Objective To study the effects of UVR or H2O2 on the expression of p53 in human melanocytes,and that of nutlin-3 and PFT-α on the DNA oxidative damage,and to investigate the role of p53 in the antioxidative stress.Methods The effect of UVR,H2O2,nutlin-3 and PFT-α on the expression of p53 of human melanocytes was detected by Western blot analysis,and that of nutlin-3 and PFT-α on UVR or H2O2 DNA damage assessed by single cell electrophoresis (comet assay).Determination of the effect of nutlin-3 on H2O2 DNA damage was detected by γ-H2AX immunofluorescence.Results UVR and H2O2 could induce p53 protein expression,accompanied by increased phosphorylation of p53 on serine 15 residue,and nutlin-3 and PFT-α could induce and inhibit p53 protein in human melanocytes respectively; nutlin-3 decreased the tail moment of DNA oxidative damage of UVR or H2O2 in human melanocytes,but PFT-α increased the tail moment of DNA oxidative damage of UVR or H2O2 in human melanocytes,and there were significant differences among the control and exposed groups; nutlin-3 decreased expression of γ-H2AX.Conclusions p53 plays a very important role in the antioxidative stress in melanocyte exposed to UV or H2O2.

Objective To estimate the effect of a cis-imidazoline derivative,nutlin-3,on the biological behavior of A375 human melanoma cells,and to investigate its mechanism.Methods Cultured A375 cells were divided into several test groups treated with nutlin-3 at different concentrations (2.5,5,10 μmol/L) for 24,48 and 72 hours,and a control group treated with dimethyl sulfoxide (DMSO) only.Then,methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate cellular proliferative activity,Western blot to measure the expression of p53 protein,flow cytometry to estimate cell cycle phase distribution and apoptosis rate,and Transwell assay to evaluate migratory activity,of A375 cells.Statistical analysis was carried out by repeated-measures analysis of variance (ANOVA).Results After treatment with nutlin-3 of 2.5,5 and 10 μmol/L for 24,48 and 72 hours,significant differences were observed among different time points at each concentration and among different concentrations at the same time point in proliferation inhibition rate (F =67.43,135.58,respectively,both P ＜ 0.01),p53 protein expression level (F =1255.00,9196.00,respectively,both P ＜ 0.01),percentage of cells at G2 phase (F =831.38,267.99,respectively,both P ＜ 0.01),apoptosis rate (F =809.45,723.83,respectively,both P ＜ 0.01),migration inhibition rate (F =1100.00,1667.00,respectively,both P ＜ 0.01).The influence of nutlin-3 on cellular proliferative activity increased with the increase in its concentration,and that on percentage of cells at G2 phase,apoptosis rate and migratory activity increased with the increase in its concentration and treatment duration.There was a significant interaction between the treatment duration and concentration of nutlin-3 for p53 protein expression level in (F =826.79,P ＜ 0.01),percentage of cells at G2 phase in (F =21.602,P ＜ 0.01),apoptosis rate in (F =44.48,P ＜ 0.01),migratory activity of (F =313.09,P ＜ 0.01),and cellular proliferative activity of (F =26.95,P ＜ 0.01),A375 cells.Conclusion Nutlin-3 may inhibit the proliferation and migration of,but promote cell cycle arrest and apoptosis in,A375 cells,through accumulation of p53 protein.

AIM　The purpose is to examine the chemical constituents in the fruits of Terminalia chebula. METHODS　Using combined chromatographies over silica gel, Diaion HP-20, Toyopearl HW-40 and MCI gel CHP -20P to purify the constituents of Terminalia chebula, and identifying their structures on the basis of spectroscopic and chemical evidence were purified. RESULTS　Twenty one hydrolyzable tannins and related polyphenols were characterized, here reports eight of them: 2,3-(S)-HHDP-D-glucose, 3,6-di-O-galloyl-D-glucose, 6-O-galloyl-D-glucose, (-)-shikimide 4-O-gallate, (-)-shikimic acid 3-O-gallate+(-)-shikimic acid 5-O-gallate, methyl gallate and 1,2,6-tri-O-galloyl-β-D-glucose were reported. CONCLUSION　The above eight polyphenols were obtained from myrobalans for the first time.

