AIM: To investigate the effects of nitric oxide(NO) and caspase-3 on dopamine-induced apoptosis in PC12 cells. METHODS: Flow cytometric assay was used to quantify the apoptotic cells. The morphological of apoptotic cells was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL). Nitrite was quantified by Griess reaction. Inducible nitric oxide synthase(iNOS) mRNA was identified by semiquantitative reverse transcription polymerase chain reaction(RT-PCR). Caspase-3 activity was determined by fluorescent spectrofluorometer. RESULTS: Dopamine induced PC12 cells apoptosis in a concentration-dependent manner (0.15-0.60 mmol/L), with positive TUNEL staining. During the development of apoptosis, the expression of iNOS mRNA and the levels of NO increased markedly, so did caspase-3 activity(P

In order to clarify material basis of effective parts of liangxue tongyu prescription, blood-heat and blood-stasis rat model induced by dry yeast was established. The changes of rectal temperature, blood viscosity and plasma viscosity were used to evaluate the cooling-blood and activating-blood effects of liangxue tongyu prescription and its parts. Compared with the model group, the extract from liangxue tongyu prescription, its volatile oil and n-butanol part could significantly reduce rectal temperature (P<0.01), and also reduce blood viscosity and plasma viscosity to various degrees (P<0.01 or P<0.05). So volatile oil and n-butanol part were primarily identified as effective parts of liangxue tongyu prescription. By using GC-MS with normalization method of area to analyze volatile oil of liangxue tongyu prescription, 70 compounds were identified, accounting for about 92.54%, mainly as β-asarone, paeonol, α-asarone and shyobunone. 42 compounds such as peony glycosides, tannins, and iridoid glycosides were identified by HPLC-MS techniques and standard comparison. The study determined the effective parts of liangxue tongyu prescription and clarified the chemical composition providing the foundation for further studies on material basis of liangxue tongyu prescription.
Acetophenones ; chemistry ; Animals ; Anisoles ; chemistry ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; chemistry ; Gas Chromatography-Mass Spectrometry ; Oils, Volatile ; chemistry ; Rats ; Tannins ; chemistry

Objective To study the relationship between Couagen Ⅰ,MMP-2,TIMP-2 gene expression and atrial fibrosis during heart failure(HF)in dog.Methods Fourteen dogs were used and randomized into HF induced by ventricular tachypacing and control group.Burst atrial pacing was used to induce atrial fibrillation(AF).And the mRNA and protein level of collagen Ⅰ,MMP-2 and TIMP-2 were detected by RT-PCR and immunohistochemical technique.Tissue samples were stained with Mallory trichrome.Results Left ventricular ejection fraction (LVEF) decreased from (67.4? 6.0)% to (29.2?7.8)%,the inducible rate of AF(7/7 vs 2/7) and sustained AF(5/7 vs 0/7) increased and duration of AF stabeatrial fibrillation(SAF) [(462.12?181.43)s vs(0.57?0.57) s] prolonged significantly in HF group.Atrial fibrous tissue content and atrial size of HF group were significantly greater than the controls dogs(268.8% in lefe atria and 190.3% in right atria).The mRNA and protein level of collagen Ⅰ(56.2% and 132.2% in lefe atria,37.4% and 78.0% in right atria)and MMP-2 (100.0% and 115.7% in lefe atria,65.7% and 96.8% in right atria) increased evidently in both lefe atria and right atria,TIMP-2 mRNA decreased 46.3% in lefe atria and had no change in right atria and that its protein had no change in both atrium,whereas the ratio of MMP-2/ TIMP-2 of mRNA and protein increased markedly in both lefe atria (285.3% and 148.8%)and right atria (106.1% and 134.7%)of HF group.SAF had a positive correlation with fibrosis and the gene level of collagen Ⅰ in lefe atria,the ratio of MMP-2/TIMP-2 had a positive correlation with fibrosis and collagen Ⅰ gene level in lefe atria during HF.Conclusions The changes of collagen Ⅰ,MMP-2 and TIMP-2 gene expression appear to be a molecular mechanism of AF, and the molecular remodeling of collagen Ⅰ induced by regulation unbalance of MMP-2/TIMP-2 appears to be an important mechanism of atrial fibrosis during HF.

