To investigate the distribution of the genes of two major metallo-β-lactamases (MBL; i.e., IMP and VIM) and class 1 integrons (intI) in the clinical imipenem-resistant Pseudomonas aeruginosa, a total of 65 isolates, from a university hospital in Sichuan between December 2004 and April 2005 were screened for MBL genes by PCR using primers specific for bla_IMP-1, bla_VIM and bla_VIM-2 genes. The MBL-positive isolates were further assessed for class 1 integrons by PCR using specific primers. The nucleotide sequences of several PCR products were also determined. The results revealed that the bla_VIM gene was found in 81.5% (53/65) of all isolates, bla_VIM-2 gene was found in only 1 isolate and the intI gene was observed in 45.3% (24/53) of bla_VIM-positive isolates. One isolate carried simultaneously both bla_IMP-1 and intI genes, and to the best of our knowledge this is the first report of such isolate in southwest China. These observations highlight that the genes for VIM β-lactamase and class 1 integrons were predominantly present among the imipenem-resistant P. aeruginosa tested, confirming the current widespread threat of imipenem-resistant, integron-borne P. aeruginosa.

Mouse blastocysts were exposed to doses of 0, 1 and 10 μmol/L retinoic acid (RA) for 24 h and the cytotoxic effect of RA on the mouse blastocysts in vitro was observed. FITC-labeled terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL-FITC) assay was employed to stain apoptotic cells and immunohistochemical S-P staining method was used to detect the expression of Fas protein in mouse blastocysts in vitro. The results showed that RA could induce apoptosis and increase the expression of Fas proteins of trophectoderm (TE) and inner cell mass (ICM) cells in blastocysts. Compared with the findings for the control blastocysts, exposure to RA (10 μmol/L) resulted in a more significant apoptosis and higher expression level of Fas proteins (P<0.01). It was concluded that RA could induce apoptosis, which may result in a significant reduction in the average number of total cells and the trophectoderm/inner cell mass in blastocysts and an increased expression of Fas protein, suggesting that RA had a cytotoxic effect on the growth and development of early embryos in mice.

The activity of nano carbon fullerene lipidosome (NCFL) against influenza virus H1N1 in vitro was studied by observing the cytotoxicities and its activity rendered by different intensities of lighting with various periods of time. Rimantadine hydrochloride was used as the positive control drug. By using microcultural technique, the morphological changes of cells were observed and by using the gentian violet staining, antiviral activity of the NCFL against influenza virus was assayed. The results showed that: (1) The maximal concentration of the NCFL was 7 μg/mL and the 50% toxic concentration (TC₅₀) was 13.54 μg/mL respectively; (2) NCFL had a significant activity of directly killing the influenza virus, while the activities in antiadsorption and antireplication were not obvious; (3) There was a dose-activity relationship between the dosages of NCFL and the direct killing effect against the influenza virus, and the periods of lighting-time could influence the activity partly. It was concluded that NCFL had a significant activity of directly killing the influenza virus.

The X gene of HBV encodes a 17-kD protein, termed HBx, which has been shown to function as a transcriptional trans-activator of a variety of viral and cellular promoter/enhancer elements. The aim of this study was to investigate the effect of HBx on gene expression of interleukin (IL)-1 and IL-6, and proliferation of rat mesangial cells in vitro. The X gene of HBV was amplified by PCR assay, and inserted into the eukaryotic expression vector pCI-neo. The structure of recombinant pCI-neo-X plasmid was proved by restrict endonuclease digestion and sequencing analysis. pCI-neo-X was transfected into cultured rat mesangial cell line in vitro via liposome. HBx expression in transfected mesangial cells was detected by Western blot. The IL-1β and IL-6 mRNA expression in those cells was assayed by semiquantitative RT-PCR. Mesangial cell proliferation was tested by MTT. The results showed that HBx was obviously expressed in cultured mesangial cell line at 36th and 48th h after transfection. The expression of IL-1β and IL-6 mRNA was simultaneously increased. The cell proliferation was also obvious at the same time. It was concluded that HBx gene transfection could induce IL-1β and IL-6 gene expression and mesangial cell proliferation. HBx may play a critical role in mesangial cell proliferation through upregulation of the IL-1β and IL-6 gene expression.

