OBJECTIVETo study the apoptosis inducing effects of bufalin on various human osteosarcoma cells and the concerning molecular mechanisms.

METHODMTT assay was used to detect the growth inhibition rates of osteosarcoma cells U-20S, U-20S/MTX300, SaOS-2, IOR/OS9 treated with bufalin in different concentrations and times. The apoptosis of cells was observed flow cytometry 48 h following bufalin treatment. The proteomic techniques were used to separate and compare the treated and control groups 48 h after bufalin-incubation. Then, the proteomic results were validated by western blot.

RESULTBufalin inhibited the growth of human osteosarcoma cells U20S, U20S/MTX300 (methotrexate resistant cells), SAOS2, IOR/OS9 in a dose- and time-dependent manner. The 72 h IC50 were (37.43 +/- 4.1), (32.24 +/- 5.3) nmol x L(-1) in U20S,U20S/MTX300 cells,respectivly. Flow cytometry showed that the apoptosis cells were increased following bufalin treatment. The protein expression profile showed 24 differentiated expression proteins. Among these proteins, the level of an anti-apoptotic protein, heat shock protein 27 (Hsp27) decreased significantly and the result was then validated by western blot. Ectopic expression of Hsp27 could reduce the bufalin-induced apoptosis remarkably in U20S and U20S/MTX300 cells.

CONCLUSIONBufalin could inhibit the cell growth and induce apoptosis on human osteosarcoma cells. The effect of bufalin may be related to the joint intervention with multiple protein targets. Among them, downregulation of Hsp27 plays a critical role in the bufalin-induced apoptosis in human osteosarcoma cells.

Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Bufanolides ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Drug Screening Assays, Antitumor ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Osteosarcoma ; pathology ; Proteomics

OBJECTIVETo produce neutralizing antibodies against envelope protein domain III (EDIII) of dengue virus serotype I (DENV-1) and evaluate the nonstructural protein 1 (NS1) antigen capture enzyme-linked immunosorbent assay (ELISA) for identification of antibody neutralizing abilities.

METHODSFive BALB/c mice and one New Zealand Rabbit were immunized with recombinant EDIII protein of DENV-1 for the production of hybridomas and hyperimmune sera. Indirect ELISA, immunofluorescence assay (IFA) and Western Blot analyses were applied to identify specificity of antibodies. Comparing to plaque reduction neutralization test (PRNT), the new established DENV-1 specific NS1 antigen capture ELISA was used for detecting the neutralizing abilities of these antibody.

RESULTSFour strains of monoclonal antibodies (mAbs) named 1A1, 1B3, 3D3 and 9D6 and one hyperimmune serum of rabbit were obtained, all of which were approved to have neutralizing abilities to DENV-1 with the PRNT titer of 1:1024, 1:512, 1:256, 1:4096 and 1:4096. MAb 3D3 with the lowest neutralization titer in PRNT had not shown neutralizing ability to DENV-1 in NS1 antigen capture ELISA, while MAbs 1A1, 1B3 and 9D6 and the rabbit hyperimmune serum could protect the C6/36 from being infected by DENV-1 with the neutralization titer of 1:32, 1:32, 1:128 and 1:128 in this assay.

CONCLUSIONNS1 antigen capture ELISA could be used to identify antibody neutralizing abilities to DENV, it was a faster and more convenient way to screen antibodies with high neutralization titer and might also be used as one of the methods to evaluate the effects of vaccines.

Animals ; Antibodies, Neutralizing ; immunology ; Antibodies, Viral ; immunology ; Dengue Virus ; immunology ; Enzyme-Linked Immunosorbent Assay ; methods ; Female ; Mice ; Mice, Inbred BALB C ; Neutralization Tests ; Rabbits ; Viral Envelope Proteins ; immunology ; Viral Nonstructural Proteins ; immunology

OBJECTIVETo establish a long-term culture system for mouse spermatogonial stem cells (SSCs) and to discuss the key factor that supports mouse SSC self-renewal and proliferation.

