AIM: To establish a method for the simultaneous determination of hesperidin and baicalin in Qingqi Huatan Pill (Pericarpium Citri Reticulatae, Radix Scutellariae, etc.). METHODS: RP-HPLC was adopted with a mobile phase of acetonitrile-water-1% phosphoric acid (20∶80∶0.2), flow rate of 0.8 mL?min -1. The detection wavelength was at 280 nm. RESULTS:The linear range of hesperidin was 0.04 ?g-0.37 ?g, the average recovery was 97.5% and RSD was 1.0%. The linear range of baicalin was 0.13 ?g-1.19 ?g. The average recovery was 97.5%and RSD was 1.2%, respectively. CONCLUSION: This method is simple, practical, accurate and suitable for the quality control of Qingqi Huatan Pill.

Objective To evaluate the effect about medication compliance for patients with chronic heart failure in outpatients using nursing intervention model based on Omaha system.Methods 100 patients were randomly divided into observation group(50 patients)and control group(50 patients).The two groups of patients were given routine nursing intervention,the observation group also used the Omaha system to develop care programs on this basis, and was given the implementation about continuity of care.Results On the point of the two or three months after the patients were discharged,the AHFKT -V2 questionnaire scores in the observation group[(17.690 ±1.892)points, (20.900 ±2.052)points]were significantly higher than the control group[(14.080 ±2.374)points,(18.450 ± 1.781)points],the differences were statistically significant (t =-8.488,-6.442,all P <0.05).However,the same as the points after the patients were discharged,Morisky questionnaire scores in the observation group[(1.036 ± 0.780)points,(0.487 ±0.260)points]were significantly lower than the control group[(1.54 ±1.182)points, (0.920 ±0.804)points],the differences were statistically significant(t =3.420,4.965,all P <0.05).Conclusion The use of Omaha system to develop the targeted continuity of care,can improve the patients medication compliance.

Objective To evaluate the clinical effect of acupuncture combined with estazolam for the treatment of primary insomnia with heart and spleen deficiency type. Methods Seventy qualified patients were randomized into treatment group and control group, 35 in each group. Both groups received oral use of estazolam before bed time for 6 continuous weeks, and acupuncture group was additionally given acupuncture for regulating governor vessel and tranquilizing spirit on acupoints of Baihui(GV20), Yintang(GV29), Shenting(GV25), Anmian (EX-HN22), Shenmen(H7), Sanyinjiao(SP6), 3 times a week and lasting for 6 continuous weeks. The sleep state of the patients was estimated according to Pittsburgh Sleep Quality Index (PSQI), Epworth Sleepiness Scale (ESS) and Athens Insomnia Scale(AIS) before treatment, on the third and the sixth week of treatment, and one month after the completion of treatment. Results (1) Five cases in the treatment group and 3 cases in the control group were dropped out for not completing the treatment timely. In the end , 30 cases in the treatment group and 32 cases in the control group finished the trial. (2) The total effective rate in the treatment group was up to 90.0%, and was 71.9% in control group, the difference being significant(P<0.01).(3) Compared to the control group, the scores of PSQI, ESS and AIS were much decreased in treatment group on the third and the sixth week of treatment , and one month after treatment(P<0 . 05 or P<0 . 01). Conclusion Acupuncture for regulating governor vessel and tranquilizing mind combined with oral use of estazolam exerts certain therapeutic effect for the treatment of heart and spleen deficiency type insomnia, and the effect was superior to that of estazolam alone.

0.05). Conclusion The technique of PEP-MN-PCR from single-cell for simultaneous detection of HLA-A, B, DR types were efficient and accurate. It was possible to apply the techniques of PEP-MN-PCR to detect HLA-A, B, DR typing for preimplantational diagnosis, preliminary selection of HLA matching embryos and the patient sibling who needs cord blood stem cell transplantation.