Objective To observe the effects of a motor relearning programme (MRP) combined with different early acupuncture interventions on muscle tension and motor function recovery after cerebral infarction.MethodsA total of 90 patients with cerebral infarction who met the inclusion criteria were divided into three groups at random:a YANGMING meridian acupuncture and MRP group ( group A),an anti-spasm acupuncture and MRP group ( group B),and an MRP group ( group C ).All of the patients in all three groups were treated with routine medication.The National Institute of Health stroke scale (NIHSS),the composite spasticity scale (CSS),Fugl-Meyer assessment (FMA),the Fugl-Meyer balance scale (FM-B) and the modified Barthel index (MBI) were used to measure performance before treatment and after 4 weeks of treatment.Another comparison was intra-group between before and after treatment. ResultsThere were significant differences in the assessment results in all of the groups after treatment compared with those before treatment.After treatment,group B was superior to group C only in terms of NIHSS scores.There was no significant NIHSS score difference between groups A and C.The FMA,CSS and MBI results revealed significant differences among all three groups,with the scores of group A consistently the highest.The average FMA score in group B was significantly higher than in group C but there was no statistically significant difference in FM-B scores among the three groups. ConclusionMRP therapy combined with early acupuncture intervention can improve motor function and muscular tension after cerebral infarction.Anti-spasm acupuncture can improve motor function and control muscular tension effectively at the same time,making it beneficial for MRP training.

Objective The aim of this study is to investigate the role of angiotensin-converting enzyme (ACE)gene susceptibility to systemic lupus erythematosus(SLE)by familial studies.Methods PCR-based re- striction fragment length polymorphism(RFLP)was applied to genotype single nucleotide polymorphism(SNP) G261T of the ACE gene.A total of 119 patients with SLE from 119 families were recruited.In addition,316 family members of these patients were also genotyped.A family-based association study was carried out to ex- plore the association between gene polymorphism and SLE.We studied the SNP encoding non-synonymous substitution in the ACE gene with respect to genetic susceptibility to SLE.Results Among 119 SLE patients. the frequency of ACEG261TG,T alleles was 44.8%.55.2% respectively,the frequency of ACEG261T GG,GT and TT genotypes was 13.9%,62.0%,24.1% respectively,Univariate(single-marker)family-based association test(FBAT)demonstrated that variant alleles at the SNP,rs4303,exon 5 of ACE gene were significantly asso- ciated with genetic susceptibility to SLE in Additive Model(Z=2.877,P=0.004),Dominant Model(Z=2.557, P=0.011).Recessive Model(Z=2.202,P=0.028).Transmission-disequilibrium test(TDT)and sib transmission -disequilibrium test(STDT)showed an excess of the allele of T from heterozygous parents to affected offspring or higher frequency of the allele of T in the patients than their normal siblings(X~2=11.66,P=0.001).Conclu- sion Our findings suggest that the ACE gene may he the susceptible gene to SLE in Chinese population,and the individuals carrying ACE-261T allele is significantly associated with susceptibility to SLE.

OBJECTIVETo observe the effect of acupuncture on proliferation and differentiation of neural stem cells in brain tissues of rats with traumatic brain injuny.

METHODSThirty SD rats were randomly and equally allocated to the sham-operated, the model and the acupuncture groups. The traumatic brain injury model was established by the free drop method. For the rats in the acupuncture group, acupuncture was applied once a day for 7 days. Brain histotomy was carried out when treatments were completed. Immunohistochemical techniques were adopted to detect the cells that express nestin, neurofilament proteins (NF)-200 and glial fibrillary acidic proteins (GFAP), the markers of neural stem cells, neurons, astrocytes respectively.

RESULTSCompared to the sham-operated group, the number of nestin-positive cells and NF-200-positive cells in brain tissues was decreased significantly in the model group (P < 0.01), whereas the number of GFAP-positive cells was significantly increased P<0.01). Compared to the model group, the positive cells of nestin, NF-200, GFAP in brain tissues in the acupuncture group were increased obviously (P<0.01).

CONCLUSIONSAcupuncture can significantly increase the number of nestin-positive cells, NF-200-positive cells and GFAP-positive cells, indicating the significant increase of neural stem cells, neurons and astrocytes in number. Acupuncture can improve neuranagenesis by promoting the proliferation and differentiation of neural stem cells in brain tissues. This might be one of the mechanisms for acupuncture to treat traumatic brain injury and to promote the repair of nervous function.