METHODSTestis cells from 4-6 days postpartum male transgenic BALB/c mce were collected by a modified two-step enzymatic digestion method and plated on 0. 2% elatin-coated tissue culture plates. The germ cells were enriched by differential adherence selections after respectively incubated for 1, 5 and 24 h and then plated on the mitomycin C-inactivated mouse embryonic fibroblast (MEF) feeder layer. The basal culture medium was StemPro-34 SFM supplemented with other 15 nutrient factors. The 20 ng/ml Glial cell line-derived neurotrophic factor (GDNF), 10 ng/ml basic fibroblast growth factor (bFGF) and 200 ng/ml GDNF-family receptor alpha 1 (GFRalpha1) were added to the serum-free medium to promote SSC proliferation. Several important surface markers and special genes were examined by immunocytochemical staining and RT-PCR analysis.

RESULTSAfter 3-4 days culture on the MEF feeder, SSCs proliferated continuously and formed typical colonies. SSCs from the BALB/c mice could be cultured in a steady state for 3 months. Immunocytochemical staining showed that Oct4 was specifically expressed in the cultured SSC nucleus and GFRalpha1 strongly expressed on the surface of the membrane. RT-PCR confirmed that the cultured SSCs expressed Oct-4, GFRalpha1, Sox2 and several other special genes resembling undifferentiated spermatogonia.

CONCLUSIONSSCs from BALB/c mice could be cultured in the improved culture system for 3 months. This culture system could help further understand the regulating mechanism of SSCs and might provide an opportunity for the treatment of male infertility by SSC transplantation.

Animals ; Cell Culture Techniques ; methods ; Male ; Mice ; Mice, Inbred BALB C ; Spermatogonia ; cytology ; Stem Cells ; cytology

BACKGROUNDSubthalamic nucleus deep brain stimulation (STN DBS) is effective against advanced Parkinson's disease (PD), allowing dramatic improvement of Parkinsonism, in addition to a significant reduction in medication. Here we aimed to investigate the long-term effect of STN DBS in Chinese PD patients, which has not been thoroughly studied in China.

METHODSTen PD patients were assessed before DBS and followed up 1, 3, and 5 years later using Unified Parkinson's Disease Rating Scale Part III (UPDRS III), Parkinson's Disease Questionnatire-39, Parkinson's Disease Sleep Scale-Chinese Version, Mini-mental State Examination, Montreal Cognitive Assessment, Hamilton Anxiety Scale and Hamilton Depression Scale. Stimulation parameters and drug dosages were recorded at each follow-up. Data were analyzed using the ANOVA for repeated measures.

RESULTSIn the "off" state (off medication), DBS improved UPDRS III scores by 35.87% in 5 years, compared with preoperative baseline (P < 0.001). In the "on" state (on medication), motor scores at 5 years were similar to the results of preoperative levodopa challenge test. The quality of life is improved by 58.18% (P < 0.001) from baseline to 3 years and gradually declined afterward. Sleep, cognition, and emotion were mostly unchanged. Levodopa equivalent daily dose was reduced from 660.4 ± 210.1 mg at baseline to 310.6 ± 158.4 mg at 5 years (by 52.96%, P < 0.001). The average pulse width, frequency and amplitude at 5 years were 75.0 ± 18.21 μs, 138.5 ± 19.34 Hz, and 2.68 ± 0.43 V, respectively.

CONCLUSIONSSTN DBS is an effective intervention for PD, although associated with a slightly diminished efficacy after 5 years. Compared with other studies, patients in our study required lower voltage and medication for satisfactory symptom control.

Aged ; China ; Deep Brain Stimulation ; methods ; Female ; Follow-Up Studies ; Humans ; Male ; Middle Aged ; Parkinson Disease ; therapy ; Quality of Life ; Subthalamic Nucleus ; Treatment Outcome

OBJECTIVETo detect the presence of endothelial injury in patients with severe acute respiratory syndrome (SARS) via enhanced levels of tissue-type plasminogen activator (t-PA) and soluble thrombomodulin (sTM).

