From original concept and literature of acupoint, the concept and clinical significance of ashi method is discussed, which clarifies that the essence of ashi method is to locate the acupoints by patients' sensitivity on force. The clinical application of heat-sensitive moxibustion has illustrated that positioning method of this therapy is based on the appearance of heat-sensitive moxibustion sensation. Although both types are based on patients' feeling, positioning method of heat-sensitive moxibustion stands on a new angle and uses a new method to locate acupoint. Therefore, it is believed that the positioning method of heat-sensitive moxibustion is the inheritance and development of ashi method.
Acupuncture Points ; China ; History, Ancient ; Humans ; Medicine in Literature ; Moxibustion ; history ; methods ; Sensation

Objective To explore the effeteness of autologous transplantation of bone marrow-derived endothelial progenitor cells in promoting the neovascularization and improving the neurological functional recovery in atherosclerotic rats with acute cerebral infarction. Methods Male Sprague-Dawley rat models of atherosclerosis were established by fat-rich diet feeding. Endothelial progenitor cells (EPCs) were obtained from bone marrow of all rats; the cells were cultured in vitro in Ml99 with VEGF, bFGF and EGF in it Assays were used to detect the expression of FLK-1 and CD34. on the 7th d, middle cerebral artery occlusion (MCAO) rat models were established by the method of thread thrombus. Three h after MCAO, all of the animals were randomized into experimental group (the autologous endothelial progenitor cells labeled with BrdU were injected into the carotid vein) and control group (same volume of PBS were injected into the carotid vein). Behavioral tests (modified neurological severity scale, mNSS) were performed 6 h and 1, 3, 7, 10 and 14 d after MCAO. Besides, immunohistochemical examinations were employed to observe the distribution of EPCs (labeled by BrdU) in the brain tissue and to measure the microvessel density. Results EPCs from bone marrow were isolated, induced and cultured successfully in vitro, which positively stained for FLK-1 by immunocytochemistry and partly positively expressed CD34 by immunofluorescence. The cells of FITC labeled UEA adsorption and DiL-acLDL internalization were positive under fluorescence confocal microscopy. These cells possessed robust proliferative potential and their number reached 5×106. On the 14th d, the neurological function recovery in the experimental group (mNSS scores: 6.13±0.30) was significantly improved as compared with that in the control group (mNSS scores: 8.50±0.46, P<0.05). On the 28th, some positive EPCs stained by BrdU were found in the experimental group and the numbers of blood vessels in the experimental group (16.87±5.52) were obviously larger than those in the control group (12.76±4.94,P<0.05). Conclusion EPCs from bone marrow can be autologously transplanted into the brain tissue of cerebral ischemia and survive. Cerebral ischemia can improve neurological functional recovery after MCAO, which were probably by promoting the neovascularization.

OBJECTIVETo investigate the level of urokinase-type plasminogen activator (uPA) and urokinase-type plasminogen activator receptor (uPAR) in synovial fluid of patients with temporomandibular disorders and to analyze their relation with temporomandibular disorders (TMD).

METHODSSynovial fluid was obtained from 64 sides of 56 TMD patients and from 16 sides of 10 asymptomatic healthy volunteers (control). The concentrations of uPA and uPAR in the synovial fluid were measured by ELISA. Forty-eight sides of TMD were divided into 3 groups: arthrosis, structure disorder and osteoarthrosis, each including 16 sides.

RESULTSThe levels of uPA and uPAR were significantly higher in the synovial fluid of TMD patients than that in the control group (P < 0.05), and the level of uPA and uPAR in osteoarthrosis group was significantly higher than that in arthrosis and structure disorder group (P < 0.05). However, there was no difference in expression of uPA and uPAR between arthrosis and structure disorder groups (P > 0.05).

CONCLUSIONSuPA and uPAR in the synovial fluid may play a role in the pathogenesis of TMD, and the level of uPA and uPAR in synovial fluid of TMD could be used as a biochemical markers to reflect pathological degree of TMD.

Adolescent ; Adult ; Aged ; Case-Control Studies ; Female ; Humans ; Male ; Middle Aged ; Receptors, Urokinase Plasminogen Activator ; metabolism ; Synovial Fluid ; metabolism ; Temporomandibular Joint Disorders ; metabolism ; Urokinase-Type Plasminogen Activator ; metabolism ; Young Adult

OBJECTIVETo construct the localization system involving anti-TfR monoclonal antibody (McAb) and AFP promoters and assess its effect on human hepatoma cell lines.

