Objective To isolate and identify exosomes from human serum , explore the feasibility of detecting exosomal miRNA in human serum.Methods Retrospective study.Serum samples from 10 healthy individuals in January 2013 were randomly selected.Besides, from January 2013 to December 2014, serum samples from prostate cancer(PCa) patients (n=20), benign prostatic hyperplasia(BPH) patients ( n=20 ) and healthy controls ( n=20 ) were selected.Exosomes were isolated from these serum samples using ExoQuick , and then identified by using transmission electron microscopy , NanoSight nano particle analyzer and Western Blot for morphology and molecular phenotype.The quality of exosomal RNA was analyzed using Agilent 2100 Bioanalyser.Then quantificational real-time polymerase chain reaction ( qRT-PCR) was carried out to detect miRNAs in different components of human serum ,and nonparametric tests were used for difference analysis.Results Exosomes isolated from human serum showed round or oval vesicles, mainly in diameter 40-100 nm, and with maximum peak distribution of 58 nm.Moreover, they expressed HSP70 and four transmembrane protein CD 63.Agilent 2100 Bioanalyzer results showed that the major RNA component of exosome was about 25nt small RNA.qRT-PCR confirmed that 4 normal miRNAs were expressed in human serum exosome , and the expression of miRNAs in exosome pellets were higher than the whole serum (miR-21, U =16,P =0.007 2; miR-16, U =3,P<0.000 1; miR-20a, U =2,P <0.000 1;let-7a, U=13,P=0.003 2) and exosome-depleted supernatant ( miR-21, U=15,P=0.006 5;miR-16, U=2,P<0.000 1;miR-20a, U=1,P<0.000 1;let-7a, U=10,P=0.002 8).miR-141, the molecular marker of prostate cancer ,were analyzed by qRT-PCR in whole serum samples and serum exosome pellets isolated from the same serum in a cohort of 20 PCa patients , 20 BPH patients and 20 healthy control people.The results showed that , in three groups , exosomal miR-141 expression were all significantly higher than serum circulating miR-141 (Control group, U=66,P=0.000 3; BPH group, U=83,P=0.001 6;PCa group, U=54,P<0.000 1).In addition, the expreession of exosomal miR-141 in PCa patients was significantly higher than BPH patients or healthy controls (3.85 fold, U=74,P=0.000 7 and 4.06 fold, U=70,P=0.000 5).Conclusion Exosome can be efficiently isolated from human serum.Compared with the whole serum , isolation of serum exosome may helpful to improve the detection of circulating miRNA.

BACKGROUND: It has been widely reported that the most types of cancer are probably originated from cancer stem cells (CSCs) which are subpopulations of tumor cells. Recent studies also suggested that lung cancer could arise from CSCs. However, the phenotypic characteristics of CSCs in lung cancer have not been precisely described.OBJECTIVE: To systematically analyze the expression of the most common CSC markers in cell line A549.METHODS: A549 cells were cultured for 1 week under the condition of conventional high glucose. After that, flow cytometry was used to assess the expression of putative stem cell markers, including CD133, CD24, CD44 and ABCG2.Cells were then sorted according to the expression of CD44 and CD24 markers by fluorescence-activated cell sorting (FACS) and characterized using their sphere-forming capacity in serum free medium supplemented with several growth factors.RESULTS AND CONCLUSION: A549 cells expressed the CSC markers CD44 and CD24 at 64.23％ and 58.62％,whereas the expression of both ABCG2 and CD133 was around 0.9％. Double-positive CD44/133 populations were rare.CD44+/CD24+ and CD44+/CD24- subpopulations respectively exhibited 54.64％ and 23.38％ expression. CD44+/CD24+ and CD44+/CD24- subsets were sorted by FACS. Both isolated subpopulations formed spheres in the serum free medium supplemented with basic fibroblast growth factor and epidermal growth factor. However, there was no significant difference in the sphere formation efficiency among CD44+/CD24+ and CD44+/CD24- subsets as well as A549 cells. Our findings suggest that CD44 and CD24 cannot be considered as potential markers for isolating lung CSCs in cell line A549, and further investigation using in vivo assays is required.

