Objective:To establish an UHPLC-MS/MS method to determine the contents of baicalin,scutellarin,wogonin,caffeic acid,ephedrine,belamcandin,irisflorentin and baicalein in Shegan mixtures. Methods:The chromatographic separation was performed on a ZORBAX SB-C18 column(2. 1 mm × 50 mm,1. 8 μm)with the mobile phase consisting of acetonitrile and water(0. 1% formic acid),the flow rate was 0.3 ml·min-1,and the column temperature was 35℃. Electrospray ionization source(ESI)was used with multiple reaction monitoring( MRM)combined with positive and negative scanning switch. üegative ion mode detection was used for caf-feic acid,belamcandin and scutellarin ,while positive ion mode detection was used for baicalin,wogonin,irisflorentin,baicalein and e-phedrine. Results:The quantification limit of baicalin,scutellarin,wogonin,caffeic acid,ephedrine,belamcandin,irisflorentin and ba-icalein was 1. 44 × 10-4 ,4. 20 × 10-3 ,2. 95 × 10-4 ,7. 80,4. 90 × 10-3 ,4. 6 × 10-2 ,3. 18 × 10-4 ng·ml-1 and 4. 85 ng·ml-1 . The detection limit was 4.32 ×10-5,1.3 ×10-3,8.84 ×10-5,0.77,2.90 ×10-4,3.33 ×10-4,9.5 ×10-5 ng·ml-1 and 1.46 ng· ml-1 ,respectively. The correlation coefficients( R2 )were all higher than 0. 992 3 within the linear ranges. Both intra- and inter-day RSD were less than 5%. The average recoveries of the eight components were within the range of 80%-120%. Conclusion:The method is simple,rapid,sensitive and accurate. It can be used to determine the contents of baicalin,scutellarin,wogonin,caffeic acid,ephed-rine,belamcandin,irisflorentin and baicalein in Shegan mixtures for the quality control.

Objective:To clone the mice interleukin-23(mIL-23) and express it in Pichia efficiently.Methods:Two subunits of IL-23 were amplified by PCR from pcDNA3/mIL-23,and ligated together with adaptor by over-PCR.The IL-23 cDNA confirmed by sequencing was inserted into expressing vector pPICZαA and expressed in Pichia X-33 strain.IL-23 protein expression was induced by methanol and ammonia.The recombinant IL-23 was identified by immunoblot and its biological activity was analyzed.Results:DNA sequencing confirmed that cloned cDNA was identical to the published sequence of mIL-23.The recombinant plasmid pPICZαA/mIL-23 was electroprated into X-33.An expected 72 kD protein of mIL-23 was founded both in the induced pellets and supermatants by SDS-PAGE and coomassie blue staining.The 72 kD protein could be recognized in Western blot.While we could detect bands at 72 kD in cell pellets induced more than 24 h by Western blot.Detected by Western blot using anti-His antibody,we could see positive band at 72 kD from supernatants induced 24 and 36 h.While we could detect band at 72 kD in cell pellets induced between 24 h and 96 h.Meanwhile,we could see obviously proliferation of mice peripheral blood mononuclear cells(PBMC) treated with the induced pellets and supermanants.Conclusion:We have successfully expressed mIL-23 protein in Pichia and the expressed product has its bioactivity in promoting the proliferation on PBMC.

Objective Percoll density gradient centrifugation and Ficoll-Hypaque density gradient cen-trifugation, which are frequently-used methods for separation of tumor-associated macrophages (TAMs) from solid carcinoma were compared, in order to find an effective way to separate TAMs from colorectal carcinoma (CRC). Furthermore, we studied the best adherence time of separating macrophage among mononuclear cells. Methods specimens were collected from CRC patients , after digesting into single cells , TAMs were separated from the same specimen by 100% Ficoll, 35% percoll and 25% combined with 65% percoll respectively. After these pre-liminary separation, the collected cells were purified a second time by adherence separation. The purity of TAMs were detected by immunofluorescence. Results TAMs purity from Ficoll-Hypaque density gradient centrifugation was 80.18%, statistically higher than that from Percoll density gradient centrifugations (54.33% and 10.93% re-spectively). Conclusion Compared to Percoll density gradient centrifugation, Ficoll-Hypaque density gradient centrifugation is a more effective and simple way to isolate TAMs from colorectal carcinoma , suggesting it can be wildly used in clinical and basic medical research. 2-4 hours is the best adherence time for isolating macrophage.

