AIM: To provide experimental evidence for gene therapy of thrombophilia disease, we constructed the eukaryotic expression plasmid with human thrombomodulin (hTM) gene and observed the alteration of hTM expression on the surface of human umbilical vein endothelial cells (HUVECs) with and without the reconstructive plasmid. METHODS: The whole expressive fragment of hTM gene was amplified by PCR from human genome. Both hTM gene and pcDNA3.1(+)/neo empty vector was digested by HindⅢ and EcoRⅠ. Two digested fragments were ligated into pcDNA3.1/hTM with T_4DNA ligase. After identifying, the reconstructive plasmid transfected into HUVECs using lipofectin. The hTM antigen on the HUVECs was detected by immunohistochemistry. RESULTS: The hTM reconstructive plasmid was confirmed by double endonuclease redigesting and sequencing. About 10% HUVECs were transfected by pcDNA3.1/hTM plasmid with lipofectin and the high-level hTM was detected on the transfected cells. CONCLUSION: We constructed the pcDNA3.1/hTM plasmid successfully, and it could be expressed on the HUVECs. [

A comparative study on the glycolic acid oxidase (GO) of the domestic Spirulina(Arthrospira) platensis (S_(1)) from alkaline lake in Erdos Plateau and the imported S. (A.) platensis (S_(2)) and S. (A.) maxima (S_(3)) as well is made with colorimetric method. The results show that activity of GO (25℃, pH 8.0) of S_(1), S_(2 )and S_(3) is 70.9 U/gFW, 59.6 U/gFW and 80.9 U/gFW respectively; the GO's optimum temperature of S_(1)、S_(2) and S_(3 )is 30℃; the GO's optimum pH value of S_(1 )is 8.6,while that of S_(2 ) 8.2 and that of S_(3) 8.4; the GO of S_(1 )is stable from 0℃ to 35℃ and from pH 7.6 to pH 10.0, while that of S_(2) from 0℃ to 30℃ and from pH 8.0 to pH 9.0 and that of S_(3) from 0℃ to 35℃ and from pH 8.0 to pH 8.6. Adaptive range of S_(1) GO for temperature and pH is wider, and activity at low and high temperature and under strong acidand alkali conditions is higher than that of the imported species.

目的探讨脊髓损伤患者尿路感染常见细(真)菌及其对不同抗生素的敏感性和耐药情况。方法取脊髓损伤患者中段尿做尿培养及药敏试验,共计586例次。结果共培养出细(真)菌586株,其中革兰氏阴性杆菌475株(81.1%)、革兰氏阳性球菌83株(14.1%)、真菌28株(4.8%);产ESBL大肠埃希氏菌12株(2.05%)、产ESBL肺炎克雷伯氏菌5株(0.85%)。结论大肠埃希氏菌是导致本组患者尿路感染的最主要细菌,且细菌耐药现象较为严重,应引起临床医生高度重视。

OBJECTIVETo investigate the effects of the plasma containing Qianlongtong Capsule (QLT)-containing plasma on the expression of the Smad4 gene in prostate stromal cells in vitro and provide some experimental evidence for the treatment of benign prostatic hyperplasia (BPH) with Chinese medicinal compound.

METHODSFifteen cases of BPH were equally randomized to three groups to be treated with QLT at a high dose (6 capsules once), a medium dose (3 capsules once), and a low dose (1.5 capsules once), tid, for 7 days consecutively. QLT-containing plasma was collected from the patients. Prostate stromal cells were identified by immunofluorescence when they became monolayered and cultured in the QLT-containing plasma for 24 hours, followed by detection of the expression of the Smad4 gene by real-time quantitative PCR and that of the Smad4 protein by Western blot.

RESULTSAfter treatment with the QLT-containing plasma, the expression of the Smad4 gene in the stromal cells was significantly increased in a dose-dependent manner as compared with the blank control and no-QLT groups (P < 0.01). The expression of the Smad4 protein was also markedly elevated after treatment. The differences were statistically significant between the blank control and medium-dose groups (P < 0.01), low-dose and medium-dose groups (P < 0.05), and high-dose and the other groups (P < 0.01), but not between the blank control and low-dose groups (P > 0.05).

CONCLUSIONQLT-containing plasma could inhibit the proliferation and improve the apoptotic index of prostate stromal cells in vitro, which was related to the elevation of the mRNA and protein expressions of Smad4.

Apoptosis ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Male ; Prostate ; drug effects ; metabolism ; RNA, Messenger ; genetics ; Smad4 Protein ; genetics ; metabolism ; Stromal Cells ; drug effects ; metabolism

OBJECTIVETo investigate the methylation status of zonula occludens-1 (ZO-1) gene promoter and its clinical significance in children with stage IV non-Hodgkin lymphoma (NHL) and to provide a basis for further etiological study and early diagnosis of this disease.

