Objective To establish the method of determination for rhein, emodin, chrysophanol and physcion in Niuhuang Qinghuo Pills. Methods Sample was hydrolyzed by 20% H2 SO4 , and anthraquinones were extracted by soxhlet extraction with acetone as solvent, and were determined by HPLC with Kromasil C18 as column, methanol-0.5%H3 PO4 (75:25,V/V) as mobile phase at the flow of 1.0 mL/min, 230 nm as the detection wavelength.Results The linear relationship of rhein, emodin, chrysophanol and physcion were 1.50-150, 0.950-95.0, 1.30-130 and 1.15-115 mg/L. The anthraquinones were seperated completely, the recovery of 4 anthraquinones were 98.14% -101.3%.Conclusion This method is simple, accurate, steady, and could be used for the quality control of Niuhuang Qinghuo Pills.

AIMN-Glycans in recombinant human erythropoietin (EPO) are essential to in vivo biological activity. This paper is to develop a method for mapping sialyated or asialyated N-glycans of EPO.

METHODSAt first, N-glycans linked to asparagines in glycoprotein EPO were released by peptide N-glycosidase F. To map asialyated N-glycans, sialic acid in N-glycans were removed by incubating N-glycans with sialidase. Oligosaccharides were labeled with a sensitive fluorescent dye 8-aminopyrene-1,3,6-trisulfonate (APTS), and all of the labeled oligosaccharides released from EPO were mapped by capillary gel electrophoresis with laser-induced fluorescence. The relationship between N-glycans and bioactivity of EPO was investigated on the basis of N-glycan mapping spectra.

RESULTSN-Glycans of seven different batches of EPO were mapped. Each sample was analysed twice, with and without sialidase treatment. The results showed that N-glycans of each sample were approximately the same. But when the expression vector was different, the types of N-glycans and their relative content were quite different. In case of asialyated N-glycan mapping, the retention time of each oligosaccharide delayed greatly, and most importantly, the resulted sialic acid peak can be used as a quantitative standard to determine sialic acid content in N-glycans of EPO. In addition, the difference of N-glycan mapping was observed when the in vivo biological activity of EPO was different.

CONCLUSIONThe approach in this article for determining N-glycan mapping can be applied to determine the source of EPO and the difference between each batch. It is also a suitable tool for routinely controlling the inner quality of EPO by coupled with peptide mapping.

Electrophoresis, Capillary ; methods ; Erythropoietin ; chemistry ; Fluorescence ; Lasers ; N-Acetylneuraminic Acid ; analysis ; Peptide Mapping ; Polysaccharides ; analysis ; chemistry ; Recombinant Proteins

OBJECTIVETo investigate the methods and solve the technical bottlenecks in the preparation of recombinant human protein hZP3 using the baculovirus expression system and pave the technical ground for the production and application of recombinant hZP3.

METHODSThe recombinant vector pFASTBAC HTa-hZP3 was constructed and transferred to competent E. coli cells carrying bacmid to produce recombinant bacmid by homologous recombination. Sf9 cells were transfected with the recombinant bacmid to produce recombinant baculovirus. Full-length recombinant hZP3 (amino acids 1-424) and truncated recombinant hZP3 (amino acids 23-348) were expressed in the sf9 cells by infection with the recombinant baculovirus. The expression time of hZP3 was determined by Western blot and its purification was explored.

RESULTSThe recombinant bacmid and baculovirus were successfully constructed for expressing both the full-length and truncated hZP3. The maximal expression of recombinant hZP3 in the sf9 cells was achieved at 72-96 hours after baculovirus infection. Some of the recombinant hZP3 with His-tag could bind affinity matrix and got purified but most of the solubilized hZP3 passed through and the reasons remained unknown. Purified recombinant hZP3 labeled with Dylight Dye488 was able to bind human sperm.

CONCLUSIONIt is feasible to express recombinant hZP3 in insect cells using the baculovirus system though the yield of hZP3 needs to be optimized. The methods for efficient enrichment and purification of recombinant hZP3 require further exploration.

Baculoviridae ; genetics ; metabolism ; Blotting, Western ; Egg Proteins ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Humans ; Membrane Glycoproteins ; genetics ; metabolism ; Receptors, Cell Surface ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Transfection ; methods ; Zona Pellucida Glycoproteins

OBJECTIVETo investigate the in vivo effect of modified platelet factor 4 (PF4)-p17-70 cDNA on tumor angiogenesis in nude mice.

