In order to construct recombinant baculovirus carryingSchistosoma japonicum 26 ku glutathione S-transferase gene (Sj26), and observe the expression ofSj26 in mammalian cells, theSj26 gene was amplified with plasmid pGEX-3X as template by PCR, and then recombined into T vector for sequencing.Sj26 gene was inserted into the downstream of CMV promoter of donor plasmid pFBDGC, and the recombinant donor plasmid pFBDGC-Sj26 transformed into DH10Bac, then the recombinant bacmid AcCMVSj26 was isolated and transfected into Sf9 cells. The recombinant baculovirus was harvested and final titer of vAcCMVSj26 was measured. BHK cells were transducted with recombinant baculovirusin vitro. By using Western blot, the expression of 26 ku glutathione S-transferase (GST) was detected. The results showed that after enzyme digestion and sequencing, the donor plasmid was successfully constructed. PCR confirmed that pFBDGC-Sj26 and Bacmid homologous recombination occurred inE. coli. After transfection of Sf9 cells with recombinant Bacmid, recombinant baculovirus was replicated in Sf9 cells and expressed green fluorescent protein. PCR further revealed recombinant baculovirus containedSj26. The titer of the harvested baculovirus was 1.24×10⁸. Western blot demonstrated that recombinant baculovirus could express 26 ku GST in BHK cells. It was concluded thatSj26 recombinant baculovirus was successfully constructed, and the 26 ku GST was expressed in mammalian cells.

Protective effect and mechanism of electroacupuncture (EA) on acute reperfusion ventricular arrhthmia was investigated. Ventricular arrhythmia was induced by occlusion of the proximal left anterior descend (LAD) branch of coronary artery for 5 min and followed with 15 min reperfusion. EA on acupoint “Neiguan”, “Jianshi” was performed at 30 min before ligation and continued another 5 min during ischemia. Isoprenaline (20, 30 and 50 μg/kg) or atropine (1 mg/kg) was intravenously injected at 5min before ischemia. The results showed that EA significantly decreased the incidence of ischemia/reperfusion (I/R) induced ventricular tachycardia (VT), ventricular fibrillation (VF) and mortality as compared to I/R group. Atropine partially suppressed the EAs effect of antiarrhythmia; Isoprenaline increased the incidence and severity of reperfusion arrhythmia, which was inhibited by EA, but this inhibition of EA was blocked with increasing dose of isoprenaline. The results indicated that EA treatment could prevent the occurrence of reperfusion ventricular arrhythmia in rats with myocardial ischemia, and its mechanism might be related to the regulation of EA on the β-adrenoceptors and M-cholinergic receptor activation in myocardium.

The carboxyl-terminal amino acids 272–299-truncated apoE4 (Δ272–299) is the main fragments of apoE4 hydrolysate in neurons. The effects of truncated-ApoE4 (Δ272–299) overexpression on tau phosphorylation in cultured N2a cells were investigated. The truncated-apoE4 (Δ272–299) cDNA was subcloned into pEGFP-c3 to form recombinant pEGFP-T-apoE4. pEGFP-c3, pEGFP-T-apoE4 and pEGFP-apoE4 were transfected into N2a cells respectively by lipofectamine 2000 method. After 24–48 h, tau phosphorylation was detected by Western blot assay and glycogen synthase kinase-3 (GSK-3) activity by using GSK-3 activity assay. The results showed that the overexpression of both full length-apoE4 and truncated apoE4 fragments in N2a cells induced a dramatic increase in phosphorylation of tau at Ser202 sites and the activation of GSK-3 as compared with untransfected cells, most significantly in the cells transfected with pEGFP-T-apoE4 (P<0.05). It as concluded that in vitro overexpression of truncated-ApoE4 (Δ272–299) can result in tau hyperphosphorylation in N2a cells by activating GSK-3, suggesting truncated-ApoE4 (Δ272–299) might contribute the pathogenesis of Alzheimer disease.

The different effects of capsaicin on I_A and I_K currents in pain-conduct neurons of trigeminal ganglia (TG) were investigated. In cultured TG neurons of rats, whole-cell patch clamp techniques were used to record the I_A and I_K before and after capsaicin perfused. Results revealed that 1 μmol/L capsaicin could inhibit the amplitude of I_A by 48.2% (n=10,P<0.05), but had no inhibitory effect on I_K (n=7,P>0.05). Ten μmol/L capsaicin could significantly inhibit the amplitude of I_A by 93.2% (n=8,P<0.01), but only slightly inhibit the amplitude of I_K by 13.2% (n=7,P<0.05). Neither 1 μmol/L nor 10 μmol/L capsaicin had effects on the active curve of I_A and I_K. It was concluded that capsaicin could selectively inhibit the I_A current, and this effect might involve in the analgesic mechanisms of capsaicin.

The potential of^99mTc labeled P¹, P⁴-di (adenosine-5’)-tetraphosphate (Ap4A) for imaging experimental atherosclerotic plaques was evaluated in New Zealand white (NZW) rabbits. To label the^99mTc to Ap4A, stannous tartrate solution was used.^99mTc-Ap4A was purified on a Sephadex G-25 column. The radiochemistry purities of^99mTc-Ap4A were 85% to 91%. Biodistribution study revealed^99mTc-Ap4A cleared from blood rapidly. Thirty min after^99mTc-Ap4A administrated on NZW atherosclerotic rabbits, lesion to blood (target/blood, T/B) ratio was 3.17±1.27, and lesions to normal (target/non-target, T/NT) ratio was 5.23±1.87. Shadows of atherosclerotic plaques were clearly visible on radioautographic film. Aortas with atherosclerotic plaques also could be seen on ex vivo gamma camera images. Atherosclerotic abdominal aortas were clearly visible on in vivo images 15 min to 3 h after^99mTc-Ap4A administration.^99mTc-labeled Ap4A can be used for rapid noninvasive detection of experimental atherosclerotic plaque.