METHODSThe conjugate of anti-TfR McAb and polylysine (PLL) was made by SPDP and purified by molecular screen chromatography. DNA blocking test determined that the ratio of one pEBAF/tk to six Ab-PLL was the most suitable to couple them. The pEBAF/tk recombinant plasmid bearing HSV-TK gene was coupled to Ab-PLL by noncovalent bond. The pEBAF/tk was transferred into human hepatoma cell line HepG2, SMMC7721 and pulmonary cancer cell line A549 by receptor-mediated gene delivery (Ab-PLL-DNA) and liposome procedure. The growth inhibitory rates of HepG2, SMMC7721 and A549 cells were measured by MTT assay.

RESULTSThe inhibitory rates of HepG2/tk in 100 mg/L and 1 mg/L of GCV were 60.5% and 24.3%, respectively. The inhibitory rate of GCV to SMMC7721 was 23.2% in 3 days. The pulmonary cancer cell A549, A549/tk (Ab) and A549 /tk (lipo) could not be inhibited by the addition of GCV.

CONCLUSIONThe localization system employed in this paper has high specificity, effectiveness and safety for gene therapy. It would be a promising strategy for gene therapy.

Antibodies, Monoclonal ; therapeutic use ; Carcinoma, Hepatocellular ; therapy ; Cell Line, Tumor ; Ganciclovir ; therapeutic use ; Genetic Therapy ; Humans ; Liver Neoplasms ; therapy ; Receptors, Transferrin ; immunology ; Simplexvirus ; enzymology ; Thymidine Kinase ; genetics ; alpha-Fetoproteins ; genetics

BACKGROUNDBioreactors are pivotal tools for generating mechanical stimulation in functional tissue engineering study. This study aimed to create a bioreactor that can simulate urinary bladder mechanical properties, and to investigate the effects of a mechanically stimulated culture on urothelial cells and bladder smooth muscle cells.

METHODSWe designed a bioreactor to simulate the mechanical properties of bladder. A pressure-record system was used to evaluate the mechanical properties of the bioreactor by measuring the pressure in culture chambers. To test the biocompatibility of the bioreactor, viabilities of urothelial cells and smooth muscle cells cultured in the bioreactor under static and mechanically changed conditions were measured after 7-day culture. To evaluate the effect of mechanical stimulations on the vital cells, urethral cells and smooth muscle cells were cultured in the simulated mechanical conditions. After that, the viability and the distribution pattern of the cells were observed and compared with cells cultured in non-mechanical stimulated condition.

RESULTSThe bioreactor system successfully generated waveforms similar to the intended programmed model while maintaining a cell-seeded elastic membrane between the chambers. There were no differences between viabilities of urothelial cells ((91.90 ± 1.22)% vs. (93.14 ± 1.78)%, P > 0.05) and bladder smooth muscle cells ((93.41 ± 1.49)% vs. (92.61 ± 1.34)%, P > 0.05). The viability of cells and tissue structure observation after cultured in simulated condition showed that mechanical stimulation was the only factor affected cells in the bioreactor and improved the arrangement of cells on silastic membrane.

CONCLUSIONSThis bioreactor can effectively simulate the physiological and mechanical properties of the bladder. Mechanical stimulation is the only factor that affected the viability of cells cultured in the bioreactor. The bioreactor can change the growth behavior of urothelial cells and bladder smooth muscle cells, resulting in the cells undergoing adaptive changes in mechanically-stimulated environment.

Bioreactors ; Cell Line ; Humans ; Myocytes, Smooth Muscle ; cytology ; Tissue Engineering ; methods ; Urinary Bladder ; cytology ; Urothelium ; cytology

Leukemia is a type of malignant tumors of hematopoietic system with the abnormal increased immature leukemia cells showing metastasis and invasion ability. Liver is one of the main targets of the leukemia cells spread to, where they may continue to proliferate and differentiate and cause liver function damage, even liver failure. Our previous studies showed that Angelica polysscharides (APS), the main effective components in Angelica sinensis of Chinese traditional medicine, was able to inhibit the proliferation and induced differentiation of the leukemia cells, however, its effect on the liver during the treatment remains elucidated. In the present study, the human leukemia NOD/SCID mouse model were established by implantation human leukemia K562 cells line, then the leukemia mouse were treated with APS, Ara-c or APS + Ara-c respectively by peritoneal injection for 14 days, to explore the effect and mechanism of the chemicals on the mouse liver. Compared to the human leukemia NOD/SCID mouse model group with the treatments of APS, Ara-c and APS + Ara-c, We found that severe liver damage and pathological changes of the liver were able to alleviate: First, the number of white blood cells in the peripheral blood was significantly lower and with less transplanted K562 leukemia cells; Second, liver function damage was alleviated as liver function tests showed that alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin (TBiL) were significantly reduced, while the albumin (Alb) was notably increased; Third, liver antioxidant ability was improved as the activities of the antioxidant enzymes glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) were significantly increased, and the contents of GSH and malonaldehyde (MDA) were decreased significantly in the liver; Fourth, the inflammation of the liver was relieved as the level of IL-1beta and IL-6, the inflammatory cytokines, were decreased significantly in the liver. Fifth, liver index was increased as the pathological observation showed that leukemia cells with diffused infiltration into the liver lobules were significantly reduced and with a remarkable increase of apoptotic positive cell rate by TUNEL test. Furthermore, the APS + Ara-c combined administration showed an even more significant positive effect. In conclusion, the APS, Ara-c therapy reduced the accumulation of leukemia cells within the liver, reduced the liver function damage and levels of inflammatory factors, improved antioxidant capacity of the liver tissue and thus alleviate the pathological changes of the liver. Moreover, the APS + Ara-c combination therapy may have an additive effect.
Angelica ; chemistry ; Animals ; Antineoplastic Combined Chemotherapy Protocols ; pharmacology ; Cell Line, Tumor ; Cytarabine ; administration & dosage ; Humans ; K562 Cells ; Leukemia ; drug therapy ; Liver ; drug effects ; Male ; Mice ; Mice, SCID ; Polysaccharides ; administration & dosage