METHODSThe p17-70 cDNA was cloned into the AdEasy system to transfect packing cell line 293 and produce viral particles encoding p17-70cDNA (Ad p17-70). The integration of p17-70 cDNA was confirmed by RT-PCR and the P17-40 peptide Western blot. The biological activity of purified recombinant adenovirus was determined by umbilical veinal endothelial cell proliferation assay in vitro and in vivo tumor angiogenesis suppression of nude mice bearing human head and neck carcinoma.

RESULTSp17-70 significantly inhibited in vitro proliferation of endothelial cells being 58% lower than that of empty vector and reduced tumor volume in vivo. The tumor mass was (0.086 +/- 0.054) g, (0.171 +/- 0.076) g and (0.195 +/- 0.067) g, the tumor volume was (16.7 +/- 5.2) mm(3), (36.5 +/- 23.7) mm(3) and (41.5 +/- 12.2) mm(3) in p17-70 cDNA transfected group, empty vector group and PBS group, respectively. Immunohistochemical staining demonstrated a decreased number of blood vessels in the tumors.

CONCLUSIONP17-70 peptide mediated by adenoviral vector could inhibit the endothelial proliferation in vitro and the tumor growth in vivo.

Adenoviridae ; genetics ; Animals ; Cell Proliferation ; Endothelial Cells ; cytology ; Female ; Genetic Therapy ; methods ; Genetic Vectors ; Humans ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasms, Experimental ; pathology ; therapy ; Neovascularization, Pathologic ; therapy ; Platelet Factor 4 ; genetics ; Transfection ; Umbilical Veins ; cytology

Objective To explore the detection trend of vaginal intraepithelial neoplasia(VaIN)of lower genital tract from 2013 to 2015. Methods A retrospective analysis was undertaken of colposcopy-directed biopsy of cervical, vaginal and vulvar intraepithelial neoplasia lesions include cervical intraepithelial neoplasia (CIN), VaIN and vulvar intraepithelial neoplasia (VIN) in Obstetrics and Gynecology Hospital of Fudan University from January 2013 to December 2015. Results (1) Overall data of CIN, VaIN and VIN:a total of 16732 cases were diagnosed of lower genital intraepithelial neoplasia in 3 years, accounting for 23.20% (16732/72128) of total colposcopy-directed biopsy cases. Among them, CIN, VaIN and VIN accounted for 19.48%(14053/72128), 2.67%(1923/72128), 1.05%(756/72128) of total colposcopy-directed biopsy cases of the lower genital tract, 83.99%(14053/16732), 11.49%(1923/16732), 4.52%(756/16732) of total lower genital intraepithelial neoplasia, respectively. (2) Annual data of CIN, VaIN and VIN from 2013 to 2015. The annual proportion of CIN in all intraepithelial neoplasia of lower gential tract was basically stable, consisting of 86.02%(3955/4598),83.25%(4795/5760) and 83.20%(5303/6374), respectively. The annual proportion of VaIN was gradually increasing, consisting of 8.09% (372/4598), 12.45%(717/5760) and 13.08%(834/6374), respectively. The annual proportion of VIN was gradually decreasing, consisting of 5.89% (271/4598), 4.31% (248/5760) and 3.72% (237/6374), respectively. Conclusion The increasing detection of VaIN from 2013 to 2015 might correlate with the increasing attention to inspection of the entire vaginal wall.

OBJECTIVEThe increasing number of aged patients with severe ascending aorta atherosclerosis who are undergoing coronary artery bypass graft (CABG) present high risk for ascending aortic cannulation, cross-clamping or partial occluding and proximal anastomosis. We reviewed the surgical experience in 22 patients of CABG with ascending aorta atherosclerosis and tried to find the way to minimize the complications.

METHODSTwenty-two patients with severe atherosclerotic and calcified ascending aorta underwent CABG in our hospital. Thirteen of them received CABG on beating heart. Nine patients had their CABG with extracorporeal circulation. With deep hypothermia, we reduced the flow rate and intermittently arrested the circulation for the proximal anastomosis on ascending aorta in 5 patients with neither cross-clamping nor partial occluding. The sequential grafts and "Y" type anastomosis between reversed saphenous venous grafts were employed.

RESULTSTwenty of the patients survived after surgery. One died of inhalation pneumonia in two weeks after surgery. Another died of right hemothorax in ten days after surgery. The complications include: pneumonia 4 patients (18%), angina 2 patients (9%), ventricular fibrillation 1 patients (5%), post-CABG myocardium infarction 1 case (5%) and hemothorax 1 case (5%). There is no neurologic complications or aortic dissection after CABG.