The imaging appearances of⁹⁹Tc^m-HL91, a new hypoxic imaging agent, in ischemic myocardium were studied and the value of⁹⁹Tc^m-HL91 in the evaluation of regional ischemic viable myocardium was explored. Acute myocardial ischemia models were made by coronary artery legations in 18 rats and randomly divided into 2 groups:⁹⁹Tc^m-HL91 group and⁹⁹Tc^m-MIBI group. Evan blue infusion during ischemia and TTC staining after operation were used to delineate the area of ischemic and viable myocardium. The isolated heart was sliced in the short axis and then autoradiography was performed. The electron microscopic examination was also done for the myocardial samples.⁹⁹Tc^m-HL91 and⁹⁹Tc^m-MIBI uptake activities (counts/g) were measured in the area of ischemic myocardium (T) and normal myocardium (NT) separately. The uptake ratios of⁹⁹Tc^m-HL91 and that of⁹⁹Tc^m-MIBI in ischemic myocardium were calculated as T/NT. It was found that the normal myocardium was blue and ischemic or infarct myocardium was negative with Evans blue in all experiment rats. Both the normal and ischemic myocardium was in red color with TTC staining. In the⁹⁹Tc^m-HL91 group the ischemic myocardium showed much higher uptake over normal myocardium, that was demonstrated both in the autoradiography and quantitative analysis. The ischemic/normal activity ratios were 1.634±0.354. It was suggested that⁹⁹Tc^m-HL91 might accumulate in ischemic and viable myocardium, which is helpful in the evaluation of hypoxic but viable myocardium and potentially used as a imaging agent to assess myocardial viability.

The effects and the mechanism of insulin treatment on intracellular lipid metabolism in liver of diabetic rats were evaluated. Type 2 diabetic rats were induced by injecting the streptozotocin (25 mg/kg) and fat rich food. According to the results of oral glucose tolerance test (OGTT) and glucose-induced insulin secretion test (IRT), the rats were divided into two groups; untreated group (UT) and insulin-treated group (IT). Normal rats (NC) served as controls. The treatment with either Humulin N (4–6 U/kg every day), or saline lasted for 4 weeks. Body weight, OGTT, IRT, blood lipids, intracellular lipids in liver, hepatic fatty acid oxidation and the activity of fatty acid synthase (FAS) were detected. The change of liver histology was observed. The insulin sensitivity index (ISI) was applied to assess the status of insulin resistance. The results showed that as compared with NC group, the plasma and hepatic intracellular Triglyceride (TG), total cholesterol (TC) and free fatty acids (FFAs) were increased significantly in UT group (P<0.05), and lipid droplets could be seen dispersedly in the liver specimens, the hepatic fatty acid oxidation was increased markedly (P<0.05), while the fatty acid synthase activity decreased (P<0.05). Insulin treatment resulted in a further accumulation of lipids in liver by 55.7%, 19.87% and 22.2% increase in TG, TC, FFAs respectively. The size of hepatocytes was enlarged and the cells were filled with fat drops. Plasma lipids showed little decrease and still significantly higher than those in NC group after the insulin treatment. Meanwhile, insulin treatment was companied by 20% decrease in the rate of fatty acid oxidation and 31% increase in hepatic FAS activity compared to UT group. It was concluded that treatment with insulin on type 2 diabetic rat increases hepatic intracellular lipid accumulation by inhibiting hepatic fatty acid oxidation and activating FAS.

The expression of resistin protein in normal human abdominal, thigh, pregnant women abdominal, non-pregnant women abdominal subcutaneous adipose tissue and placenta and the relationship between obesity, type 2 diabetes mellitus (T2DM), pregnant physiological insulin resistance (IR) and gestational diabetes mellitus (GDM) was investigated. The expression of resistin protein in normal human abdominal, thigh, pregnant women abdominal, non-pregnant women abdominal subcutaneous adipose tissue and placenta was detected by using Western blotting method. Fasting serum glucose concentration was measured by glucose oxidase assay. Serum cholesterol (CHOL), serum triglycerides (TG), serum HDL cholesterol (HDL-C) and serum LDL cholesterol (LDL-C) were determined by full automatic biochemical instrument. Fasting insulin was measured by enzyme immunoassay to calculate insulin resistance index (IRI). Height, weight, systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured to calculate body mass index (BMI) and body fat percentage (BF%). Resistin protein expression in pregnant women placental tissue (67 905±8441) (arbittary A values) was much higher than that in subcutaneous adipose tissue in pregnant women abdomen (40 718±3818,P<0.01), non-pregnant women abdomen (38 288±2084,P<0.01), normal human abdomen (39 421±6087,P<0.01) and thigh (14 942 ±6706,P<0.001) respectively. The resistin expression in abdominal subcutaneous adipose tissue showed no significant difference among pregnant, non-pregnant women and normal human, but much higher than that in thigh subcutaneous adipose tissue (P<0.001). Pearson analysis revealed that resistin protein was correlated with BMI (r=0.42), fasting insulin concentration (r=0.38), IRI (r=0.34), BF% (r=0.43) and fasting glucose (r=0.39), but not with blood pressure, CHOL, TG, HDL-C and LDL-C. It was suggested that resistin protein expression in human abdominal subcutaneous adipose tissue was much higher than that in human thigh subcutaneous adipose tissue. Resistin was closely related with central obesity, leading to IR, subsequently obesity and T2DM. Resistin protein expression in placental tissue was much higher than that in subcutaneous adipose tissue in normal human abdomen, pregnant abdomen, non-pregnant women abdomen and adipose tissue in normal human abdomen, pregnant abdomen, non-pregnant women abdomen and thigh. It was indicated that resistin protein could be secreted from human placental tissue. Resistin might be one of the factors that lead to pregnant physiological IR and GDM.