METHODSCase patients were from Xuanwu Hospital (Capital University of Medical Sciences, Beijing, China), and all of them met clinical criteria for SARS. Healthy controls were some of the hospital employees. Endothelial injury bio-markers tPA and sTM were detected by commercial ELISA-methods.

RESULTSClassic plasma markers of endothelial injury, tPA and sTM significantly elevated in SARS patients in comparison to controls [t-PA: 1.48 +/- 0.16 nmol/L versus 0.25 +/- 0.03 nmol/L (P<0.0001), and sTM: 0.26 +/- 0.06 nmol/L versus 0.14 +/- 0.02 nmol/L (P<0.05)]. The only patient who died had extremely high levels of these endothelial injury markers (t-PA: 2.77 nmol/L and sTM: 1.01 nmol/L). The likelihood ratio analysis indicated the excellent discriminating power for SARS at the optimal cut-point of 0.49 nmol/L for tPA and 0.20 nmol/L for sTM, respectively. Significant numerical correlations were found among these endothelial injury markers in SARS patients. The numerical coefficient of correlation Pearson r between t-PA and sTM was 0.5867 (P<0.05).

CONCLUSIONIncreased plasma concentrations of tPA and sTM in patients with SARS suggest the possibility of endothelial injury. SARS patients might need anticoagulant therapy or fibrinolytic therapy in order to reverse intraalveolar coagulation, microthrombi formation, alveolar and interstitial fibrin deposition. It may not only provide a useful treatment and prognostic index but also allow a further understanding of the pathological condition of the disease.

Adult ; Biomarkers ; blood ; Case-Control Studies ; China ; Female ; Humans ; Male ; Prognosis ; Severe Acute Respiratory Syndrome ; blood ; Thrombomodulin ; blood ; Tissue Plasminogen Activator ; blood

[Objective]To analyze the feasibility and clinical efficacy of one-stage debridement,bone grafting and in-ternal fixation for the treatment of single-segmental lumbar spinal tuberculosis with extreme lateral approach.[Methods]Thirteen patients of single-segmental lumbar spinal tuberculosis that received the surgeries from April 2013 to August 2016 were included.The operation duration and the amount of intraoperative blood loss were recorded.The VAS and ODI of the back pain,lumbar kyphosis angle,segment height restoration,and vertebral fusion rate were used to analyze the clinical efficacy.[Results]Thirteen patients were successfully followed up for 13-32 months(mean,20.3 months);the operation duration was 160-280 min(average,214±96)min;the amount of intraoperative blood loss was 150-350 mL, average(average,263±63)mL. At the final follow-up,ESR and CRP were normal and lower back pain(VAS)and Oswestry disability index(ODI)were significantly reduced(7.2±1.6 vs 2.5±1.2 and 63.3±5.4 vs 31.9±3.7,respectively)compared to preoperative values;there were no significant difference in the lumbar kyphosis angle,segment height resto-ration between preoperation(segmental lordosis,7.1°±4.7°;segmental height,64.8 mm±9.3 mm)and the values at final follow-ups(segmental lordosis,5.2°±3.5°;segmental height,69.4 mm±10.5 mm;P>0.05). All cases acquired good lumbar interbody fusion with no internal fixation failure or recurrence of tuberculosis.[Conclusions]Under systemic and routine antituberculosis chemotherapy,one-stage extreme lateral approach debridement,bone graft and internal fixation is effective and feasible for single-segmental lumbar spinal tuberculosis.

Deep brain stimulation(DBS) can not only significantly improve the motor symptoms of Parkinson's disease(PD), but also effectively reduce the refractory motor complications caused by levodopa. During the past 20 years, DBS has turned into one of the most successful treatment strategies in advanced stages of the PD. Careful patient selection, accurate electrode implantation during surgery, and individualized programming and drug adjustment are the guarantees for optimal efficacy and minimal side effects of DBS. New technologies such as directional deep brain stimulation and visual programming modes will further optimize the efficacy of DBS.

