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.

BACKGROUNDThere are few researches for the healing of metaphyseal fractures; moreover, the animal models to study the metaphyseal fractures are usually made by the oscillating saw osteotomy without reliable fixation, which is not in accordance with our current clinical practice. In this study, we established a new model to observe the healing process of metaphyseal fractures.

METHODSEighteen New Zealand rabbits were used in the study. The fracture model was created by splitting the medial tibial plateau in rabbits, then reset, and fixed with compression screws. At 1, 2, 3, 4, 6, and 8 weeks postoperatively, the tibial specimens were collected; firstly, a general observation and an X-ray examination of the specimens was done, and then they were embedded in methylmethacrylate and cut into sections with hard tissue slicer. The sections were stained with Giemsa reagent and examined under light microscopy.

RESULTSThere was no fracture displacement in the tibial specimens of all time points, except for one showing a collapse. No external callus formation could be observed by X-ray and general examination. After 1 week of the operation, the fracture gap was filled by mesenchymal tissue; 2 weeks postoperatively, a large number of woven bones were formed; from the third week onwards, the woven bone began to turn into lamellar bone, and new trabecular structure began to form. In all of the slices, no obvious chondrocytes formed in fracture areas; thus, there was no endochondral ossification.

CONCLUSIONSThis model was an ideal fracture animal model and suitable for the study of metaphyseal fracture healing. The X-ray and histological images demonstrated that metaphyseal fracture healing was a process of direct bone healing through intramembranous bone formation under the conditions of minor trauma, good reduction, and firm fixation.

Animals ; Fracture Healing ; physiology ; Fractures, Bone ; diagnostic imaging ; pathology ; Rabbits ; Radiography

OBJECTIVETo investigate the effects of the over-expression of hypoxia-inducible factor-1alpha (HIF-1alpha) on the epithelial-mesenchymal transition (EMT) and invasive potency of LNCaP cells in vitro and in vivo.

METHODSWe cultured LNCaP cells stably expressing HIF-1alpha (LNCaP/HIF-1alpha) and LNCaP cells, identified the over-expression of HIF-1alpha, determined the proliferation of the two cell lines by MTT assay and the level of PSA in the supernatant of culture medium, and detected the anchorage independent growth by soft-agar colony formation assay. A subcutaneous tumor model was established in nude mice by injecting LNCaP/HIF-1alpha and LNCaP cells followed by observation of the tumor growth. Tumor specimens were obtained for immunohistochemistry.

RESULTSThe over-expression of HIF-1alpha was confirmed in the LNCaP/HIF-1alpha cells by immunofluorescence staining and Western blotting. The level of PSA was obviously decreased in LNCaP/HIF-1alpha as compared with that in LNCaP cells. MTT assay identified the increased proliferation of LNCaP/HIF-1alpha cells. The cell colony forming ability of the LNCaP cells was significantly lower than that of the LNCaP/HIF-1alpha cells. The rate of tumorigenesis was increased and its time shortened in the LNCaP/HIF-1alpha group. Immunohistochemistry revealed an up-regulated expression of vimentin and a down-regulated expression of E-cadherin in the tumor specimens.

CONCLUSIONThe overexpression of HIF-1alpha can up-regulate the expression of vimentin and down-regulate the expression of E-cadherin, which may enhance the invasive potency of LNCaP cells by inducing EMT.

Animals ; Cell Hypoxia ; Cell Line, Tumor ; Epithelial-Mesenchymal Transition ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Male ; Mice ; Mice, Nude ; Prostatic Neoplasms ; metabolism ; pathology

OBJECTIVETo evaluate the effect of hypoxia-inducible factor-1 alpha (HIF-la) on angiogenesis in human prostate cancer cells.

METHODSHuman prostate cancer cells of the line LNCaP were cultured and transfected by the recombinant plasmid pcDNA3. 1(-)/HIF-1alpha containing the gene HIF-1alpha with the Lipofectamine 2000 system. The positive clone cells were selected by G418 and confirmed by Western blotting and immunofluorescence (LNCaP/HIF-1alpha cells). The expressions of VEGF, iNOS and Ang-2 were detected by Western blotting.

RESULTSThe expression of HIF-1alpha in the LNCaP/HIF-1alpha cells was distinctly higher than that in the LN-CaP cells. Compared with the LNCaP cells, the expressions of VEGF and iNOS were up-regulated, whereas Ang-2 remained unchanged in the LNCaP/HIF-1alpha cells.

CONCLUSIONThe over-expression of HIF-1alpha can induce an increase in angiogenesis proteins and improve the angiogenesis potency of prostate cancer.

