A UPLC-ESI-MS/MS method was used to determinate the main active fractions gallic acid, protocatechuic acid, myricetrin, hyperoside and quercitrin in Polygonum capitatum extracts by in situ intestinal perfusion models; the absorption rate constants and cumulative penetration rate of absorption were calculated. The effect of different drug concentrations, different intestine segments, bile and P-gp inhibitors on the absorption mechanism of Gallic acid and other compositions in P. capitatum extracts. The experimental results showed that gallic acid, protocatechuic acid, myricetrin and quercitrin were observed saturated at high concentration (P < 0.05). Bile had significant inhibition effect on protocatechuic acid absorption and had promotion effect on myricetrin and hyperoside absorption (P < 0.05). P-gp inhibitor verapamil could significantly enhance the absorption of Protocatechuic acid (P < 0.05). The overall trend for absorption of various compositions was that small intestine > colon. This indicated that the absorption mechanism of P. capitatum extracts in rat intestine was in line with fist-order kinetics characteristics. The composition could be absorbed in all of the different intestinal segments, and the absorption was mainly concentrated in small intestine. The protocatechuic acid may be the substrate of P-gp.
Animals ; Drugs, Chinese Herbal ; chemistry ; metabolism ; Female ; Intestinal Absorption ; Intestines ; chemistry ; metabolism ; Male ; Polygonum ; metabolism ; Rats ; Rats, Sprague-Dawley

Objective To analyzed the expression and clinical characteristics of CD 20 marker in children with B-lineage acute lymphoblastic leukemia ( B-ALL) and evaluated its medical significance in assessing the prognosis of disease.Methods From November 2008 to July 2012,125 cases of children with B-lineage acute lymphoblastic leukemia were collected from Shanghai Children ′s Hospital,including 79 males and 46 females, aged between 2 months to 14 years old.Flow cytometry based immunophenotyping and Minimal Residual Disease ( MRD) screening were applied to these children when newly diagnosed ,and MRD monitoring was again carried out after 35 days of induction remission therapy to those bears the MRD markers.These 125 patients were divided into CD20-positive group and CD20-negative group, and the corresponding clinical characteristics ,stage of immunophenotype ,MRD,risk stratification,and overall survival rates were recorded and compared.Data were statistically analyzed by using SPSS 16.0 software including χ2 test,t-test,standard deviation test and survival test.Results A total of 125 children with ALL-B,the group of CD20-positive were 48 while CD20-negative groups were 77,with a median age of 6 years old,and the median follow-up time of 30 months.Multivariate Cox regression Analysis showed that there was no clear correlation between CD20 expression level with age ,sex,white blood cell count at diagnosis ,fusion-gene,the stage of immunophenotype as well as risk stratification.The MRD-positive incidence at 35 days in the CD20 positive group was 35.4%,much higher than that of the CD20-negative group (16.9%),which is statistical significance (χ2 =5.236,P<0.05),while the overall survival rate (OS) for the CD20 positive group is 75.0%,much lower than that of the CD20 negative group (84.4%,χ2 =4.160,P<0.05).Conclusions CD20 positive expression level in children with B-lineage acute lymphoblastic leukemia at diagnosis demonstrates negative correlation with the overall survival rate of the patient ,indicating its usefulness as an additional joint marker for the current regimens to incorporate CD 20-targeted monoclonal therapy.

Objective To evaluate the practicability of using flow cytometry analysis for diagnosis of non-hodgkin′s lymphoma ( NHL) among children with serous effusion.Methods Twelve children who were diagnosed with malignant lymphoma from February 2011 to November 2013 at Shanghai Children′s hos-pital were recruited in this study.Pleural effusion and ascites samples were collected from those children who showed serous effusion as initial symptoms and analyzed by using flow cytometry based immunophenotyping. The antibodies used for immunophenotyping included CD45, CD10, CD33, CD7, CD1a, MPO, cCD3, CD79a, CD22, CD19, CD20, CD5, CD3,κ,λ,αβ,γδ,CD56 and other common markers for T, B and NK cells.Anti-CD30 antibody was used when necessary.Results All of the twelve cases with serous effusion were diagnosed with aggressive NHL.Six out of the twelve children including five cases with ascites and one case with pleural effusion showed high expression of CD20 and were classified as NHL-B type by flow cytom-etry.Three children with pleural effusion and one child with both pleural effusion and ascites were typed as NHL-T as characterized by monoclonal expression of αβorγδ.The other two children with pleural effusion were diagnosed with anaplastic large cell lymphoma with positive expression of CD30 and morphological het-erogeneity.Conclusion Flow cytometry analysis based immunophenotyping could be used as an auxiliary method for rapid and accurate diagnosis of lymphoma in children with serous effusions.

