently cell proliferation, but seemed to have little impact on NF-κB activity. Our results indicate that CpG-ODN stimulates tumor cell proliferation through TLR-9-mediated AP-1-activated cyclin D1 expression in OSCC HB cells. Pharmacologic inhibition of the TLR-9/AP-1/cyclin D1 pathway may be a new therapeutic approach for prevention as well as treatment of OSCC.

Increased expression of Toll-like receptor-9 has close relation with tumour cell proliferation in oral squamous cell carcinoma.

OBJECTIVE: Toll-like receptor-9 (TLR-9), a new member of the interleukin-1 receptor superfamily, was recently found to have a high level of expression in many carcinoma specimens. The objective of this study was to examine the TLR-9 expression and its role in tumour cell proliferation in oral squamous cell carcinoma. MATERIALS AND METHODS: Western blot and immunohistochemistry were used to detect TLR-9 protein in oral squamous cell carcinoma (OSCC) Tca-8113 cell lines and clinical specimens (n=60). The relationship between TLR-9 expression and clinicopathologic features was analysed. Cell proliferation and inflammatory chemokines secretion were tested by MTT and ELISA methods respectively. RESULTS: Results showed that TLR-9 expression level was higher in OSCC tissues than in paired adjacent normal tissues (P<0.01), and the expression level of TLR-9 was significantly associated with tumour size (P=0.001), tumour clinical stage (P=0.003) and Ki-67 expression (P<0.01). In vitro results also suggested that stimulation of Tca-8113 cells with TLR-9 agonist CpG-ODN could significantly increase tumour cell proliferation as well as subsequent IL-1α and IL-6 secretions (P<0.01), which could be partially inhibited by usage of anti-TLR-9 protein. CONCLUSIONS: It was therefore hypothesized that increased expression of TLR-9 may be of great value in assessing the development of OSCC, and could be used as a new target for OSCC prevention and therapy in future.

Oral and maxillofacial desmoid-type fibromatoses in an eastern Chinese population: a report of 20 cases.

OBJECTIVE: Primary desmoid-type fibromatoses arising from the oral and maxillofacial region are uncommon and hold the character of local invasion and regional recurrence. The purpose of this study was to analyze the clinicoepidemiologic characteristics of this rare disease in an eastern Chinese population. STUDY DESIGN: Epidemiological and clinicopathological data on 20 patients were reviewed from the hospital records and analyzed accordingly. RESULTS: Twelve males and 8 females with a ratio of 3:2 were involved. The mandible was the most common site of presentation. Suspected malignant change was found in 6 cases and an extremely high recurrence rate was also found in up to 53%. Abnormally high blood levels of alkaline phosphatase were found in 65% of patients, which had a close relation with tumor malignant change. CONCLUSION: Chinese people are more susceptible to desmoid-type fibromatoses and are at an extremely high risk of regional malignant change. Elevated serum alkaline phosphatase levels may be taken as an indicator for malignant transformation.

Study on MRI sequences in the diagnosis of articular cartilage injury of the knee joint / 中国基层医药

ObjectiveTo compare the accuracy of the four sequences of MRI in the diagnosis of articular cartilage injury of the knee and to investigate the advantage and shortage of the four sequences. In order to evaluate the most adequate sequence in detecting articular cartilage injury of the knee. Methods36 knee joints of 31 patients,underwent MRI scanning prior to arthroscopy included T1WI-TSE,T2 WI-TSE-SPIR, PDW-TSE-SPIR,3D-WATSC. Divided each knee side of every cartilage into five parts. Investigated and diagnosed the 180 articular cartilage surface according to Recha criteria by detecting and compared with arthroscopic examination respectively. The accurate criteria of the four sequences of MRI was sensitivity, specificity, accuracy. The criteria of correlation was the value of Kappa.ResultsCompared with the arthroscopic result which was the gold standard, the sensitivity, specificity, accuracy and Kappa value of the T1 W-TSE sequence was 63.8％ ,95.3％ ,78.8％ and 0. 403 respectively;The sensitivity,specificity ,accuracy and Kappa value of the T2WI-TSE-SPIR sequence was 56.3％ ,97.6％ ,76.1％ and 0. 353 respectively.The sensitivity,specificity,accuracy and Kappa value of the PDW-TSE-SPIR sequence was 79. 8％, 93.0％ ,86. 1％and 0. 642 respectively; The sensitivity, specificity, accuracy and Kappa value of the 3D-WATSC was 96. 8％,88. 3％ ,92.8％ and 0. 812 respectively. ConclusionThe accuracy and Kappa values of Fat-suppressed three-dimensiunal fast spoiled gradient-echo sequence (3D-WATSC)was superior to other sequences in the diagnosis of articular cartilage injury of the knee. The correlation between 3D-WATSC and arthroscopy in detecting cartilage injury was remarkable. 3D-WATSC had been deemed the most adequate sequence in detecting articular cartilage injury of the knee.