A Comparative Study of Pressureless Sintered Nanostructured Hydroxyapatite/TiO2 Composites Prepared by Different TiO2 Addition Methods.

Two methods of TiO2 addition were applied to prepare hydroxyapatite/TiO2 (HA/TiO2) composite, i.e., in-situ hydrolysis TiO2 in HA powders (N-HA/TiO2) and mixing commercial nano-sized HA and TiO2 powder (C-HA/TiO2). Effects of TiO2 addition methods and sintering temperatures on phase, microstructure and microhardness were investigated for pressureless sintered HA/TiO2 composites, and pure HA was investigated for comparison. Results show that TiO2 from both in-situ hydrolysis and mixing commercial powder presented similar effects on phase structures and composition, and trended to chemically react with HA in the HA/TiO2 composites at high sintering temperature. Weight loss for different composites was investigated by thermal analysis. Sintering behavior for two different composite was also discussed. The TiO2 from in-situ hydrolysis can effectively enhance the TiO2 distribution and densification for the N-HA/TiO2 composites. Both two different composites showed typical grain growth and pore formation with the increase of sintering temperature. The N-HA/TiO2 composite had a lower porosity, higher shrinkage and microhardness than that of C-HA/TiO2 composite at sintering temperature from 700 °C to 1100 °C.

A novel data processing method CyC* for quantitative real time polymerase chain reaction minimizes cumulative error.

Quantitative real-time polymerase chain reaction (qPCR) is routinely conducted for DNA quantitative analysis using the cycle-threshold (Ct) method, which assumes uniform/optimum template amplification. In practice, amplification efficiencies vary from cycle to cycle in a PCR reaction, and often decline as the amplification proceeds, which results in substantial errors in measurement. This study reveals the cumulative error for quantification of initial template amounts, due to the difference between the assumed perfect amplification efficiency and actual one in each amplification cycle. The novel CyC* method involves determination of both the earliest amplification cycle detectable above background ("outlier" C*) and the amplification efficiency over the cycle range from C* to the next two amplification cycles; subsequent analysis allows the calculation of initial template amount with minimal cumulative error. Simulation tests indicated that the CyC* method resulted in significantly less variation in the predicted initial DNA level represented as fluorescence intensity F0 when the outlier cycle C* was advanced to an earlier cycle. Performance comparison revealed that CyC* was better than the majority of 13 established qPCR data analysis methods in terms of bias, linearity, reproducibility, and resolution. Actual PCR test also suggested that relative expression levels of nine genes in tea leaves obtained using CyC* were much closer to the real value than those obtained with the conventional 2-ΔΔCt method. Our data indicated that increasing the input of initial template was effective in advancing emergence of the earliest amplification cycle among the tested variants. A computer program (CyC* method) was compiled to perform the data processing. This novel method can minimize cumulative error over the amplification process, and thus, can improve qPCR analysis.

Effects of connective tissue growth factor (CTGF) gene silencing on the radiosensitivity of glioblastoma.

The effects of connective tissue growth factor (CTGF) gene silencing on the radiosensitivity of glioblastoma cells (GBM) were investigated. The lentivirus-mediated short hairpin RNA (shRNA) expression vector targeting CTGF was constructed and transinfected into U87MG human GBM cell line. The CTGF gene expression in U87MG cells was significantly down-regulated. After irradiation with 6 MV X-rays at a dose rate of 2.5 Gy/min, the clonogenicity, proliferation and migration of U87MG cells were assayed in vitro. The survival, proliferation and migration of U87MG cells were all remarkably inhibited by CTGF silencing (p < 0.05 vs control). Our results demonstrate that CTGF is important for GBM and CTGF gene silencing can be a potential tool to enhance the sensitivity of GBM to radiotherapy.

[Chemokine receptor CXCR4 gene silencing with shRNA inhibits breast cancer metastasis to the lung in nude mice].