Blotting, Western ; Cell Line, Tumor ; Fluorescent Antibody Technique ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Lipids ; chemistry ; Male ; Nitric Oxide Synthase ; metabolism ; Plasmids ; chemistry ; genetics ; Prostatic Neoplasms ; blood supply ; metabolism ; pathology ; Transfection ; methods ; Vascular Endothelial Growth Factor A ; metabolism

OBJECTIVETo establish an animal model of tumor-associated depression and observe their biological behaviors and biochemical indices.

METHODSFour groups of SD rats kept in separate cages were subjected to tumor cell inoculation with or without chronic unpredictable moderate stress administered before or after the inoculation. The depressive behaviors of the rats were examined by open-field test, and the concentration of 5-HT in the hippocampus was measured by spectrophotofluorometry. The body weight of the rats and volume of the implanted tumor were monitored and sugar water test was performed.

RESULTSThe rats subjected to chronic stress displayed significant depression, manifested by reduction in movement in the central area and total movement distance with prolonged resting time and shortened time of activity. These rats maintained high levels of depression even 12 days after withdrawal of chronic stress. Compared with the control group, the depressive rats showed obviously reduced sugar water consumption and hippocampal 5-HT level. Tumors of different sizes were observed in all rats in the 4 groups.

CONCLUSIONA rat model of tumor-associated depression is established, and the tumor-bearing rats exhibit obvious depressive behaviors and reduced level of neural substance (5-HT), which provides a good basis for studying the association of depression with tumorigenesis,progression and prognosis of tumor.

Animals ; Carcinoma 256, Walker ; complications ; psychology ; Depressive Disorder ; etiology ; Disease Models, Animal ; Female ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Stress, Psychological ; complications

OBJECTIVETo analyze the impact of efforts of community-based organizations (CBO) in HIV testing mobilization and case finding among men who have sex with men(MSM).

METHODSResults of HIV testing mobilization among MSM through CBOs in 15 program areas were collected and compared with corresponding HIV case reporting data to demonstrate the contribution of CBO-based HIV testing in HIV case finding among MSM from July 2008 to December 2011. Meanwhile,the proportion of screened HIV positives who received testing results notification,confirmatory test, following up and CD4 cell tests were analyzed and compared with those identified in medical institutions.

RESULTSA total of 196 075 HIV tests were performed for MSM, as a result of mobilization efforts of CBOs. Cumulatively 7704 new HIV cases were identified, accounting for 51.7% (7704/14 914) of all newly diagnosed HIV cases infected via homosexual sex in the program areas.Among the newly diagnosed MSM HIV infections in the program areas,the proportion of infections detected through the mobilization of CBOs increased from 35.4% (609/1722) in 2008 to 63.7% (2371/3722) in 2010, and 58.3% (3024/5189) in 2011. Compared with those identified through medical institutions, newly diagnosed MSM infections detected though CBOs testing mobilization have higher rates of receiving screening testing results notification (97.3% (4441/4563) vs 92.8% (13 140/14 153)) , (84.6% (2559/3024) vs 79.8% (5589/7002)) and CD4 cell tests (66.1% (1999/3024) vs 52.9% (3705/7002)), and a lower rate of receiving confirmatory test (78.6% (3588/4563) vs 85.6% (12 115/14 153)).

CONCLUSIONCBOs can take their advantages in mobilizing MSM to receive HIV test, and MSM HIV cases detected through CBOs have become the main source of MSM HIV case finding in program areas.

Community Health Services ; HIV Infections ; prevention & control ; HIV Seropositivity ; Health Promotion ; Homosexuality, Male ; Humans ; Male ; Mass Screening

OBJECTIVETo detect the expression and distribution of metallothionein 2 (MT-2) in middle ear cholesteatoma, and to explore the relationship of MT-2 with interleukin 1α (IL-1α) and interleukin 6 (IL-6), and well as to explore the role of MT-2 in the pathogenetic mechanism of middle ear cholesteatoma.

METHODSUsing the immunohistochemistry and reverse transcription polymerase chain reaction to examine the expressions of MT-2, IL-1α and IL-6 protein and MT-2 mRNA in twenty-five middle ears' cholesteatoma and seven normal external ears' canal skin. The influence of cholesteatoma debris on the MT-2 mRNA and protein of HaCaT cell were further analyzed.

RESULTSAccording to immunohistochemistry research, the expressions of MT-2, IL-1α and IL-6 were extremely higher in middle ear cholesteatoma than those in normal external ears' canal skin (P < 0.05). The result of RT-PCR showed that there was significant difference between the expression of MT-2 mRNA of and middle ear cholesteatoma than those in normal external ear canal skin (t = 15.38, P < 0.05). There was a positive correlation between the expression of MT-2 and that of IL-1α (r = 0.856, P < 0.05). There was also positive correlation between the expression of MT-2 and that of IL-6 (r = 0.714, P < 0.05). MT-2 mRNA and protein of HaCaT cell significantly increased when exposed to cholesteatoma debris in a dose dependent manner.