Objective To establish a PCR sequencing-based typing (PCR-SBT) method for simultaneous genotyping of human platelet antigen HPA-1 to HPA-16w.Methods All DNA polymorphism sites of HPA-1 to HPA-16w were obtained from the immuno polymorphism database.The specific primers were designed using Primer Premier 5.0 software to amplify nucleotide acid fragments encompassing each HPA polymorphism site.The primer sequence and PCR condition were optimized to obtain specific and single amplification product.The PCR product was purified and then sequenced to determine the HPA genotypes.Two standard DNA samples were detected using the HPA PCR-SBT method to examine the accuracy d this method.Sixteen reference samples (including 6 interference samples with HPA gene mutations) provided by 14th platelet immunology workshop of international society of blood transfusion (ISBT) in 2008 were also tested by this home-brew HPA PCR-SBT method.Results Total eleven pairs of primers were designed to amplify and sequence the sixteen HPA systems.The HPA genotypes of two standard samples were 1aa/2aa/3ab/4aa/5ab/6aa/7aa/8aa/9aa/10aa/11aa/12aa/13aa/14aa/15aa/16aa and 1aa/ 2aa/3aa/4aa/5aa/6aa/7aa/8aa/9aa/10aa/11aa/12aa/13aa/14aa/15aa/16aa,respectively.The 256 HPA genotypes of 16 reference samples were clear.128 genotypes among them were completely accordance with the results provided by ISBT report.Conclusions The PCR-SBT assay combining high-throughput DNA sequencer established in the study provides a simple,rapid and accurate method for HPA-1 to HPA-16w systems genotyping.The assay is suitable for routine clinical HPA genotyping and shows a broad prospect in further applications.

Objective To study the gene expression profiles of megakaryocytes(MKs) from human cord blood CD34+ cells in vitro expanded and to understand megakaryopoiesis at the molecular level. Methods CD34+ cells were isolated using density gradient centrifugation and magnetic activated cell sorting. The cells were cultured and stimulated with recombinant human TPO ( 100 ng/ml). After 12 days, the MKs fraction was separated using an anti-CD41 monoclonal antibody by immunomagnetic sorting. The gene expression profiles of MKs, non-MKs as well as meg-01 cells were studied by gene chip assay. THBSI, HOX A9,β-actin, lL-8,Annexin A6, FGF-8 were selected to validate the gene chip results by RT-PCR. Results A total of 116 genes between MKs and non-MKs cells were significantly different, 52 genes were up-regulated and 64 genes were down-regulated. In addition, 158 genes between MKs and meg-01 cells were significantly different, 71 genes were up-regulated and 87 genes were down-regulated. THBSI showed higher expression in MKs than in non-MKs. HOXA9 showed lower expression in MKs than in non-MKs. The expression of β-actin did not show any significant difference in MKs and non-MKs. IL-8 showed higher expression in MKs than in meg-01 cells, while ANXA6 showed lower expression in MKs than in meg-01 cells. The expression of FGF-8 did not show any significant difference between MKs and meg-01 cells. Conclusions MKs, non-MKs and meg-01 cells show different gene expression profiles. The regulatory genes include stress response genes,immune related genes, DNA synthesis and repair genes, metabolism genes, pro-onco genes and tumor suppressor genes.

Objective To analyze the molecular genetic basis of novel allele HLA-B * 9534 and establish the allele group specific primer PCR method. Methods Genomic DNA was extracted from whole blood by commercial DNA extraction kit. The HLA-B exons 1 to 8 coding sequences of the proband were am-plified by PCR and the amplification product was purified with double enzymes digestion and both strands of exons 2, 3 and 4 were sequenced. The exon 2-4 amplification of the HLA-B * 9534 was performed with al-lele group specific primers PCR and the PCR product was directly sequenced for exon 2 to 4. Results The proband has two HLA-B alleles. The result was assigned for HLA-B * 1518 and B * 4601 combination with a mismatch in 593A/G heterozygote by DNA sequencing of exon 2 to 4 with loci primers. After separating the two alleles of the proband with allele group specific primers polymerase chain reaction method, HLA-B * 4601 and HLA-B * 9534 alleles were identified after sequencing. The HLA-B * 9534 is identical to HLA-B * 1518 except for one nucleotide substitutions in exon 3 at position 593 A→G, this results in amino acid substitution at cedon 174 from Asn to Ser. The sequences of the novel allele have been submitted to GenBank (EU046491) and the allele has been officially nominated by the WHO Nomenclature Committee. Conclusion Identification of a novel HLA-B * 9534 allele and allele group specific primer PCR for HLA-B * 9534 was re-liable.

