Nerve growth factor is one of the most important factors playing an important role in regulating the growth, development and survival of the neuron. The purified NGF from human placenta has been reported, the tissue from which can be isolated the NGF is very limited. It is important for basic research and clinic application to expression hNGF by genetic engineering. By polymerase chain reaction,gene fragment encoding the mature part of ?-NGF was amplified using the DNA of human placenta as template. The fragment was sequenced and inserted into expression vector pET-15b, and the recombinant expression vector pET15b-NGF was transformed into E.coli BL21(DE3)pLysS. After inducing with IPTG the NGF was higher expressed up to 25% of the total cell proteins. The expression product was purified with metal chelate affinity chromatography on Ni-IDA agarose under denaturing condition. The purity of rNGF was higher than 90% and yield of rNGF was 4.56mg/L expressing bacteria. SDS-PAGE revealed the NGF expression product had a Mr 16kDa. Western-blot displayed the recombinant product had strong immunological activity with rabbit anti-human ?-NGF polyclonic antibodies. The expression products were dealed with solubilizing inclusion bodies and refolding protein. The test of nerve fiber growth of chicken embryo DRG neurons displayed rNGF had biological activity.

Objective: To analyze the efficiency of ureteroscope and biopsy in the diagnosis of tumor grade, muscle-invasiveness and multifocality in suspected upper tract urinary carcinoma (UTUC) patients in order to find out whether it can be used in the risk stratification of UTUC patients.Methods: A retrospective study of 76 UTUC patients who underwent preoperative ureteroscope and/or biopsy and received radical nephroureterectomy in Peking University Third Hospital during January 2014 to December 2016 was undertaken.Results: In this study, 76 patients were included.There were 31 males (40.8%), and 45 females (59.2%).The median age was 64.5 years (31-88), and 51 patients had the symptom of hematuresis.The tumor was located in renal pelvis in 39 patients, and in ureter in 37 patients.Post-operative pathology confirmed that all the 76 patients included in this study suffered from UTUC, of whom 21 (21.6%) were of low-grade, 51 (67.1%) were of high-grade, 4 (5.3%) were undetermined, and 47 (61.9%) patients were muscle-invasive, and 27 (35.5%) were not, and 2 (2.6%) were undetermined.Among the 50 patients, in whom the grade of the tumor could be diagnosed by biopsy, the sensitivity, specificity, accuracy, positive predictive value and negative predictive value for low-grade tumor was 88.2%, 69.7%, 76.0%, 60.0% and 92.0%, respectively.Among the 27 patients, in whom the muscle-invasiveness could be diagnosed by biopsy, 5 patients were diagnosed with muscle-invasiveness, all confirmed by pathology after surgery and 22 patients were diagnosed with none muscle-invasiveness, turned out to be 50% muscle-invasive and 50% none-muscle invasive after surgery.The accuracy was 59.3%.The accuracy of ureteroscopic biopsy to diagnosis multifocality was 61.0%.On univariate ana-lysis, biopsy grade was associated with postoperative pathology (P=0.001), while gender, age, side, body mass index (BMI), hematuresis, preoperative estimated glomerular filtration rate (eGFR), hydronephrosis, tumor size, location, multifocality and sessile were not associated with postoperative pathology grade.Biopsy grade (P=0.02), preoperative eGFR<90 mL/(min·1.73 m2)(P=0.025) and tumor located in pelvis (P=0.049) were associated with muscle invasiveness.Gender, age, side, BMI, hematuresis, hydronephrosis, tumor size, multifocality and sessile were not significantly associated with muscle invasiveness.Conclusion: Ureteroscope and biopsy can assist risk stratification in upper tract urothelial carcinoma patients.

OBJECTIVETo observe the effect of Chinese herbal therapy on T-lymphocyte subsets in patients with IgA nephropathy (IgAN).

METHODSTotally 36 inpatients and outpatients at Department of Nephropathy, Xiyuan Hospital, China Academy of Chinese Medical Sciences, from June 2011 to June 2013 were recruited in the treatment group, while 20 volunteers were recruited as the healthy control group. Patients in the IgAN group only took Chinese herbal decoctions by syndrome typing for 3 months (except those accompanied with hypertension additionally took antihypertensive agents such as angiotensin-converting enzyme inhibitor and/or dihydropyridines calcium antagonist). No intervention was performed in the healthy control group. The values of Th1, Th2, and CD4+ CD25+ Treg, and red blood cell number in urine were detected using flow cytometry before and after treatment. 24 h urine protein was detected using inmmunoturbidimetry.

