Objective:To investigate the effect of dimethyl amiloride (DMA) on invasive activity of PGCL3 cells from a human highly-metastatic lung carcinoma cell line in vitro and elucidate its possible mechanism. Methods:The invasion and migration capacities of PGCL3 cells were measured by using Transwell chamber assay after pretreatment with DMA. The effects of DMA on the activity of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) secreted by PGCL3 cells were measured by chromogenic substrate assay. The effects of DMA on uPA, urokinase-type plasminogen activator receptor (uPAR), and PAI-1 mRNAs transcription were determined by RT-PCR. The expression levels of extracellular regulated protein kinases 2 (ERK2) and ras protein were assessed by Western blot. Results:DMA inhibited invasion and migration capabilities of PGCL3 cells in vitro, down-regulated the mRNA transcription of uPA, uPAR and PAI-1, as well as up-regulated the expression of ras protein. After 24-hour treatment, DMA reduced the activity of uPA at higher concentration, but DMA had no effects on the activity of secreted PAI-1 protein and expression of ERK2 protein. Conclusion:DMA inhibits the invasion and migration of highly-metastatic lung cancer PGCL3 cells. The mechanism might be associated with down-regulation of the expression of uPA system.

Objective: To investigate the effect of amiloride on in vitro invasive activity and uPA (urokinase-type plasminogen activator)system of human highly metastatic lung carcinoma cell line PGCL3. Methods: At 6 hours after treatment with amiloride at the concentrations of 25μmol/L，50μmol/L and 100μmol/L for PGCL3 cells，Transwell Chamber assay was performed to detect the effect of amiloride on the invasive and migratory capacity of PGCL3 cells.Effect of amiloride on the activity of uPA and PAI-1(plasminogen activator inhibitor-1)secreted by PGCL3 cells were measured by chromogenic substrate assay after PGCL3 cells were incubated with amiloride for 24 hours.RT-PCR was used to analyze the effect of amilorede on mRNA levels of uPA，uPAR(urokinase-type plasminogen activator receptor)and PAI-1.The expression levels of uPA，ERK2(extracellular regulated protein kinases 2)and ras protein were assessed by Western blot. Results: The number of cells through membrane was significantly decreased in invasion and migration test in vitro.The inhibitory rates of invasion and migration after treatment with amiloride of 100μmol/L were 37.7%±4.1%and 64.9%±4.9%.respectively,with a significant difference from those in the control group(P<0.01).At 24 hours after amiloride treatment，the chromogenic substrate assay showed direct inhibition of the activity of uPA and PAI-1 secreted by PGCL3 cells.No effect on the expression of uPAR in mRNA level was observed,but the expression of PAI-1 in mRNA level was significantly inhibited.Amiloride of 100μmol/L dramatically inhibited the expression of uPA mRNA.The expression level of uPA protein was decreased with the increase of the concentration of amiloride,but no effect was observed on the expression of ERK2 and ras in protein level.Conclusion: Amiloride can inhibit the invasion and migration of PGCL3 cells,through inhibiting the expression and activity of uPA and PAI-1.Amiloride is a potential agent to inhibit cancer invasion and metastasis.

Objective To investigate the clinical values of NT-proBNP in left ventricular enlargement(LVE) and left ventricular dysfunction(LVD) of the patients with essential hypertension(EH) to provide a diagnostic basis for their diagnosis .Methods Plas-ma NT-proBNP concentrations in 120 patients with EH and in 29 normal controls were measured ,then the echocardiography exami-nation was performed to determine the left ventricular ejection fraction ( LVEF) ,left ventricular end-diastolic diameter(LVEDD) , left atrium(LA) and left ventricular systolic diameter(LVSDD) .The correlation between plasma NT-proBNP with LVE and LVD was analyzed .The diagnostic accuracy of NT-proBNP was analyzed by using receiver operating characteristic (ROC) curve .Results There were no statistically significant differences in the aspects of age ,sex and serum creatinine between the EH group and con-trol group ,but plasma NT-proBNP level of the former was significantly higher than that of the latter .The NT-proBNP level in the patients with LVE was significantly higher than that in the patients with normal left ventricle (P<0 .05) .The NT-proBNP level in the LVD patients was significantly higher than that in the patients with normal left ventricular function (P<0 .05) .The plasma NT-p roBNP level was positively correlated with LA ,LVEDD and LVSDD(r=0 .518 ,0 .58 ,0 .48 ,P<0 .01) while negatively crrelated with LVEF(r= -0 .61 ,P<0 .01) .The ROC curve showed that when the NT-proBNP was set at 380 pg/mL ,the sensitivity and specificity of NT-proBNP for diagnosing LVE were 80 .6% and 72 .4% ;which for diagnosing LVD were 80 .8% and 77 .4% ,re-spectively .Conclusion NT-proBNP is closely correlated with multiple ultrasonic indicators reflecting the left ventricular function and its level can reliably reflect the left ventricular contraction function ,which can serve as the marker for screening LVE and LVD in the patients with EH .