CONCLUSIONCABG on beating heart with pedicel arterial grafts is the best approach to performing the surgery without touching the diseased ascending aorta. Ventricular fibrillation under mild hypothermia cardiopulmonary bypass and left ventricular suction were employed for quiet and bloodless field while distal anastomosis had no cross-clamping the ascending aorta. Also deep hypothermia and intermittently circulatory arrest offer quiet and bloodless field for the proximal anastomosis on ascending aorta without cross-clamping or partial-occluding. Distal sequential anastomosis and proximal "Y" type anastomosis are the effective approach to minimizing the proximal anastomosis on the ascending aorta.

Aged ; Aged, 80 and over ; Aortic Diseases ; complications ; surgery ; Atherosclerosis ; complications ; surgery ; Coronary Artery Bypass ; methods ; Coronary Artery Disease ; complications ; surgery ; Female ; Humans ; Male ; Retrospective Studies ; Treatment Outcome

Objective:To compare the accuracy of two methods in reducing leg length discrepancy(LLD)during hip hemiarthroplasty.Methods:We retrospectively analyzed 89 patients of hip hemiarthroplasty who suffered from femoral neck fracture.There were 47 patients in the new method group(NM), and 42 patients in the traditional method group(traditional method, TM)which comparing the position of the greater trochanter tip and the center of the femoral head.In the NM group, the distance from the center of femoral head to the lesser trochanter(L)and the diameter of femoral head(D)of the healthy side hip were measured on preoperative anteroposterior pelvic X-ray film, and the ratio(R)of D to L was calculated.During operation, the diameter of the femoral head(d)was measured with a caliper, and the distance should be obtained from the center of the femoral head prosthesis to the lesser trochanter according to the ratio R of the healthy side.The difference of postoperative LLD between the two groups and the incidences of |LLD| in each range were compared.Results:In the NM group, the maximum LLD was 11.10 mm and the minimum LLD was -4.0 mm, with an average of(4.4±3.2)mm, 80.9%(38/47)| LLD | < 6 mm, 93.6%(44/47)| LLD | < 10 mm, 6.4%(3/47)| LLD | ≥ 10 mm.In the TM group, the maximum LLD was 13.2 mm and the minimum LLD was -8.3 mm, with an average of (6.2±5.1)mm, 42.9%(18/42)|LLD|<6 mm, 69.0%(29/42)|LLD| <10 mm, 31.0%(13/42)|LLD|≥10mm.The differences of patients of the mean postoperative LLD and the incidences of |LLD| in each range between two the groups were statistically significant( t=-2.036、 χ2=14.629, P=0.046、0.001). Conclusions:The new method is simple, convenient, more accurate and can obtain a more satisfactory LLD compared with the traditional method which refers to the relative position of the great trochanter tip and the center of the femoral head.

OBJECTIVETo describe the prevalence and disability of stroke as well as the stroke-related diseases among elderly in urban and rural regions of Beijing.

METHODSIn 2002, three communities were selected from urban, suburb and rural regions from Beijing areas, respectively. Twenty percent of the elderly were randomly selected from three communities. The information about history of stroke, hypertension, heart diseases and diabetes, self-rated health (SRH), activity of daily living (ADL) and instrumental ADL (IADL), smoking and drinking habits, knowledge about cardiovascular diseases prevention were collected.

RESULTSA total of 2487 elderly were interviewed and the prevalence of stroke was 12.9% (321/2481). Eighty-seven of the stroke patients were diagnosed by CT/MRI. 19.9% of stroke patients had experienced 2 or more attacks. The highest prevalence of stroke was in the urban region and the lowest in the rural region (16.9% vs. 8.5%, P for trend < 0.01) while it was higher in males than in females (P < 0.05). The prevalence of stroke tended to increase with age in urban and 34.6% of the stroke patients had recovered completely. The proportions of poor SRH, ADL and IADL dependence, as well as the prevalences of hypertension, heart diseases and diabetes were higher among the elderly with stroke than those without. Although rates of awareness and treatment of hypertension were at the high levels among the elderly with stroke , the control rate was low, especially in the rural region (as low as 4.3%). The level of knowledge on the prevention of cardiovascular diseases, and the rates of smoking and drinking were similar between the elderly with or without stroke.

CONCLUSIONThe prevalence of stroke had increased dramatically during the past decade in Beijing. The proportion of poor SHR, ADL and IADL dependence, prevalence rates of stroke related diseases were higher among the elderly with stroke than those without. Secondary prevention of stroke among Beijing elderly called for urgent action.