In adipose tissue of rats on long-term high fat diet, the inflammatory changes the roles of angiotensin receptor blocker (ARB) in pimelitis and insulin resistance (IR) were observed. IR rat model was established by feeding high calorie and high fat diet. The change in insulin sensitivity was detected by euglycemic-hyperinsulinemic clamp technique 8 weeks after intervention by valsartan. The expression levels of CD68 and MCP-1 mRNA and proteins in adipose tissue were examined by RT-PCR and immunohistochemistry respectively. The parameters of blood glucose, insulin and blood lipid were analyzed. The results showed that in high fat diet group intra-abdominal obesity developed, the content of visceral fat and the number of inflammatory cells in local adipose tissue were significantly increased (P<0.01), the levels of serum triglyceride, free fatty acids and fasting serum insulin were markedly increased, the insulin sensitivity was significantly lowered (P<0.01), and the expression of CD68 and MCP-1 was significantly increased as compared with control group (P<0.01). In ARB interventional group, the content of visceral fat, the number of inflammatory cells and the expression of CD68 and MCP-1 in local adipose tissue were significantly reduced (all P<0.01), but the insulin sensitivity was significantly enhanced (P<0.01) as compared with high fat diet group. There were pimelitis and IR in rats with obesity induced by long-term high calorie and high fat diet. The ARB can significantly inhibit the infiltration of macrophages and the expression of MCP-1 in adipose tissue, thereby attenuating the inflammation and improving IR in rats.

The effects of berberine on the expression of hepatocyte nuclear factor-4α (HNF-4α) in liver of rats with fructose-induced insulin resistance and the molecular mechanism of berberine preventing insulin resistance were investigated. The experimental animals were divided into two groups of 16 animals each. The control group received a control routine diet containing 60% carbohydrate, and the study group a high-fructose diet containing 60% fructose as the sole source of carbohydrate. At the end of 6 weeks these were each subdivided into two groups. One was administered with berberine [187.5 mg/(kg·d) in 5 g/L carboxymethyl cellulose] by intragastric intubation and the other group was treated with a vehicle (5 g/L carboxymethyl cellulose). The rats were fed on the same dietary regimen for the next 4 weeks. After the experimental period of 10 weeks, plasma glucose, insulin and triglyceride levels were measured. HOMA insulin resistance index (HOMA-IR) was assayed. Immunohistochemistry, semiquantitative RT-PCR and western blot were used to detect the expression of HNF-4α in liver. Compared with control diet, fructose feeding induced hyperinsulinemia, HOMA-IR and increased triglyceride (all P<0.01). Berberine prevented the rise in plasma insulin (P<0.01), HOMA-IR (P<0.01) and triglyceride (P<0.05) in the fructose-fed rats. No change in plasma glucose was seen among these groups. The mRNA and protein expression of HNF-4α was decreased in the fructose-fed rats, but berberine could promote its expression. It was concluded that berberine could prevent fructose-induced insulin resistance in rats possibly by promoting the expression HNF-4α in liver.

In order to screen potential mRNA locations of P gene in which targeting siRNAs can effectively inhibit HBV expression, 5 recombinant plasmids containing 4 targeting-specific siRNA fragments and a control were prepared and transfected into 2.2.15 cells respectively. The expression levels of HBx mRNA, HBs mRNA and HBc mRNA were detected by RT-PCR. The concentrations of the hepatitis B virus antigens, including HBsAg and HBeAg harvested from the culture supernatant of transfected 2.2.15 cells, were measured by ELISA. X protein was tested by Western blot. The results showed that four siRNAs against distinct mRNA locations of HBV polymerse gene had different inhibitory effects on their targeted mRNA. The plasmid-derived psiRNA1 and psiRNA2 could effectively inhibit the transcription and translation of HBs gene, whereas the inhibitory efficiency of psiRNA3, psiRNA4 for HBe gene was much higher than that of psiRNA1 and psiRNA2. In comparison to the rest of psiRNAs in this study, psiRNA4 was the most effective to suppress the transcirption and translation of HBx. It is suggested that siRNA can be considered as a powerful therapeutic agent for reducing HBV expression. The siRNAs against HBV polymerase are effective largely depending on the location of targeted sites. To enhance inhibitory efficiency, hunting for high effective target in polymerase gene is necessary and feasible.

In current study, the effect of angiotensin receptor blocker Micardis on the localization and expression of aquaporin-2 (AQP2) was investigated in the renal medullary collecting duct of mice with diabetic nephropathy (DN). Mice were divided into three groups: normal group, DN group and Micardis-treated group. Six weeks after establishment of STZ-induced DN model in mice, the expression of AQP2 in renal medulla was detected measured by semiquantitative immunofluorescence histochemistry and Western blot techniques, and the localization of AQP2 by confocal immunofluorescence laser scanning microscopy. The results showed that the urinary osmolality was decreased in DN group as compared with normal group (2.39±0.11 vs 3.16±0.16, P<0.05). Although the localization of AQP2 on the renal medulla was unchanged, the expression of AQP2 was increased significantly in DN group as compared with normal group. Micardis could partly attenuate above changes. It was concluded that treatment with Micardis could partly rectify the abnormal expression of AQP2 in renal medulla of DN mice, which suggested that rennin-angiotensin system (RAS) is implicated in the pathogenesis of DN by regulating the expression of AQP2.