The effects of mycophenolic acid (MPA) on high glucose-induced expression of transforming growth factor-β (TGF-β) and connective tissue growth factor (CTGF) in mesangial cells (MC) were investigated. Rat MC were cultured in the presence of different concentrations of MPA (1.0 and 10.0 μmol/L) or MPA plus high glucose for 72 h. The expression of TGF-β and CTGF was detected by Western blot. The results showed that high glucose could induce the expression of TGF-β and CTGF in MC, but MPA could inhibit this effects. MPA did not influence the expression of TGF-β and CTGF in normal glucose. It was concluded that MPA might prevent the progression of diabetic nephropathy by inhibiting the expression of TGF-β and CTGF in MC.

A potential pathological role of angiopoietins (Ang) in glomeruli following podocyte injury-induced progressive glomerulosclerosis was explored. Eighty male Wistar rats were randomly allocated into sham operation group (Sham,n=25), Uninephrectomy group (UPHT,n=25) and Uninephrectomy+Daunorubicin group (DRB,n=30). In DRB group, daunorubicin (5 mg/kg) was injected via tail vein on the 7th and 14th day after uninephrectomy. At week 1, 2, 4, 6 and 8 respectively following establishment of the animal model, 5 rats in Sham group and UPHT group, and 6 in DRB group were taken respectively for determining 24-h urinary protein excretion rate (24hUPER), blood urea nitrogen (BUN) and serum creatinine (Scr). The sections of kidneys were examined by an electric microscope, PAS staining, immunohistochemical staining and in situ hybridization histochemistry. The results showed that 24hUPER, BUN and Scr in DRB group were more than those in Sham group and UPHT group at the same time points, and there was a trend towards an increase on level of GSI in DRB group from week 2 to week 8. Electric microscopy revealed that podocyte injury presented in DRB group. The expression of Angl mRNA and protein in glomeruli of DRB group was decreased, while the expression of Ang2 protein in glomeruli of DRB group increased, Meanwhile, the expression of Angl mRNA had a negative correlation with expression of Ang2 mRNA, and the expression of Angl protein had a positive correlation with the expression of Ang1 mRNA, and had a negative correlation with 24hUPER, BUN, Scr, glomerular sclerotic index (GSI), the expression of Ang2 protein and CoIV protein. The expression of Ang2 protein had a positive correlation with the expression of Ang2 mRNA, and had a positive correlation with 24hUPER, BUN, Scr, GSI, the expression of CoIV protein. It was concluded that podocyte injury might lead to an alteration in the expression of Ang1 and Ang2 within glomeruli. Ang2 may get rid of inhibition from Angl for downregulation of the Ang1 expression, which facilitate upregulation of the Ang2 expression in glomeruli to promote progressive glomerulosclerosis in the rats.

In order to establish state high expression cell lines, the eukaryotic expression vector pIRES2EGFP and recombinant plasmid pIRES2EGFP-TIM-3 were transfected into mammalian cells CHO by Lipofectamine. The transfected cells were cultivated under selective growth medium including G418 and green fluorescent protein (GFP) positive cells were sorted by FACS. Simultaneously, growing transfectants were selected only by G418 in the medium. The GFP expression in stably transfected cells was detected by FACS. Under selective growth conditions with G418, the percent-age of GFP positive cells was reduced rapidly and GFP induction was low. In contrast, the percent-ages of GFP positive cells were increased gradually after FACS. By 3 rounds of GFP selection, the stable high expression cell lines were established. Furthermore, using FACS analysis GFP and the target protein TIM-3 co-expression in the stable transfectants cultured in nonselective medium was detected. Theses results demonstrated that the stably transfected cell lines that express high titer of recombinant protein can be simply and fleetly obtained by using GFP and selective growth medium.

To investigate the effects of TGF-β1 on the gelatinases (MMP-2 and MMP-9), and their roles in lung remodeling after irradiation-induced lung injury. Expressions of TGF-β1 were measured with western blot, and expressions of MMP-2 and MMP-9 were analyzed with zymography in a TGF-β1 transgenic mouse model after thoracic irradiation with 12 Gy. We found expressions of TGF-β1 in the lung from the transgenic mice were three folds as compared to those from control mice. With densitometrical analysis, we found a significant decrease in MMP-9 activity in lung homogenates from the transgenic mice as compared with those from non-transgenic control mice 8 weeks after sham-irradiation (relative MMP-9 activity: C: 1.000±0.1091; TG: 0.4772±0.470 (n=8,P<0.05). But MMP-2 was constitutively expressed in the lung homogenates from the transgenic mice as compared to those from control mice 8 weeks after sham-irradiation (relative MMP-2 activity 8 weeks after sham-irradiation: C: 1.000±0.1556, TG: 1.0075±0.1472). Eight weeks after thoracic irradiation with 12 Gy, we observed a significant increase of MMP-2 and MMP-9 activity in lung homogenates from both transgenic and normal mice. In TGF-β1 transgenic mice relative MMP-9 activity was increased to 1.5321±0.2217 folds 8 weeks after thoracic irradiation with 12 Gy as compared to those after sham-irradiation (1.000±0.2153), and relative MMP-2 activity was increased to 1.7142±0.4231 folds. Our results show that TGF-β1 itself down-regulates activity of MMP-9, thereby decreases ECM degradation in lungs of TGF-β1 transgenic mice. Also we find that ionizing irradiation upregulates both MMP-2 and MMP-9 activity. Over-expressions of MMP-9 and MMP-2 after lung irradiation are involved in the inflammatory response associated with radiation-induced lung injury, and maybe further in radiation-induced lung fibrosis.

