[Serum inflammatory related cytokines and circulating endothelial cells in patients with acute coronary syndrome].

OBJECTIVE: To study the number of CECs in patients with acute myocardial infarction (AMI) and unstable angina (UA), and to investigate its relationship with inflammatory related cytokines. METHODS: 37 patients with AMI, 12 patients with UA, and 42 health controls were studied. CECs were isolated from peripheral blood by using of immunomagnetic beads coated with antibodies against CD146. Their endothelial origin was confirmed by the positive labelling of von Willebrand Factor (vWF), CD31 and electron microscope. Annexin V-FITC/PI kit was used to measure the apoptosis of CECs. Inflammatory related cytokines were analyzed turbidimetrically or ELISA using of commercially available testing kit. RESULTS: CECs number was significantly higher in AMI and UA patients [medians (interquartile range) were 52 (28 approximately 81.5) cells/ml and 29 (18 approximately 61) cells/ml respectively] compared with health control [10.5 (6-16.5)cells/ml, P < 0.001]. After excluding diabetes patients, the number of CECs and CRP in AMI and UA group (n = 26) were still significantly higher than controls. The necrotic rate of CECs in AMI and UA was significantly higher than controls (P < 0.01). Correlation analysis revealed a significant positive correlation between CECs and CRP, or IL-6 (r = 0.677, 0.316, P = 0.000, 0.002). The multivariate linear regression analysis showed that CRP and Diabetes increased the number of CECs significantly (OR = 0.620, 0.164, 95% CI 3.985-6.751, 0.301-21.877, P = 0.000, 0.044). CONCLUSION: The mechanism responsible for the increase of CECs in acute coronary disease may be due to the vessel injury caused by inflammation.

[Transfection and expression of hRI gene on human umbilical blood stem cells and gene therapy for mouse melanoma].

In order to explore the transfection and expression of hRI gene on human umbilical blood stem cells, and observe it's effect on the tumor growth. After enriching human umbilical cord blood CD34+ cells with a high-gradient magnetic cell sorting system (MACS), transfected them with supernatant of retrovirus containing human Ribonuclease inhibitor (hRI) cDNA. Hematopoietic progenitor clonogenic assay and PCR were used to evaluate transfection efficiency, and Western-blot and immune fluorescence were used to evaluate the expression quantity of hRI gene after transfection. Observe the effect of RI on the growth of melonoma in B16C57BL mice. The results showed that human umbilical blood CD34+ cells were highly purified by MACS, which made the purity of human umbilical blood CD34+ cells average 96.15%. hRI can be transfected on umbilical blood CD34+ cells, and the transfection efficiency was 35%. The positive expression of hRI gene on transfected CD34+ cells is identified by Western-blot and immune fluorescence assay. Mice injected with transfected CD34+ cells show a significant restraint of the tumor growth, a lower efficiency of tumor formation, a lower weight of the tumor and a longer incubation period of tumor formation with respect to the control groups. The results demonstrated the capacity of RI to inhibited the tumor growth by blocking the vasculature in tumor.

Effects of DNA methylation inhibitor on down-regulation of ribonuclease inhibitor expression in cancer cell lines.