Acupuncture Therapy ; Animals ; Brain ; pathology ; Brain Injuries ; pathology ; therapy ; Cell Differentiation ; Cell Proliferation ; Cerebral Cortex ; pathology ; Glial Fibrillary Acidic Protein ; metabolism ; Intermediate Filament Proteins ; metabolism ; Male ; Nerve Tissue Proteins ; metabolism ; Nestin ; Neural Stem Cells ; metabolism ; pathology ; Neurofilament Proteins ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the psychological stress in the survivors of the May 12, 2008 Wenchuan earthquake transferred to Guangzhou for treatment, and explore the association between the psychological stress and the physical injuries related to the earthquake to promote both the physical and psychological recoveries of the survivors.

METHODSFifty-five earthquake survivors, including 27 with 28 without physical injuries, were transferred in our hospital 1 week after the earthquake. The Posttraumatic Stress Disorder (PTSD) Checklist-Civilian version (PCL-C) and the Self Reporting Questionnaire (SRQ) were used to investigate the psychological stress in these survivors.

RESULTSThe earthquake caused serious psychological stress in these survivors, and 43.63% of the survivors had high PCL-C scores (50 or above) and 78.18% had high SRQ scores (over 7), with 41.82% having high scores for both PCL-C and SRQ. The survivors with physical injuries generally had high PCL-C and SRQ scores (49.780-/+14.503 and 12.110-/+4.619, respectively), which, however, were not statistically different from the scores of the survivors without injuries (44.460-/+13.206 and 10.460-/+5.000, respectively, P>0.05). The injured survivors showed high scores for the subscales of persistent avoidance symptoms and of anger and irritability, in which the item scores for "efforts to avoid activities or places that arouse recollections of the earthquake" and "feeling of detachment or estrangement from others" were significantly different between the survivor with physical injuries and those without (P=0.034 and 0.018, respectively).

CONCLUSIONThe Wenchuan earthquake causes serious psychological stress in the survivors, and the wounded are more inclined to have persistent avoidance symptoms and enhanced irritability. In addition to the care of the physical injuries, psychological counseling and social support may help ameliorate the psychological trauma in the survivors and reduce the incidence of PTSD.

Adolescent ; Adult ; Aged ; China ; Earthquakes ; Female ; Humans ; Male ; Middle Aged ; Stress, Psychological ; psychology ; Surveys and Questionnaires ; Survivors ; psychology ; Wounds and Injuries ; psychology ; Young Adult

OBJECTIVETo separate the cell subpopulation with high tumorigenic ability and study the biological characteristics of this subpopulation in laryngeal carcinoma cells.

METHODSHuman laryngeal carcinoma cells were obtained by primary tissue culture technique. CD44 and CD133 molecules were used as markers to isolate the CD44(+), CD133(+), CD44(+)CD133(+) and CD44(+)CD133(-) cell subpopulations from the laryngeal carcinoma cells by flow cytometry. A nude mouse tumor xenograft model was developed for the study of the tumorigenic effects of the different cell populations. 1 x 10(6), 1 x 10(5), 1 x 10(4) and 1 x 10(3) cells were injected into the left flank of the mice, respectively. The mice were observed for palpable tumor formation and were sacrificed at 4 weeks later to assess the tumor formation rate, tumor volume and tumor weight. Boyden chamber migration assay was used to determine the migration ability and immunochemistry was used to detect the expression of stem cell antigen SCA-1 and beta1-integrin. Semi-quantities RT-PCR and Western blot analysis were performed to detect the expression level of Bmi-1 in the different cell subpopulations.

RESULTSThe growth of subcutaneous tumors in nude mice showed that a tumor can be generated with 1 x 10(3) CD44(+)CD133(+) cells. When the same dose of 1 x 10(6) CD44(+)CD133(+) cells was injected into the mice, both the average weight and volume of the tumors were significantly higher than those generated from other cell subpopulations. Boyden chamber migration assay showed that the invasion ability of CD44(+)CD133(+) cells was significantly higher than that of other cell subsets. The results of immunochemical analysis showed an abundant expression of stem cell antigen SCA-1 and beta1-integrin in the CD44(+)CD133(+) cells. Semi-quantitative RT-PCR and Western blot analysis provided strong evidence that the Bmi-1 expression in CD44(+)CD133(+) and CD133(+) cells was very significantly higher than that in CD44(+), CD44(+)CD133(-) and control cells (P < 0.01).