In order to investigate the effects of short hairpin RNA (shRNA) on the expression of Survivin, cell cycle and cell proliferation in MCF-7 cells, using a pEGFP vector which contained a U6 promoter shRNA plasmid targeted against survivin was constructed and transfected into MCF-7 cells. The change of the expression of Survivin and cell proliferation rates were detected by semiquantitative reverse transcription polymerase chain reaction (RT-PCR) and MTT methods respectively. The change of cell cycle after transfection was analyzed by flow cytometry. The results indicated that the recombinant plasmid containing Survivin shRNA was constructed successfully, which could suppress the expression of Survivin at mRNA and protein level. The growth of MCF-7 cells was arrested in G1 phase of the cell cycle and the proliferation activity was suppressed after transfection. It was concluded that Survivin shRNA plasmid could knock down the expression of Survivin in MCF-7 cells specifically. In addition, Survivin shRNA plasmid could lead to G1 arrest and inhibit the proliferation of MCF-7 cells, which suggested that Survivin shRNA might be used as a new therapeutic method for breast cancer.

The expression of the cytokines IL-2, IL-10, TNF-α and their roles in mice with herpes simplex viral encephalitis (HSE) were studied. By using semiquantitative reverse transcription polymerase chain reaction (RT-PCR), the expressions of IL-2, IL-10, and TNF-α mRNA in control group, HSE group and acyclovir (ACV)-treated group were detected and the pathological changes of brain were observed. It was found that after HSV1 infection, the cerebral lesions of haemorrhage and necrosis in mice were observed under the microscopy, and the levels of IL-2, IL-10 and TNF-α were increased remarkably. After treatment with ACV after HSV1 infection, the cerebral lesions in mice were improved, the level of IL-2 maintained stable, IL-10 was increased consistently, and TNF-α was decreased significantly as compared with those in HSE group. In acute HSE, many cytokines are upregulated, including IL-2, IL-10 and TNF-α to eliminate virus and TH1 type response is dominant. In convalescence, there is a shift in the cytokine expression profile from TH1 profile to TH2 profile and the shift can inhibit the overexpression of immune response in animals. ACV has remarkable effects in the treatment of HSE.

The difference of gene expression profile changes in Barrett’s esophagus (BE) and cardia intestinal metaplasia (CIM) epithelium was studied and the novel associated genes were screened in the carly stage by cDNA microarray. The cDNA retro-transcribed from equal quantity mRNA from BE and CIM epithelial tissues were labeled with Cy3 and Cy5 fluorescence as probes. The mixed probe was hybridized with three pieces BiostarH-40s double dot human whole gene chip. The chips were scanned with a ScanArray 4000. The acquired images were analyzed using GenePix Pro 3.0 software. It was found a total of 141 genes were screened out that exhibited differentially expression more than 2 times in all three chips. It was identified that in gene expression profiles of BE, 74 genes were up-regulated and 67 down-regulated as compared with CIM. The comparison between the difference of gene expression profile changes in BE and CIM epithelia revealed that there existed the difference between BE and CIM at gene level. 141 genes with the expression more than two time were probably related to the occurrence and development of BE and the promotion or progress in adenocarcinoma.

The effects of in vivo local expression of recombined human tissue-type plasminogen activator (t-PA) gene on the thrombosis and neointima formation of vein grafts were explored. Jugular vein-to-artery bypass grafting was performed on 72 New Zealand white rabbits. The rabbits were divided into 3 groups according to the different processing methods: transfected t-PA gene group (n=24), vector group (n=24) and blank control group (n=24). Samples of vein grafts were harvested at different time points after surgery. The expression of t-PA gene in vein graft was detected by RT-PCR and the synthesis of t-PA protein by Western-Blot assay. The t-PA activity was measured by chromogenic substrate assay. The Cr51 labeled platelets accumulation in vein grafts was counted. The histopathological changes were compared in intima hyperplasia index among the three groups after operation. The results showed that at the 2^nd, 5^th, 14^th and 28^th day after operation, RT-PCR and Western-blot confirmed the expression of t-PA mRNA and protein at the site of gene transfer. The t-PA activity detected on the 2^nd, 5^th, 14^th and 28^th day in experimental group was 370.63±59.44, 344.13±48.47, 252.87±51.80 and 161.75±68.94 U/g respectively, and disappeared on the 60^th day and undetected in the control groups. The number of platelets accumulated in the vein grafts in gene group, vector group and blank control group was (85.04±21.58) 10⁶, (225.87±85.13) 10⁶ and (211.57±78.02) 10⁶ respectively. The number of platelets accumulated in gene group was signficantly fewer than that in the control groups. Morphometric analysis revealed that intimal hyperplasia was markedly reduced in the t-PA gene group as compared with that in the control groups. It was suggested that the local expression of t-PA gene in vein graft significantly inhibited the accumulation of platelets, thrombosis and concomitant intimal hyperplasia, by which stenosis of bypass graft could be prevented effectively.

The enhancing effects of Smac gene on the mitomycin C-induced apoptosis of the bladder cancer cell line T24 were investigated. The Smac gene was transfected into bladder cancer cell line T24 under the induction of liposome. The intrinsic Smac level was detected by using immunohistochemistry and RT-PCR. The in vitro cellular growth activities were assayed by MTT colorimetry. Apoptosis was assayed by the flow cytometry. The results showed that as compared with the control cells, the apoptosis rate of T24 cells induced by mitomycin C was enhanced by transfected Smac gene. Flow cytometry revealed that, the apoptosis rate was 18.84% and 33.52%, and 10.72% and 26.24% respectively in blank and transfected cells treated with 0.05 or 0.005 mg/mL mitomycin C (P<0.05). It was concluded that Smac could enhance the apoptosis of T24 by mitomycin C, which could provide a useful experimental evidence for bladder cancer therapy.

To discuss and evaluate the method and effect of using calcaneal anatomic plate in treatment of intra-articular fractures of the calcaneus with assistant of arthroscope, 86 intra-articular fractures of the calcaneus in 78 patients were reduced by open reduction, and rigid fixation was made with calcaneal anatomic plate under assistant of arthroscope. The average follow-up duration was 18 months (range 12–30 months). The effect of treatment was evaluated according to AOFAS and X-ray before and after operation. The results showed that 86 patients have obtained satisfactory reduction according to X-ray, and there was significant difference before and after operation (P<0.01), the total excellent and fine rate was 91.86%. Treating intra-articular fractures of the calcaneus with calcaneal anatomic plate under arthroscope may provide more chance to achieve anatomical reconstruction, which can lead to satisfied recovery of function and few complication.

