Objective To screen polymorphisms and mutations in the promoter region of WD gene.Methods DNA from peripheral blood was obtained from 71 subjects of 36 family (48 WD patients,23 patients first-degree relatives) and 20 healthy people from Feb.2001 to Feb.2004.DNA sequence of the genes was analyzed by PCR amplification and direct genomic sequencing.Results There were three polymorphisms at positions-190,-78,+260(transcription start site as +1) of the promoter region of WD gene.Normal controls,WD patients and patients’ first-degree relatives all showed the polymorphisms;three of 48 WD patients presented C→T base substitution mutations at the same position -183:two were homozygous mutation,while the other was heterozygous.Normal control subjects and patients' relatives didn’t show this kind of mutation.Conclusion It suggests that the mutation of the promoter region is one of WD pathogenesis.

Objective 　To study the sequence and structure of intron 8 in WD gene in order to further understand the relationship between intron 8 and WD. Methods　We utilized polymerase chain reaction (PCR) to the amplification of exon 8-intron 8-exon 9 which were then sequenced by a dideoxy chain termination methon in 10 normal controls and 32 members of 11 families(20 WD patients and 10 of their relative). The results were analyzed by the computer. Results　The sequence of intron 8 was 703 bp with the G　+　C content of 42.7%. There were one short tandom repeats, 7 direct and inverted repeats in it. An open reading frame coded with 82aa was found at 323 base pairs of downstream of a TATAbox. There were two DNA polymorphisms at 408 and 487 nucleotides. The sequence analysis showed that the 5end has the sequence of 5-GTAAC, 3end has the sequence of CCTAG-3, and branchpoint of 5-TTTCGA-3.Conclusions　The sequences and structures of intron 8 in WD familiess members are not different from normal controls. Our data suggest that the WD gene intron 8 might not play an important role in the pathogenesis of WD.

Objective To observe the role of chronic inflammation in the development of atherosclerosis (AS) by analyzing the expression of TLR4 and NF-κB in artery endothelium. Methods To construct the atherosclerotic animal model, the balloon catheter was used to injure common carotid artery and rabbits were fed the high cholesterol diet. All the rabbits were divided into three groups: control group with the normal diet, high cholesterol diet-fed group and model group (balloon-injured common carotid artery and the high cholesterol diet fed rabbits). The rabbits were sacrificed after 8 weeks and their tissues were collected. Then morphological changes of rabbit common carotid artery were observed by light microscope. The expression of TLR4 and NF-κB in endarterium was displayed using immunochemistry method. Results Both hyperlipidemia and exterior inflammatory stimulation promoted the expression of TLR4 and NF-κB in vascular endothelium (P ＜ 0.01 ). And when both of them were present, the level of TLR4 and NF-κB expression would get higher even than that affected by one of them( P ＜ 0.01 ). Conclusion Both hyperlipidemia and chronic inflammatory process can improve the expression of TLR4 and NF- κB in vascular endothelium in different degree; the inflammatory stimulation would promote the atherosclerosis to some extent; TLR4/ NF-κB would play a role as a bridge between the internal environment changes and the arterial morphological changes.

Objective: To explore the relationship among somatostatin (SS), neuron-specific enolase (NSE) and early vascular dementia.Methods: A total of 40 patients with early vascular dementia treated in our hospital were selected as vascular dementia group, another 40 inpatients with cerebral infarction (CI) treated during the same period were enrolled as CI group.Plasma NSE and SS levels were compared between two groups during different periods.Results: Compared with CI group at onset, one month and three months after CI, there was significant rise in plasma NSE level[(22.08±7.05) ng/ml vs.(26.39±6.80) ng/ml, (23.92±4.25) ng/ml vs.(28.12±4.06) ng/ml, (25.55±4.72) ng/ml vs.(30.10±4.33) ng/ml], and significant reduction in plasma SS level[(1084.50±133.00) ng/ml vs.(748.30±129.10) ng/ml, (836.40±160.20) ng/ml vs.(624.25±140.50) ng/ml, (690.25±146.30) ng/ml vs.(432.70±151.00) ng/ml]in vascular dementia group, P<0.05 or <0.01.Plasma NSE level gradually rose and SS level gradually reduced along with the time went by(P<0.05 or <0.01).Conclusion: Early dynamic detection of somatostatin and neuron-specific enolase levels in patients with cerebral infarction may help to early diagnosing and treatment of vascular dementia.