Aconiti Radix is a commonly used traditional Chinese medicine( TCM) herb in clinic,with the effects in expelling wind and removing damness,warming menstruation and relieving pain. With a long medicinal history and high medicinal value,it was used for anemofrigid-damp arthralgia,arthralgia,cold hernia and anesthesia analgesia. Modern pharmacological studies have shown that Aconiti Radix has a good therapeutic effect on rheumatoid arthritis,neuropathic pain and hypertension. As a well-known toxic TCM herb,its main pharmacodynamic and toxic components are alkaloids,which can lead to neurotoxicity and cardiotoxicity while exerting anti-inflammatory,analgesic,anti-tumor and other pharmacodynamic effects. Therefore,it is often processed to reduce its toxicity or combined with Paeoniae Radix Alba and Stephaniae Tetrandrae Radix to achieve the purpose of reducing toxicity and increasing efficacy in clinic.In recent years,with the deepening of the study on the incompatibility of TCM represented by " eighteen incompatible herbs",there have been new findings about TCM incompatibility. It has been found complementary effect,rather than no obvious toxic and side effects after the combination with incompatible herbs of Aconiti Radix. To provide the basis for further study and clinical application of Aconiti Radix,this paper reviewed chemical components,pharmacological action,toxicity and compatibility of Aconiti Radix by consulting relevant literatures published in recent years at home and abroad. Meanwhile,this paper also described the relationship between chemical constituents,as well as anti-inflammatory,analgesic,anti-tumor and other pharmacological effects and toxicity.
Aconitum ; chemistry ; Alkaloids ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Medicine, Chinese Traditional ; Plant Roots ; chemistry

OBJECTIVETo understand the infection status and epidemiological features of HBV in permanent residents of Shenzhen city.

METHODSA multi-stage stratified random sampling method was performed for questionnaire survey to permanently-registered residents of 1-59 years old in Luohu and Baoan district of Shenzhen in 2010, and blood samples of the subjects were collected. Hepatitis B virus-related surface antigen (HBsAg) and hepatitis B virus surface antibody (anti-HBs) were detected with ELISA.

RESULTSThe total 3771 studied population showed 252 HBsAg positive and 2712 anti-HBs positive residents with the standardization prevalence as 9.73% and 72.83% , respectively. The difference of the prevalence of HBsAg and anti-HBs between males and females were not statistically significant (P > 0.05). The prevalence of HBsAg was reduced with increasing age. The differences of the prevalence of HBsAg between Shenzhen permanent registered and non-permanent registered population were not significant, but the prevalence of anti-HBs in Permanent registered residents (78.32%) was higher than in non-permanent (66.03%, χ(2) = 41.613, P < 0.001). The prevalence of HBsAg was significantly different in various occupational and educational levels. Peasants had the highest prevalence (24.13%) and medical workers had the highest prevalence of anti-HBs (89.10% ). People with junior high school education had the highest prevalence of HBsAg (12.76%) and the lowest of anti-HBs (62.45%). Population with high-level education had the highest prevalence of anti-HBs(81.00% average). The prevalence of HBsAg was over 10% in people who were born in Shenzhen and Guangdong province, and the anti-HBs was the highest in Shenzhen population with the prevalence as 74.48% and 76.47% , respectively.

CONCLUSIONIn the Shenzhen resident population, the overall prevalence of HBV was lower than the average level of Guangdong province, but higher than the national wide.