Objective To observe the changes of immunological parameters and the effects of intravenous immunoglobulin (IVIG) in children with idiopathic thrombocytopenic purpura (ITP),schonlein-Henoch purpura(HSP) and mucocutaneoas lymph node syn-drome(kawasaki disease, KD).Methods T cell subsets and serum immunoglobulins and complements were measured with the flow cytometry and enhanced trubidimetric immunoassay. Routine therapy combined with IVIG. Results CD3+ CD4+ T cells were de-creased in children with ITP accompanied by increased serum IgG;Serum IgA and CD3+ T cells were increased in children with HSP; CD3+ CD8+ T cells were decreased in children with KD. Clinical features were markedly improved after treatment with IVIG. It was noted that the incidence of damage to coronary artery decreased after the use of IVIG. Conclusions T cell subsets, serum immunoglobulins should undergo the clinical routine examinations in ITP,HSP and KD. Early administration of IVIG might be important to improve disease prognosis and shorten its course of treatment in children with three kinds of immunology related diseases.

ObjectiveTo explore the related factors influencing effect of community-based rehabilitation (CBR) on activities of daily living (ADL) in stroke patients in.Methods202 stroke patients were randomly divided into the CBR group (n=103) and control group (n=99). The patients of the CBR group treated with rehabilitation and following-up including risk factors controlled with drug, rehabilitation, health education and mental treatment, those of the control group only treated by following-up. Before and five months after treatment, patients of two groups were assessed with Barthel Index (BI), clinical never function limitation score, cognitive item of Functional Comprehensive Assessment (FCA). Multielement regression analysis was applied, the ADL score of last assessment was taken as dependent variable, group information, location, smoking, sex, age, course of diseases, education, drinking, sleeping, the first scores of FCA and BI were taken as independent variable.ResultsThe improvement of ADL of stroke patients was related with group information, drinking, course of diseases, the first scores of FCA, BI, and etc.ConclusionEarly CBR can significantly improve the ADL of the stroke patients. Cognitive deficit also has influence on ADL.

Heterotrimeric G proteins are classes of signal-transducing proteins that bind to guanine nucleotides and possess GTP hydrolase activity. G proteins are composed of three subunits α, β, and γ, and are considered as the "molecular switch" in the GPCR signaling pathway. The abnormal activation of G protein is strongly related to diseases such as uveal melanoma, asthma, et al., making directly targeting G protein as an promising strategy for combating diseases. In this review, the classification and physiological functions of G protein are briefly described, and the research progress of G proteins in diseases, G protein modulators are reviewed.

Objective To investigate the pharmaeokinetics of levofloxacin in rat's pancreatic tissue. Methods Pancreatic tissue and blood were sampled in vivo by microdialysis simultaneously. The concentrations of levofloxacin in beth blood and tissues were measured by high performance liquid chromatography. All date were analyzed by WinNonlin software. Results The maximum concentration of free levofloxacin in blood and pancreatic tissue were (65.23 ± 12.9) μg/ml at 10min and (30.56±3.22) μg/ml at 20 min, respectively, then beth continuously decreased. Concentration of free levofloxacin in pancreatic tissue was higher than that in blood from 20min to 100min, then returned to similar level. The area under the concentration curve(AUC)of unbound levofloxacin was(2465.11±258.56)min·μg~(-1)·ml~(-1) in pancreas,and (2914.38±205.73)min·μg~(-1)·ml~(-1) in blood.Conclusions Microdialysis with reversed phase high performance liquid chromatography established in this essay could be used to determine the pharmacokinetics of levofloxacin objectively. High concentration of levofloxacin in pancreatic tissue and blood was observed.

The 29 medical records about acupuncture and moxibustion in Zhenjiu Dacheng are involved in the internal, surgical, gynecological, pediatric and five sense organs diseases and symptoms. Analysis on characteristics of these medical records and absorbing experiences of predecessors can better guide clinical application. Analysis of results indicates that these medical records have the characteristics of combination of acupuncture, moxibustion and medicine, less and better point selection, and stressing reinforcing-reducing of manipulation, at the same time, it reflects YANG's train of thought in clinical diagnosis and treatment of acupuncture and moxibustion.
Acupuncture ; methods ; Humans ; Medical Records ; Medicine in Literature ; Medicine, Chinese Traditional ; methods ; Moxibustion ; methods