Objective To investigate the level of human cytomegalovirus(HCMV)specific CD8+ T lymphoeytes in peripheral blood and the immune reaponae of HCMV reactivation after kidney transplantation.Methods Thirty-eight HCMV seropesitive HLA-A*0201 kidney transplant recipients(9 with HCMV infection and 29 without HCMV infection)and 54 healthy individuals were enrolled.The levels of total HCMV specific CD8+ T cells were measured using HLA-A2 pentamer folded with HCMV-peptide NLVPMVATV.The levels of IFN-γ secreting CD8+ T cells were measured by intracelluhr IEN-γ staining pulsed with the same peptide.Results The median levels of pentamer stained CD8+ T cells were 1.19%(0-19.42%),1.20%(0-18.40%)and 3.2%(0.51%-18.90%)in healthy group,negative HCMV group and positive HCMV group(H=5.34,P＞0.05),respectively.The median levels of IFN-γ secreting CD8+ T cells were 0.72%(0-0.70%),0.47%(0-5.61%)and 0.67%(0.07%-4.00%),respectively(H=0.58,P＞0.05).However,the mean proportions of IFN-γ secreting pentamer stained T cells relative to total HCMV specifc CIL were(60.18±19.16)%,(39.19±17.22)% and(20.02±13.26)%,respectively.There were significant differences among the groups(P＜0.01).Condusiorm There was no significant difference of levels of HCMV specific CD8+ T lymphocytes in peripheral blood between the kidney transplant recipients and healthy individuals.However,the proportion of HCMV-specific IFN-γ producing CD8+ T cells in pentamer stained cells was reduced in the kidney transplant recipients especially in those with active HCMV infection,which may contribute to the inability to control HCMV reactivation.

Objective To investigate the impact of subclinical hypothyroidism (SCH) on macrovascular complications in elderly type 2 diabetic patients.Methods A total of 1170 hospitalized elderly patients with type 2 diabetes mellitus were enrolled in the study through systematic sampling and underwent testing for blood biochemical indicators, thyroid function and C peptide.Parameters for macro-vascular complications, including the ankle/brachial index (ABI), transcranial Doppler vascular ultrasound (TCD), electrocardiogram (ECG), ejection fraction (EF), history of coronary heart disease, and hypertension grading were also monitored.Results All the subjects were divided into two groups based on the thyroid stimulating hormone (TSH) level: the euthyroid group (4 mU/L≥TSH＞0.4 mU/L) and the SCH group (TSH＞4 mU/L), and the latter was further sub-grouped into the mild SCH group (10 mU/L≥TSH＞4 mU/L) and the severe SCH group (TSH＞10 mU/L).ABI was significantly decreased in SCH (R/L: 0.86/0.92, P＜0.01).Levels of basal C-peptide (CP0) and post glucose-challenge C-peptide (CP1-3) were higher in the SCH group than in the euthyroid group [(2.16±0.93)pg/L vs.(1.56±1.05)pg/L, (0.53±0.25)pg/L v, (0.38±0.37),(0.72±0.23) pg/L vs.(0.56 ±0.32) pg/L, (6.21± 2.69) pg/L vs.(4.46 ± 2.62) pg/L,respectively, P＜0.01 for all].EF was higher in the SCH group than in the euthyroid group[(70.87± 6.66)％ vs.(65.10 ± 8.08％), P＜ 0.01].There were no significant differences in other biochemical indicators, ECG, TCD, history of coronary heart disease, hypertension grading and intervention treatment (P＞0.05 for all).Conclusions Lower extremity atherosclerotic disease has a higher incidence in elderly type 2 diabetic patients with SCH and occurs earlier than other macrovascular complications.Elevated TSH levels and insulin resistance may be the major causes.