CONCLUSIONSMT-2, IL-1α and IL-6 may play an important role in the pathogenetic process of middle ear cholesteatoma. Cholesteatoma debris may involve in the proliferation of middle ear cholesteatoma by activation of MT-2.

Adolescent ; Adult ; Case-Control Studies ; Cholesteatoma, Middle Ear ; metabolism ; Female ; Humans ; Interleukin-1 ; metabolism ; Interleukin-6 ; metabolism ; Male ; Metallothionein ; metabolism ; Middle Aged ; Young Adult

OBJECTIVETo investigate the influence of tinnitus frequency on medication and prognosis in patients with chronic subjective tinnitus.

METHODSSeventy-two patients (Ninety-three ears) diagnosed as chronic subjective tinnitus were studied from October 2010 to March 2011. All cases were divided into low frequency(twenty-three ears), medium frequency(fourteen ears) and high frequency (fifty-six ears) according to tinnitus matching test. All cases were treated with microcirculation promotion and steroid therapy (5% glucose 250 ml + ginkgo biloba extract 87.5 mg + dexamethasone 10 mg intravenous drip). Curative effect was evaluated and the factors of prognosis were analyzed after three weeks.

RESULTSAfter medication, results were acquired as follows: recovery in 0 ear (0%), excellent in 0 ear (0%), effective in 18 ears (19.4%), invalid in 75 ear (80.6%). The effective percentage was 39.1%, 35.7% and 7.1%, respectively. There was significant difference between these groups, but no significant difference between low frequency and medium frequency. Logistic regression analysis showed that the difference of frequency was significant prognostic factors for medication.

CONCLUSIONSMicrocirculation promotion and steroid therapy had a poor treatment effect in patients with chronic subjective tinnitus. The prognosis of chronic low-medium frequency tinnitus was better than chronic high frequency tinnitus. The difference of frequency retained significant influence on effects and prognosis of medication.

Adult ; Aged ; Chronic Disease ; Female ; Humans ; Male ; Middle Aged ; Prognosis ; Tinnitus ; diagnosis ; drug therapy ; Treatment Outcome

Chronic kidney disease (CKD) is a major public health problem that affects about 10% of the general population. Current approaches to characterize the category and progression of CKD are normally based on renal histopathological results and clinical parameters. However, this information is not sufficient to predict CKD progression risk reliably or to guide preventive interventions. Nowadays, the appearance of systems biology has brought forward the concepts of "-omics" technologies, including genomics, transcriptomics, proteomics, and metabolomics. Systems biology, together with molecular analysis approaches such as microarray analysis, genome-wide association studies (GWAS), and serial analysis of gene expression (SAGE), has provided the framework for a comprehensive analysis of renal disease and serves as a starting point for generating novel molecular diagnostic tools for use in nephrology. In particular, analysis of urinary mRNA and protein levels is rapidly evolving as a non-invasive approach for CKD monitoring. All these systems biological molecular approaches are required for application of the concept of "personalized medicine" to progressive CKD, which will result in tailoring therapy for each patient, in contrast to the "one-size-fits-all" therapies currently in use.
Computational Biology ; Gene Expression Profiling ; Genome-Wide Association Study ; Humans ; Renal Insufficiency, Chronic ; genetics ; metabolism ; Systems Biology ; methods

OBJECTIVETo investigate the common nasal bleeding points and the management of intractable epistaxis.

METHODSThe bleeding points and its correlation with age distribution, surgical techniques as well as its effects were studied retrospectively in 92 patients, in whom the bleeding points were not found by routine nasal endoscopy and the hemorrhage was not controlled with standard nasal packing.

RESULTSThe bleeding points were found in the following different sites: superior wall of inferior nasal meatus (56.5%, 52/92), olfactory cleft of nasal septum (27.2%, 25/92), posterosuperior wall of middle nasal meatus (8.7%, 8/92) and uncertain (7.6%, 7/92). The results showed that the bleeding points had correlation with age. Epistaxis was well controlled by electrocoagulation in 83 cases, gelfoam packing in 8 cases, and transcatheter maxillary artery embolization in 1 case. There were no complications during a followed-up for 1 - 3 months after management. Among the 92 cases, the numbers of treatment needed to stop bleeding were 82 cases (89.1%) after 1 time of treatment, 9 cases (9.8%) after 2 times and in one case (1.1%) after 4 times.

CONCLUSIONSEndoscopy combined with displacement of the middle and inferior turbinate gives good visualization and direct management of the deeply-sited bleeding points, which were difficult in localization. The combined method provides an effective and safe way to control intractable epistaxis.

Adolescent ; Adult ; Aged ; Electrocoagulation ; Endoscopy ; Epistaxis ; diagnosis ; pathology ; therapy ; Female ; Humans ; Male ; Middle Aged ; Treatment Outcome ; Young Adult