Aged ; Cardiovascular Diseases ; complications ; China ; epidemiology ; Diabetes Complications ; epidemiology ; Female ; Health Knowledge, Attitudes, Practice ; Humans ; Hypertension ; complications ; Life Style ; Male ; Middle Aged ; Quality of Life ; Residence Characteristics ; statistics & numerical data ; Rural Population ; statistics & numerical data ; Stroke ; complications ; epidemiology ; Urban Population ; statistics & numerical data

The aim of this paper was to observe the effect of triptolide( TP) on cardiovascular function and its possible mechanism by intraperitoneal injection of bacterial lipopolysaccharide in rats with endotoxemia. Sixty male Sprague-Dawley rats were randomly divided intonormal group( NC group),endotoxemia model group( LPS group),TP low concentration intervention group( LPS + TP-L group,25 μg·kg~(-1)),TP middle concentration intervention group( LPS+TP-M group,50 μg·kg~(-1)),TP high concentration intervention group( LPS+TP-H group,100 μg·kg~(-1)) and polymyxin B group( LPS+PMX-B group,0. 2 mg·kg~(-1)). 10 mg·kg~(-1) LPS was injected intraperitoneally for 6 h to replicate the endotoxemia rat model. The rats in TP intervention groups were pre-treated 15 min before intraperitoneal injection of LPS. Rats in each group underwent total arterial intubation to measure hemodynamic parameters: heart rate( HR),left ventricular diastolic pressure( LVDP),the maximum rate of the increase/decrease of left ventricular pressure( ±dp/dtmax). The levels of BNP,CK-MB and c Tn-Ⅰ in serum and levels of TNF-α and IL-6 in plasma were detected by ELISA. The contents of p65 protein in myocardium and contents of p65,TLR4,i NOS and e NOS protein in thoracic aorta were detected by Western blot. As compared with NC group,the hemodynamic indexes in LPS group were significantly decreased; the contents of BNP,CK-MB and c Tn-Ⅰ in serum,TNF-α and IL-6 in plasma,p65 in myocardium,i NOS,e NOS,TLR4 and p65 in vascular tissues were significantly increased. As compared with LPS group,the hemodynamic indexes were significantly improved in LPS+TP-M group,LPS+TP-H group and LPS+PMX-B group; the contents of BNP,CK-MB and c Tn-Ⅰ in serum,TNF-α and IL-6 in plasma,p65 in myocardium,i NOS,e NOS,TLR4 and p65 in vascular tissues were significantly decreased in each treatment group. Triptolide has a protective effect on cardiovascular damage in a dose-dependent manner in endotoxemia rats,probably through TLR4/NF-κB p65 signaling pathway to improve endothelial function.
Animals ; Diterpenes/pharmacology* ; Endothelium ; Endotoxemia ; Epoxy Compounds/pharmacology* ; Lipopolysaccharides ; Male ; NF-kappa B ; Phenanthrenes/pharmacology* ; Protective Agents/pharmacology* ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Toll-Like Receptor 4/metabolism* ; Tumor Necrosis Factor-alpha

adrenocorticalcarcinoma；cortisolhypersecretion；hypokalemia；prognosis 【Objective】To analyze the prognostic determinants of adreno cortical carcinoma（ACC）inadults.【Methods】Alladult patients who were admitted to SunYat-sen Memorial Hospital，SunYat-sen University from December 2011 to March 2017 and pathologically diagnosed ACC were included in this study.Thec linical data and preoperative laboratory examinations of those patients were analyzed retrospectively. Overall survival or disease-free survival was calculated and survivalcurves were plotted by Kaplan-Meier and compared by log-rank test. Harzardratios（HRs） with their 95% confidenceintervals（CIs） were calculated by univariate and multivariate Coxregression model.【Results】The study included 20 adult patients with ACC， with a median follow-up of 13months （6~73 months）. The mean survival time of those patients was 49.2 months（6~73months），with a 1-year survival rate of 70.0%. The results of multivariate Coxregression analysis revealed that the presense of cortisol hypersecretion（HR=23.60，95%CI：2.49-223.79，P=0.006） and hypokalemia（HR=23.60，95%CI：2.49-223.79，P=0.006）were predictors of poorprognosis of ACC. Moreover，in 18 patients with completely resected ACC，the presense of hypokalemia resulted in a worse disease-free survival.【Conclusion】The presense of cortisol hypersecretion and hypokalemiaare independent risk factors associated with poorprognosis of ACC in adults.​