The recombinant defective adenovirus vector carrying human PTEN tumor suppressor gene was constructed by using AdEasy-1 system and its expression was detected in human breast cancer cell line MDA-MB-468. Human PTEN cDNA was cloned into adenovirus shuttle plasmid pAdTrack-CMV to generate a recombinant plasmid pAdTrack-CMV-PTEN, then homologeous recombination was carried out in the E. coli BJ5183 by contransforming linearized shuttle vector with adenovirus backbone plasmid pAdEasy-1. The newly recombined defective adenovirus vector Ad-PTEN containing green fluorescent protein (GFP) was packaged and propagated in 293 cells. After being purified by cesium chloride gradient centrifugation, the adenovirus was transfected into human breast cancer cell line MDA-MB-468 in vitro. The expression of PTEN mRNA and protein in infected human breast cancer cell line MDA-MB-468 was detected by RT-PCR and Western blot respectively. The recombinant defective adenovirus vector carrying PTEN gene was constructed successfully. The viral titer of purified adenovirus was 2.5×10¹⁰ pfu/mL, and about 70% breast cancer cells were infected with Ad-PTEN when multiplicity of infection (MOI) reached 50. The exogenous PTEN mRNA and protein were expressed in MDA-MB-468 cells infected with Ad-PTEN by RT-PCR and Western blot. The recombinant defective adenovirus vector of PTEN gene was constructed successfully using AdEasy-1 system rapidly, which paved a sound foundation for gene study of breast cancer.

Circulating vascular endothelial growth factor-C (VEGF-C) and vascular endothelial growth factor (VEGF) levels in patients with colorectal carcinoma were determined in order to assess their clinical significance as a diagnostic tool for monitoring lymph node metastasis. In 66 patients with colorectal carcinoma and 30 healthy controls, circulating VEGF-C and VEGF levels were assessed by using enzyme-linked immunosorbent assay (ELISA). Serum VEGF-C and VEGF levels were higher in patients with colorectal carcinoma than in healthy controls. Patients with lymph node metastasis had higher serum VEGF-C and VEGF levels than those without lymph node metastasis. The levels of VEGF-C and VEGF were higher in the invasion group than in the non-invasion group. Serum VEGF-C levels reached a sensitivity of 81% and a specificity of 76% with a cutoff value of 1438.0 pg/mL, whereas VEGF levels reached 72% sensitivity and 74% specificity at 240.2 pg/ mL If 66 patients were divided into 4 groups according to the combined determination of VEGF-C and VEGF levels, the positive predictive value was 85.3%, the negative predictive value was 94.6% and accuracy was 93.7%. It was suggested that circulating VEGF-C levels might provide additional information for distinguishing the absence from presence of lymph node metastasis in patients with colorectal carcinoma. The combined determination of VEGF-C and VEGF levels could be used as an important index for preoperatively clinical stage of colorectal carcinoma.

The focal hepatic lesion caused by local injection of absolute alcohol in rats was evaluated with ultrasonic contrast agent and pathologic examination. Twenty adult Wistar rats weighing about 200 g were injected with absolute alcohol (0.05–0.1 mL each one) on the exterior left lobe of the liver under the monitoring of ultrasound. Pulse inversion harmonic imaging was used to evaluate the focal lesion after bolus injection of ultrasonic contrast agent (0.05 mL/200 g) through caudal vein. Seven days later, the focal lesion was studied again as before. The exterior left lobe of liver with focal lesion was incised and underwent pathologic examination. The results showed that all of the focal lesions could be defined clearly after bolus injection of the ultrasonic contrast agent under the mode of pulse inversion harmonic imaging. There was good correlation between the size of the focal lesion measured by ultrasound on the 7th day after the “ablation” under the mode of pulse inversion harmonic imaging and that gotten by pathologic examination (P=0.39). The focus size measured by ultrasound right after the ablation was larger than that gotten by pathologic examination (P=0.002). It was concluded that ultrasonic contrast agent plus pulse inversion harmonic imaging could be used to assess the size of the focal hepatic lesion caused by local injection of absolute alcohol in rats.

The value of tissue strain imaging (SI) in regional myocardial systolic and diastolic function assessment was studied. In 18 patients with nonobstructive hypertrophic cardiomyopathy (HCM) and 20 age-matched healthy subjects, regional myocardial longitudinal peak systolic strain in eject time (represented by εet) was measured at basal, mid and apical segments of septal, lateral and posterior walls of the left ventricle (LV) and compared between groups. εet had no significant difference between segments in control group (P>0.05), which displayed a decreasing trend from basal segments to apical ones, εet in the HCM group was significantly decreased (P<0.05) as compared with that in the healthy group. In the HCM group, εet in the midseptum was significantly less than at the basal and apical septum, and was also less than at the rest LV walls in the same group (P<0.01). The systolic reversed εet was noticed in 35% of the hypertrophic segments in HCM group. Significantly negative correlation existed between the absolute value of εet and wall thickness in the midseptum (r=−0.83). The post-systolic strain (PSS) segment number the and amplitudes in healthy group were significantly less than those in HCM group (P<0.05). Both regional myocardial systolic and diastolic functions were impaired in hypertrophic or non-hypertrophic segments in patients with the HCM, especially in hypertrophic segments. Strain imaging technique is a sensitive and accura tool in myocardial dysfunction assessment.

