Kallistatin (Kal) is a negative acute phase endogenous protein which can inhibit tumor angiogenesis, growth and metastasis effectively. To express and purify recombinant human kallistatin (rHKal), and characterize its biological activity, P. pastoris was transformed with pPIC9-Kal/GS115 (His4) to express rHKal. The fermentation was carried out in a 7.5 L bioreactor with high density cell culture. 1%-2% methanol was added to the medium to induce the expression of rHKal. The secretion was purified with phenyl sepharose, G-25 sepharose, heparin sepharose and Sephacryl S-100 chromatography. The biological activity of purified bulk rHKal on HUVEC was evaluated with MTT and tube formation assays. The final expression of rHKal in the supernatant reached 50 mg x L(-1), the purity of bulk rHKal after purification was above 98%. A dose-dependent inhibition of rHKal on HUVEC proliferation was observed, however, a U-shaped dose-response curve of rHKal on capillary formation of HUVEC was revealed. The described protocol provides an effective means for preparing rHKal that could be used for anti-angiogenesis therapy in the future.

ObjectiveTo explore the hemodynamic characteristics of parotid glands before and after acid stimulation test in patients with Sjogren syndrome.MethodsTwenty female patients with Sjogren syndrome were enrolled as case group,and 20 healthy women were selected in the same period as control group.The blood flow spectrums of bilateral superficial temporal artery and intra-parotid small artery before and after acid stimulation were detected by color Doppler ultrasonography.Peak systolic velocity (PSV),end-diastolic minimum velocity(EDV) and resistance index( RI ) were obtained and analyzed.ResultsPSV and EDV of intra-parotid small artery in case group were significantly higher than those in control group before acid stimulation test,and RI was obviously lower than that in control group [ ( 16.01 ± 6.18 ) cm/s vs.( 14.00 ± 5.23 ) cm/s,( 5.96 ± 3.00 ) cm/s vs.( 3.54 ± 2.03 ) cm/s,0.64 ± 0.07 vs.0.76 ± 0.06 ] ( P ＜ 0.05 ).In control group,PSV and EDV of superficial temporal artery and intra-parotid small artery increased and RI reduced after acid stimulation test,which had significant differences before and after acid stimulation test (P ＜ 0.05).There was no statistical significance in the hemodynamic indexes of case group before and after acid stimulation test(P＞ 0.05 ).Conclusions The reactivity of parotid artery to acid stimulation test in patients with Sjogren syndrome reduce,especially the intra-parotid small artery.Color Doppler ultrasonography can contribute to show the hemodynamic changes of the parotid lesions efficiently.

BACKGROUND:At present bone marrow mesenchymal stem cel s act as the main seed cel s in bone tissue engineering, but only 0.001%-0.01%cel s are in the bone with difficulty in cel separation and purification. OBJECTIVE:To explore the biological characterization of human placenta derived mesenchymal stem cel s and biocompatibility with three-dimensional porous hydroxyapatite ceramic scaffold. METHODS:Human placenta derived mesenchymal stem cel s were morphological y observed and identified usingflow cytometry, fol owed by osteogenic, adipogenic, chondrogenic induction for 3 weeks. Afterwards, the potential of multi-directional differentiation was identified by alizarin red S, oil red O and toluidine blue staining. DAPI staining was used to observe the adhesion of cel s on the surface of the hydroxyapatite ceramic scaffold under scanning electron microscope. RESULTS AND CONCLUSION:The human placenta derived mesenchymal stem cel s showed long spindle shape and uniform size under the microscope;they highly expressed CD29 and D90, but did not express CD45 and CD106. Fol owing induction, mineralized nodules were observed by alizarin red S staining, lipid droplets by oil red O staining and blue-dyed toluidine blue staining. These cel s adhered wel to the scaffold surface, indicting they are suitable for bone tissue engineering.