RESULTSCompared with the healthy control group, the CD4+ CD25+ Treg level obviously decreased in the IgAN group, showing statistical difference (P < 0.01). In the IgAN group, Th1, 24 h urine protein, and urine red blood cell counts were obviously lower after treatment, showing statistical difference when compared with before treatment (all P < 0.05).

CONCLUSIONChinese herbal therapy could reduce urine erythrocyte number and 24 h urine protein of IgAN patients, and down-regulating Th1 expression might be its mechanism.

Adult ; Case-Control Studies ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Glomerulonephritis, IGA ; drug therapy ; Humans ; Male ; Middle Aged ; Phytotherapy ; T-Lymphocyte Subsets ; drug effects ; Young Adult

Objective To investigate the imaging characteristics of both intra- and extrathoracic sarcoidosis on 18F-FDG PET/CT.Methods From 2007 Aug.to 2009 Nov.,22 patients( 10 males,12 females) with sarcoidosis,confirmed by pathological study and clinical follow-up,underwent 18 F-FDG PET/CT imaging.The imaging patterns of intrathoracic and extrathoracic lesions were analyzed.The patterns were classified as the typical or atypical ( symmetrical or asymmetrical FDG accumulation and enlargement of hilar lymph nodes) based on PET and CT separately.Nonparametric McNemar test,independent t-test and Fisher exact test were applied for statistical analysis.Results For typical pattern vs atypical pattem identification,PET was significantly different from CT ( 18 and 4 vs 12 and 10,P =0.031 ).In those with atypical pattern demonstrated by CT alone at hilar region,PET showed either symmetrical or asymmetrical accumulation of FDG.Except for mediastinal lymph nodes involvement,lung parenchyma was the second common site ( 19/22,86.4％ ),followed by lymph nodes at abdomen and (or) pelvis ( 12/22,54.5％ ).Conclusion The imaging characteristics of both intra- and extrathoracic sarcoidosis on 18F-FDG PET/CT may be helpful for the diagnosis of atypical sarcoidosis on CT image alone.

Tumors of synchronous benign and malignant in unilateral salivary glands have rarely been reported. A case of 21-year-old girl who was diagnosed as synchronously adenoid cystic carcinoma of the left parotid and pleomorphic adenoma of the left submandibular gland. The classification, clinic pathology, diagnosis, possible mechanism were discussed based on similar literatures.
Adenoma, Pleomorphic ; Carcinoma, Adenoid Cystic ; Female ; Humans ; Salivary Glands ; Young Adult

OBJECTIVETo extract tumor interstitial fluid (TIF) from MKN-45 gastric cancer which is similar to "muddy phlegm" in Chinese medicine and observe influences of MKN-45 tumor interstitial fluid (MKN-45 TIF) intervention on metastasis of gastric cancer and on the expressions of vascular endothelial growth factor (VEGF), kinase insert domain containing receptor (KDR), epithelial-cadherin (E-cad), cyclooxygenase-2 (COX-2), intercellular adhesion molecule-1 (ICAM-1) and telomerase genes and proteins in primary tumor tissue.

METHODSAn MKN-45 tumor-bearing model was established in 50 nude mice. The modeled animals were equally randomized to 5 groups: the simple tumor-bearing group (model group), the normal saline (NS) via tail vein injection (i.v.) group (NS i.v. group), MKN-45 TIF i.v. group (TIF i.v. group), NS intraperitoneal injection (i.p.) group (NS i.p. group), and MKN-45 TIF i.p. group (TIF i.p. group). The TIF and NS intervention groups received injection (i.p. or i.v.) of MKN-45 TIF or NS twice a week, 0.2 mL at a time. After 8 weeks, the primary tumors were removed, weighed and HE stained to observe tumor metastasis. The primary tumor tissues were analyzed by immunohistochemistry and real-time quantitative PCR to detect expressions of VEGF, KDR, E-cad, COX-2, ICAM-1, and telomerase genes and proteins in different groups.

RESULTSThere were significant differences in tumor weight between TIF intervention groups and the model and NS intervention groups. Tumor metastasis was observed in all 5 groups, but the tumor metastasis rate in TIF intervention groups was significantly higher than those in the model and NS intervention groups. The gene and protein expressions of gastric cancer-related factors VEGF, KDR, COX-2, ICAM-1 and telomerase were unregulated while the gene and protein expressions of E-cad were downregulated in TIF intervention groups.

CONCLUSIONSTIF promotes tumor growth, invasion and metastasis of gastric cancer. These findings provide preliminary experimental clues for verifying the hypothesis of "tumor-phlegm microenvironment".

