[Changes of rabbit IL-1 and TNF-alpha, etc cytokines in response to acute normovolemic hemodilution with HAES-balanced solution as diluting agent].

AIM: To observe effect of acute normovolemic hemodilution(ANH) with HAES-balanced solution as diluting agent on levels of cytokines including IL-1, IL-2, IL-6 and TNF-alpha in rabbit serum so as to provide theoretical basis for clinical application. METHODS: A total of 20 healthy adult rabbits were enrolled in the study and randomly divided into two groups (10 rabbits per group), i.e., control group (Group C) and HAES group (Group H). Under anesthesia of the rabbits, we performed incision of trachea, high-frequency jet ventilation and liberation of femoral artery and femoral veins. Group C was free from hemodilution. Group H was injected with dilution (2-fold of blood letting volume) via femoral veins during blood letting of the femoral artery. 6% HAES-steril plus compound solution of sodium lactate, with crystal/gel ratio of 2:1, blood letting volume = TBV x (Ho-Hf)/Hav. All blood was transfused back 60-120 min after blood letting. Venous blood was collected before blood letting (T0) and 30 min (T1), 60 min (T2), 120 min (T3) and 24 h(T4) after blood letting to detect Hb and Hct and measure level of IL-1, IL-2, IL-6 and TNF-alpha in serum. RESULTS: In Group H, levels of IL-1, IL-2, IL-6 and TNF-alpha in serum were increased from T1 after ANH, reached peak at T3 but showed decrease at T4, with significant difference compared with Group C at T1, T2, T3 and T4 (P < 0.01) and significant difference compared with those before ANH (P <0.01). In Group C, there was no significant difference upon IL-1, IL-2, IL-6 and TNF-alpha in serum at different time points. CONCLUSION: ANH with HAES-balanced solution as diluting agent can up-regulate the levels of cytokines IL-1, IL-2, IL-6 and TNF-alpha in rabbit serum. In the meantime, ANH may arouse eustress with low intensity and short action time, which exerts effect of enhancing immune function of the organisms.

[Effect of decreased GSH on sensitivity of breast cancer cells to ADM].

OBJECTIVE: Glutathione(GSH) maintains an optimum cellular redox potential. Elevated levels of GSH render some types of cancer cells resistant against anti-cancer drugs. The aim of this study was to determine the effect of a thiol-depleting agent, diethylmaleate (DEM), on the sensitivity of human breast cancer cells to ADM. METHODS: The ADM-resistant human breast cancer MCF-7/ADM cell lines and ADM-sensitive MCF-7/S cell lines were treated by thiol-depleting agent DEM for 3 h respectively. The changes of sensitivity to ADM were then measured by MTT assay. The intracellular GSH contents were examined by fluorescent-spectrophotometry and the correlation between the changes of sensitivity to ADM and the intracellular GSH content was analyzed. RESULTS: Treatment of MCF-7/ADM and MCF-7/S cells by 0.1 micromol/L DEM for 3 h decreased 37.4% and 29.7% of the intracellular GSH content respectively (P < 0.01). ADM also decreased intracellular GSH content in a ADM-concentration-dependent manner. The combined use of DEM and ADM depleted the intracellular GSH content in both cells significantly more than the sum of single use of ADM and DEM alone. The sensitivity of both cells to ADM increased with the decline of intracellular GSH content. CONCLUSION: The depletion effect of DEM on the intracellular GSH could be enhanced by ADM and such depletion may be involved in the changes of the sensitivity of MCF/7 cells to ADM.

[Gene therapy for melanoma by bifidobacterium infantis-mediated transfer of CD and UPRT genes with 5-FC in vitro].

