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 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 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

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.

Objective To study the effect of ganoderma lucidum polysaccharides combined with metformin on oxidative stress of type 2 diabetic rats. Methods SD rats were fed with high fat diet for 4 weeks and injected with streptozotocin (30 mg·kg-1 ) to produce type 2 diabetic model. The diabetic rats were randomly divided into diabetes model group, ganoderma lucidum polysaccharides group (600 mg·kg-1 ), metformin group (600 mg·kg-1 ), combination group (ganoderma lucidum polysaccharides 300 mg·kg-1+ metformin 300 mg·kg-1 ), After 12 weeks of treatment, the level of fasting blood glucose was determined, and the activity of superoxide dismutase ( SOD), malondialdehyde ( MDA), catalase ( CAT), glutathione peroxidase (GSH-Px), total cholesterol (TC) and triglyceride (TG) were detected. Results The levels of fasting blood glucose in the treatment groups were significantly lower than that in the diabetes model group (P<0. 01). Furthermore, fasting blood glucose in the combination group was significantly lower than that in ganoderma lucidum polysaccharides group and metformin group (P<0. 01). Compared with diabetes model group, serum TC and TG in the treatment groups were significantly lower (P<0. 05, P<0. 01). Serum TC and TG were significantly lower in the combination group than in ganoderma lucidum polysaccharides group and metformin group (P<0. 05, P<0. 01). Compared with diabetes model group, serum SOD levels in the treatment groups were significantly higher (P<0. 01). Compared with ganoderma lucidum polysaccharides group and metformin group, serum SOD levels in the combination group was significantly higher (P<0. 05). Compared with diabetes group, serum MDA levels in the treatment groups were significantly lower (P<0. 01). Serum MDA in the combination group was significantly lower than that in ganoderma lucidum polysaccharides group and metformin group ( P<0. 05). Compared with diabetes model group, serum CAT and GSH-Px in the treatment groups were significantly higher (P<0. 05, P<0. 01). Serum CAT and GSH-Px in the combination group were significantly higher than those in ganoderma lucidum polysaccharides group and metformin group (P<0. 05). Conclusion Ganoderma lucidum polysaccharides combined with metformin could effectively inhibit oxidantion stress in type 2 diabetic rats. The effect was better than ganoderma lucidum polysaccharides or metformin used alone. The possible mechanism may be related to increased activity of SOD, CAT, GSH-Px in vivo and regulation of dyslipidemia.

Aim To investigate the effects and mecha of ganoderma lucidum polysaccharides and metformin on pathological changes of thoracic aorta in diabetic ratsandthemechanisms.Methods SDratswerefed with high fat diet for 4 weeks and injected with strepto-zotocin ( 30 mg · kg-1 ) to replicate type 2 diabetic model. The diabetic rats were randomly into diabetes group, ganoderma lucidum polysaccharides group ( 600 mg·kg-1 ) ,metformin group(600 mg·kg-1 ) ,combi-nation group ( ganoderma lucidum polysaccharides 300 mg· kg-1 + metformin 300 mg · kg-1 ) and normal control group. After 12 weeksˊ treatment, the levels of fasting serum glucose, the activity of catalase(CAT), glutathione peroxidase ( GSH-Px ) , total cholesterol (TC)and triglyceride(TG) in serum were detected. Pathological changes of thoracic aorta were observed by HE staining. Immunohistochemy and Western blot were used to detect thoracic aorta VEGF protein expression. Results Combination group could lower fasting serum glucose and blood fat significantly, meanwhile the ac-tivity of CAT and GSH-Px in serum was improved. The expression of VEGF in thoracic aorta was repressed. The result of HE staining suggested that the lipid de-posits in aortic endothelium in combination group were lessthanthoseinthemodelgroup.Conclusions Ga-noderma lucidum polysaccharides combined with met-formin has an obvious prevention on pathological chan-ges of thoracic aorta in diabetic rats. The possible mechanism may be related to repressing oxidative stress of thoracic aorta, regulating the dyslipidemia, and the down regulation of the expression of VEGF in thoracic aorta.

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

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