BACKGROUND: Chronic myeloid leukemia (CML) is characterized by the clonal expansion of hematopoietic stem cells. Without effective treat ment, individuals in the indolent, chronic phase (CP) of CML will undergo blast crisis (BC), the prognosis for which is poor. Therefore, it is important to clarify the mechanism underlying CML from a whole-genome perspec tive. OBJECTIVE: To investigate the gene expression profile of bone marrow mononuclear cells from CML with Applied Biosystems Expression Array System.DESIGN: Observation and controlled analysis.SETTING: Institute of Gene Engineering, Southern Medical University PARTICIPANTS: Samples of two cases of bone marrow (a chronic myeloid leukemia patient and a healthy person).METHODS: This experiment was conducted at the Institute of Gene Engineering, Southern Medical University from October 2004 to September 2005.The total RNAs were extracted and purified from bone marrow mononuclear cells derived from a CML patient and a healthy person. mRNAs were purified using an oligo (dT)-cellulose mRNA purification kits and labeled using reverse transcription, in vitro transcription (RT-IVT), then hybridized with microarray. Gene expression differentiation of the bone marrow mononuclear cells were examined by ABI 1700 Chemiluminescent Microarray Analyzer. Reproducibility of microarray results was assessed by comparing data sets obtained from the same sample and analyzed by two different arrays.MAIN OUTCOME MEASURES: ①Assessment of quality of total RNA and labled cRNA. ②Reproducibility of microarray. ③ Hybridization of array.④Results of semi-quantitative reverse transcription-polymerase chain reaction RESULTS: ①Using statistical data analysis tools, we identified 6 706 genes that were up- or down-regulated in CML patient compared with the healthy person. In these genes, we found that 17 genes were up-regulated while 51 genes were down-regulated among 68 genes closely related to CML. ②most differentially expressed genes in C/EBPalpha mediated path way and CD40L signaling pathway had reduced expression. ③Good repro ducibility of microarray was confirmed by analysis of correlation and detection concordance in technical replicates. The correlation coefficient of the detectable probe in technical replicates was 0.991 for the CML patient and 0.988 for the healthy person. ④The results of semi-quantitative RT-PCR experiments supported the reliability of our microarray analysis.CONCLUSION: By comparing expression patterns of CML with those of the healthy person, we identified a large number of genes that, were up- or down-regulated in CML patients. These data should provide useful information for finding candidate genes whose products might serve as molecular targets for treatment of CML patients.

Objective To report a new method of fluorescent labeling technique in microarray studies: universal primer U2 labeling( UPL). The efficiency was compared of the UPL with that of random primer, restriction display labeling method and the reverse transcription coupled random primer spiking labeling method(RT-PSL). Methods Influenza viral RNA was labeled with both UPL and the conventional random primer labeling method as well as two other more laborious labeling methods( RD-direct and RD-incorporate), and hybridized with influenza virus oligonucleotide microarrays. The signals extracted from the microarrays were analyzed using SPSS 10.0 software. Results The fluorescent intensity, signal-to-noise ration(SNR), true positive ratio(TPR) of probes and labeling reproducibility of UPL were demonstrated to be higher than those of the Random primer approaches.Conclusion These results established that UPL is a valid new labeling protocol, which may have wide applications in the research and development of the microarray technology.