Objective To analyze Chinese oncology papers included by Chinese biomedical literature database (CBM) and science citation index (SCI),and to comprehensively and objectively reflect the current situation of Chinese oncology literatures published in domestic and international journals.Methods Chinese oncology papers were retrieved by using CBM and SCI.A comparison analysis was made from the respects of subject distribution,district distribution,organization distribution and high citation situation.Results 67 071 papers of 2012 were retrieved from CBM database,while 9226 papers were indexed by SCI.In terms of district distribution,the first three districts were Jiangsu (6707),Guangdong (4897) and Beijing (2214) in CBM database,while in SCI database,they were Beijing (2214),Shanghai (2008) and Guangdong (1393).The institute with the most amount of literature is university,respectively 9725 and 5755.Moreover,the mostly funded project is National Natural Science Foundation (4710,35.38％ ; 3290,35.68％).Conclusion The 2012 Chinese oncology literatures published in domestic journals has advantage with respect to quantity,compared with the international published journals.However,there exists a bigger difference in terms of quality.Through the comparative analysis,the medical researchers in our country will have some implications in the research orientation.

Objective To investigate the feasibility of non-invasive quantitative evaluation of portal pressure (Pp) by contrast enhanced ultrasonography (CEUS).Methods 18 portal hypertension patients (PTH group) were performed with CEUS within one week before splenectomy and pericardial devascularization,and 20 healthy volunteers as controls were recruited in this study (control group).Hepatic-right kidney sectionwas chosen to calculate the area under curve of portal vein/hepatic artery (Qp/Qa) and the perfusion intensity of portal vein/hepatic artery (Ip/Ia) through time intensity curves (TIC) of liver parenchyma generated from CEUS images.Pp was measured by intra-operative mesenteric vein catheter,and the correlation betweenPp and Qp/Qa,Ip/Ia were analyzed by Pearson correlation test.Results The levels of Qp/Qa and Ip/Ia in the PTH group were 2.28 ± 0.66 and 0.35 ± 0.14 respectively,which were both significantly declined than that in the controlgroup (5.72 ± 3.69 and 1.97 ± 0.17).In the PTH group,the correlation coefficient were-0.747 and-0.617,and the linear regression equations were Y =-83 X + 5.013 andY =-15X + 0.837,which indicated that Qp/Qa and Ip/Ia had significant correlation with Pp.Conclusions CEUS parameters,including Qp/Qa and Ip/Ia,are significantly correlated to Pp in portal hypertension patients,which indicate that CEUS could be a new non-invasive clinical method for evaluating Pp.

Objective To investigate the diagnostic value of the acoustic radiation force impulse (ARFI) technology and AST/PLT ratio index (APRI） for the assessment of the liver fibrosis in chronic hepatitis C patients.Methods 107 patients with chronic hepatitis C were included,the subjects were underwent liver biopsy,liver function,blood count,as well as real-time acoustic elastography examination.The APRI was calculated according the following formula,APRI =AST (ULN)/PLT (109/L).ARFI and APRI were compared by correlation with liver fibrosis stage in chronic hepatitis C.Referring to the histologic fibrosis stage on liver biopsy,all the ARFI and the APRI value were assessed by using ROC curve analysis.The corresponding cut-off values,sensitivity and specificity were also calculated and compared.Results The mean values of ARFI and APRI were (1.26 ± 0.27)m/s and 0.30 ± 0.46 for the patients with S1,(1.45 ± 0.51)m/s and 0.29 ± 0.21 for those with S2,(2.03 ± 0.54) m/s and 0.59 ± 0.56 for those with S3,(2.29 ± 0.82) m/s and 0.63 ± 0.35 for those with S4,respectively.ARFI (r =0.61,P ＜0.001) had a better correlation with liver fibrosis stage in chronic hepatitis C than APRI (r =0.49,P ＜0.001).Cut-off points of ARFI and APRI were 1.529 m/s and 0.170 for S≥2,1.780 m/s and 0.277 for S≥3,1.780 m/s and 0.446 for S =4,respectively.Accordingly,the areas under the ROC curves for ARFI and APRI were 0.779 and 0.724 for S≥2,0.866 and 0.786 for S≥3,0.790 and 0.779 for S=4,respectively.Conclusions As a non-invasive technology,ARFI is more accurate when applied to evaluate liver fibrosis in patients with chronic hepatitis C than APRI.ARFI technology has potential value for quantitatme evaluation of the liver fibrosis for chronic hepatitis C.

