Proteomic profiling of osteosarcoma cells identifies ALDOA and SULT1A3 as negative survival markers of human osteosarcoma.

Osteosarcoma (OSA) is the most common primary malignancy of bone. Molecular mechanism underlying OSA remains to be fully elucidated. It is critical to identify reliable diagnostic and prognostic markers for OSA at the molecular levels. This study is designed to investigate possible molecular mechanisms behind OSA development and to identify novel prognostic markers related to OSA survival. We conduct a comprehensive proteomic profiling analysis of human OSA cell lines with differential metastatic potential. Through comprehensive combinatorial analyses of the proteomic data and the previously obtained cDNA microarray results, we identify 37 candidate proteins which are differentially expressed in OSA sublines. Among them, ALDOA and SULT1A3 are selected for further investigation. The expressions of protein are confirmed by Western blotting analysis. We further analyze the expression levels of ALDOA and SULT1A3 from 40 clinical cases of OSA. The results demonstrate that the expression of ALDOA and/or SULT1A3 is significantly higher in patients with worse survival time than patients with better survival time. Five-year survival analysis shows there is a statistically significant difference between two patient populations. The data strongly suggest that ALDOA and/or SULT1A3 expression level in biopsy samples may predict the clinical outcomes of OSA patients. Furthermore, the biological functions of ALDOA and SULT1A3 may be implicated in OSA development and/or progression.

[Coexpression of Schwann cell marker GFAP and myoepithelial cell marker alpha-SMA in salivary adenoid cystic carcinoma].

OBJECTIVE: To observe the expression of Schwann cell marker GFAP and myoepithelial cell marker alpha-SMA in salivary adenoid cystic carcinoma (ACC), and to evaluate the relationship of GFAP, alpha-SMA and perineural invasion in ACC. METHODS: Immunohistochemical SABC method, double-label immunofluorescence and CLSM were used to detect the expression of GFAP and alpha-SMA proteins in salivary ACC tissue samples. RESULTS: In salivary ACC tissue samples, both GFAP and alpha-SMA proteins were positive, which were coexpressed in cytoplasm of the same onco-myoepithelial cells. CONCLUSIONS: There may be Schwann cell differentiation in onco-myoepithelial cell of salivary ACC, and it may be the pathological base of perineural invasion in salivary ACC.

[Effects of simulated weightlessness on ALP, ACP, collagen I, and biomechanics of hind-limb bones in rats].

OBJECTIVE: To investigate effects of 3 weeks simulated weightlessness on biomechanical parameters, alkaline phosphatase (ALP), acid phosphatase (ACP) and collagen I of hind-limb bones in tail-suspended rats. METHOD: Fourteen male SD rats were divided equally into control group (CON) and experimental group; tail-suspended (TS) was used to simulate weightlessness. After 3 weeks tail-suspension, biomechanical parameters of femur were measured; ALP, ACP and collagen I of tibia were observed. RESULT: Elastic load, maximum load, and bending rigidity coefficient decreased significantly (P<0.01), while the maximum deformation and bending toughness coefficient increased markedly (P<0.01 or P<0.05). Morphological results showed that ALP declines significantly in TS group, ACP and type I collagen increased significantly in TS group. CONCLUSION: After tail-suspension for 3 weeks, growth of rat's weight bearing bones are suppressed, biomechanical capability declines, and collagen I metabolism becomes disordered.

[Preliminary study on anti-periodontitis immunization with DNA vaccine].

OBJECTIVE: To observe the protection against periodontal bone loss in the Sprague-Dawley (SD) rats periodontitis model, with the recombined plasmid pcDNA3.1+/kgpcd as DNA gene vaccine. METHODS: PcDNA3.1+/kgpcd was delivered into rats by submandibular gland-targeted injection. The anti-KGPcd sIgA in saliva was measured by indirect ELISA method. Immunohistochemistry staining was used to assess the protection in the animal model. RESULTS: The level of specific anti-KGPcd sIgA in saliva of the experimental group was significantly higher than that of control group. HE staining showed that immunization with recombined plasmid pcDNA3.1+/kgpcd could protect or minimize tissue destruction caused by subsequent P. gingivalis challenge in the rat model. CONCLUSIONS: The results indicate that pcDNA3.1+/kgpcd was a good candidate for anti-periodontitis gene vaccine and could provide protection against Porphyromonas gingivalis-caused periodontitis in rat lesion model.

[Anti-tumor immune response in vitro induced by fusion of Tca8113 cells with macrophages].

OBJECTIVE: To evaluate the therapeutic effectiveness of fusion tumor vaccine in tongue cancer treatment. METHODS: Human macrophages fused with human tongue carcinoma cell line Tca8113 cell. The fusion cells were selected by magnetic cell sorting (MACS) and cultured. The biological properties of fusion cells and anti-tumor immune response in vitro induced by fusions were observed. RESULTS: In contrast to Tca8113, the fused cells grew significantly slow in vitro. The expression of MHC I, II antigen of the fusion cells which was detected by flow cytometry (FCM) was higher than that of Tca8113. The fused cells significantly increased the proliferation of mixed lymphocyte and induced their cytotoxicity on parental Tca8113. CONCLUSIONS: The fusion tumor vaccine of macrophages and OSCC cells increase in vitro immunogenicity significantly. This indicates that fusion tumor vaccine could be a new method of anti-tumor immunotherapy, which has important potentials for effective individualized human OSCC vaccine.

[Construction of eukaryotic expression vector for KGPcd gene from Porphyromonas gingivalis and expression in mammalian cells].