0.05),but enhanced the expression of CD80,CD86 significantly(P

Objective Kangfuling granules can be used for the treatment of radiation damage , but its quality criteria has not been established .This paper aimed to establish the quality criteria of Kangfuling granules to control its quality . Methods Radix as-tragali and Radix angelicae sinensis in the formula both were identified qualitatively by TLC .The content of astragaloside IV was exam-ined by HPLC:Agilent Zorbax Extend-C18 (4.6 mm ×250 mm, 5μm) was used as the chromatographic column .The mobile phase was consisted of acetonitrile-water (32:68) with a flow rate at 1.0 mL/min.The evaporative light scattering detector was used to detect the compound.The content of astragaloside IV and ferulaic acid was examined by HPLC: Agilent Zorbax SB-C18 (4.6 mm ×250 mm, 5 μm) was used as the chromatographic column .The mobile phase was consisted of acetonitrile-0.085% phosphoric acid water (17:83) with a flow rate at 1.0 mL/min.The wave length was 320 nm and column temperature was set at 35℃. Results The same color spot was shown in the chromatogram of the test sample and the control sample in the corresponding position with no interference of negative control.The linear range for Astragaloside IV 0.030 6 mg/mL~0.612 0 mg/mL with R2 =0.999.The RSD of stability test was 2.17%.The RSD of precision test was 1.89%.The RSD of re-peatability test was 1.58%.The average recovery was 101.26%. The linear range for ferulaic acid 0.24-4.80 μg/mL with R2 =0.999.The RSD of stability test was 1.37%.The RSD of precision test was 0.83%.The RSD of repeatability test was 1.14%.The average recovery was 98.39%.This product was tentatively calculat-ed according to the dry matter , the amount of Astragaloside IV and ferulaic acid should not be less than 0.19 mg/g and 0.08 mg/g, re-spectively. Conclusion In this study, the established TLC was used for the qualitative identification of Kangfuling granules , and the content of Astragaloside IV and ferulaic acid was not less than 0.19 mg and 0.08 mg in per gram of Kangfuling granules , respectively. The established standard is suitable for the quality control of Kangfuling granules .

Objective To investigate the diagnostic value of serum FER, AFP and AFP-L3 alone or in combination for diagnosis of primary hepatic carcinoma( PHC).Methods This was a case-control study.
Serum FER, AFP and AFP-L3 were determined in 212 patients with PHC ( StageⅠ45 cases, StageⅡ78 cases, StageⅢ81 cases, StageⅣ8 cases) , 127 patients with cirrhosis, 101 patients with chronic hepatitis and 98 controls in the Beijing Youan Hospital affiliated to Capital Medical University from January 2014 to December 2014.Levels of FER, AFP and AFP-L3 were measured by chemiluminescence, while serum samples were pre-treatment with affinity adsorption before AFP-L3 detection.FER, AFP and AFP-L3 levels were analyzed using the nonparametric Wilcoxon test among all groups.Diagnostic performance were analyzed among the groups with the three biomarkers independently and combined.Logistic regression, plotted ROC curve and calculated the area under ROC curve ( AUC) were applied to assess the diagnostic value of each index.Results Serum concentration of FER in PHC, cirrhosis, chronic hepatitis groups and healthy controls were 308.45 ( 148.98 -662.80 ) , 151.70 ( 51.44 -507.40 ) , 298.20 ( 157.30 -701.80 ) , 113.50( 54.98-221.38) μg/L, respectively.The concentration of AFP were 48.50(5.25 -748.40), 3.91(1.80-17.53), 4.76 (2.29-30.56), 2.57 (0.93-3.68) μg/L in each group.The serum levels of AFP-L3 in each group were 4.75(0.61-127.95), 0.61 (0.61-2.50), 0.61 (0.61-2.85), 0.61 (0.61-0.61) μg/L.The concentration of FER, AFP and AFP-L3 differs statistically in PHC, cirrhosis, chronic hepatitis group and healthy controls (χ2 =67.66,146.31,119.02,P<0.001).The content of serum FER, AFP and AFP-L3 increased gradually as the stage level aggravating ( StageⅠ-Ⅳ) , there was significant differences among groups (χ2 =21.63,22.68,21.98, P<0.001) .When using one serum marker, FER had the highest sensitivity (75.00%) , while AFP-L3 had the highest specificity (82.52%). While using two serum markers, FER/AFP had the highest sensitivity (89.15%) , FER+AFP-L3 and AFP+AFP-L3 had a higher specificity (86.20%).The combined detection of FER/AFP/AFP-L3 improved the sensitivity of the test to 89.15%, while FER+AFP+AFP-L3 had a specificity of 86.50%.The AUC of combination of FER, AFP and AFP-L3 was 0.803 ±0.019 (95% CI:0.765-0.841), which was higher than the AUC of either FER(0.748 ±0.022,95% CI:0.705-0.790, Z=4.67,P<0.001) and AFP-L3 (0.726 ±0.024,95% CI: 0.679 -0.772, Z=3.64,P<0.001).However, there was no significant difference in AUC between AFP alone ( 0.776 ±0.021, 95% CI: 0.735 -0.818 ) and the combined detection ( Z=1.34, P=0.18 ) .Conclusions FER was a potential marker for PHC diagnosis.The combination of FER, AFP and AFP-L3 has higher value of clinical applications than one of them independently.