Objective To investigate the clinical value of the acoustic radiation force impulse imaging (ARFI) in the quantitative evaluation of fibrosis staging in chronic hepatitis B .Methods A total of 373 patients with chronic hepatitis B were grouped in accordance with the depth of the right anterior lobe and posterior lobe of the liver and underwent ARFI to measure ARFI values .Liver biopsy was performed in all patients to investigate the relationship between liver fibrosis stages and ARFI values in different groups . Results The ARFI values in different pathological grades with the same depth of the right anterior and right posterior lobe of the liver were statistically significant ( P < 0 0.01);the ARFI values between the middle and deep parts of the right anterior lobe and the same location of the right posterior lobe were statistically significant ( P <0 0.1);for the right anterior lobe ,the ARFI value of the shallow part showed statistical significance from that of the middle and the deep part ( P < 0 0.5 ) .Receiver operating characteristic (ROC) curve analysis for different sampling locations showed that the largest area under the ROC curve ,which was 0 8.18 ,existed in the middle part of the right anterior lobe ,indicating that it could be the optimal sampling location for the measurement of ARFI values .Liver stiffness at ARFI imaging was significantly correlated with liver fibrosis stage in chronic hepatitis B and spearman coefficient of correlation was 0 5.30 ( P <0 0.01) .The areas under the ROC curves for the chronic hepatitis B patients with severe liver fibrosis (≥ S3) and early cirrhosis (= S4) were 0 8.18 and 0 8.60 ,respectively .Conclusions ARFI imaging was promising for clinical application and could be utilized as a noninvasive method for the quantitative evaluation of fibrosis staging in chronic hepatitis B .

Objective To investigate the clinical efficacy of acupuncture plus TDP in treating stageⅡ-Ⅲ pressure sore.Methods Thirty-three patients with pressure sore meeting the inclusion criteria were randomly allocated to treatment and control groups, 17 cases each. Both groups were first given routine clean care. The control group received routine surgical asepsis dressing change and the treatment group, fire needling, surrounding electroacupuncture and TDP irradiation to the affected part. The pressure sore area was observed and the PUSH score was recorded in the two groups before and after treatment. The clinical therapeutic effects were compared between the two groups of patients. Results The total efficacy rate was 88.0% in the treatment group and 71.0% in the control group; there was no statistically significant difference between the two groups (P>0.05). The total efficacy rate was 88.0% in the treatment group and 71.0% in the control group; there was no statistically significant difference between the two groups (P>0.05). The cure and marked efficacy rate was 58.8% in the treatment group and 23.5% in the control group; there was a statistically significant difference between the two groups (P<0.05). There was a statistically significant pre-/post- treatment difference in the pressure sore area in the two groups at one, two and three weeks after treatment (allP<0.05). There was a statistically significant difference in the pressure sore area between the two groups at two and three weeksafter treatment (bothP<0.05). There was a statistically significant pre-/post-treatment difference in the PUSH score in the two groups at two and three weeks after treatment (bothP<0.05). There was a statistically significant difference in the PUSH score between the two groups at three weeks after treatment (P<0.01).Conclusions Acupuncture plus TDP can markedly relieve the clinical symptoms and accelerate the sore healing in treating stageⅡ-Ⅲ pressure sore.