Hypermethylation in the promoter region is an important epigenetic mechanism for the transcriptional repression of a number of cancer-associated genes, and over-expression and/or increased activity of DNA methyltransferases are considered to be the main cause of promoter hypermethylation. In order to explore the roles of two methyltransferase members (DNMT1 and DNMT3b) in the cholangiocarcinoma tumorigenesis, antisense eukaryotic expression plasmid of DNMT1 and DNMT3b gene was constructed respectively, and were co-transfected into the human cholangiocarcinoma cell line QBC-939 to observe their biological effects on the cell growth and proliferation ability, apoptosis, cell cycle alteration, and the tumorigenesis ability in the subcutaneous tissue of nude mouse. The results demonstrated that co-transfection with antisense eukaryotic expression plasmid of DNMT1 and DNMT3b gene and single transfection with antisense eukaryotic expression plasmid of DNMT1 gene can suppress the growth and proliferation of QBC-939, block the cell cycle at G1 phase, increase the apoptosis rate, minimize the tumor size in the subcutaneous tissue of nude mouse. The suppressing biological effect of co-transfection is stronger than single transfection with antisense DNMT1. Meanwhile, single transfection with antisense eukaryotic expression plasmid of DNMT3b gene has no effects on the biological characteristics of QBC-939. This study suggests that DNMT1 gene plays a key role in DNA methylation and DNMT3b gene may act as an accessory to support its function in inactivation of tumor suppressor genes. Combination DNMT1 and DNMT3b will increase their biological effects and have the synergistic effect on suppressing the growth of human cholangiocarcinoma cell line QBC-939.

In order to study the effects of ethyl pyruvate on cardiomyocyte apoptosis following ischemia/reperfusion (I/R) in vitro and the expression of Bcl-2 and Bax proteins, isolated rat hearts were perfused in a Langendorff model. Twenty-four rats were randomly divided into 3 groups (n=8 in each group): control group was perfused for 120 min. In the I/R group, after 30 min stabilization the injury was induced by 30 min global ischemia followed by 60 min reperfusion. Ethyl pyruvate (EP) group was set up with the same protocol as I/R group except that it was supplied with 2 mmol/L EP 15 min before ischemia and throughout reperfusion. Myocardial malonaldehyde (MDA) content was measured. Myocardial apoptotic index (AI) was tested by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) method. The expression of anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax in cardiac myocytes was detected by immunohistochemistry. As compared with control group, the content of MDA, myocardial AI and the expression of Bcl-2, Bax proteins were increased significantly in I/R group, but the content of MDA, myocardial AI and the expression of Bax protein were decreased obviously and the expression of Bcl-2 protein was up-regulated in EP group (P<0.05). These results demonstrate that EP could inhibit apoptosis of cardiac myocytes possibly via alleviating oxidative stress, up-regulating Bcl-2 and down-regulating Bax proteins.

The value of color Doppler flow imaging (CDFI) and intravenous contrast-enhanced ultrasound (CEUS) for assessing the transplanted liver and early diagnosing complications by examining hemodynamic changes was discussed. Seventy-five patients with orthotopic liver transplantation (OLT) underwent CDFI. The following parameters were measured: peak systolic velocity (PS), resistance index (RI) and Doppler perfusion index (DPI) of the hepatic artery (HA), time average velocity (TAV) of portal vein (PV) and velocity of hepatic vein (HV) in different stages postoperation. And 11 patients of them received CEUS. Thirty healthy subjects were enrolled as controls. The results showed that: (1) In 23 patients without obvious complications, TAV of PV within 15 days post-operation was significantly higher than in controls (P<0.05), PS and DPI of HA within 7 days postoperation were lower, but RI was higher than in controls (P<0.05); (2) When the hepatic artery thrombosis (HAT) occurred, PS and DPI of HA were obviously decreased, but TAV of PV significantly increased like a high saw-tooth wave; (3) While rejection occurred, both TAV of PV and PS of HA were decreased with the increase in RI of HA, and the triphasic wave of HV disappeared and displayed as saw-tooth wave; (4) The incidence of biliary complications in liver transplantation was increased when DPI was reduced; (5) Seven cases of hepatic carcinoma relapse after OLT demonstrated hyperecho in the arterial phase and hypoecho in the portal and later phase on CEUS; (6) In 2 cases of HA thrombus, there was no visualized enhancement in arterial phase of CEUS, but enhancement during the portal vein and parenchymal phase. It was concluded that the hemodynamic changes of PV, HA and HV in the transplanted liver are valuable for assessing the transplanted liver and early diagnosing complications on CDFI and CEUS.