OBJECTIVE: To determine the sensitivity of cetuximab induced apoptosis in laryngeal squamous carcinoma cells Hep-2, and to evaluate the synergistic killing effects and regulation mechanism of cetuximab alone or cetuximab in combination with chemotherapeutic agents (cisplatin) or radiation means on Hep-2 cells. METHOD: To investigate the cytotoxicities of cetuximab, cisplatin and radiation, cell counting kit-8 (CCK-8) assay was used for the detection of cell growth inhibition ratio, and fluorescence activated cell sorter FACS for the apoptotic rate and cell cycle distribution. RESULT: Cetuximab had inhibitive effect on Hep-2 cells within a certain range of concentration in a time- and dose-dependence manner. The inhibition concentration 50% (IC50) of cetuximab on Hep-2 cells for 24 h was 1 036.84 microg/ml. For application of cisplatin and radiation, the apoptotic rate of Hep-2 cell was higher by combining with cetuximab than their single or combined administration. Moreover, the cell cycle arrested at G0/G1 phase. CONCLUSION Laryngeal cancer Hep-2 cells was sensitive to the cetuximab induced apoptosis. Cetuximab combined with cisplatin and/or radiation can increase the antiproliferative effects on Hep-2 cells. These findings suggest the synergistic combination of cetuximab and cytotoxic agents was sequence depended.
Antibodies, Monoclonal, Humanized ; pharmacology ; Apoptosis ; drug effects ; radiation effects ; Cell Line, Tumor ; Cetuximab ; Cisplatin ; pharmacology ; Combined Modality Therapy ; Humans

OBJECTIVE: To determine an approach enriching cancer stem cells from laryngeal cancer cell line. To investigate whether laryngeal cancer stem cells in chemoradiotherapy have the characteristic of resistance. METHOD: CD133+ cells and CD133- cells was detected and isolated from Hep-2 cell line by fluorescence activated cell sorting technology. The cytotoxicities of cisplatin and radiation were investigated by cell counting kit-8(CCK-8) assay. The apoptosis and cell cycle was analyzed with flow cytometry. RESULT: CD133+ cells accounted for a fraction of (2.43 +/- 0.77)% in Hep-2 cell line. CD133+ cells have a more obvious characteristics of cancer stem cells. Different cisplatin and radiation concentrations of for two cell have inhibition, in a certain concentration range and the dosage dependence. Cisplatin and radiation had synergistic inhibitory effects with CD133- cells on the growth of two cell. Moreover, cell cycle arrest at G0/G1 phase and more apoptosis was induced by synergistic combination. Different concentrations of cetuximab for Hep-2 cells have inhibition, in a certain concentration range and time and the dosage dependence. The half maxial inhibitory concentration (IC50) of cetuximab to Hep-2 cells on 24 h was 1 036.84 microg/L. Cisplatin and radiation had synergistic inhibitory effects with cetuximab on the growth of Hep-2 cell line. Moreover, cell cycle arrest at G0/G1 phase and more apoptosis was induced by synergistic combination. CONCLUSION Compared with CD133- cells, CD133+ cells subpopulation exhibited extraordinary cancer stem.
AC133 Antigen ; Antibodies, Monoclonal, Humanized ; pharmacology ; Antigens, CD ; analysis ; Antineoplastic Agents ; pharmacology ; Apoptosis ; Cell Cycle ; Cell Line, Tumor ; Cetuximab ; Chemoradiotherapy ; Cisplatin ; pharmacology ; Drug Resistance, Neoplasm ; Flow Cytometry ; Glycoproteins ; analysis ; Humans ; Laryngeal Neoplasms ; therapy ; Neoplastic Stem Cells ; drug effects ; radiation effects ; Peptides ; analysis ; Radiation Tolerance

Objective To prepare human prostatic carcinoma-targeted ultrasound contrast agent and assess its targeted ability in vitro. Methods Human prostatic carcinoma-specific polyclonal antibody PSM(C-15) was attached to the surface of self-made liposome microbubbles by electrostatic attraction to prepare targeted microbubbles. Immunofluorescent staining assay was used to identify the combination of PSM(C-15) with liposome microbubbles and the targeted microbubbles were added to prostatic-carcinoma cells and then observed under the light and fluorescence microscope to evaluate the targeting ability of the targeted liposome microbubbles with prostatic carcinoma cells in vitro, while the common liposome microbubbles were controls. Results Targeted microbubbles were positive in immunofluorescent straining assay. In vitro study of the targeting ability showed the targeted microbubbles could actively adhere to LNCaP cell. While the control was negative. Conclusion The targeted liposome contrast agent with highly specific biological activity was prepared successfully. The contrast agent could bind to human prostatic carcinoma cells specially and effectively in vitro.