Objective To explore the value of ultrasound-guided balloon control technique in abdominal aorta for reducing intraoperative hemorrhage in high-risk placenta previa undergoing cesarean section.Methods From Aug 2013 to Oct 2017,40 cases were admitted,among them,16 cases were treated with ultrasound-guided towed balloon prophylactic control technique of abdominal aorta (the study group) before cesarean,and 24 cases did not receive balloon occlusion (the control group) during the cesarean.Clinical data were compared between the two groups.Results The time used for uterine suture (t =10.34,P =0.01),the amount of intraoperative blood loss (t =9.51,P =0.01) and blood transfusion (t =3.41,P=0.005)in the two groups were all statistically different.While the differences in PT (t =1.02,P =0.32),ALT (t =0.54,P =0.59),AST(t =0.91,P =0.37),creatinine(t =0.75,P =0.46) were not statistically significant between the two groups.Conclusion Ultrasound-guided abdominal aortic balloon control technique can reduce the blood loss significantly in cesarean section with high-risk placenta previa.

Fluorescent Linear Labelling technique is an effective, single-tube and linear amplification method. The sample cDNA was synthesized from total RNA, and was labelled antisense cRNA from double-stranded cDNA with T7RNA polymerase; it can be used for hybridization to oligonucleotide biochip. Fluorescent Linear Labelling Method can result in 50- to 100-fold higher degrees of amplification, and has been shown to retain information on transcript abundance, thus making it an efficient, robust technique for fluorescent labelling on biochip.
Fluorescent Dyes ; chemistry ; Humans ; Oligonucleotide Array Sequence Analysis ; methods ; Staining and Labeling ; methods

Objective: Compare the detection result of blood samples of severe fever with thrombocytopenia syndrome (SFTS) patients using different detection techniques, and observe the dynamic characteristics of the virus specific RNA, IgM antibody and IgG antibody, to provide theoretical basis for selection of diagnostic methods of disease. Methods: Acute phase serum of suspected SFTS cases and convalescent serum samples of lab-confirmed cases were collected. Real-time fluorescence quantitative PCR and enzyme-linked immunosorbent assay (ELISA) were used to detect the virus specific RNA, IgM antibody and IgG antibody. The detection results of different methods, the relationship between positive results and the acquisition time, and the dynamic characteristics of viral nucleic acid and antibodies were analyzed. Results: A total of 87 serum samples of the suspected SFTS patients were collected, the positive rate of virus specific RNA, IgM antibody and IgG antibody were 53.41%, 31.03% and 3.41%, respectively. Among 55 confirmed cases of SFTS, the consistent rate of virus specific RNA and IgM antibody detection methods was 36.36%, and the difference between the two methods was significant (χ²=6.82, P=0.009), kappa=-0.257. The sampling intervals of RNA positive samples were all within 12 days, of which the positive detection rate was highest after 7-9 days, and the difference was statistically significant (χ²=10.35, P=0.016). In 34 SFTS convalescent serum samples, all the nucleic acid tests were negative, the positive rate of IgM antibody was 41.18%, which was not significantly different from the acute phase serum samples (P=1.00). The positive rate of IgG antibody was 94.12%, which was significantly higher than that of acute IgG antibody (0%). The dynamic characteristics of IgM and IgG antibody showed that IgM antibody could be detected on the second day after onset, the latest detection time was 74 days after onset, and the highest absorbance value and antibody detection rate occurred in 30-60 days. The earliest detection time of IgG antibody was 12 days after onset, and the last detection time was 100 days.The detection rate of IgG antibody and absorbance value increased rapidly after 30 days, and maintained in a high level. The detection rate of IgG antibody was 100% in 30-60 days. Conclusions Blood samples taken from SFTS suspected patients within two weeks of onset may be prioritized for detection of viral nucleic acids using Real-time fluorescence PCR or for detection of IgM antibodies by ELISA. Although IgM antibody can be detected 2 days after the onset, the peak appeared much later, so the negative result can’t rule out the diagnosis. IgG antibody has a high seroconversion rate in convalescent samples, and can be used as an auxiliary tool for disease diagnosis.