BACKGROUNDAsthma is a chronic inflammatory disease characterized by reversible bronchial constriction, pulmonary inflammation and airway remodeling. Current standard therapies for asthma provide symptomatic control, but fail to target the underlying disease pathology. Furthermore, no therapeutic agent is effective in preventing airway remodeling. A substantial amount of evidence suggests that statins have anti-inflammatory properties and immunomodulatory activity. In this study, we investigated the effect of rosuvastatin on airway inflammation and its inhibitory mechanism in mucus hypersecretion in a murine model of chronic asthma.

METHODSBALB/c mice were sensitized and challenged by ovalbumin to induce asthma. The recruitment of inflammatory cells into bronchoalveolar lavage fluid (BALF) and the lung tissues were measured by Diff-Quik staining and hematoxylin and eosin (H&E) staining. ELISA was used for measuring the levels of IL-4, IL-5, IL-13 and TNF-α in BALF. Periodic acid-Schiff (PAS) staining was used for mucus secretion. Gamma-aminobutyric acid type A receptor (GABAAR) β2 expression was measured by means of immunohistochemistry, reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting.

RESULTSRosuvastatin reduced the number of total inflammatory cells, lymphocytes, macrophages, neutrophils, and eosinophils recruited into BALF, the levels of IL-4, IL-5, IL-13 and TNF-α in BALF, along with the histological mucus index (HMI) and GABAAR β2 expression. Changes occurred in a dose-dependent manner.

CONCLUSIONSBased on its ability to reduce the inflammatory response and mucus hypersecretion by regulating GABAAR activity in a murine model of chronic asthma, rosuvastatin may be a useful therapeutic agent for treatment of asthma.

Animals ; Asthma ; drug therapy ; metabolism ; Chronic Disease ; Disease Models, Animal ; Female ; Fluorobenzenes ; pharmacology ; therapeutic use ; GABA-A Receptor Antagonists ; pharmacology ; Hydroxymethylglutaryl-CoA Reductase Inhibitors ; pharmacology ; Lung ; chemistry ; Mice ; Mice, Inbred BALB C ; Mucus ; secretion ; Pyrimidines ; pharmacology ; therapeutic use ; Receptors, GABA-A ; analysis ; Rosuvastatin Calcium ; Sulfonamides ; pharmacology ; therapeutic use

OBJECTIVETo explore the feasibility of autologous fascia as a scaffold for the reconstruction of skeletal muscle in vivo.

METHODSTwenty-eight healthy New Zealand rabbits were employed in the study. The anterior tibial muscle in both legs were divided to create a gap of 10 mm in each muscle. One leg was used in the experiment (E, n = 28), while the contralateral as self-control (C). The legs in C group were further divided into 3 groups (C1, C2 and C3). While defects in the midportion of anterior tibial muscle in the hind legs were created in all rabbits. In E group, each defect was filled with a tubule made of autologous fascia lata, and the fascial tubule was filled with tiny muscular granules (< 1 mm x 1 mm x 1 mm). In C1 group (n = 10), the defect was also filled with fascial tubule but with no muscle filling. The defect in C2 group (n = 10) was only filled with muscle granules without fascial tubule. The defect in C3 group (n = 8) received no treatment. The survival rate of the transplantation was grossly observed, and the tissue samples were harvested for histological and ultra-structural examination and immunohistochemical identification of desmin at 2, 3, 4, 6 and 9 post-operation weeks. The expression level of alpha-actin DNA in the tissue samples from the midportion of grafted fascia was assessed by RT-PCR (reverse transcription polymerase chain reaction) in E and C1 groups.