Animals ; Cadherins ; genetics ; metabolism ; Cell Line, Tumor ; Cyclooxygenase 2 ; genetics ; metabolism ; Extracellular Fluid ; metabolism ; Gene Expression Regulation, Neoplastic ; Humans ; Intercellular Adhesion Molecule-1 ; genetics ; metabolism ; Male ; Mice ; Mice, Nude ; Neoplasm Transplantation ; Stomach Neoplasms ; metabolism ; secondary ; Telomerase ; genetics ; metabolism ; Tumor Microenvironment ; physiology ; Vascular Endothelial Growth Factor A ; genetics ; metabolism ; Vascular Endothelial Growth Factor Receptor-2 ; genetics ; metabolism

OBJECTIVETo establish a gene-modified embryonic stem (ES; E14.1-2) cell line with hepatoblast differentiation reporter genes, albumin (ALB) and cytokeratin 19 (CK19), labeled to facilitate study of their potential applicability as differentiated hepatoblasts.

METHODSTwo expression vectors were constructed, one with the ALB promotor driving the enhanced green fluorescent protein (EGFP) and anti-neomycin genes (pAlb-EGFP), and the other with the CK19 promotor driving the red fluorescence protein and anti-hygromycin genes (pCK19-hCD25-IRES-tdTOMATO). The linearized vectors were electroporated into the E14.1 line, and double reporter genes-modified ES cells (E14.1-2) were selected by neomycin and hygromycin. E14.1-2 hepatoblast differentiation was induced by exposure to growth factors (BMP4 and bFGF) and evidenced by embryoid body formation. Fluorescence-activated cell sorting (FACS) and reverse transcription-polymerase chain reaction (RT-PCR) were used to confirm whether differentiated cells were hepatoblast-like and to quantify the differentiation efficiency.

RESULTSThe pAlb-EGFP and pCK19-hCD25-IRES-tdTOMATO vectors were shown to specifically activate ALB and CK19 expression. The E14.1-2 cell line with labeled ALB and CK19 was established, and shown to have pluripotency by RT-PCR detection of pluripotent markers' expression, namely Oct4 and SSEA-1. After 22 days of induction, 21.27% of the differentiated hepatoblasts were detected by FACS as positive for ALB and CK19 expression.

CONCLUSIONSA gene-modified ES cell line was generated with hepatocyte differentiation reporter genes ALB and CK19 labeled. The differentiation of the resultant E14.1-2 line was technically simple to qualify and quantify, and will likely aid future studies of hepatoblast characteristics.

Albumins ; genetics ; Animals ; Biomarkers ; Cell Differentiation ; Cell Line ; Embryonic Stem Cells ; cytology ; Genes, Reporter ; Hepatocytes ; cytology ; Keratin-19 ; genetics ; Mice ; Transfection

OBJECTIVETo investigate the dynamic changes of Th1/Th2 type cytokines in peripheral blood of patients with hepatitis B e antigen-positive chronic hepatitis B treated with telbivudine (LDT).

METHODSThe levels of IL-2, IL-4, IL-6, IL-10, TNF alpha, IFN gamma in the blood cells of HBeAg positive chronic hepatitis B patients were measured at 0, 4, 8, 12, 24, 48 weeks after LDT treatment by fluorescence activated cell sorting (FACS), the levels and dynamic changes of Th1/Th2 type cytokines in groups of complete response, partial response, non-response, virologic breakthrough were compared.

RESULTSThe levels of Th1 type cytokines in complete response group were higher than those in groups of partial response, non-response and virologic breakthrough, however, the levels of Th2 type cytokines showed an opposite trend compared with Th1 type cytokines. There were no significant differences between each group. In complete response group, the levels of IL-2, TNF alpha and IFN gamma were higher than baseline 12 weeks after LDT treatment (P < 0.05). In partial response group the level of IFN gamma was higher than baseline 24 weeks after LDT treatment (P < 0.05). In non-response group, the levels of IL-6 and IL-10 were higher than baseline at 48 weeks after LDT treatment (P < 0.05). In virologic breakthrough group, the level of IL-4 was higher than baseline 24 weeks after LDT treatment (P < 0.05), while the level of IL-6 was higher than baseline 12 weeks after LDT treatment (P < 0.05).

CONCLUSIONSThe balance of Th1/Th2 type cytokines plays an important role in the outcome of patients with hepatitis B e antigen-positive chronic hepatitis B treated with LDT. The immune response of patients with chronic hepatitis B is improved to some extent after LDT therapy.