Objective:To explore the risk factors of spontaneous rupture of hepatocellular carcinoma (HCC), and to analyze the prognosis of transcatheter arterial embolization (TAE) in the treatment of spontaneous rupture of HCC.Methods:From January 2008 to December 2018, at Tianjin Third Central Hospital, 126 patients diagnosed with spontaneous rupture of HCC (spontaneous rupture group) and in the same period 252 patients diagnosed as HCC without rupture (non-spontaneous rupture group) were retrospectively selected. Independent sample t test, Mann-Whitney U test and chi-square test were used to compare the general data, laboratory test results and imaging results between spontaneous rupture group and non-spontaneous rupture group. Multivariate logistic regression was used to analyze the independent risk factors of spontaneous rupture of HCC. Kaplan-Meier method and log-rank test were used for survival analysis of 92 patients treated with TAE and 31 patients treated with conservative treatment in patients with spontaneous rupture of HCC, and multivariate Cox proportional hazard regression was used to analyze the independent prognostic factors of patients treated with TAE. Results:The proportions of tumor maximum diameter ≥5 cm, tumors mainly located in segment Ⅱ, Ⅲ, Ⅳ and Ⅵ of the liver, tumors protruding height from the surface of liver ≥1 cm, liver cirrhosis and portal vein thrombosis (PVT) in spontaneous rupture group were all higher than those in non-spontaneous rupture group (46.8%, 59/126 vs. 35.7%, 90/252; 61.1%, 77/126 vs. 46.0%, 116/252; 73.0%, 92/126 vs. 18.7%, 47/252; 81.7%, 103/126 vs. 69.0%, 174/252; 20.6%, 26/126 vs. 11.5%, 29/252), and the level of fibrinogen is lower than that in non-spontaneous rupture group ((3.07 ±1.38) g/L vs. (3.92±1.13) g/L), and the differences were statistically significant ( χ2=4.343, 7.644, 106.780, 6.918 and 5.628, t=-3.276, all P<0.05). The results of multivariate logistic regression analysis showed that the location of tumors in segment Ⅱ, Ⅲ, Ⅳ, Ⅵ of the liver, the height of tumors protruding from the surface of liver ≥1 cm, liver cirrhosis and the fibrinogen was lower than the lower limit of the reference value were independent risk factors of spontaneous rupture of HCC (odds ratio ( OR)=1.354, 5.726, 1.152, 1.892; 95% confidence interval ( CI) 1.062 to 2.008, 1.049 to 19.575, 1.016 to 1.895, 1.267 to 3.346; all P<0.05). Among 92 patients with spontaneous rupture of HCC treated with TAE, the TAE technical success rate and clinical success rate were 100.0% (92/92) and 93.5% (86/92), respectively. Thirty-one patients received conservative treatment. The median survival time of the patients treated with TAE was 243 d (38 d, 377 d), which was about 10 times that of the patients who accepted conservative treatment (23 d (9 d, 51 d)). The 1-month, 3-month, 6-month, 1-year and 3-year survival rates of the patients treated with TAE were 72.8%, 50.8%, 46.0%, 31.1% and 13.6%, respectively, and those of patients who accepted conservative treatment were 25.8%, 17.2%, 11.5%, 5.7% and 0, respectively; and the differences in survival rates between the two groups were statistically significant ( χ2=34.606, P<0.01). Log-rank test analysis showed that initial hemoglobin <60 g/L, C grade of liver function, total bilirubin (TBil)≥50 μmol/L, complicated with portal vein tumor thrombus, hepatic lobe embolism during TAE, and tumor maximum diameter ≥10 cm were all correlated with poor survival rates of patients with spontaneous rupture of HCC after TAE ( χ2=3.752, 4.146, 22.318, 4.087, 5.685 and 7.893, all P<0.05). The results of multivariate Cox proportional hazard regression analysis showed that TBil ≥ 50 μmol/L, hepatic lobe embolism during TAE, and tumor maximum diameter ≥10 cm were independent factors of poor prognosis of spontaneous rupture of HCC treated with TAE ( OR=25.873, 8.415, 18.620; 95% CI 4.916 to 126.005, 1.136 to 27.319, 2.754 to 84.368; all P<0.05). Conclusions:In HCC patients with tumors located in segments Ⅱ, Ⅲ, Ⅳ and Ⅵ of the liver, the height of tumors protruding from the surface of liver ≥1 cm, liver cirrhosis or the fibrinogen is lower than the lower limit of the reference value, close attention should be paid to the risk of spontaneous tumor rupture. TAE is an effective treatment for acute spontaneous rupture of HCC, and the prognosis of patients treated with TAE is better than that of patients receiving conservative treatment. However, patients with TBil ≥50 μmol/L, hepatic lobe embolism during TAE and tumor maximum diameter ≥10 cm have a poor prognosis after TAE treatment.