The role of 16-slice spiral CT was evaluated in the diagnosis of coronary stenosis, with selective X-ray coronary angiography (SCA) serving as the reference standard. Sixty-five patients who were suspected of having coronary heart disease, without percutaneous transluminal coronary angioplasty or coronary bypass-grafting, were investigated using 16-slice CT. Eight patients with pre-scan heart rate of more than 80 beast/min were given β-blockers. After the retrospectively ECG-gated axial imaging reconstruction, volume redering (VR), multi-planar reconstruction (MPR), curved MPR and maximum intensity projection (MIP) were used to reconstruct. Every segment of coronary artery with a diameter ≥1.5 mm was assessed, and the presence on CT with a stenosis exceeding 50% diameter reduction was compared with that on SCA. The reasons which lead to some segments unevaluable were analysed. Compared with SCA, 93% coronary segments and 94% main branches were evaluable. Residual cardiac motion artifacts, severe calcification and poor opacification made 58%, 28% and 14% of the remaining 60 segments unevaluable respectively. Without routine administration of β-blockers, good coronary imaging quality can be acquired using 16-slice spiral CT. It is a reliable noninvasive method for detection of obstructive coronary artery disease.

An experimental animal model of malignant soft-tissue tumor was established to investigate the applied value of multi-slice spiral CT perfusion imaging preliminarily. Ten New Zealand white rabbits which were implanted with VX₂ tumor in either proximal thigh were subjected to CT plain scan and perfusion scan two weeks later respectively, the the original perfusion images were transmitted to AW4.0 Workstation. The functional maps and perfusion parameters including blood flow (BF), blood volume (BV), mean transit time (MTT) and permeability surface (PS) were computed and analyzed. All the values of BF, BV and PS in VX₂ soft-tissue tumors were obviously higher while the MTT-values were lower than those in the normal muscular tissues significantly. It was suggested that multi-slice spiral CT perfusion imaging is an accurate, convenient and relatively safe functional imaging technique, and can give a quantitative assessment to angiogenesis and blood perfusion of soft-tissue tumors.

The effect of triptolide (TP) on the expression of receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) was explored in rat adjuvant induced arthritis (AA). AA was induced in Wistar rats. Arthritis rats were treated with TP and methotrexate (MTX) at the onset (day 9) of arthritis. On the peak of arthritis (day 24), the expression of RANKL and OPG protein in the joints and RANKL mRNA in peripheral blood mononuclear cells (PBMC) was detected. TNF-α and IL-1β levels in peripheral blood were determined. Bone erosion scores were also evaluated. The results showed that bone erosion scores in TP and MTX groups were lower than in AA group (P<0.01); The expression levels of RANKL in the synovium (P<0.01) and bone (P<0.05), and OPG level in synovium (P<0.05) were lower in TP group than in AA group (P<0.05). In TP group, the expression levels of RANKL mRNA and TNF-α, IL-1β in PBMC were lower than in AA group (allP<0.01). It was concluded that TP could inhibit rat adjuvant arthritis bone erosion by suppressing the expression of RANKL.

The relationship between tumour necrosis factor-α (TNF-α) gene polymorphism and inhibitory effects of triptolide on TNF-_α production from perlpheral blood mononuclear cells (PBMC) of healthy humans was investigated. Genomic DNA from 41 healthy people was typed for TNF-_α—308 polymorphism by allele-specific polymorphism chain reaction (AS-PCR). The TNF-_α concentration in the supernatant was measured by ELISA. The results showed that the production of TNF-_α from TNF-_α—308 non-G/G genotype PBMC was higher than that from TNF-_α—308 G/G genotype PBMC after stimulated by LPS. Triptolide could lower the production of TNF-_α from G/G genotype PBMC, but had no effect on the level of TNF-_α from non-G/G genotype PBMC. It was concluded that TNF-_α gene polymorphism was related to the TNF-_α production from triptolide-inhibited PBMC culture in healthy humans.

In order to observe the effect of ursodeoxycholic acid (UDCA) in the treatment of intrahepatic cholestasis of pregnancy (ICP), 68 patients with ICP were equally divided into treatment group and control group at random. The patients in treatment group were administered with UDCA 300 mg three times every day and those in control group received a combination of 10% glucose, Vitamin C and Inosine. Itching scores, serum ALT and total bile acids (TBA) were measured before, during and after treatment. The results showed that as compared with those before treatment, itching scores, serum ALT and TBA were significantly reduced after treatment (P<0.05). The occurrences of premature labor, fetal asphyxia and meconium staining in amniotic fluid were significantly lower in treatment group than in control group (P<0.05). It was suggested that UDCA was an effective drug in the treatment of ICP.

Taking the mouse as a model, the experimental method of observing the morphology of meiotic spindles and chromosomes in mature oocytes were investigated in order to evaluate the effects of various interventions on the quality of oocytes accurately and rapidly. Laser scanning confocal microscope (LSCM) was used to examine the meiotic spindles and chromosomes by the technologies of optical section and three-dimensional (3D) image reconstruction. The results showed that the configurations of meiotic spindles and chromosomes could be observed clearly by LSCM. The normal rate of meiotic spindles and chromosomes was 82% and 86% respectively. It was concluded that the LSCM was a valid instrument to observe the meiotic spindles and chromosomes of mature oocytes and could be used as a valid method to evaluate the quality of M II oocytes.

The expression of endothelial nitric oxide synthase traffic inducer (NOSTRIN) in the placenta of the patients with pregnancy induced hypertension (PIH) was detected and its role in the pathogenesis of PIH was studied. The pathological changes in placental vessels were observed by HE staining, NO₂₋/NO₃₋, the stable metabolic end products of NO, was measured with nitrate reductase. The eNOS activity in placental tissues was assayed by spectrophotometry. Western blot analysis was applied to detect NOSTRIN expression. The incidence of thickening and fibronoid necrosis of placental vessels was significantly higher in women with PIH than in the normal group (P<0.01). The levels of placental NO₂₋/NO₃₋ in PIH patients (27.53±7.48 μmol/mg) were significantly lower than in normal group (54.27±9.53 μmol/mg,P<0.01). The activity of eNOS was significantly decreased in PIH group (12.826±3.61 U/mg) as compared with that in normal group (21.72±3.83 U/mg,P<0.01). Western blot analysis revealed that both groups expressed 58 kD NOSTRIN, but the protein level was significantly higher in women with PIH than in the normal group (P<0.01). A significant negative correlation existed between the expression of NOSTRIN protein and the activity of eNOS in placental tissue of women with PIH (r=−0.57,P<0.01). It was concluded that the level of NOSTRIN expression in placenta of women with PIH was increased, which may play an important role in the pathogenesis of PIH.