OBJECTIVE12-0-tetradecanoylphorbol-13 acetate (TPA) plays an important role in precipitating cell differentiation for various tumor cells, especially leukemic cells. Changes of many genes may be involved in this process. The purpose of this study was to observe the relationship between the EGR1mRNA expression and cell differentiation during TPA-induced K562 cell differentiation.

METHODSIncubation of human K562 cells in vitro was applied to cultivate K562 cells. The cells were treated in two different ways. K562 cells of experiment group were treated with TPA and those of control group were treated without TPA. Using morphology (Wright's staining and NSE staining) and flow cytometry (FCM), the investigators observed the differentiation characteristics of K562 cells, cell-cycle and the differentiation antigen expressions of CD33 and CD14 on cell membranes. RT-PCR was carried out to assay EGR1 mRNA expression.

RESULTSAfter treated with TPA for 7 d, the morphology of K562 cells obviously tended to mature differentiation, like monocytes. The differentiation rate of induced K562 cells was up to 95% in experiment group and 4.5% in control group, respectively. Using SPSS software, the above result showed statistical significance (P < 0.01). Using NSE staining, K562 cells showed positive reaction. Some of them were densely stained. The positive rate was up to 86%. More than half of the positive cells could be inhibited by NaF. The inhibiting rate of NaF was up to 58.72%, showing statistical difference when compared with that of control group. FCM analysis showed that most of K562 cells stimulated by TPA underwent G1/S phase cell-cycle arrest. The composing rate of cell-cycle in TPA-treated group showed that (53.7 +/- 1.25)% of cells were at G0 + G1 phase and (44.3 +/- 1.32)% were at S phase (P < 0.05). The level of CD33 expression on cell membranes was mildly decreased from 0.997% to 0.893% (P > 0.05). However, the level of CD14 expression was significantly increased from 0.049% to 0.387% (P < 0.05).

CONCLUSIONK562 cells could express EGR1mRNA during TPA-induced differentiation, which suggested that EGR1mRNA might participate in the process of K562 cells differentiating into monocyte/macrophages, and might play an important role in precipitating and maintaining cell differentiation for leukemic cells.

Antigens, CD ; metabolism ; Antigens, Differentiation, Myelomonocytic ; metabolism ; Carcinogens ; pharmacology ; Cell Cycle ; drug effects ; genetics ; Cell Differentiation ; drug effects ; genetics ; Cell Division ; drug effects ; genetics ; Cell Membrane ; chemistry ; drug effects ; DNA-Binding Proteins ; genetics ; Early Growth Response Protein 1 ; Flow Cytometry ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Immediate-Early Proteins ; genetics ; K562 Cells ; Lipopolysaccharide Receptors ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Sialic Acid Binding Ig-like Lectin 3 ; Tetradecanoylphorbol Acetate ; pharmacology ; Transcription Factors ; genetics

To explore a rapid and easy method to detect labile iron of pool (LIP) in cells, HL-60 and K562 cells were cultured at a concentration 1 x 10(6)/ml in RPMI 1640 containing 10% heat-inactivated fetal bovine serum. The iron deprivation was induced by adding desferrioxamine (DFO) 10 - 100 micromol/L for 0 - 48 hours. The intracellular LIP was measured by probe calcein-AM. Calcein fluorescence was monitored in 1420 multilabel counter. The results indicated that when HL-60 and K562 cells were incubated with different concentrations of DFO, the calcein fluorescence intensity was higher than that of control group at 12, 24 and 48 hours (P < 0.05). Fluorescence value of representing LIP in DFO groups was lower than that in the control group. In conclusion, DFO can decrease LIP in leukemia cells. The approach used in this study may provide a simple and reliable method for detection of intracellular iron homeostasis.
Cation Transport Proteins ; antagonists & inhibitors ; biosynthesis ; metabolism ; Deferoxamine ; pharmacology ; Fluoresceins ; Fluorescent Dyes ; HL-60 Cells ; Humans ; Iron ; metabolism ; Iron Chelating Agents ; analysis ; metabolism ; Iron-Regulatory Proteins ; metabolism ; K562 Cells