Objective To explore the clinical effect of Masquelet membrane induction technique combined with antibiotic coated intramedullary nail fixation in the treatment of lower limb large segment infected bone defects.Methods From June 2009 to August 2015,53 patients who have lower limb large segment infected bone defects were analyzed retrospectively,including 40 males and 13 females,aged from 23 to 61 years,with an average age of 36.2±8.4 years.37 cases were secondary to infection after fracture surgery,and 16 cases were caused by open fractures.There were 17 cases of femoral shaft defects and 36 cases of tibia diaphysis defects.All 53 cases were treated with Masquelet technique.The first stage was infection debridement,then bone defect was filled by bone cement mixed with sensitive or broad-spectrum antibiotics,and then temporary fixation was given.When the infection was controlled,debridement was given again and sensitive antibiotic bone cement was replaced to induce membrane,and antibiotic coated intramedullary nail was used for internal fixation.In the second stage,after intramedullary nailing internal fixation for 4-6 weeks,the bone cement occupying device was taken out and the autologous cancellous bone was planted in the induced membrane.Then the membrane was covered and sutured.The cure rate of infection,the time of bone healing and the related complications were observed.Results 53 patients were followed up for 24 to 63 months (with an average of 39±4.7 months).The length of tibia bone defect after debridement was 6-15 cm (average 8.7±4.9 cm).49 patients' infection were cured in 12 months after operation,and the bone defects were healed,with healing time of 5.3-9.7 months (mean 7.4±3.2 months).No refracture occurred.The healing time of tibia was 7.8±2.1 months,while the healing time of the femur was 7.2±3.9 months.1 case of femoral shaft defect had recurrence of infection 4 months after membrane induced bone grafting,and the first stage treatment was restarted which were debridement and implantation of sensitive antibiotic bone cement occupying device.After 6 weeks,the infection was controlled and the second stages continued.3 cases' s (2 cases of femoral shaft,1 case of tibial shaft) autologous cancellous bone were absorbed 3 to 6 months after operation,and no bone density increased in the bone defect area.The autologous cancellous bone was reimplanted and the bone defect was cured in 8 months.Conclusion Masquelet technology combined with antibiotic coated intramedullary nailing can effectively control infection and create a good biological and mechanical environment for bone defect repair.It has good clinical efficacy.

OBJECTIVE To explore and evaluate the value of CT in diagnosis of malignant tumor of maxillary sinus and the accuracy of the involved bone wall by comparing the preoperative CT imaging with the pathologic examination.METHODS 11 patients without maxillary sinus squamous cell carcinoma and lymph node metastasis received pathological examination and enhanced CT scan before operation,partial or total maxillary resection were implemented according to the CT features and scope.The position and azimuth of the cut bone tissue samples were marked.The specimens were routinely fixed,decalcified,embedded,sliced and HE stained to observe the bone tissue pathological changes on the bone wall under light microscope.RESULTS Nasal sinus enhancement CT scan showed that the medial wall of maxillary sinus were all resorped and invaded(4 cases lack inner wall).Anterior wall was invaded in 6 cases,superior wall in 7 cases and bottom wall in 3 cases,posterior and exterior wall in 9 cases;After ruling out the cases without internal wall of maxillary sinus,the inner wall of the maxillary sinus was invaded by cancer cells,so was the front wall and the bottom wall.Those showed bone wall erosion on preoperative CT with continuous change but without interruption and accompanied by bone wall thickening and hardening were found without tumor invasion by postoperative pathological verification.CONCLUSION Bone wall damage on preoperative CT does not mean tumor invasion,and the probability of each maxillary sinus wall invasion is different;the comprehensive analysis found that for wormhole like change of bone wallon preoperative CT with continuous bone wall thickening and hardening of the 'reconstruction of bone destruction',there was no tumor invasion by postoperative pathological validation;Routine selection analysis of bone tissue pathology can supplement the extent diagnosis of malignant tumor of maxillary sinus on preoperative CT scan,so it can accurately estimate the T staging of tumor.It may provide a more effective basis for selection of minimally invasive surgery,postoperative evaluation of surgical effect and formulating more comprehensive treatment protocol.