Adult ; Antiviral Agents ; therapeutic use ; DNA, Viral ; blood ; Female ; Flow Cytometry ; Hepatitis B e Antigens ; blood ; Hepatitis B virus ; drug effects ; immunology ; Hepatitis B, Chronic ; blood ; drug therapy ; immunology ; Humans ; Interferon-gamma ; blood ; Interleukins ; blood ; Male ; Nucleosides ; therapeutic use ; Pyrimidinones ; therapeutic use ; Th1 Cells ; immunology ; metabolism ; Th2 Cells ; immunology ; metabolism ; Thymidine ; analogs & derivatives ; Time Factors ; Tumor Necrosis Factor-alpha ; blood ; Young Adult

PURPOSE: To evaluate the practical applications of the diagnosis algorithms recommended by the American Academy of Pediatrics urinary tract infection (UTI) guideline. METHODS: We retrospectively reviewed the medical records of febrile UTI patients aged between 2 and 24 months. The patients were divided into 3 groups: group I (patients with positive urine culture and urinalysis findings), group II (those with positive urine culture but negative urinalysis findings), and group III (those with negative urine culture but positive urinalysis findings). Clinical, laboratory, and imaging results were analyzed and compared between the groups. RESULTS: A total of 300 children were enrolled. The serum C-reactive protein level was lower in children in group II than in those in groups I and III (P<0.05). Children in group I showed a higher frequency of hydronephrosis than those in groups II and III (P<0.05). However, the frequencies of acute pyelonephritis (APN), vesicoureteral reflux (VUR), renal scar, and UTI recurrence were not different between the groups. In group I, recurrence of UTI and presence of APN were associated with the incidence of VUR (recurrence vs. no recurrence: 40% vs.11.4%; APN vs. no APN: 23.3% vs. 9.2%; P<0.05). The incidence of VUR and APN was not related to the presence of hydronephrosis. CONCLUSION: UTI in febrile children cannot be ruled out solely on the basis of positive urinalysis or urine culture findings. Recurrence of UTI and presence of APN may be reasonable indicators of the presence of VUR.
C-Reactive Protein ; Child ; Cicatrix ; Diagnosis ; Humans ; Hydronephrosis ; Incidence ; Medical Records ; Pediatrics* ; Pyelonephritis ; Recurrence ; Retrospective Studies ; Urinalysis ; Urinary Tract Infections* ; Urinary Tract* ; Vesico-Ureteral Reflux

Tetramethylpyrazine (TMP), an effective component of traditional Chinese medicine Chuanxiong, is commonly used to resolve embolism. Its possible therapeutic effect against atherosclerosis has received considerable attention recently. Angiotensin II (Ang II) is highly implicated in the proliferation of vascular smooth muscle cells (VSMCs), resulting in atherosclerosis. The mechanisms of TMP in the proliferation of VSMCs induced by Ang II remain to be defined. The present study was aimed to study the effect of TMP on Ang II-induced VSMC proliferation through detection of nuclear factor-kappaB (NF-kappaB) activity and bone morphogenetic protein-2 (BMP-2) expression. Primary cultured rat aortic smooth muscle cells were divided into the control group, Ang II group, Ang II + TMP group and TMP group. Cells in each group were harvested at different time points (15, 30 and 60 min for detection of NF-kappaB activity; 6, 12 and 24 h for measurement of BMP-2 expression). NF-kappaB activation was identified as nuclear staining by immunohistochemistry. BMP-2 expression was observed through Western blot, immunohistochemistry and in situ hybridization. The results showed that: (1) Ang II stimulated the activation of NF-kappaB. Translocation of NF-kappaB p65 subunit from cytoplasm to nucleus appeared as early as 15 min, peaked at 30 min (P<0.01) and declined after 1 h. (2) TMP inhibited Ang II-induced NF-kappaB activation (P<0.01). (3) Ang II increased BMP-2 expression at 6 h but declined it significantly at 12 and 24 h (P<0.01). (4) BMP-2 expression was also kept at high level at 6 h in Ang II + TMP group but maintained at the normal level at 12 and 24 h. (5) There was no significant difference in NF-kappaB activation and BMP-2 expression between the control group and TMP group. These results indicate that TMP inhibits Ang II-induced VSMC proliferation through repression of NF-kappaB activation and BMP-2 reduction, and BMP-2 expression is independent of the NF-kappaB pathway. In conclusion, TMP has therapeutic potential for the treatment of atherosclerosis.
Angiotensin II ; antagonists & inhibitors ; Animals ; Atherosclerosis ; drug therapy ; Bone Morphogenetic Protein 2 ; Bone Morphogenetic Proteins ; analysis ; antagonists & inhibitors ; Immunohistochemistry ; Muscle, Smooth, Vascular ; cytology ; drug effects ; metabolism ; Myocytes, Smooth Muscle ; metabolism ; NF-kappa B ; analysis ; antagonists & inhibitors ; Pyrazines ; pharmacology ; therapeutic use ; Rats ; Rats, Sprague-Dawley ; Transforming Growth Factor beta ; analysis ; antagonists & inhibitors