Objective Impulse noise was adopted in adult rats to built acute deafferent animal model.Differential proteomics techniques were applied to detect the changes of protein expression in the auditory cortex before and after the noise exposure.Methods Thirty adult SD rats were divided into three groups:normal group,rats with acute noise exposure and rats 28 days recovery after noise exposure (n =10/group).All animals were exposed to impulse noise at 156 dB for 50 pulses with a rise-time of 100 μs and duration of around 0.25 ms.ABR was used to evaluate the auditory function.The two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS) were used to identified the differential protein expression.Results Compared with the normal group,ABR thresholds were found significantly increased at 2,4,8,16,32 kHz(P ＜0.05) in the acute and recovery groups.There was a 40 - 60 dBSPL ABR threshold shift at all tested frequencies immediately after impulse noise exposure.There was a partial recovery of ABR thresholds at 7 day to 28 days after impulse noise exposure.In addition,it seemed that the thresholds were rather stable and no further ABR threshold recovery was observed from 14 day to 28 days after the impulse noise exposure.Using differential proteomic techniques,36 spots containing 27 proteins were revealed and identified in auditory cortex.Those proteins are related to cytoskeleton,neurotransmission,energy supply,mitochondrial function and synaptic remolding.Conclusions Impulse noise may influence the function of microtubule transport and cell metabolism,there after affect the neurotransmission of auditory neurons.The compensatory changes such as pre- and postsynaptic or such related functional changes may also happen in auditory cortex after the deafferentation treatment.

OBJECTIVETo study the expressions of cystathionine-beta-synthase (CBS) and cystathionine-gamma-lyase (CSE) in the corpus cavernosum smooth muscle of castrated rats and their roles in erectile dysfunction after castration.

METHODSWe randomly assigned 40 eight-week-old male SD rats to groups A (2-week sham-operation), B (4-week sham-operation), C (2-week castration) and D (4-week castration). We determined the level of serum testosterone (T) and the expressions of CBS and CSE in the corpus cavernosum smooth muscle of the rats after operation using immunohistochemistry and RT-PCR.

RESULTSThe T level was significantly decreased in groups C ([11.85 +/- 6.73] nmol/L) and D ([1.96 +/- 1.23] nmol/L) as compared with A ([89.65 +/- 17.13] nmol/L) and B ([106.75 +/- 19.68] nmol/L) (P < 0.05). CBS and CSE were expressed in all groups of rats, but the relative expressions of CBS and CSE mRNA were significantly lower in groups C (0.93 +/- 0.14 and 0.87 +/- 0.20) and D (0.79 +/- 0.17 and 0.71 +/- 0.12) than in A (2.13 +/- 0.65 and 1.93 +/- 0.15) and B (2.07 +/- 0.53 and 1.89 +/- 0.45) (P < 0. 05), so were the optical density values (IA) of the CBS and CSE proteins, 130.35 +/- 23.56 and 93.56 +/- 36.64 in group C and 80.29 +/- 29.65 and 58.56 +/- 19.95 in group D, as compared with 310.57 +/- 130.56 and 269.56 +/- 116.76 in group A and 349.68 +/-112.35 and 298.35 +/- 100.76 in group B (P < 0.05). The androgen level was positively correlated with the expressions of CBS and CSE in the corpus cavernosum smooth muscle of the rats.

CONCLUSIONAndrogen regulates erectile function via the expressions of CBS and CSE.

Animals ; Cystathionine beta-Synthase ; metabolism ; Cystathionine gamma-Lyase ; metabolism ; Male ; Muscle, Smooth ; enzymology ; Orchiectomy ; Penis ; enzymology ; Rats ; Rats, Sprague-Dawley ; Testosterone ; blood

BACKGROUNDEndovascular therapy is a treatment option for localized occlusion of the subclavian artery. In this report the long-term experience with 59 patients is presented.

METHODSBetween June 1998 and September 2008, we used endovascular therapy to treat 61 subclavian arterial obstructive lesions in 59 patients (46 males and 13 females, 34 - 82 years of age with a mean age (61.9 + or - 11.0) years). Twenty patients (34%) had clinical symptoms due to vertebrobasilar insufficiency, 26 (44%) had disabling arm ischemia, and 13 (22%) had both symptoms. We performed all procedures under local anesthesia. The approaches were from the femoral artery (n = 47), brachial artery (n = 1, involving bilateral subclavian disease) or both (n = 11). Sixty stents were implanted. All patients were followed-up at 1, 3, 6, and 12 months post-procedure, and annually thereafter.