OBJECTIVE: To construct a CXCR4 specific recombinant plasmid vector and study its inhibiting effect on invasion capacity in vitro of human breast cancer MDA-MB-231 cell line and its metastatic potential to the lung in nude mice. METHODS: A CXCR4 specific recombinant plasmid vector was constructed and transfected into the cultured MDA-MB-231 cell line with lipofectamine 2000. RT-PCR and Western blot were used to detect the mRNA and protein expression of CXCR4, respectively. Invasion capability in vitro of the cells was evaluated by Boyden chamber. The cell proliferation capacity was detected by MTT method. The nude mouse model of lung metastasis was established by injection of MDA-MB-231 cells into the tail vein. The animals were sacrificed at 6 weeks after the tumor cells injection. Whole lung tissues were harvested, embedded in paraffin, sectioned serially, and the HE-stained paraffin sections were examined pathologically to evaluate the presence and number of metastatic tumors. RESULTS: The CXCR4 mRNA expression rate was 29.5% +/- 3.8% in the CXCR4-shRNA group, significantly lower than that of the control group (69.7% +/- 2.6%, P < 0.01) and mock-control group (67.8% +/- 3.5%, P < 0.01). The CXCR4 protein expression rate was 15.4% +/- 1.1% in the CXCR4-shRNA group, significantly lower than that of the control group (39.0% +/- 2.4%, P < 0.01) and mock-control group (35.9% +/- 3.9%, P < 0.01). Silencing of CXCR4 by shRNA lead to a significant decrease in breast cancer cell invasion and proliferation capacity in vitro. Furthermore, tumor cells with CXCR4 shRNA permanent transfcetion had a much lower lung metastatic potential in nude mice than control cells and mock control cells in vivo. CONCLUSION: CXCR4 shRNA can inhibit the expression of CXCR4 and decrease the invasion and lung metastatic potential of human breast cancer cells.

[Expression of thymidine phosphorylase in cancer].

OBJECTIVE: To study the thymidine phosphorylase (TP) expression in different types of cancer and its correlation with tumor microvessel density (MVD). METHODS: The expression of TP and MVD was detected by immunohistochemistry method. In a series of 251 cancer patients there were 48 patients with gastric cancer, 53 with colorectal cancer, 47 with breast cancer, 56 with cervical cancer, 47 with lung cancer. Normal gastric (n = 25), colorectal (n = 25), cervical (n = 17) and lung (n = 25) tissues around the cancer were also examined. RESULTS: The TP expression rate was 64.6% in gastric cancer, 67.9% in colorectal cancer, 80.9% in breast cancer, 82.1% in cervical cancer, and 63.8% in lung cancer, which was significantly higher than that in normal tissues (P = 0.0000). TP expression was positively correlated with MVD in gastric, colorectal, breast, and cervical cancers. The correlation was not statistically significant in lung cancer. CONCLUSION: This study indicates that TP overexpression in cancer may be associated with tumor angiogenesis.

[Diallyl disulfide-induced G2/M arrest of human gastric cancer MGC803 cells involves activation of p38 MAP kinase pathways].

BACKGROUND & OBJECTIVE: p38 MAP kinase signal transduction pathway was thought to be involved in the G(2)/M arrest,however,the involved mechanisms were not clear. This study was designed to determine the role of p38 MAP kinase signal transduction pathways in diallyl disulfide (DADS)-induced G(2)/M arrest in human gastric cancer MGC803 cells and its related molecular mechanisms. METHODS: MGC803 cells growth inhibition was measured by MTT assay. Phase distribution of cell cycle was analyzed by flow cytometry. Expression of Cdc25C, p38, phosphorylation of p38 (pp38) were determined by Western blot analysis. RESULTS: MTT assay showed that SB203580, a specific p38 MAPK inhibitor, blocked DADS-induced growth inhibition. Flow cytometry analysis revealed that treatment of MGC803 cells with 30 mg/L DADS for 24 hours increased in the percentage of cells arrested in the G(2)/M phase from 9.3% to 39.4%(P< 0.05). Whereas in the presence of SB203580 the percentage decreased nearly one time, from 39.4% to 21.2% (P< 0.05). Western blot analysis showed that phosphorylation of p38 was increased 3.52-folds following treatment of MGC803 cells with 30 mg/L DADS for 20 minutes. At the same time, the total p38 abundance did not change. DADS treatment of MGC803 cells for 24 hours decreased the level of Cdc25C by 68%, and pretreatment of MGC803 cells with SB203580 partially reversed the downregulation of Cdc25C level by DADS (P< 0.05). CONCLUSION: DADS- induced G(2)/M arrest of MGC803 cells involves the activation of p38 MAP kinase pathway. Decreased Cdc25C protein expression by p38 played a crucial role in G(2)/M arrest after the treatment with DADS.