BACKGROUND & OBJECTIVE: Human ribonuclease inhibitor (RI) could effectively block angiogenin-induced angiogenesis, and inhibit growth of transplant solid tumors in animals. However, its exact molecular mechanism of antitumor has not been totally ascertained. Many tumor suppressor genes occur loss of expression by aberrant methylation in promoter region. Demethylation, treated with methylation inhibitor 5-aza-2'-deoxycytidine (5-Aza-CdR),can restore the gene expression. To further explore functions of RI, and investigate relationship between RI and tumorigenesis, the study was designed to explore effects of 5-Aza-CdR on expression of RI in cancer cell lines. METHODS: Human breast cancer cell line MCF-7, human gastric cancer cell line BGC-823, human prostate cancer cell line DU-145, and human colon cancer cell line HT-29 were treated with 5-Aza-CdR. Expression of RI was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot, immunofluorescence, and immunocytochemistry. RESULTS: Expression of RI significantly elevated by 5-Aza-CdR at both mRNA and protein level in MCF-7, BGC-823, and DU-145 cells (P< 0.01). Compared with control cells, mRNA levels of RI in MCF-7, BGC-823, and DU-145 cells treated with 5-Aza-CdR were increased by percentages of 37.2%, 46.0%, and 32.4%, respectively; protein levels of RI were increased by percentages of 26.4%, 20.9%, and 24.4%, respectively; but no obvious change observed in HT-29 cells. CONCLUSION: RI gene may be involved in tumorigenesis of gastric, prostate, and breast cancer.

[Construction and identification of a tumor-specific expression vector driven by human telomerase reserve transcriptase gene promotor].

OBJECTIVE: To construct the tumor-specific expression vector driven by human telomerase reserve transcriptase gene promotor. METHODS: The fragment of the enhanced green fluorescent protein (EGFP) gene was PCR amplified from the pEGFP-N1 plasmid and cloned into the multiple cloning site of pLNCX vector, and the recombinant was named as pLNCX-EGFP. The fragment of human telomerase reserve transcriptase gene promoter was amplified from the human genome by using the human telomerase reserve transcriptase gene specific primers, and cloned into the pLNCX-EGFP vector, where the cytomegalovirus promoter was previously removed using restriction enzymes, in sense orientation relative to the green fluorescent protein coding sequence. Then the expression vector pLNT-EGFP under the control of the human telomerase reserve transcriptase gene promoter, containing green fluorescent protein reporter gene, was successfully constructed. To detect the transcriptional activity of the human telomerase reserve transcriptase gene promoter, transient transfection of this specific expression vector into HLF cell lines with high telomerase activity and WI38 cell lines without telomerase activity was performed. RESULTS: The expression vector proven by restriction enzymes digestion and sequencing was in correspondence with the design. The results of transient transfection showed that the pLNT-EGFP vector could highly expressed green fluorescent protein reporter gene in telomerase-positive cells, but not in telomerase-negative cells. CONCLUSION: A tumor-specific expression vector driven by human telomerase reserve transcriptase gene promotor has been successfully constructed.

The antioxidant effects of ribonuclease inhibitor.

Ribonuclease inhibitor (RI) is an acidic cytosolic glycoprotein with molecular weight of about 50 kDa, which contains 32 cysteine residues. It is possibly that RI may have antioxidant effect by thiol-disulfide exchange reaction. We studied the effects of RI over-expression on the rat glial cell line C6 injured with H2O2. The transfected C6 cells with RI cDNA (C6') had higher viability, less LDH leakage and MDA contents, but more GSH contents compare that in the control C6 cells. In transfected C6 cells, the activities of CAT and GST were higher than that in the control C6 cells. Without H202 stress, the activities of CAT and GST in the C6' cells were 1.73 and 3.62 times that in the control C6 cells, respectively; With 1.00 mmol/L H2O2 stress, the activities of CATand GSTin the C6' cells were 3.38 and 2.11 times that in the C6 cells, respectively. These results suggest that the over-expression RI has antioxidant activity and it is able to protect cells from per-oxidative injuries. Moreover, we investigated whether RI has a protective role against mouse hepatic damage in vivo. The mice pretreated with different doses of human RI were injected by CC14. The results show that the SOD activities of therapy groups were significantly higher than that of the control group (p < 0.01), while the contents of MOD and activities of ALT and AST in blood were remarkably lower than that of the control group (p < 0.01). Pathological examination shows that the degree of damage was alleviated with RI therapy. These results suggest that RI has the protective role against mouse hepatic damage induced by CC14. The anti-oxidative effects of RI may play an important role in cell protection from per-oxidative injuries.