Adolescent ; Adult ; Child ; Child, Preschool ; China ; epidemiology ; Female ; Hepatitis B ; blood ; epidemiology ; Humans ; Infant ; Male ; Middle Aged ; Seroepidemiologic Studies ; Surveys and Questionnaires

BACKGROUNDEpidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) targeted treatment has been a standard therapy for advanced non-small cell lung cancer (NSCLC), but it is not tolerated well by all patients. In China, some studies have reported that traditional Chinese medicinal herbs (TCMHs) may increase efficacy and reduce toxicity when combined with EGFR-TKI, but outside of China few studies of this kind have been attempted.

OBJECTIVEThis study is intended to systematically review the existing clinical evidence on TCMHs combined with EGFR-TKI for treatment of advanced NSCLC.

SEARCH STRATEGYPubMed, the Cochrane Library, the Excerpta Medica Database (EMBASE), the China BioMedical Literature (CBM), and the China National Knowledge Infrastructure (CNKI) and web site of the American Society of Clinical Oncology (ASCO), the European Society for Medical Oncology (ESMO), the World Conference of Lung Cancer (WCLC) were searched; the search included all documents published in English or Chinese before October 2013.

INCLUSION CRITERIAWe selected randomized controlled trials based on specific criteria, the most important of which was that a TCMH plus EGFR-TKI treatment group was compared with an EGFR-TKI control group in patients with advanced NSCLC.

DATA EXTRACTION AND ANALYSISThe modified Jadad scale was used to assess the quality of studies. For each included study, patient characteristics, treatment details, therapeutic approach and clinical outcomes were collected on a standardized form. When disagreements on study inclusion or data extracted from a study emerged, the consensus of all coauthors provided the resolution. The clinical outcome metrics consisted of objective response rate (ORR; complete response + partial response divided by the total number of patients), disease control rate (DCR; complete response + partial response + no change divided by the total number of patients), survival rate, improved or stabilized Karnofsky performance status (KPS), and severe toxicity. RevMan 5.0 software was used for data syntheses and analyses. Risk ratio (RR) and 95% confidence interval (CI) were calculated; if the hypothesis of homogeneity was not rejected (P>0.1, I(2)<50%), the fixed-effect model was used to calculate the summary RR and the 95% CI. Otherwise, a random-effect model was used.

RESULTSIn this review, 19 studies were included based on the selection criteria. Of them, 13 studies were of high quality and 6 studies were of low quality, according to the modified Jadad scale. When the TCMH plus EGFR-TKI treatment groups were compared with the EGFR-TKI control groups the meta-analysis demonstrated a statistically significant higher ORR (RR 1.34; 95% CI 1.15 to 1.57; P=0.000 2), DCR (RR 1.18; 95% CI 1.09 to 1.27; P<0.000 1), one-year survival rate (RR 1.21; 95% CI 1.01 to 1.44; P=0.04), 2-year survival rate (RR 1.91; 95% CI 1.26 to 2.89; P=0.002) and improved or stable KPS (RR 1.38; 95% CI 1.26 to 1.51; P<0.000 01). Severe toxicity for rash was decreased (RR 0.55; 95% CI 0.32 to 0.94; P=0.03), as were nausea and vomiting (RR 0.17; 95% CI 0.04 to 0.72; P=0.02) and diarrhea (RR 0.46; 95% CI 0.24 to 0.89; P=0.02). Sensitivity analysis indicated that findings of the meta-analysis were robust to study quality. In the funnel plot analysis, asymmetry was observed, and publication bias was indicated by Egger's test (P=0.03).

CONCLUSIONTCMH intervention can increase efficacy and reduce toxicity when combined with EGFR-TKI for advanced NSCLC, although this result requires further verification by more well designed studies.

Antineoplastic Agents ; administration & dosage ; Carcinoma, Non-Small-Cell Lung ; drug therapy ; enzymology ; Drug Therapy, Combination ; Drugs, Chinese Herbal ; administration & dosage ; Humans ; Lung Neoplasms ; drug therapy ; enzymology ; Protein Kinase Inhibitors ; administration & dosage ; Randomized Controlled Trials as Topic ; Receptor, Epidermal Growth Factor ; antagonists & inhibitors ; metabolism