Objective 　Determination of Wilson disease gene mRNA expression in human fibroblast cell strain (Me32aT22/2L) by reverse transcription-polymerase chain reaction (RT-PCR). Methods　Using lipofection reagent, the plasmid vector carrying the Wilson disease gene (pRc/CMV-WD) was transferred into Me32aT22/2L cultured in serum free complement medium. RT-PCR was used to determine WD mRNA expression in Me32aT22/2L. Results　 Wilson disease gene expression was detected in Me32aT22/2L, while no specific signals were detected in untransfected fibroblast. Conclusions　It demonstrated that Me32aT22/2L strain could express the Wilson disease gene, suggesting that Wilson disease gene transfer might develop a new approach to study Wilson disease.

] AIM: Lysophosphatidic acid (LPA) is a bioactive phospholipid known to have growth factor-like activity on fibroblasts, and is involved in cardiovascular diseases. Besides direct effects, usually, LPA can work together with other bioactive factors to regulate cardiovascular homeostasis by induction of their expression and production, or increase in their activity. Among variety of bioactive factors, adrenomedullin (ADM) is a multifunctional peptide with an important cytoprotective effect against cardiovascular damage, but the interaction between ADM and LPA on adventitia remains unknown. METHODS: The experiment was performed on the bath of isolated rat aortic adventitia, ADM produced and secreted from adventitia stimulated by LPA was detected by using radioimmunoassay, proliferation in adventitia cells was evaluated by the level of [3H]-thymine incorporation, and prepro ADM gene expression was measured by semi-quantitative reverse transcriptase polymerase chain reaction. RESULTS: It was found that LPA stimulated aortic adventitia to secrete ADM and express its mRNA in a concentration-dependent manner. ADM inhibited LPA-induced proliferation in adventitial cells, and attenuated the activity of mitogen activated protein kinase (MAPK) stimulated by LPA. In contrast, the treatment with specific antagonists of ADM receptor potentiated the LPA-induced proliferation in adventitial cells. CONCLUSION: LPA stimulates adventitia to produce and secrete ADM, and in turn, ADM produced by adventitia regulates the vascular biological effects of LPA. [

Objective To explore the methods and the effect of the surgical treatment of patients with chronic ischemia of extremities.Methods In recent 6 years,the clinical data of 75 patients,including 7 upper(extremities) and 71 lower extremities with chronic ischemia,treated surgically were reviewed retrospectively.The surgical treatment included: traditional operation in 65 cases,interventional surgery in 4 cases and stem cell transplantation in 6 cases.Results Sixty-three patients(66 extremities) were followed up from 2 to 56 months after the surgical treatment,and the result showed that 9 extremities of 8 patients were amputated(within) 1 year(13.6%).In the other cases(83.3%),the ischemic symptoms were relieved or(disappeared),the ulcer healed or the amputating level had lowered(3.0%).The effective rate of the surgical treatment was 86.3%.Conclusions The appropriate surgical treatment of chronic ischemia limbs should be choiced based on the ischemic state of the limb,and a satisfactory result can be obtained in most patients.

Objective:To investigate the relationship between caspase recruitment domain protein 9 (CARD9) level and inflammatory response in cerebral tissue of ischemic brain injury mice.Methods:Totally 24 SPF BALB/c male mice were randomly(random number) divided into 4 groups: sham operated group, ischemia 3 h group, ischemia 6 h group, and ischemia 12 h group, 6 mice in each group. The permanant middle cerebral artery occlusion (pMCAO) model in the ischemia groups was established by using line embolism to block blood flow. Mice in each group were sacrificed at the predetermined time point after operation. CARD9 and p-p65NF-κB levels were detected by Western blot, and the inflammatory factors mRNA and protein including TNF-ɑ, IL-lβ and IL-6 were detected by RT-PCR and ELISA, respectively. The data were analyzed by SPSS 21.0 software, the comparison of measurement data between each two groups was analyzed by independent sample t test, and the correlations between CARD9 and inflammatory factors were analyzed by Pearson analysis. Results:Compared with the sham operated group, the CARD9 levels in the ischemia 3 h, 6 h and 12 h groups were increased significantly [(0.325±0.011) vs. (0.462±0.019), P=0.036; (0.735±0.036), P=0.003; (0.903±0.024), P=0.001], the p-p65NF-κB levels in the ischemia 3 h, 6 h and 12 h groups were increased significantly [(0.227±0.016) vs. (0.316±0.017), P=0.041; (0.445±0.021), P=0.016; (0.671±0.039), P=0.008], the TNF-ɑ levels in the ischemia 3 h, 6 h and 12 h groups were significantly increased [(0.53±0.06) vs. (1.06±0.10), P=0.009; (1.47±0.15), P=0.004; (2.78±0.18), P=0.001], the IL-lβ levels in the ischemia 3 h, 6 h and 12 h groups were significantly increased [(0.55±0.07) vs. (1.01±0.11), P=0.009; (2.13±0.16), P=0.003; (3.09±0.18), P=0.001], and the IL-6 levels in the ischemia 3 h, 6 h and 12 h groups were significantly increased [(1.99±0.18) vs. (4.10±0.41), P=0.006; (8.54±0.84), P=0.002; (11.56±0.96), P=0.001]. Pearson analysis showed that CARD9 was positively correlated with the p-p65NF-κB and TNF-ɑ, IL-lβ, IL-6 ( r=0.894, P=0.001; r=0.747, P=0.008; r=0.810, P=0.001; r=0.773, P=0.007). Conclusions:A positive correlation exists between CARD9 and inflammatory responses in the early stage of ischemic brain injury in mice