Objective To investigate the serological characteristics and molecular basis of the B (A)phenotype in ABO blood group and provide the data for clinical transfusion of individuals with B(A) phenotype.Methods The ABO group antigens on red cells of the proband,family members and donors were identified by monoclonal antibodies and the ABO antibodies in sera were detected by the standard A,B,O cells.The compatibility testing for the proband and donors was detected by salted test,polybrene test and antiglobulin test.The coding region of exon 6 to exon 7 in ABO gene was amplified by polymerase chain reaction(PCR) and the PCR products were sequenced.The haplotypes of proband were analyzed by cloning and sequencing.Results It was showed that both A and B antigens were detected on red cells of the proband and her two family members,and there was anti-A_1 antibody in their sera.The serological phenotype of the samples are identified as the A_2B.DNA sequencing showed 261 G/del,297A/G,526C/G,657C/T,700C/G,703G/A,796C/A,803G/C,930G/A heterozygotes in exon 6 to exon 7.It can be deduced that genotype in the proband is B(A)_(02)/O_(01).The genotypes of her mother and grandmother-in-law were B(A)_(02)/B_(101) and B(A)_(02)/O_(01),respectively.After cloning and sequencing,two alleles B(A)_(02) and O_(01) in proband was showed.B(A)_(02) has snigle nucleotide change(700 C>G),which resets replacement of proline with alanine at position 234.Two donors with phenotype A_2B were identified as genotype B(A)_(02)/O_(01) and A_(208)/B_(101),respectively.The results of crossmatch testing is in accordane between the proband and two donors and there was no clinical adverse reaction after transfusion.Conclusions 700C>G in α-1,3galactosyltransferase allele(B allde)can result in B(A)phenotype in individuals with the phenotype of A_2B.The donors in the transfusion for the individuals with B(A) phenotype should include individuals with A_2B phenotype.

OBJECTIVETo observe the effects and duration of electroacupuncture on the mechanical pain threshold induced by paclitaxel and explore its analgesic mechanism.

METHODSSixty-four C57BL/6J male mice were randomly divided into 4 groups, a normal+sham EA group, a normal+EA group, a medicine+sham EA(Med+ sham EA) group, a medicine + EA (Med + EA) group, 16 cases in each group. The model of chemotherapy-induced peripheral neuropathy was established with paclitaxel intraperitoneal injection on the 1st, 3rd, 5th, 7th day in the Med + sham EA group and the Med + EA group. EA of 30 min was used on bilateral "Zusanli (ST 36)" on the 9th, 11th, 13th, 16th, 18th, 20th, 23rd, 25th, 27th, 30th day in the EA groups, 2 Hz/100 Hz and 1~ 1.5 mA. Acupuncture was applied on the same acupoint at the same times in the sham EA groups. Mechanical pain thresholds were tested by VonFrey before and after model establishment, namely on the 8th, 14th; 21st and, 28th day. The heart blood of 8 mice was drawn quickly to collect serum in every group on the 31st day, and the contents of tumor necrosis factor α (TNF-α), interleukin-1α (IL-1α), interleukin-1β (IL-1β) in proinflammatory cytokine were examined by ELISA. Mechanical pain thresholds were tested by VonFrey for the rest 8 mice of each group until there was no apparent difference in the two paclitaxel groups once a week,namely on the 35th, 42nd, 49th day.

