Biomedical subjects

Fei-yue Xing

Publications and source records attributed to Fei-yue Xing.

7 recordsLinked to original sources

PubMed2006

[Real-time quantitative PCR for evaluating murine thymic function].

OBJECTIVE: To establish a real-time quantitative PCR method for detecting the levels of the signal joint T cell receptor excision circles (sjTRECs) in murine thymocytes and spleen lymphocytes for determining the amount of naive T cells and evaluating the thymic function. METHODS: The genomic DNA was extracted from murine thymocytes and splenocytes for PCR amplification of the target fragments. After purification of the PCR product, the recombination-activating gene 2 (RAG(2)) fragment was cloned into pGEMT-Easy vector to construct the standard plasmid. After PCR optimization, the standard curve was obtained and the samples (thymocytes and splenocytes of BALB/c and C(57)BL/6 mice) were detected for sjTRECs by real-time quantitative PCR. RESULTS: The standard plasmid was correctly constructed, and the standard curve with high reliability was obtained. No statistical difference was observed in sjTREC contents in the T lymphocytes between the two mouse strains. CONCLUSIONS: Real-time quantitative PCR for sjTREC analysis is established successfully, which offers an important means for thymic function analysis and a reliable model establishment for study the thymus.

Animals↗

PubMed2006

[Change of mitochondria during apoptosis of HL-60 cells induced by camptothecine].

AIM: To study the changes of mitochondrial mass and membrane potential (deltapsi(m)) during apoptosis of human promyelocytic leukemia cells (HL-60) induced by camptothecine(CPT). METHODS: HL-60 cells were stimulated with 4x10(-6) mol/L CPT. Apoptosis and necrosis of HL-60 cells were detected by annexin V-FITC/PI staining. Mitochondrial mass and membrane potential (deltapsi(m)) were measured by NAO and DiOC(6)(3) stanings, respectively. RESULTS: After 12 hour treatment with 4x10(-6) mol/L CPT, the early apoptotic rate of HL-60 cells was significantly increased compared with the control group (12.75+/-4.61)% vs (2.88+/-2.49)%,(P<0.01), and the necrotic rate was significantly elevated too, (3.48+/-1.67)% and (0.71+/-1.10)%, (P<0.01). The result of PI staining showed that the apoptotic rate of HL-60 cells at late phase (12 h) in CPT group was (3.52+/-1.07)%, whereas that in control group was (0.46+/-1.06)%. At the same time, we observed that the cells in G(2)/M phase were arrested. The percentage of cells in G(2)/M phase in CPT group and the control was (13.45+/-1.91)% and (22.46+/-2.19)% (P<0.01) respectively. The percentage of cells with low mitochondrial mass in CPT and control groups was (25.74+/-2.09)% and (4.53+/-1.26)%, respectively (P<0.01). Mitochondrial membrane potential in CPT group and control group was (17.71+/-5.23)% and (1.64+/-2.00)%, respectively. CONCLUSION: During CPT-induced apoptosis of HL-60 cells, mitochondrial mass and membrane potential drop significantly, but its depolarization heightens.

Annexin A5↗

PubMed2006

[The changes of the thymocyte mitochondria mediated by nitric oxide in thymocyte apoptosis].

AIM: To study the changes of mitochondrial potential (delta psi m) and cardiolipin (CL) content during thymocyte apoptosis mediated by nitric oxide (NO). METHODS: SNAP was used as NO's donor to induce thymocyte apoptosis in mice and dexamethasone (DEX) was used as positive control drug. Three experiment groups were set, which were blank control group, SNAP group and DEX group. The ectropion of cell phosphatidylserine (PS) was detected by flow cytometry after stained with FITC-anti-annexin V mAb/PI. The changes of delta psi m and CL content during cell apoptosis were detected by DiOC6(3)/PE-anti-annexin V mAb and NAO/PE-anti-annexin V mAb, respectively. RESULTS: 6 hours after SNAP treatment, the thymocytes exhibited typical cell apoptotic characteristics and crenation appeared in most annexin V-positive cells. In DEX group, the delta psi m decreased and the rate of non-apoptotic cells was significantly higher than that in blank control group, but there was no significant difference between SNAP and control groups. 40%-50% of DiOC6(3)-negative cells in each group had the same size as normal cells. The percentage of the cells with reduced CL content in SNAP group was significantly higher than that in blank control group (P<0.01). Non-apoptotic cells with reduced CL content were not found. In blank control group and SNAP group, percentage of normal-sized cells with reduced CL was (48.32+/-3.96)% and (43.64+/-4.90)%, respectively. CONCLUSION: The process of thymocyte apoptosis mediated by NO in mice includes successively PS ectropion, mitochondrial depolarization, CL oxidation and cell crenation. As compared with DEX group, the mitochondrial change mediated by NO is a later event in cell apoptosis.

Animals↗

PubMed2006

[The effect of p38 on the cycloheximide-induced HL-60 cell death through mitochondria pathway].