RESULTSWe achieved technical success in 58 (95.1%) arteries, all of which were stented. There were three technical failures; two were due to the inability to cross over an occlusion, necessitating the switch to an axillo-axillary bypass, and the third was due to shock after digital subtraction angiography and prior to stenting. Arterial stenosis pre- and post-stenting was (83.6 + or - 10.8)% and (2.5 + or - 12.5)% (P < 0.01). Clinical success was achieved in 55 of the 59 patients (93.4%). Of the four clinical failures, three were technical and the remaining patient had a stent thrombosis. Systolic blood pressure difference between the two brachial arteries was (44.7 + or - 18.5) vs. (2.2 + or - 3.9) mmHg (P < 0.01). Primary patency was 98% at 12 months, 93% at 24 months, and 82% at 5 years. Five patients were lost to follow-up by 12 months post-stenting. Significant recurrent obstruction developed in five patients with resumption of clinical symptoms. The overall survival rate was 98.2% at 12 months, 89.5% at 24 months, and 84.5% at 5 years.

CONCLUSIONSEndovascular therapy for proximal subclavian arterial obstructive lesions is effective and successful. This minimally invasive treatment may be the first choice of treatment for proximal subclavical arterial obstructive lesions.

Adult ; Aged ; Aged, 80 and over ; Arterial Occlusive Diseases ; pathology ; therapy ; Female ; Humans ; Male ; Middle Aged ; Stents ; Subclavian Artery ; pathology ; Subclavian Steal Syndrome ; pathology ; therapy ; Vertebrobasilar Insufficiency ; pathology ; therapy

Objective To explore the effects of radiofrequency catheter ablation(RFCA)on the blood coaguable states and the clinical value of perioperative plasma D-dimer.Methods The plasma level of D-dimer was assayed by enzyme-linked immunosorbent assay(ELISA)in blood samples of 30 children who were undertaken RFCA.Blood samples were consecutively obtained before cannulating,after electrophysiologic(EP)study,immediately after RFCA,the second day and the seventh day after RFCA.The centrifuged spead was 3 000 r/min,keep it for 10 minutes to obtain the upper plasma,and the crvopreserve.Results The plasma levels of D-dimer was highest at the time point when RFCA was successfully accomplished and restored to preoperative level in the seventh day after RFCA.There were statistically significant difference in the paried values at different time points(Pa

Objective: To analyze the characteristic mutations of epitopes in HBV Pre-S/S region in HIV/HBV co-infected patients’ peripheral blood to provide basic data for studying the pathogenesis of HIV/HBV co-infection. Methods: The chronic hepatitis B infected patients admitted to the Infectious Disease Center of the Eighth People′s Hospital of Guangzhou from January 2009 to December 2011 were enrolled into HIV/HBV co-infected group and HBV mono-infected group according to the result of HIV antibody detection respectively before treatment. HBV DNA in serum was extracted and Pre-S/S region of HBV DNA was amplified by nested-PCR. After sequencing of the obtained PCR products (direct sequencing), ContigExpress software was used for sequence splicing and BioEdit software was used for sequence alignment. With reference to the standard sequence of the matched genotype HBV, mutants of HBV Pre-S/S region in HIV/HBV co-infected group and HBV mono-infected group were analyzed respectively. Statistical analysis was performed by chi-square test with SPSS19.0 statistical analysis software. Results: HBV Pre-S/S fragments were successfully amplified from 150 patients, including 90 cases of HIV/HBV co-infected group and 60 cases of HBV mono-infected group, with matched gender, age, genotype, HBeAg status, alanine aminotransferase (ALT), aspartate aminotransferase (AST). The result of analyzing mutants of HBV Pre-S/S region indicated that the incidence of mutation in all epitopes for cytotoxic T cells (CTL cells) was higher in the HIV/HBV co-infected group, and Pre-S2 aa1-15 epitope was significantly higher (χ²=6.964, P=0.008). The incidence of deletions in PreS2 aa1-15 epitope in HIV/HBV co-infected group (11.1%) was higher than HBV mono-infected group (3.3%) (χ²=2.959, P=0.085). In the B cell epitopes, the incidence of mutations in Pre-S2 aa1-26 in the HIV/HBV co-infected group was significantly higher than HBV mono-infected group (χ²=6.924, P=0.010), and there was no statistical significance between two groups in other B cell epitopes. No differences in helper T cell (Th cell) epitopes were found between the two groups. Conclusions Co-infection with HIV increased the CTL cell epitopes’ mutations in the HBV Pre-S/S region, especially the 5′ end epitope mutations in Pre-S2 region, which indicated that HBV mutation is related to the host immune status, and showed guiding information for further study on the pathogenesis of HIV/HBV co-infection