METHODSFifty-five children with a confirmed diagnosis of stage IV NHL (40 cases of T-NHL and 15 cases of B-NHL) were selected as the case group, and 20 children with diseases other than hematologic malignancies were selected as the control group. Bone marrow samples were collected from these subjects. Methylation-specific PCR (MS-PCR) was applied to evaluate the methylation status of ZO-1 gene promoter, and the integrated optical density (IOD) was determined. RT-PCR was used to measure the mRNA expression of ZO-1.

RESULTSMS-PCR showed that the methylated bands of ZO-1 gene promoter were found in 39 (70.9%) of 55 patients in the case group before treatment, while no ZO-1 gene promoter methylation was detected in the control group. With close tracking of 47 cases in the study group, consisting of 32 cases of T-NHL and 15 cases of B-NHL, the rates of ZO-1 gene promoter methylation prior to treatment were 72% and 67%, respectively, (P>0.572). The cases of T-NHL and B-NHL showed no significant changes in methylation rate in the early and middle phases of chemotherapy (P>0.05), but they showed significant changes in methylation rate in the late phase of chemotherapy (P<0.05). RT-PCR showed that the NHL cases carrying methylated ZO-1 gene had no mRNA expression of ZO-1, while all children in the control group had mRNA expression of ZO-1. There was no linear relationship between the total number of peripheral blood leukocytes and ZO-1 gene IOD (r=0.093, P=0.575); a positive correlation was found between the number of malignant cells in bone marrow and ZO-1 gene IOD (r=0.669, P<0.001).

CONCLUSIONSZO-1 gene shows a hypermethylation status in children with NHL, and the methylation level is positively correlated with the number of malignant cells in bone marrow. ZO-1 may be used as a novel molecular marker in early diagnosis, outcome assessment, prognostic evaluation, and detection of minimal residual disease.

Adolescent ; Child ; Child, Preschool ; DNA Methylation ; Female ; Humans ; Infant ; Lymphoma, Non-Hodgkin ; genetics ; Male ; Promoter Regions, Genetic ; Zonula Occludens-1 Protein ; genetics

OBJECTIVETo develop a RP-HPLC method for the determination of the content of macrozamin in Rhizoma Heterosmilacis Japonicae.

METHODA Century C18 AQ column (4.6 mm x 250 mm, 5 microm) was used with the mobile phase consisted of water (4:96). The flow rate was 1.0 mL x min(-1). The detection wavelength was set at 215 nm, and the column temperature was 35 degrees C.

RESULTThe calibration curve was linear (r = 0.999 8) in the range of 19.12 - 382.4 microg x mL(-1) for macrozamin, the average recovery of the method was 99.5%, with RSD 2.1% (n = 9).

CONCLUSIONThis method can be used for the quality study of Rhizoma Heterosmilacis Japonicae.

Chromatography, High Pressure Liquid ; methods ; Liliaceae ; chemistry ; Methylazoxymethanol Acetate ; analogs & derivatives ; analysis ; Plants, Medicinal ; chemistry ; Reproducibility of Results ; Rhizome ; chemistry

Aim To discuss the effects and mechanism of Ganoderma lucidum polysaccharides and metformin on myocardial structure and hemodynamics in type 2 diabetic rats.Methods High fat diet combined with intraperitoneal injection of low dose streptozotocin 30 mg· kg -1 was applied to establish rat model of type 2 diabetes mellitus .The diabetic rats were randomly into normal control group ,diabetes group , ganoderma lucid-um polysaccharides group (600 mg· kg -1 ) , metformin group ( 600 mg · kg -1 ) , combination group ( ganoder-ma lucidum polysaccharides 300 mg · kg -1 +metform-in 300 mg· kg -1 ) .After 12 weeks′treatment,the lev-els of fasting serum glucose were determined and the hemodynamic parameters (LVSP,LVEDP,dp/dtmax,-dp/dtmax ) were determined.Collagen volume fraction ( CVF ) was detected by Van Gieson . Immunohisto-chemical method and Western blot were used to detect myocardial tissue MMP-2 protein expression .Results The fasting blood glucose was significantly decreased in the combined treatment group .Combined medication could significantly improve hemodynamic parameters in diabetic rats: reduced LVEP and raised LVEDP , dp/dtmax and -dp/dtmax .CVF was significantly decreased in combination group .The expression of MMP-2 in my-ocardial tissue was significantly inhibited .Conclusions The combination of Ganoderma lucidum polysaccha-ride and metformin can significantly improve the hemo-dynamic parameters in type 2 diabetic rats, and have a preventive effect on diabetic cardiomyopathy . The mechanism may be related to the down regulation of the expression of MMP-2.