Objective: To investigate the community promotion feasibility of superficial needling plus club swing for post-stroke motion impairment of the shoulder joint. Methods:A total of 180 cases (duration <1.5 years) with post-stroke motion impairment of the shoulder joint were recruited from three community health centers in Changning District, 60 from each community. They were randomly allocated into an observation group (n=90) and a control group (n=90). Patients in both groups received standard internal and rehabilitation care. Patients in the observation group received additional superficial needling plus club swing. The visual analogue scale (VAS) was conducted before and 60 d after the treatment to evaluate the severity of shoulder pain. The active movement of the shoulder joint and activities of daily living (ADL) were also observed. Results:There were no between-group statistical differences before the treatment (allP>0.05). After a 60-day treatment, the shoulder pain severity, active range of motion of the shoulder joint and ADL in the observation group were significantly improved than those in the control group (allP<0.01). In addition, no adverse events were reported by participants in the observation group. Conclusion:Superficial needling plus club swing plays a positive role in improving post-stroke motion impairment of the shoulder joint. This safe, reliable and economical therapy has good patient compliance and is suitable for community promotion.

OBJECTIVETo investigate the expressions of gamma aminobutyric acid transporter 1 (GAT-1) and glutamate decarboxylase 65 (GAD65) mRNA in different brain regions at brain propofol uptake equilibrium in dogs.

METHODSEighteen 12- to 18-month-old healthy hybrid dogs were randomized equally into control group (group C), low dose group (group L), and high dose group (group H). In groups L and H, anesthesia was administered by intravenous injection of 5.5 and 7.0 mg/kg propofol followed by propofol infusion at a constant rate of 55 and 70 mg·kg(-1)·h(-1) for 50 min, respectively. Blood samples were taken from the internal carotid artery and jugular vein to measure plasma propofol concentrations, and the brain tissues of the hypothalamus, sub thalamus, dorsal thalamus, hippocampus, pons, parietal lobe and frontal lobe were examined for GAT-1 and GAD65 mRNA expressions using quantitative real-time PCR.

RESULTSIn groups L and H, propofol infusion at a constant rate for 50 min resulted in comparable plasma propofol concentrations between the internal carotid artery and jugular vein (P>0.05), but the concentrations differed significantly between the two groups (P<0.01). GAT-1 mRNA levels in the hypothalamus and hippocampus were significantly higher in groups L and H than in group C (P<0.05 and P<0.01), but comparable between the former two groups. The variations of GAT-1 mRNA levels between the hypothalamus and hippocampus were similar in both group L [(61.26∓7.17)% and (79.34∓39.95)%, P>0.05] and group H [(74.64∓19.63)% and (97.12∓32.31)%, P>0.05]. GAT-1 mRNA levels in other brain regions showed no significant difference among the 3 groups. GAD65 mRNA levels were similar between group L and group H, but both significantly higher than that in group C (P<0.01). GAD65 mRNA in other brain regions had no significant difference among the 3 groups.

CONCLUSIONGAT-1 mRNA in the hypothalamus and hippocampus and GAD65 mRNA in the dorsal thalamus are upregulated when propofol uptake reaches an equilibrium in the brain of dogs.

Animals ; Brain ; drug effects ; metabolism ; Dogs ; GABA Plasma Membrane Transport Proteins ; genetics ; metabolism ; Glutamate Decarboxylase ; genetics ; metabolism ; Hippocampus ; drug effects ; metabolism ; Hypothalamus ; drug effects ; metabolism ; Propofol ; pharmacology ; RNA, Messenger ; genetics ; Thalamus ; drug effects ; metabolism