Objective To investigated the value of intraoperative ultrasonography in guiding the surgical approach,improving the total resection rate of the tumor,and the survival time of patients who underwent resection of cerebral gliomas.Methods One hundred and ninteen patients with cerebral gliomas (pathologically confirmed) comprised the study groups.Patients who underwent intraoperative ultrasoundguided surgery were followed up for 6-60 months.In addition,randomly selected low and high grade cerebral glioma patients (all 30 cases of which were without intraoperative ultrasonography guidance) served as the control groups.Follow up included survival time,and the differences in survival time between the study groups and control groups were statistically analyzed.Results Complete follow-up data were obtained for 101 of 119 cases (84.9％) for a follow-up time of 6 60 months.Total removal of the lesion was achieved in 68 cases (67.3％) and subtotal removal in 33 cases (32.7 ％).Sixty-five cases had partial recurrence (64.4％) and 60 patients died due to tumor recurrence.Survival in patients with low grade gliomas (LGG) was significantly higher than that in patients with high grade gliomas (HGG).Patients with total tumor resection had a longer survival time than patients who had subtotal tumor resection.In the control LGG group,6-month survival was 96.7％,1-year survival was 73.3％,2-year survival was 53.3％,4-year survival was 36.7％ and 5-year survival was 26.7％.In the control HGG group,survival at 6 months,1 year,2 years,4 year and 5 years was 83.3％,36.7％,13.3％,3％,0 respectively.When comparing survival between the control groups and study groups,there was no significant difference at 6 months (P ＞0.05),but survival at 1 year,2 years,4 year and 5 years was different (P ＜0.05).Conclusions Intraoperative ultrasonography-guided resection of cerebral gliomas helps the surgeon to understand the relationship between the lesion and the surrounding structures,helps to protect normal brain tissue and increase the safety of surgery.It is of value in improving the total resection rate of tumors and the patient's survival time.

Objective To investigate the value of intraoperative contrast-enhanced ultrasound(CEUS) in evaluating pathological grades of cerebral gliomas. Methods Intraoperative CEUS was performed in 33 patients of cerebral gliomas of different pathological grades. Real-time blood perfusion and enhance characteristics of these tumors were observed,quantitative parameters from the automatically derived time-intensity curve (TIC) were obtained, and compared with the tumor microvessel density (MVD) by immunostaining with anti-CD34. Results The time to peak was significantly shorter and the MVD was significantly higher in the high grade cerebral gliomas compared with the low grade ones ( P <0. 05). The time to peak was negatively correlated with the MVD by immunostaining (r = -0.79, P < 0. 05). Conclusions Intraoperative CEUS could be used to observe microvascular perfusion in real-time, and could indirectly reflect the information of MVD in cerebral gliomas, which is of help to grade cerebral gliomas and guide surgical resections.