Objective: To study the pharmacological action and clinical curative effect of Linglongqingre microenema. Methods: The actions of relieving fever and calming hypnotism. anti convulsions of Linglongqingre Microenema on rabbit, mouse and rat were observed. Also children's fever illess was treated to judge preliminary curative effect. Results: Linglongqingre Microenema has obvious action of relieving fever, more effective than Chaihu injection, as same as Antipyrine (80mg/kg) on subcutaneous injection. It was used to treat children's feverillness on 38 cases, markedly effective rate 60.53%, total effective rate 86.84%. Among them body temperature of one third case fell over 1.5?C in two hours. Conclusion: Linglongqingre microenema is a new quick effective pharmaceutical preparation which is suitable for emergency treatment of traditional Chinese medicine

OBJECTIVE To explore the resistance of Pseudomonas aeruginosa isolated in clinic against five antiseptics involving in povidone iodine(Iodophor),glutaraldehyde,chlorhexidine,symclosene(trichloroisocyanurate) and benzalkonium bromide.METHODS The susceptibility test of P.aeruginosa was determined by PhoenixTM-100 system.Minimun inhibitory concentration(MIC) of povidone iodine,glutaraldehyde,chlorhexidine,symclosene and benzalkonium bromide was detected by liquid dilution method.RESULTS The resistant rates of ampicillin/sulbactam,chloramphenicol,tetracycline and trimethoprim-sulfamethoxazole in 190 isolates of P.aeruginosa were all more than 98.0%.However,P.aeruginosa was to imipenem and meropenem were 15.3% and 6.8%.It was found that P.aeruginosa possessed the most resistant to glutaraldehyde and symclosene with its MIC50 being 32 ?g/ml and 64 ?g/ml.But the MIC50 of chlorhexidine and benzalkonium bromide were only 1 ?g/ml and 2.4 ?g/ml.Meanwhile,time-kill assays indicated that chlorhexidine could still produce rapid and powerful bactericidal effects at a concentration of 1MIC after 10 min treatment.CONCLUSIONS There are distinct differences in P.aeruginosa against povidone iodine,glutaraldehyde,chlorhexidine,symclosene and benzalkonium bromide.It is very important that antiseptics should be used rationally.Measurements should be taken to decrease dissemination of resistant bacteria and prevent nosocomial infection.

OBJECTIVE:To optimize the extraction technology of asiaticosides from Centella asiatica. METHODS:Using to-tal amounts of asiaticoside and hydroxy asiaticoside as investigation indexes,single factor and orthogonal test were used to investi-gate the enzyme amount,enzymolysis time,enzymolysis temperature,ethanol volume fraction,liquid material ratio and ultrasonic extraction time and optimize the extraction technology of asiaticosides from C. asiatica by ultrasonic-enzyme method,and verifica-tion test was conducted. RESULTS:Optimal extraction technology was as follow as cellulose dosage of 12 mg/g,10-fold liquid ma-terial ratio added into 60% ethanol,enzyme hydrolysis for 60 min at 60 ℃,ultrasonic assisted extraction for 50 min. Average ex-traction rate of total asiaticosides was 1.92%(RSD=1.83%,n=3)in verification test. CONCLUSIONS:Ultrasonic-enzyme meth-od is stable and feasible for the extraction technology of asiaticosides from C. asiatica.