RESULTS(1) Survival rate of the transplantation: In E group, it was 93.33% with near normal tissue contour in the grafting area. The muscle defects were not completely repaired in C1, C2 and C3 groups. (2) Under light and electronic microscopy, marked proliferation of muscular cells surrounding fibrous tissue could be discerned at 2 and 3 post-operation weeks in E group, while only necrotic tissue and fibrosis were observed in C1 and C2 groups, and no definite tissue could be discernible in C3 group. (3) Immunohistochemical staining revealed that over 85% of the cells were positive for desmin in E group, while only less than 25% in C1 group. (4) The expression level of alpha-actin DNA was significantly higher in E group than that in C2 group (P < 0.05).

CONCLUSIONThese results suggested that autologous fascia as a scaffold is beneficial for skeletal muscle reconstruction in vivo.

Animals ; Disease Models, Animal ; Fascia ; transplantation ; Muscle, Skeletal ; surgery ; Rabbits ; Soft Tissue Injuries ; surgery ; Tissue Culture Techniques ; Tissue Scaffolds ; Transplantation, Autologous

Objective To establish the test way of latex enhanced immune turbidimetric method for the concentration detection of urine free hemoglobin on the automatic biochemical analyzer,and evaluated the detection performance.Methods Prepared the detection reagents of latex enhanced immune turbidimetry method for detection of urine free hemoglobin with materials such as hemoglobin HBA0 antibody (8.5 mg/ml),80 nm carboxylic olystyrene latex microspheres (estapor,nanometer microspheres) and so on.Then,seted the project parameters on TBA120 automatic biochemical analyzer and proceeded the calibrations.Evaluated the detection performance by accuracy,precision,linear range,sensitivity,specificity and other indicators.Lastly,established the biological reference interval for urine free hemoglobin of healthy people in the region and verified it.Results To seted the project parameters on TBA120 automatic biochemical analyzer and passed the calibration.The calibration fitting curve had a smooth trend and no obvious deviation with each measuring point.In the recovery tests,added standard liquid with different concentrations of 100 mg/L and 500 mg/L to the samples with low values,the recovery rate was 95.3％ and 102.7％ respectively.Same to the samples with high values,the recovery rate was 104.2％ and 103.5％ respectively.In the precision test,samples with low and high value had a precision CV of 6.52％ and 4.18％ respectively.The linear range was 0 ～ 1 100 mg/L,analysis sensitivity was 2.8 mg/L.In the interference test,found that,samples had no obvious disturbance to the test with lower concentrations of free bilirubin,combined with bilirubin,vitamin C,animal hemoglobin lower than 342 μmol/L,342 μmol/L,0.03 g/L,500 mg/L respectively,while had significant interference when the concentration of chyle was higher than 870 FTU.Their study had established the biological reference interval of healthy people,male was 0～ 13.3 mg/L and female 0 ～ 17.1 mg/L,there was no significant difference between them by a t test (P＞0.05).Conclusion The latex enhanced immune turbidimetric method for the detection of urine free hemoglobin has the performance of testing clinical urine specimen,which not only solved the lackage problem of specificity for occult blood in the urine dry chemistry test and realized batch automation quantitative detection.The experimental data of this study provides a theoretical basis for the application of the method in other types of clinical samples.

OBJECTIVETo construct the multi-probe ribonuclease protection assay (RPA) template set to be used for detecting expression patterns of MD-2, TLR4, CD14 mRNAs in human peripheral blood mononuclear cells.

METHODSThe designed cDNA fragments of the three genes were generated by polymerase chain reaction (PCR) using specific primers and directionally cloned into EcoR I and Hind III sites of expression plasmid pSP72 containing the T7 promoter, the linearized plasmids was used as template to synthesize anti-sense RNA probes. Then we extracted total RNA from peripheral blood mononuclear cells (PBMC) and detected the dynamic expression patterns of the three genes with RPA method.

RESULTSThe proper sequence and orientation of the template set were confirmed by sequencing and the template set was successfully used to assay TLR4, MD-2 and CD14 mRNAs in human PBMC. The results showed that the three detected genes decreased transiently 1-3 hours after 100 ng/ml LPS stimulation.

CONCLUSIONSThese new RPA multi-probe set provided valuable tool for the simultaneous quantitative determination of expression of TLR4, CD14 and MD-2 mRNAs in both constitutive and inducible types.

Antigens, Surface ; analysis ; Base Sequence ; Biological Assay ; Cells, Cultured ; DNA ; analysis ; genetics ; Gene Expression Profiling ; methods ; Humans ; Lipopolysaccharide Receptors ; analysis ; Lymphocyte Antigen 96 ; Membrane Glycoproteins ; analysis ; Molecular Probe Techniques ; Molecular Sequence Data ; Monocytes ; metabolism ; RNA Probes ; analysis ; genetics ; Receptors, Cell Surface ; analysis ; Receptors, Immunologic ; analysis ; Ribonucleases ; Toll-Like Receptor 4 ; Toll-Like Receptors