RESULTSThe pain thresholds of each group were not statistically different before model establishment (P > 0.05). After model establishment (on the 8th day), thresholds of the paclitaxel groups were lower than those of the normal groups (all P < 0.05). After EA, the mechanical pain thresholds of the Med + EA group were higher than those of the Med + sham EA group at all the time points, and there was statistical difference on the 14th, 21st and 28th day (all P < 0.05). The analgesic effect was lasting to the 49th day. The contents of TNF-α, IL-1α, IL-1β of the Med + EA group were decreased than those of the Med+sham EA group in different degree, with statistical significance of IL-1α (P < 0.05).

CONCLUSIONEA can effectively treat paclitaxel-induced peripheral neuropathy,and the analgesic mechanism is probably related to decreasing the proinflammatory cytokine.

Acupuncture Points ; Animals ; Antineoplastic Agents ; administration & dosage ; adverse effects ; Electroacupuncture ; Humans ; Interleukin-1beta ; genetics ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Neoplasms ; drug therapy ; Peripheral Nervous System Diseases ; etiology ; genetics ; metabolism ; therapy ; Tumor Necrosis Factor-alpha ; genetics ; metabolism

Objective To investigate the protective effect of agmatine (AGM) against peritoneal inflammatory response and neutrophil (PMN) infiltration induced by zymosan (ZYM) in mice. Methods Thirty-six adult male C57BL/6 mice were randomly divided into sham group, model group, and AGM treatment group. Peritonitis model was reproduced by intra-peritoneal injection of 1 mg/mL ZYM (0.5 mL), while equivalent phosphate buffer saline (PBS) was given to sham group. 200 mg/kg AGM was injected into peritoneal cavity after ZYM challenge in AGM treatment group. Six mice in each group were sacrificed at 2 hours and 6 hours, respectively, after reproduction of the model. Blood sample and peritoneal lavage fluid (PLF) were collected. The levels of keratinocyte-derived chemokine (KC), macrophage inflammatory protein 2 (MIP-2), tumor necrosis factor-α (TNF-α), interleukins-6 (IL-6) in serum and PLF were determined by enzyme linked immunosorbent assay (ELISA). The number of leukocytes and PMN in PLF were determined by hemocytometer and flow cytometry, respectively. Results Compared with sham group, all serum and PLF levels of KC, MIP-2, TNF-α and IL-6 were greatly elevated at 2 hours after ZYM injection in model group, while AGM treatment could dramatically reduce the levels of the above-mentioned cytokines in serum and PLF as compared with those of the model group [serum KC (ng/L): 990.7±137.9 vs. 2 053.2±262.7, MIP-2 (ng/L): 642.2±124.4 vs. 1 369.7±146.5, TNF-α (ng/L): 608.6±38.1 vs. 1 044.7±101.0, IL-6 (ng/L): 1 058.2±129.1 vs. 1 443.3±190.1; PLF KC (ng/L): 7 462.3±839.6 vs. 12 723.5±1 515.7, MIP-2 (ng/L): 1 570.8±193.4 vs. 3 471.4±384.7, TNF-α (ng/L): 1 115.8±156.7 vs. 1 499.2±231.2, IL-6 (ng/L): 2 646.5±223.2 vs. 3 126.7±291.4; all P < 0.05]. The expressions of KC, MIP-2 and TNF-α at 6 hours were significantly lower than those at 2 hours in model group and AGM treatment group, but IL-6 levels were further increased. The levels of KC and MIP-2 in serum and PLF at 6 hours were decreased to the levels of sham group. At 6 hours after the reproduction of the model, the number of total inflammatory cells and PMN of PLF in the model group was significantly higher than those of the sham group. In contrast, AGM notably lowered the number of inflammatory cells and PMN in peritoneal fluid after ZYM attack [total inflammatory cells (×109/L): 14.7±1.1 vs. 2.0±0.4, 10.1±1.2 vs. 14.7±1.1; PMN (×109/L): 11.37±1.22 vs. 0.18±0.05, 7.69±0.57 vs. 11.37±1.22, all P < 0.05]. Conclusion AGM can effectively alleviate acute peritoneal inflammatory injury induced by ZYM, mainly through reducing the secretion of inflammatory mediators and chemokines, and inhibiting the infiltration of leukocytes and neutrophils.