OBJECTIVE: This study aimed at constructing secretory eukaryotic expression vector of KGPcd gene encoding whole amino acid residues of mature KGPcd from Porphyromonas gingivalis and investigating the transcription and expression of recombined plasmid VR1020/KGPcd in mammalian cells. METHODS: Eukaryotic expression plasmid VR1020/KCPcd was constructed by using molecular cloning methods. Then, the VR1020/KGPcd was transfected into mammalian cell COS7 with Lipofectamine 2000 according to the manufacturer's instruction. The transcription of VR1020/KGPcd was assayed by reverse transcription polymerase chain reaction (RT-PCR). The expression product of VR1020/KGPcd was analyzed by using indirect immunofluorescence. The protein secretion in cultural medium was detected by ELISA method. RESULTS: It proved that the VR1020/KGPcd could be transcribed and translated into transfected COS7 cells. The expressed targeted protein could be secreted into cultural supernatant and could be detected by ELISA. CONCLUSION: The eukaryotic expression plasmid of VR1020/KGPcd was constructed successfully and its product can be expressed in mammalian cells. The results indicated that the recombinant plasmid has antigenicity and may be acted as candidate gene vaccine. This laid a basis for its use as gene vaccine candidates in the development of anti-periodontitis and paved the way for further study.

[Changes of osteocalcin in bone and bone marrow in tail suspended rats].

OBJECTIVE: To study osteocalcin [correction of osteocakin] (OC) changes in bone and marrow and calcium deposition in bone and cartilage under simulated weightlessness. METHOD: Twenty SD rats were randomly divided into 14 d and 28 d tail suspension group and 2 corresponding control groups. Histological samples were in situ hybridized and trichrome stained. RESULT: OC expression of bone and marrow of rats were lower in tail suspended rats than that in the control (P<0.05). OC expression in 14 d tail suspended rats were higher than that in 28 d tail suspended group (P<0.05). Mineralization was inhibited, and demineralization of femur [correction of furmer] and cartilage mineralized matrix was prominent. Demineralization was more prominent in 28 d group. CONCLUSION: OC levels in bone and marrow of rats were lower after tail suspension. Calcium deposition was inhibited in bone and cartilage. Demineralization was prominent after long term hindlimb unloading.

[Changes of bone morphogenesis proteins and transforming growth factor-beta in hind-limb bones of 21 d tail-suspended rats].

OBJECTIVE: To investigate changes of bone morphogenesis proteins (BMP), transforming growth [correction of grouth] factors-beta (TGF-beta) in tibia and the growing of femur in tail-suspended rats after 21 d simulated weightlessness. METHOD: Fourteen male SD rats were randomly divided into control group (CON) and tall-suspension group (TS). After 21 d tail-suspension, basic physical parameters of the femur were measured; changes of BMP, TGF-beta in tibia were assayed by immunohistochemical method. RESULT: During 21 d tail-suspension, all rats grew well without apparent stress reaction. After 21 d weightlessness simulation, wet weight, dried weight, ash, diameter and density of femur in TS group declined significantly (P<0.01). Immunohistochemical results of tibia showed that both BMP and TGF-beta declined in tissues from all regions of the tibia. CONCLUSION: After 21 d tall-suspension, the growth of rat's weight bearing bones were suppressed, production and secretion of BMP and TGF-beta was holdback.

[Construction and expression of eukaryotic expression vector containing hVEGF165 gene in rat bone marrow stroma cell].

AIM: To construct the eukaryotic expression vector containing human vascular endothelial growth factor 165(VEGF165) gene and express it in rat bone marrow stroma cells(rMSCs). METHODS: The recombinant plasmid pSP73-VEGF165 was digested with BamH I and Xho I. Then the hVEGF165 gene segment obtained was again cloned into pcDNA3.1 to construct recombinant eukaryotic expression vector pcDNA3.1-VEGF165. Then the recombinant vector was identified by enzyme digestion analysis and sequencing. The rMSCs were transformed by recombinant vector and positive clones were screened with G418. The expression of hVEGF165 gene in the transformed cells was detected by immunocytochemical staining. RESULTS: Enzyme digestion analysis and sequencing showed that target gene had been cloned into recombinant vector. The expression of hVEGF165 gene in the transformed cells had been demonstrated by immunocytochemical staining. CONCLUSION: The recombinant eukaryotic expression vector has been constructed and expressed successfully in the transformed cells. Therefore, it is possible to use the rMSCs expressing hVEGF165 gene as seed cells in the bone tissue-engineering.

[A study of injectable autogenous tissue-engineered bone].

OBJECTIVE: To investigate the utilization of carrier for delivering osteoblasts and creating autogenous bone tissue in ectopic site of animal via injection. METHODS: Bone marrow cells harvested from iliac bone of New Zealand rabbits were induced to differentiate into marrow stromal osteoblasts. The osteoblasts were mixed with 1.5% alginate sodium solution to generate osteoblasts/alginate composites with final cellular density of 4 x 10(9)/L. Calcium chloride was used as cross-linking agent to gel aqueous alginate solution. The marrow stromal osteoblasts/alginate composites were injected into the dorsal subcutaneous tissue of rabbits with autogenous cells transplantation. The samples were examined with X-ray and histological analysis. RESULTS: Four, eight and twelve weeks after injection, the hard knobbles were easily palpated under the dorsal skin of animals. On X-ray photograph the samples showed calcified image with more density than surrounding soft tissue, new bone formation was observed in the osteoblasts/alginate composites in histological analysis. The osteogenesis was in association with regenerated hematopoietic bone marrow. CONCLUSIONS: These results demonstrate that new bone tissue could be created through the injection of alginate sodium treated with autogenous marrow stromal osteoblasts.