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 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 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 metabolite changes in systemic lupus erythematosus (SLE) patients with and without neuropsychiatric symptoms using magnetic resonance spectroscopy (MRS) and explore the associations between image findings and clinical variables. Methods Twenty-two SLE patients with neuropsychiatric symptoms (NPSLE), twenty-one SLE patients without neuropsychiatric symptoms (non-NPSLE) and twenty healthy controls (HCs) underwent routine MRI scan and multivoxel magnetic reson-ance spectroscopy (MVS). The absolute metabolite concentrations were measured bilaterally in the posterior cingulate gyrus (PCG), dorsal thalamus (DT), lentiform nucleus (LN) and posterior paratrigonal white matter (PWM) using LCModel and SAGE software. The relationships between metabolite con-centrations and cognitive function scores were analyzed by Spearman rank correlation. Single-factor Chi-square analysis and t-test were used for analysis. Results ① Compared to control subjects, NPSLE patients had significantly lower N-acetylaspartate (NAA) values in bilateral PCG and DT, with the mean differences of -1.504 [95% confidence interval ( CI) (-2.335, -0.672), P=0.001], -1.460 [95%CI (-2.349, -0.570), P=0.002], -1.259 [95%CI (-1.894, -0.625), P=0.000] and -1.022[95%CI (-1.688, -0.356), P=0.003] for RPCG, LPCG, RDT and LDT, respectively. The concentration of total creatinine were observed to decline in RPCG and RDT, with the mean differences of-1.094 [95%CI (-1.845, -0.342), P=0.003], -0.955 [95%CI (-1.630, -0.280), P=0.006], -1.259 [95%CI (-1.894,-0.625), P=0.006] respectively. Glutamine and glutamate-values decreased significantly in RDT [mean difference=-2.586, 95%CI (-4.139, -1.033), P=0.002]. ② Compared to non-NPSLE patients, NPSLE patients had a lower NAA level in LPCG [mean difference=-1.256, 95%CI (-2.146, -0.367), P=0.006]. Positive correlations between mini-mental state examination scores [RPCG: rs=0.312, P<0.05; LPCG: rs=0.355, P<0.01], Montreal cognitive assessment scores (RPCG: rs=0.362, P<0.01; LPCG: rs=0.285, P<0.05) and NAA values in bilateral PCG were detected. Conclusion Both NPSLE and non-NPSLE patients may have metabolite dysfun-ctions in different brain regions. The cognitive disorder in SLE patients may be interpreted by neuronic damage of PCG.

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 study the detection rate of contrast-enhanced ultrasound (CEUS) with different perfusion methods on rabbit VX2 small hepatocellular carcinoma less than 10.0 mm. Methods VX2 tumor cells were inoculated into the subcutaneous tissue of New Zealand rabbit′s thigh. Then the plant tumor were cut into small blocks under sterile conditions and transplanted into hepatic parenchyma in 30 New Zealand rabbits. The contrast media was injected through peripheral vein by single and double perfusion methods. The detection rate of two methods were compared. Results There were 41 hepatocellular carcinoma lesions in the 30 rabbits. There were 15 lesions with size between 3.0 mm and 5.0 mm, and 26 lesions between 5.0 mm and 10.0 mm in diameter. On CEUS, the VX2 tumor presented fast-in and fast-out pattern. In arterial phase, the lesion was enhanced rapidly. In portal venous phase, contrast began to wash out from the carcinoma. In delay phase, the enhancement of lesion was signiifcantly lower than the surrounding normal liver parenchyma. A total of 32 lesions were detected by single perfusion method, including 7 lesions ranging 3.0-5.0 mm and 25 lesions ranging 5.0-10.0 mm. A total of 39 lesions were detected by double perfusion method, including 13 lesions ranging 3.0-5.0 mm and 26 lesions ranging 5.0-10.0 mm. The detection rate of micro-hepatocellular carcinoma by single and double perfusion method was 78% and 95% respectively. The difference was statistically signiifcant (χ2=5.150, P=0.023). The detection rate of 3.0-5.0 mm lesions by single and double perfusion method was 47%and 87%, respectively. The difference was statistically signiifcant ( χ2=5.400, P=0.025). The detection rate of 5.0-10.0 mm lesions by single and double perfusion method was 96% and 100%, respectively. There was no statistically signiifcant difference (χ2=1.020, P=0.500). Conclusion The double perfusion method greatly promotes the detection of micro hepatocellular carcinoma, especially for the lesions less than 5.0 mm in diameter.

Objective To summarize the imaging features of hepatic tumors contained lipid by contrast enhanced ultrasound (CEUS), and compare with MRI ifndings. Methods From July 2010 to December 2012, 17 patients of hepatocellular carcinoma containing lipid component in the 302nd Hospital of the People′s Liberation Army underwent dual-echo sequence MRI imaging. Fifteen patients were conifrmed by enhanced MRI and contrast-enhanced CT, while the other 2 patients were confirmed by pathology. The features of ultrasonography, CEUS and MRI were analyzed. Kappa analysis test was analyzed for the consistency of CEUS and MRI ifndings. Results In the two-dimensional ultrasound of 17 lesions, 9 were hypoechoic heterogeneous lesions, 5 were hypoechoic lesions;13 lesions with clear boundaries, 4 lesions with unclear boundaries;peripheral blood lfow signal in 5 lesions and rich blood lfow in 1 lesion were observed by color Doppler ultrasound, 11 lesions had no signiifcant blood lfow signal;15 lesions showed fast high enhancement in CEUS arterial phase, 1 lesion with synchronization enhancement, 1 lesion with mild enhancement;10 lesions showed portal clearance, 1 lesion showed an enhanced synchronization, 1 lesion with mild enhancement;16 lesions showed clearance in delay phase, 1 enhanced lesion showed isoenhancement;6 lesions presented intra-lesion or surrounding patchy, irregular non-enhancement areas. CEUS diagnostic accuracy was 82%(14/17), the diagnostic accuracy of MRI was 88%(15/17). The Kappa consistency test was 0.628, while CEUS and MRI results were in good agreement. Conclusions Performance of hepatocellular lipid-containing ultrasound contrast arterial phase shows non-enhancement areas. Contrast enhanced ultrasound features of lipid-containing hepatocellular carcinoma are valuable for the clinical diagnois.

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.

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.