Expression of Wnt/ß-catenin related genes after skeletal muscle contusion.

BACKGROUND: It was aimed to determine expressions of genes related to Wnt/ß-catenin signaling for evaluating time duration after skeletal muscle contusion. METHODS: Pathological change of skeletal muscle was observed after H-E staining. mRNA of respective genes was quantified with real-time quantitative PCR. Expression of ß-catenin was further characterized with immunostaining and quantified as intensity/area and further immune blotting and quantified as grey intensity normalized to loading control (GADPH). RESULTS: After injury, skeletal muscle exhibited prominent inflammatory response, hyperplasia and regeneration. Infiltration of inflammatory cell, formation of myotube and maturation of skeletal muscle fiber were observed under HE staining. Expression of FZD4, Myo D, Myf5 changed during early stages after injury and could serve to evaluate injury within 24 h; Expression of SFRP5 and Fra1 changed during early-to-intermediate stages after injury and could serve to evaluate injury within 12-48 h; Expression of MRF4 changed during intermediate stages after injury and could serve to evaluate injury within 36-48 h; Expression of ß-catenin changed during intermediate stages after injury and could serve to evaluate injury within 36 h-3 d; Expression of MyoG changed during late stages after injury and could serve to evaluate injury within 48 h-7 d. Immunostaining experiments showed that 36 h after injury, membrane ß-catenin decreased while nucleus ß-catenin increased. CONCLUSION: Wnt/ß-catenin related genes are involved in regeneration of skeletal muscle after contusion. The sequential changes of gene expression can be used for evaluating the duration after contusion.

Distinct patterns of ALDH1A1 expression predict metastasis and poor outcome of colorectal carcinoma.

PURPOSE: Aldehyde dehydrogenase 1A1 (ALDH1A1) has been proposed as a candidate biomarker for colorectal carcinoma (CRC). However, the heterogeneity of its expression makes it difficult to predict the outcome of CRC. The aim of this study was to evaluate the diagnostic and prognostic value of this molecule in CRC. METHODS AND RESULTS: In this study, we examined ALDH1A1 expression by immunohistochemistry including 406 cases of primary CRC with corresponding adjacent mucosa, with confirmation of real-time PCR and Western blotting. We found that the expression patterns of ALDH1A1 were heterogeneous in the CRC and corresponding adjacent tissues. We defined the ratio of ALDH1A1 level in adjacent mucosa to that in tumor tissues as RA/C and found that the capabilities of tumor invasion and metastasis in the tumors with RA/C < 1 were significantly higher than those with RA/C ≥ 1. Follow-up data showed the worse prognoses in the CRC patients with RA/C < 1. For understanding the underlying mechanism, the localization of ß-catenin was detected in the CRC tissues with different patterns of ALDH1A1 expression from 221 patients and ß-catenin was found preferentially expressed in cell nuclei of the tumors with RA/C < 1 and ALDH1A1(high) expression of HT29 cell line, indicating that nuclear translocation of ß-catenin might contribute to the increased potentials of invasion and metastasis. CONCLUSION: Our results indicate that RA/C is a novel biomarker to reflect the distinct expression patterns of ALDH1A1 for predicting metastasis and prognosis of CRC.

Effects of simulated carbon dioxide and helium peumoperitoneum on proliferation and apoptosis of gastric cancer cells.

AIM: To investigate the effects of carbon dioxide (CO(2)) and helium insufflation administered at different pressures on the growth and apoptosis of cultured human gastric cancer cells. METHODS: The gastric cancer cells MKN-45 were exposed to a CO(2) and helium environment maintained at different pressures (0, 5, 10 and 15 mmHg). The cells were exposed to simulated pneumoperitoneum environment for 4 h, and pH of the culture media was measured after it was moved to normal conditions for 0, 2, 4, 6 and 8 h. Proliferation viability of MKN-45 was examined by 3-[4,5Dimethylthiazol-2-yl],5-diphenyltetrazolium bromide or triazolyl blue (MTT) assay after it was moved to normal conditions. Apoptotic ratio was measured by Annexin V-FITC/PI double labelled staining. RESULTS: The pH of media was acid and recovered to normal after 4 h in the CO(2) group while it was basic in the helium group. There was no difference between CO(2) groups (under 10 mmHg ) and control group (P > 0.05) in the proliferative viability of the cells. The cultured cells exposed to 15 mmHg CO(2) environment grew more slowly than control group from 4 to 7 d (P < 0.01 ) while there was no difference from 1 to 3 d (P > 0.05). The proliferative viability in helium group was not obviously different from the control group (P > 0.05). The apoptotic ratio of the cultured cells was markedly higher than that of the control group (P < 0.01) at 10 and 15 mmHg CO(2) insufflation pressure. In helium group, the apoptotic ratio was not obviously different from the control group (P > 0.05). CONCLUSION: There is no obvious effect in the proliferation and apoptosis of MKN-45 cells under 10 mmHg CO(2) insufflation pressure and helium in any pressure. Fifteen mmHg CO(2) insufflation pressure can inhibit the proliferation of the cells and improve apoptosis.

Cloning, expression and bio-activity assay of chimeric fusion protein sTNFRII-IgG Fc / 生物工程学报

Tumor necrosis factor (TNF) is a key cytokine in immunology system and is related to many human diseases. In order to inhibit the activity of TNF, cDNA coding for soluble TNF receptor II (sTNFRII) and human IgG Fc were linked using a flexible hinge. This gene was expressed in E. coli as a chimeric protein and purified by metal chelate chromatography. The results show that the fusion protein exists in the physiological form as a dimer, has the ability to bind with TNF and inhibits the cytotoxicity of TNF on L929 cells. Contrasting to monomer sTNFRII, the chimeric protein has an improved bioactivity, and displays potential prospects for research and application.