OBJECTIVE: This study was conducted by using Bifidobacterium Infantis as a delivery system to transport suicide gene CD and UPRT to tumors in an attempt to assess the UPRT-enhanced antitumor effect of CD/5-FC. METHODS: The recombinant plasmid pGEX-UPRT was constructed and transfered into Bifidobacterium Infantis by electroporation and then identified. The synergistic antitumor effect of coexpression CD and UPRT was determined by MTT method. And the morphologic changes of B16-F10 cells were observed. RESULTS: Recombinant Bifidobacterium Infantis could express UPRT correctly. The suvival rate of cells administrated CD+ UPRT and 5-FC was significantly lower than that of control (P<0.01), and the 5-FC sensitivity (IC50 = 0.015 micromol/mL) exhibited a 8. 5-fold increase when compared with that of cells administrated CD alone (IC50 = 0.127 micromol/mL). The cells treated with CD+UPRT were remarkably damaged morphologically, and the growth of cells was significantly inhibited as compared with that of other groups. CONCLUSION: Recombinant Bifidobacterium Infantis with UPRT gene can significantly enhance the killing effect of CD/5-FC suicide gene system on melanoma B16-F10 cells of mice.

[Hypermethylation in promoter area of LDLR gene in atherosclerosis patients].

To investigate the methylation pattern of 12 CpG dinucleotide sites in promoter region of LDLR gene in atherosclerosis patients, peripheral blood DNA samples were prepared from 61 atherosclerosis patients and 28 healthy subjects. The methylation status of CpG islands in LDLR gene promoter region was measured by using a modified coordinative method with nested-PCR, Touchdown-PCR and methylation-sensitive-single-strand conformation analysis (MS-SSCA). Three types of methylation patterns were detected in promoter region of LDLR gene in peripheral blood genome of atherosclerosis patients: full-methylation, part-methylation and none-methylation. 2 cases were full-methylation and 13 cases were part-methylation, the methylation frequency was 24.59% in atherosclerosis patients. While in 28 healthy control, only 1 half-methylation sample was found, the methylation frequency was 3.57%, which is much lower than that in atherosclerosis patients (P < 0.05). A significantly higher methylation degree in promoter region of LDLR gene was found in peripheral blood genome of atherosclerosis patients compared with the controlled healthy subjects, which may suggest an involvement of aberrant methylation of LDLR gene in initiation and development of AS. The modified coordinative method with nested-PCR, Touchdown-PCR and MS-SSCA manifests merits of convenience, cheap and high efficiency, which lowers the requirement for the DNA template and increases the sensitivity and specificity.

Dysfunction of endothelial NO system originated from homocysteine-induced aberrant methylation pattern in promoter region of DDAH2 gene.

BACKGROUND: Hyperhomocysteinemia (HHcy)-mediated dysfunction of endothelial NO system is an important mechanism for atherosclerotic pathogenesis. Dimethylarginine dimethylaminohydrolase (DDAH) is the key enzyme for degrading asymmetric dimethylarginine (ADMA), which is an endogenous inhibitor of endothelial nitric oxide (NO) synthase (eNOS). This study was designed to investigate whether the dysfunction of endothelial NO system originates from HHcy-mediated aberrant methylation modification in promotor region of DDAH2 gene. METHODS: Human umbilical vein endothelial cells (HUVECs) were cultured to the third generation and treated with homocysteine (Hcy) at different concentrations (0, 10, 30, 100, and 300 micromol/L) for 72 hours. The methylation pattern in promoter region CpG island of DDAH2 gene was analyzed by nested methylation-specific PCR (nMSP). The mRNA expression of eNOS gene and DDAH2 gene was detected by semi-quantitative RT-PCR. The activity of DDAH2 and eNOS in cells, and the concentrations of ADMA and NO in culture medium were assayed respectively. RESULTS: Mild increased concentration of Hcy (10 and 30 micromol/L) induced hypomethylation, while high concentration of Hcy (100 and 300 micromol/L) induced hypermethylation in the promoter CpG island of DDAH2 gene. The mRNA expression of DDAH2 increased in mild enhanced concentration of Hcy, and decreased in high concentration of Hcy correspondingly. The inhibition of DDAH2 activity, the increase of ADMA concentration, the reduction of eNOS activity and the decrease of NO production were all consistently relevant to the alteration of Hcy concentration. CONCLUSION: The increased concentration of Hcy induced aberrant methylation pattern in promotor region of DDAH2 gene and the successive alterations in DDAH/ADMA/NOS/NO pathway, which showed highly relevant and dose-effect relationship. The results suggested that the dysfunction of endothelial NO system induced by HHcy could be partially originated from Hcy-mediated aberrant methylation in DDAH2 gene.