ObjectiveTo evaluate the clinical safety and efficacy of SilverHawk directional atherectomy for femoropopliteal occlusive lesions. MethodsEighteen ischemia occlusive lesions in 11 patients of the lower extremity were treated with SilverHawk directional atherectomy.The mean lesion number was 1.6 ± 1. 1 per patient. The mean lesion length was ( 3.4 ± 2. 2 ) cm. The average degree of diameter stenosis was 96％ ± 14％. 9 lesions were totally occlusive. Clinical symptoms included claudication in 4 cases ( Rutherford classes: 3) and critical limb ischemia ( Rutherford classes: 4) in 7 cases. Lesions characteristics were divided by TASC classification: TASC B in 7 cases; TASC C in 1 case (in-stent occlusion); TASC D in 3 cases.Mean ABI was 0. 5± 0.4. Patency was evaluated with color duplex sonography or CTA besides clinical examination during follow-up.ResultsNine totally occlusive lesions were recanalizated successfully via intraluminal approach. 18 lesions achieved technical success (residual stenosis ＜50％ ) leaving 15％ ±7％ mean residual stenosis in mean (8 ±3)min, predilation was needed in one lesion ( in-stent occlusion) prior to atherectomy. Clinical symptoms improved or disappeared with mean ABI 1.07 ±0. 12 and Rutherford grades: 0 (n =9) and 1 (n =2). Patency rate was 100％ with mean 0. 93 ± 0. 14 ABI and Rutherford grades remain unchanged after follow-up of mean ( 9 ± 4 ) monthes.ConclusionsSilverHawk directional atherectomy is safe and effective for the treatment of lower extremity ischemia.

ObjectiveTo evaluate the clinical application of endovascular treatment for severe Takayasu's arteritis (TA).MethodsIn this study,35 target lesions in 32 patients [28 women,mean age (30 ±8) years] with severe Takayasu's arteritis were treated with endovascular merthod.The average length of lesion was 3.1 cm( range 2.7 -5.3).The overall average degree of diameter stenosis was 90％ ± 11％ (range 70- 100)in which 15 lesions were completely occlusive.There were 10 patients whose ESR were higher than 20 mm/h( range 25 -37).Follow-up included physical examination and patency evaluated by color duplex souography/computed tomography angiography/angiography at 6 months and then annually.ResultsRecanalization was unsuccessful in 3 completely occlusive lesions,with a successful rate of 80％(12/15).There was one case in which embolization leading to acute thrombogenesis developed during interventional procedure and resulting in severe stroke.The technical successful rate ( residual stenosis ＜ 50％ ) was 88.6％ ( 31/35 ).The transient cerebral ischemia attack ( TIA ) symptoms disappeared in 31 cases.26 cases were followed up for an average of (19 ± 10) months (range 13 -40).Occipital infarction following severe in-stent restenosis developed 13 months later in one case.Symptomatic in-stent restenosis18monthslaterwasfoundin2cases. Patencyratewas88.5％( 23/26 ).ConclusionsEndovascular treatment is safe and effective for severe TA.Strict indication and accurate targeting the lesions help ensure the success of management.

Aim To study the effects of ganoderma lu-cidum polysaccharides and metformin on myocardial fi-brosis of type 2 diabetic rats and its mechanism. Methods SD rats were fed with high fat diet for 4 weeks, and then were injected with streptozotocin (30mg·kg-1 ) to replicate type 2 diabetic model. The diabetic rats were randomized into normal control group,diabetes group, ganoderma lucidum polysaccha-rides group ( 600 mg · kg-1 ) , metformin group ( 600 mg·kg-1 ) , and combination group( ganoderma lucid-um polysaccharides 300 mg·kg-1 +metformin 300 mg ·kg-1 ) . After 12 weeks’ treatment,the levels of fast-ing serum glucose were determined and the extent of myocardial fibrosis was observed by Picro-sirius red staining. The contents of AGEs in serum were deter-mined by fluorescence spectrophotometer. The activities of CAT and GSH-Px in myocardium were detected. Im-munohistochemical method and Western blot were used to detect myocardial tissue AGEs and CTGF protein ex-pression. Results Combination group could repress patho-proceeding of myocardial fibrosis efficiently, im-prove the activity of CAT and GSH-Px in myocardium and lower the concentration of AGEs in serum, as well as reduce the expression of AGEs and CTGF in myo-cardium. Conclusions Ganoderma lucidum polysac-charides and metformin could prevent myocardial fibro-sis. The possible mechanism may be related to repress-ing oxidative stress of myocardium, lowering serum AGEs and down regulating AGEs and CTGF of myocar-dium.