OBJECTIVE: To study the effect of p38 on the cycloheximide (CHX)-induced HL-60 cell death through mitochondria pathway. METHODS: Inhibition of p38 pathway was by SB203580 (SB). Four groups were set up: control, SB only, CHX only and SB + CHX. Sub-diploid cell ratio was detected by PI staining flow cytometry at 6, 9, 12, 18, 24 h time points, and apoptotic cell ratio by Annexin V-FITC/PI double staining flow cytometry at 6 h and 18 h time points. High J-aggregate cells were evaluated by the J-aggregate contents, measurement of the J-aggregate (FL2) and J-monomer (FL1) by JC-1 flow cytometry, calculation of the delta psi m by FL2/FL1 and analysis of the delta psi m changes at 18 h time points. RESULTS: The sub-diploid cell ratio in CHX group was significantly higher than that in control group at 6 h time point, and the ratio in SB + CHX group was significantly higher than that in CHX group at 9 h time point. At 18 h time point the apoptotic cell ratios in both CHX and SB + CHX groups were significantly higher than those in control group (P < 0.01). There was no significant difference of apoptotic cell ratio between CHX group and SB + CHX group (P > 0.05). At 18 h time point the necrotic cell ratios in both CHX and SB + CHX groups were significantly higher than that in control group (P < 0.01); and that in SB + CHX group was significantly higher than that in CHX group (P < 0.01). The high J-aggregate cell ratios in CHX and SB + CHX groups were significantly lower than that in control group (P < 0.05), and that was signficantly lower in SB + CHX group than in CHX group (P < 0.01). For the FL2/FL1 value (delta psi m) CHX group (0.17 +/- 0.01) and SB + CHX group (0.05 +/- 0.003) were significantly higher than control group (0.38 +/- 0.02) (P < 0.01), and SB + CHX group was significantly lower than CHX group (P < 0.01). CONCLUSION: CHX can induce HL-60 cell apoptosis and the cell mitochondria depolarization, and the latter was intensified by inhibition of the p38 pathway. p38 pathway may related to the cell necrosis in the cycloheximide-induced HL-60 cell apoptosis model. s

Apoptosis↗

PubMed2004

[Effect of astrocyte-conditioned medium on tert-butyl hydroperoxide-induced apoptosis of PC12 cells].

OBJECTIVE: To investigate the effect of astrocyte-conditioned medium (ACM) on PC12 cell apoptosis induced by reactive oxygen species tert-butyl hydroperoxide (tbOOH). METHODS: PC12 cells attacked by tbOOH were cultured with Sprague Dawley rat cerebral cortex ACM, and the cell apoptosis was observed by fluorescent microscopy and transmission electron microscopy. The cell apoptosis rate was evaluated by flow cytometry and malondialdehyde (MDA) concentration measured using thiobarbituric acid (TBA)-reacting substances (TRABS). RESULT: TbOOH induced PC12 cell apoptosis, and ACM significantly decreased the apoptosis rate and MDA concentration in tbOOH-treated cells. CONCLUSION: ACM may increase the anti-oxidation capacity of PC12 cells and inhibit cell apoptosis induced by tbOOH.

Animals↗

PubMed2004

[Effect of total Panax notoginseng saponins on the activity of human endothelial nitric oxide synthase gene promoter].

OBJECTIVE: To study the mechanism of total Panax notoginseng saponin (tPNS) in regulating the transcription activity of human endothelial nitric oxide synthase (heNOS) gene promoter. METHODS: With gene recombination technique, we subcloned the heNOS gene promoter sequence (from -1 to -1 600 bp) into the BglII/HindIII sites of the firefly luciferase reporter gene vector, pGL2-Basic (Promega), to yield the recombinant plasmid designated as peNOS-Luc. With lipofectamine- mediated co-transfection technique, peNOS-Luc, pGL2-Basic and pCMV-beta were cotransfected into NIH3T3 cells, which were treated with lipopolysaccharide (LPS), tPNS and transforming growth factor beta1 (TGFbeta1) respectively. The relative activities (Luc/beta-gal) were subsequently determined in the cell lysates to evaluate the effects of these 3 factors on the activity of heNOS gene promoter. RESULTS: Double restriction enzyme digestion and sequencing both confirmed that the recombinant plasmid, peNOS-Luc, was constructed correctly, which could be effectively expressed in NIH3T3 cells. Upon LPS stimulation, the luciferase activity was obviously decreased, contrary to the results of tPNS and TGFbeta1 treatment, and between the latter two agents, TGFbeta1 produced higher transcription activity. CONCLUSIONS: A firefly luciferase reporter gene vector containing heNOS gene promoter sequence has been constructed correctly. tPNS can up-regulate the activity of heNOS gene promoter in NIH3T3 cells.

Animals↗

PubMed2003

Construction of signal peptide-FRET fusion expression vectors and their expressions in NIH3T3 cells.

OBJECTIVE: To construct the fluorescence protein vector pairs with toll-like receptor 4 (TLR4) signal peptide, namely pECFP-C1-SP and pEYFP-C1-SP, therefore to make the application of fluorescence resonance energy transfer (FRET) possible in the study of the membrane protein interaction during lipopolysaccharide (LPS) recognition. METHODS: pECFP-C1-SP and pEYFP-C1-SP were constructed by means of site-directed mutagenesis, and the constructed plasmids were transiently transfected into NIH3T3 cells via lipofectamin to observe their intracellular expressions under a fluorescence microscope. RESULTS: DNA sequence analysis attested the validity of the constructed fluorescence vectors with signal peptide for FRET, and the expression of the vectors was located principally on the cell membrane as observed under fluorescence microscope. CONCLUSION: The constructed vectors TLR4 signal peptide are valid and capable of expressing on the cell membrane, therefore they can be effectively used in the study of the interaction between the membrane proteins.

3T3 Cells↗