CONCLUSIONOur findings demonstrate that CD44(+)CD133(+) subset cells in laryngeal carcinoma posses some biological characteristics of tumor stem cells, which may be the original cells of laryngeal carcinoma and may become a new target of tumor therapy.

AC133 Antigen ; Animals ; Antigens, CD ; analysis ; Antigens, Ly ; metabolism ; Cell Adhesion ; Gene Expression Regulation, Neoplastic ; Glycoproteins ; analysis ; Humans ; Hyaluronan Receptors ; analysis ; Integrin beta1 ; metabolism ; Laryngeal Neoplasms ; immunology ; metabolism ; pathology ; Male ; Membrane Proteins ; metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Nuclear Proteins ; genetics ; metabolism ; Peptides ; analysis ; Polycomb Repressive Complex 1 ; Proto-Oncogene Proteins ; genetics ; metabolism ; RNA ; metabolism ; Repressor Proteins ; genetics ; metabolism ; Tumor Burden ; Tumor Cells, Cultured

OBJECTIVETo evaluate a retro-grade auricular flap for repairing large alar defects.

METHODSTwenty-nine adult cadavers were anatomically used for vascular investigation of the frontal, nasal and temporal regions by injecting a dye into the main vessels. Based on the anatomical study in these regions, a retro-grade auricular flap was designed for repairing alar defects in 16 patients.

RESULTSThe blood supply of the auricle could be nourished by the inner carotid artery system from the supratrachlear artery and supraorbital artery through the frontal arterial anastomotical network into the frontal branch of the superficial temporal artery. It is then passing the main trunk of the superficial temporal artery into the ear area through the auricular branches of the superficial temporal artery. The retro-grade auricular island flap could be formed by basing the supratrachlear artery and the supraorbital artery through the vascular network between the superficial temporal artery and the supratrachlear artery or the supraorbital artery. Sixteen patients with large alar defects and half-sized nasal defects were successfully repaired by this technique.

CONCLUSIONSThe retro-grade auricular island flap, based on the inner carotid artery system, could be a good and safe flap for repairing a large alar defect or half-sized nasal reconstruction.

Adult ; Arteries ; Cadaver ; Carotid Artery, Internal ; Ear Auricle ; blood supply ; Humans ; Nose Deformities, Acquired ; surgery ; Ophthalmic Artery ; Reconstructive Surgical Procedures ; Surgical Flaps ; blood supply ; transplantation ; Temporal Arteries

OBJECTIVETo clone pgmA gene of Porphyromonas gingivalis, to construct the expression vector of the gene and to identify immunity of the fusion protein.

METHODSThe pgmA genes from ATCC 33277 and 47A-1 strains of P.gingivalis were amplified by high fidelity PCR. The nucleotide of the target DNA amplification fragments were sequenced after T-A cloning. The pET32a expression vectors inserted with pgmA gene fragments were constructed. PgmA fusion protein was expressed in E.coli strain BL21DE3 induced by IPTG with different dosages. Western blot test by using rabbit antiserum against the fusion protein was applied to determine immunity of the fusion protein. ELISA was applied to determine the immunoreaction of antibody against PgmA fusion protein and 65 strains of P.gingivalis isolates.

RESULTThe nucleotide sequence homology of the cloned pgmA gene fragments from ATCC 33277 and 47A-1 strains was 100%. In comparison with the reported corresponding sequences, the homologies of the nucleotide sequences of the cloned pgmA gene fragments were 98.98%, while the homologies of their putative amino acid sequences were 99.18%. The expression output of PgmA fusion protein in pET32a-pgmA-BL21DE3 system was approximately 50% of the total bacterial proteins. PgmA fusion protein was able to induce rabbit to produce specific antibody that could combine with PgmA protein. 92.3% of P. gingivalis isolates (60/65) were able to react with the antibody against PgmA fusion protein.

CONCLUSIONAn expression system of P.gingivalis pgmA gene with high efficiency was established successfully. The expressed PgmA fusion protein possesse satisfied immunogenicity and immunoreactivity,which can be used as a candidate antigen in detection of P.gingivalis and possible development of corresponding vaccine.

Animals ; Bacterial Outer Membrane Proteins ; genetics ; immunology ; Base Sequence ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; Molecular Sequence Data ; Rabbits ; Recombinant Fusion Proteins ; genetics