In order to investigate the roles of MTA2 in the pathogenesis of ovarian epithelial cancer, the expression of MTA2 in 4 ovarian cell lines were detected by semi-quantitative RT-PCR and Western-blot assays. MTA2 expression in normal, borderline, benign and malignant epithelial ovarian tissues was immunohistochemically examined. The expression of MTA2 mRNA and protein was detected in all of 4 cell lines of ovarian epithelial cancer. The expression of MTA2 mRNA and protein was higher in strong migration cell lines than in weak migration ones. In borderline and malignant ovarian tissues tested, MTA2 staining was dramatically stronger than in normal and benign tissues (P<0.01). The expression levels in malignant ovarian tissues were significantly higher than that in borderline epithelial ovarian tissues (P<0.01). The expression of MTA2 was correlated with clinical stage, histopathological grade and lymph node metastasis. It was concluded that the high expression of MTA2 was associated with more aggressive behaviors of epithelial ovarian cancer. MTA2 provides a novel indicator of ovarian cancer.

In order to investigate the effects of TGF-β₁ on the expression of MMP-2, −9 and TIMP-1 in human retinal pigment epithelial (RPE) cells, the third-sixth passage cultured RPE cells were treated with TGF-β₁ at different concentrations (0. 01, 0. 1, 1. 0, 10 ng/mL), the expression of MMP-2, −9 and TIMP-1 mRNA was detected by semi-quantitative RT-PCR assays. MMP-2, −9 and TIMP-1 mRNA were expressed in the cultured RPE cells. The values of MMP-2/β-actin in the cells treated with 0. 1, 1. 0, 10 ng/mL TGF-β₁ were 1.04±0.04, 1.07±0.02 and 1.11±0.03, respectively, significantly higher than in the control group (0.96±0.03,P<0.05–0.01). The expression of MMP-2 mRNA could be up-regulated by TGF-β₁, in a dose-dependent manner. The expression of MMP-9 mRNA in the cultured RPE cells was slightly up-regulated by various TGF-β₁ concentrations treatment. The values of TIMP-1/β-actin in the cells treated with 0.01 and 0.1 ng/ mL TGF-β₁ were 0.85±0.01 and 0.97±0.02 respectively, significantly lower than in the control group (1.07±0.04,P<0.01), indicating that the expression of TIMP-1 mRNA was down-regulated by TGF-β₁ at low concentrations. But along with the increase of TGF-β₁ concentrations (1.0 and 10 ng/mL), the expression of TIMP-1 mRNA was slightly up-regulated, not significantly different from that in the control group (P>0.05). It was concluded that TGF-β₁ might play an important role in the up-regulation of the expression of MMP-2 in RPE cells and result in a directional shift in the balance between MMP and TIMP. This may be facilitated for RPE cells to migrate in the pathogenesis of vitreoretinopathy.

In order to investigate the effects of mouse CTLA4Ig gene-modified dendritic cells (DCs) on the survival of the corneal allografts in rats, the plasmid PG/CTLA4Ig was transfected into DCs of F344 rats mediated by Lipfectamine^TM 2000. The expression of CTLA4Ig was detected by immunofluorescent microscopy. The effects of donor DCs on the proliferation of T cells in Lewis rats (recipients) were tested by by CCK8. Corneal transplantation was performed from F344 rats to Lewis rats. The DCs modified with CTLA4Ig gene were injected into the Lewis rats on the day 0 and 3 after transplantation. The movement of the DCs after modification in vivo was observed by immunofluorescent microscopy, and the survival of corneal allografts was evaluated by Holland criterion. The results showed that the CTLA4Ig-modified DCs could restrain the proliferation of allogenetic T cells. The CTLA4Ig-modified DCs prolonged survival of corneal allografts. (P<0.01). It was suggested that the injection of CTLA4Ig gene-modified DCs could obviously inhabit the allograft rejection and prolong the survival of corneal allografts.

In order to elucidate the effect of dexamethasone on the expression of transforming growth factor-betal (TGF-β1) in ciliary pigment epithelial (CPE) cells cultured in vitro, rabbit CPE cells were cultured in vitro, treated with DMEM medium containing 0,1×10⁻⁸, 5×10⁻⁸, 10×10⁻⁸ and 50×10⁻⁸ mol/L dexamethasone respectively for 5 days. The TGF-β1 expression was detected by immunohistochemistry Supervision methods and analyzed semi-quantitatively by HMIAS-2000 image system. As opposed toin vivo, rabbit CPE cells expressed TGF-β1 under cultured circumstance in vitro. The gray scales of the positive yellow staining in the groups of 1×10⁻⁸, 5×10⁻⁸, 10×10⁻⁸ and 50×10⁻⁸ mol/L dexamethasone were 136.57±4.43, 140.20±6.10, 142.98±2.99, 146.80±1.68 and 150.05±1.94 respectively. When the concentrations of dexamethasone were equal to or higher than 5×10⁻⁸ mol/L and, the expression of TGF-β1 was inhibited. 10⁻⁷ mol/L dexamethasone showed a significant inhibition. It was suggested that CPE cells possess the potential ability of synthesizing and expressing TGF-β1. The inhibition of TGF-β1 expression by dexamethasone may be beneficial to the treatment of proliferative vitroretinopathy, also exert some influence on the secretion of aqueous humor and ciliary inflammation.