Objective To investigate the potential application of human transforming growth factor-beta-1 (hTGF-β1) gene mediated by type 2 recombinant adeno-associated virus (rAAV2) vector inducing chondrogenic differentiation of canine mesenchymal stem cells (MSCs) in vitro. Methods Canine MSCs from bone marrow were isolated and cultured in vitro by density gradient centrifngation and adherence screening methods. The morphology of MSCs was observed by inverted phase contrast microscope and Giemsa stain. Flow eytometry was used to detect surface antigens of MSCs, The third generation of MSCs were transfected by rAAV2-hTGF-β1 with or without MOI of 1 ×105 v.g./cell or 5×105 v.g./cell. The expression of hTGF-β1 was detected by Western blot after 10 days, and TGF-β1 synthesis was determined by ELISA at 3, 6 and 9 day, respectively. After 2 weeks of culturing, mRNA expressions of type Ⅱ collagen and aggrecan were determined by RT-PCR and the collagen Ⅱ protein was detected by immunocytochemistry. Results The MSCs appeared to be morphologically spindle-shaped and showed active capability of proliferation both in primary and passage generations. Flow cytometry analysis indicated that MSCs were universally positive for CD29, CD44 and CD105, but negative for CD34 and CD45. TGF-β1 expression can be observed by Western blot after 10 days in two transfection groups, MOI of 5 × 105 group and MOI of 1× 105 group. With the extension of time, the contents of hTGF-β1 increased in the two groups detected by ELISA, while there was a significant difference between them two (P < 0.01). After 2 weeks of transfection of MSCs by rAAV2-hTGF-β1, the expression of collagen Ⅱ and Aggreacan mRNAs were positive. It also showed positive of collagen Ⅱ detected by immunocytochemistry. Conclusion Canine MSCs show chondrogenesis differentiation after induction by Type 2 rAAV mediated transfer of TGF-β1 gene. The process is a potential application for cartilage tissue engineering.

To study the effects of rCBF and brain function in the patients with cerebral infarction byearly rehabilitation training. 89 cases were randomized into rehabilitation and control groups and were ex-amined rCBF by 133Xe inhalation method and BEAM. Total effect rate was 93.9%in rehabilitationgroup,to the control 77.5%(X2=3. 95,P<0.05). The rCBF rised up in two groups,but it was higher inthe foriner,to the contro1,t=4. 99,P<0. 01. BEAM improve rate was 95.9%,to the control,77.5%(X2=5. 30,P<0. 05). So we confirmed that early rehabilitation training may promote rCBF and improve brainfunction of patients with cerebral infarction.

Objective To investigate the optimal conditions for establishing insulin-resistant 3T3-L1 adipocytes. Methods Dexamethason (DEX), 3-isobutyl-methylxanthine (IBMX) and different concentrations of insulin (10-8, 10-7, and 10-6 mol · L-1) were used to induce 3T3-L1 preadipocytes into mature adipocytes identified by oil red O staining. We established insulin-resistant 3T3-L1 adipocytes cell model (IR-3T3-L1) by exposing the cells to 1μmol· L-1 DEX, and the changes of glucose concen-tration in the cell culture were determined by glucose oxidase-peroxidase (GOD-POD) assay. Results Treatment of 3T3-L1 cells with DEX, IBMX and 10-6 mol· L-1 insulin for 9 days resulted in the differentiation of>90%of the cells into mature adipocytes. IR-3T3-L1 cells cultured for 96 h in the culture media containing 1 μmol · L-1 DEX showed significantly increased glucose consumption (P=0.0003) as compared with the control group at 36 h (P<0.001). Conclusion 3T3-L1 cells can be induced into mature adipocytes by exposure to 1μmol· L-1 DEX, 0.5 mmol· L-1 IBMX and 10-6 mol· L-1 insulin. A 96 h exposure to 1μmol· L-1 DEX can induce 3T3-L1 adipocytes to acquire insulin resistance that can be maintained for 36 h.

Objective To investigate the optimal conditions for establishing insulin-resistant 3T3-L1 adipocytes. Methods Dexamethason (DEX), 3-isobutyl-methylxanthine (IBMX) and different concentrations of insulin (10-8, 10-7, and 10-6 mol · L-1) were used to induce 3T3-L1 preadipocytes into mature adipocytes identified by oil red O staining. We established insulin-resistant 3T3-L1 adipocytes cell model (IR-3T3-L1) by exposing the cells to 1μmol· L-1 DEX, and the changes of glucose concen-tration in the cell culture were determined by glucose oxidase-peroxidase (GOD-POD) assay. Results Treatment of 3T3-L1 cells with DEX, IBMX and 10-6 mol· L-1 insulin for 9 days resulted in the differentiation of>90%of the cells into mature adipocytes. IR-3T3-L1 cells cultured for 96 h in the culture media containing 1 μmol · L-1 DEX showed significantly increased glucose consumption (P=0.0003) as compared with the control group at 36 h (P<0.001). Conclusion 3T3-L1 cells can be induced into mature adipocytes by exposure to 1μmol· L-1 DEX, 0.5 mmol· L-1 IBMX and 10-6 mol· L-1 insulin. A 96 h exposure to 1μmol· L-1 DEX can induce 3T3-L1 adipocytes to acquire insulin resistance that can be maintained for 36 h.