[Effects of pregnancy on the ROS, NO, cytokine levels and lymphocytes activation from mouse peripheral blood].

OBJECTIVE: To investigate the influence of pregnancy on the production of reactive oxygen species (ROS) from mouse peripheral blood neutrophils (PMN), the levels of NO and cytokines from serum, the activation of T lymphocytes, and initially find the immune regulation effects of pregnancy on the mouse peripheral blood lymphocytes. METHODS: Take the BALB/c mice which were at the mid trimester of pregnancy (day 14) as the object, full blood staining using ROS probe 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) combing with flow cytometry was used to test the levels of ROS from PMN. The production of NO from peripheral blood serum were analyzed by Griess kit while the soluble cytokines interleukin (IL) 6, IL-10, monocyte chemotactic protein 1 (MCP-1), interferon γ (IFN-γ), tumor necrosis factor α (TNF-α) and IL-12 were detected by liquid protein quantitative technology cytometric bead array (CBA) using flow cytometry. The activation of peripheral blood lymphocytes at early, middle and later phases which marked with CD69, CD25 and CD71 respectively were tested by flow cytometry and two-color fluorescent staining. RESULTS: Comparing to the normal non-pregnant mouse, pregnancy obviously promoted the production of ROS from PMN (101.1±2.2 versus 134.5±10.3, P<0.05). Comparing to the normal non-pregnant mouse, pregnancy obviously promoted the secretion of NO [(22.7±0.7) versus (36.3±1.2) µmol/L, P<0.01]. In normal non-pregnant mouse, the serum levels of IL-6, IL-10, MCP-1, IFN-γ, TNF-α and IL-12 were (9.3±0.5), (26.7±0.9), (21.2±1.6), (14.5±1.8), (22.6±1.6) and (8.4±1.2) pg/ml, while in pregnancy group the levels were (26.5±1.0), (40.4±2.5), (25.1±0.7), (457.4±17.9), (93.2±4.3) and (7.5±0.9) pg/ml correspondingly; the levels of IL-6, IFN-γ, TNF-α from peripheral blood serum (P<0.01), while had no effects on the production of IL-10 and MCP-1 (P>0.05). About the CD+3 T lymphocytes activation, in normal non-pregnant mouse, the CD69, CD25 and CD71 expression rate were (0.43±0.15)%, (5.13±0.25)% and (0.37±0.11)%, while in pregnancy group the CD69, CD25 and CD71 expression rate were (0.40±0.10)%, (6.17±0.40)% and (6.10±0.31)%. The levels of middle and later phases markers as CD25 and CD71 were highly up-regulated (P<0.05), while the early phase action CD69 had no obvious variation (P>0.05). CONCLUSION: The mid trimester of pregnancy promoted the production of ROS from PMN, the levels of NO, IL-6, IFN-γ, TNF-α from peripheral blood serum, and the middle- and later-phase activation of T lymphocytes.

Exploitation of phosphorescent labelling reagent of fullerol-fluorescein isothiocyanate and new method for the determination of trace alkaline phosphatase as well as forecast of human diseases.

A new phosphorescent labelling reagent consisting of fullerol, fluorescein isothiocyanate and N,N-dimethylaniline (F-ol-(FITC)(n)-DMA) was developed. The mode of action is based on the reactivity of the active -OH group in F-ol with the -COOH group of FITC to form an F-ol-(FITC)(n)-DMA complex containing several FITC molecules. F-ol-(FITC)(n)-DMA increased the number of luminescent molecules in the biological target of WGA-AP-WGA-F-ol-(FITC)(n)-DMA (WGA and AP are wheat germ agglutinin and alkaline phosphatase, respectively) which improved the sensitivity using solid substrate room temperature phosphorimetry (SSRTP) detection. The proposed method provided high sensitivity and strong specificity for WGA-AP. The limit of detection (LD) was 0.15 ag AP spot(-1) for F-ol and 0.097 ag AP spot(-1) for FITC in F-ol-(FITC)(n)-DMA, which was lower than the method using single luminescent molecules of F-ol-DMA and FITC-DMA to label WGA (0.20 ag AP spot(-1) for F-ol-DMA and 0.22 ag AP spot(-1) for FITC-DMA). Results for the determination of AP in human serum were in good agreement with those obtained by enzyme-linked immunosorbent assay. The mechanism of F-ol-(FITC)(n)-DMA labelling of WGA was discussed.