Changes of corneal properties induced by laser in situ keratomileusis (LASIK) results in low inaccurate intraocular pressure (IOP) readings by Goldmann applanation tonometry (GAT). Before and after LASIK, the applied value of IOP, measured by dynamic contour tonometry (DCT) in comparison to GAT, was evaluated. Before and 1, 4 weeks after LASIK, the IOP in 30 cases (60 eyes) was measured by GAT and DCT respectively. The obtained results were statistically processed by SPSS11.5 statistical software. The results showed that central corneal thickness (CCT) could affect GAT measurements but not DCT measurements. The comparison of IOP one and 4 weeks after LASIK revealed that the readings from GAT was separately decreased by 5.00±1.12 and 5.45±1.13 mmHg as compared with those before LASIK, while those from DCT had no significant difference. It was concluded that LASIK-induced changes of CCT could influence the accuracy of GAT measurements, but had no influence on those from DCT. DCT was more beneficial to the measurements of IOP in normal eyes and those subject to LASIK surgery.

The distribution of the Na−K−2Cl co-transporter (NKCC1) in the cochlear K⁺ cycling pathway in cochlea and cochlear histological changes in the NKCC1 knockout mice were investigated. By using immunohistochemistry and toluidine blue staining, the localization of NKCC1 in cochlea of the C57BL/6J mice and the cochlear histological changes in the NKCC1 knockout mice were observed. It was found that the NKCC1 was expressed mainly in the stria marginal cells and the fibrocytes in the inferior portion of the spiral ligament in the adult C57BL/6J mice. Subpopulation of the fibrocytes in the suprastrial region and the limbus was also moderately immunoreactive. While in the cochlea of the NKCC1 knockout mice, Reissner’s membrane was collapsed and scala media disappeared, accompanied with the loss of inner hair cells, outer hair cells and the support cells. The tunnel of Corti was often absent. All the findings suggested the localization of NKCC1 in the cochlea was closely correlated with cochlear K⁺ cycling. Loss of NKCC1 led to the destruction of the cochlear structures, and subsequently influenced the physiological function of cochlea.

To observe the expression of CD40/CD40L on peripheral blood mononuclear cells (PBMC) in patients with condyloma acuminatum (CA), flow cytometry was employed to examine the expression of CD40 and CD40L on PMBC in 36 patients with CA and 20 healthy controls. Our results showed that mean level of CD40 expression in CA patients was significantly lower than that in the controls (6.58%±2.74% vs 14.81%±6.12%,t=5.703,P<0.05); the average level of CD40L in CA patients was also significantly lower than that in the controls (0.73%±0.54% vs 2.67±2.43%,t=3.532,P<0.05). Our results suggest that the reduced costimulatory interaction of CD40 and CD40L in CA patients may be one of the important factors responsible for the low cellular immunity.

In order to identify family factors obviously relevant to aggression, and offer a theoretical foundation for the prevention of aggression, 4010 students from primary and secondary schools in 5 different areas in Hubei province were surveyed. The Child Behavior Checklist “parents’ form” (Chinese version) and the four scales of Family Environment Scale were used. A multiple logistic regression was used to identify risk factors of children’s and adolescents’ aggressive behavior. The results showed that maternal education, paternal occupation, family type, parental child-rearing attitude and patterns, students’ interpersonal relationship were significantly associated with the children’s and adolescents’ aggression. The risk factors of aggression were parental child-rearing patterns, peer relationship, teacher-student relationship, and family conflicts.

In order to explore the value of combined detection of atypical lymphocytes (ATL) and transaminase (alanine aminotransferase, ALT; asparate aminotransferase, AST) in the diagnosis of infectious mononucleosis (IM), The data of blood routine and liver function were collected from 54 IM patients, 34 acute hepatitis (AH) patients, 44 upper respiratory infection (URI) patients in Union Hospital during March 2002 to March 2005. Same data were also collected from 40 healthy children as normal control. These data were analyzed retrospectively. Both proportion of atypical lymphocytes and enzyme activity of transaminase were elevated simultaneously (ALT>40 IU/L, AST>45 IU/L) in 57.4% (31.54) IM patients. There was significant difference (P<0.01) between IM group and the other groups. Combined detection of atypical lymphocytes and transaminase can be regarded as a diagnostic marker of infectious mononucleosis.

The distinction of antimicrobial resistance of clinical bacteria isolated from county hospitals and a teaching hospital was investigated. Disc diffusion test was used to study the antimicrobial resistance of isolates collected from county hospitals and a teaching hospital. The, data was analyzed by WHONET5 and SPSS statistic software. A total of 655 strains and 1682 strains were collected from county hospitals and a teaching hospital, respectively, in the year of 2003. The top ten pathogens were Coagulase negative staphylococci (CNS), E. coli, Klebsiella spp., S. areus, P. aeruginosa, Enterococcus spp., Enterobacter spp., otherwise Salmonella spp., Proteus spp., Shigella spp. in county hospitals and Streptococcus spp., Acinetobacter spp., X. maltophilia in the teaching hospital. The prevalence of multi-drug resistant bacteria was 5% (4/86) of methicillin-resistant S. areus (MRSA), 12% (16/133) and 15.8% (9/57) of extended-spectrum β-lactamases producing strains of E. coli and Klebsiella spp., respectively, in county hospitals. All of the three rates were lower than that in the teaching hospital and the difference was statistically significant (P<0.01). However, the incidence of methicillin-resistant CNS (MRCNS) reached to 70% (109/156) in the two classes of hospitals. Generally, the antimicrobial resistant rates in the county hospitals were lower than those in the teaching hospital, except the resistant rates of ciprofloxacin, erythromycin, clindamycin, SMZco which were similar in the two classes of hospitals. There were differences between county hospitals and the teaching hospital in the distribution of clinical isolates and prevalence of antimicrobial resistance. It was the basis of rational use of antimicrobial agents to monitor antimicrobial resistance by each hospital.