In order to evaluate the left ventricular remodeling in patients with myocardial infarction after revascularization with intravenous real-time myocardial contrast echocardiography (RT-MCE), intravenous RT-MCE was performed on 20 patients with myocardial infarction before coronary revascularization. Follow-up echocardiography was performed 3 months after coronary revascularization. Segmental wall motion was assessed using 18-segment LV model and classified as normal, hypokinesis, akinesis and dyskinesis. Myocardial perfusion was assessed by visual interpretation and divided into 3 conditions: homogeneous opacification=1; partial or reduced opaciflcation or subendocardial contrast defect=2; constrast defect=3. Myocardial perfusion score index (MPSI) was calculated by dividing the total sum of contrast score by the total number of segments with abnormal wall motion. Twenty patients were classified into 2 groups according to the MPSI: MPSI≤1.5 as good myocardial perfusion, MPSI>1.5 as poor myocardial perfusion. To assess the left ventricular remodeling, the following comparisons were carried out: (1) Comparisons of left ventricular ejection fraction (LVEF), left ventricular end-systolic volume (LVESV) and left ventricular end-diastolic volume (LVEDV) before and 3 months after revascularization in two groups; (2) Comparisons of LVEF, LVESV and LVEDV pre-revascularization between two groups and comparisons of these 3 months post-revascularization between two groups; (3) Comparisons of the differences in LVEF, LVESV and LVEDV between 3 months post-and pre-revascularization (ΔLVEF, ΔLVESV and ΔLVEDV) between two groups; (4) The linear regression analysis between ΔLVEF, ΔLVESV, ΔLVEDV and MPSI. The results showed that the LVEF obtained 3 months after revascularization in patients with MPSI>1.5 was obviously lower than that in those with MPSI≤1.5. The LVEDV obtained 3 months post-revascularization in patients with MPSI>1.5 was obviously larger than that in those with MPSI≤1.5 (P=0.002 and 0.04). The differences in ΔLVEF and ΔLVEDV between patients with MPSI>1.5 and those with MPSI≤1.5 were significant (P=0.002 and 0.001, respectively). Linear regression analysis revealed that MPSI had a negative correlation with ΔLVEF and a positive correlation with ΔLVESV, ΔLVEDV (P=0.004, 0.008, and 0.016, respectively). It was concluded that RT-MCE could accurately evaluate the left ventricular remodeling in patients with myocardial infarction after revascularization.

The myocardial viability after myocardial infarction was evaluated by intravenous myocardial contrast echocardiography. Intravenous real-time myocardial contrast echocardiography was performed on 18 patients with myocardial infarction before coronary revascularization. Follow-up echocardiography was performed 3 months after coronary revascularization. Segmental wall motion was assessed using 18-segment LV model and classified as normal, hypokinesis, akinesis and dyskinesis. Viable myocardium was defined by evident improvement of segmental wall motion 3 months after coronary revascularization. Myocardial perfusion was assessed by visual interpretation and divided into 3 conditions: homogeneous opacification; partial or reduced opaciflcation or subendocardial contrast defect; contrast defect. The former two conditions were used as the standard to define the viable myocardium. The results showed that 109 abnormal wall motion segments were detected among 18 patients with myocardial infarction, including 47 segments of hypokinesis, 56 segments of akinesis and 6 segments of dyskinesis. The wall motion of 2 segments with hypokinesis before coronary revascularization which showed homogeneous opacification, 14 of 24 segments with hypokinese and 20 of 24 segments with akinese before coronary revascularization which showed partial or reduced opaciflcation or subendocardial contrast defect was improved 3 months after coronary revascularization. In our study, the sensitivity and specificity of evaluation of myocardial viability after myocardial infarction by intravenous real-time myocardial contrast echocardiography were 94.7% and 78.9%, respectively. It was concluded that intravenous real-time myocardial contrast echocardiography could accurately evaluate myocardial viability after myocardial infarction.

In order to investigate the application of proton magnetic resonance spectroscopy (¹H-MRS) and computerized tomography (CT) in the quantitative diagnosis of nonalcoholic fatty liver disease (NAFLD) and evaluation of therapeutic effects, 22 patients with NAFLD were selected according to the Chinese Medical Association’s (CMA) standard of the NAFLD in comparison with 20 healthy volunteers (as control group). Blood samples for biochemistry were collected. The severity of hepatosteatosis was evaluated by ¹H-MRS scan and CT scan of liver. The intrahepatic content of lipid (IHCL) and CT value ratio of liver to spleen were calculated. The patients in NAFLD group were treated with Ganzhixiao Capsule for 8 weeks. The changes in IHCL and CT value ratio of liver to spleen were observed before and after treatment. In NAFLD group serum ALT, TG, IHCL calculated by ¹HMRS were increased and CT value ratio of liver to spleen decreased significantly as compared with control group. After treatment for 8 weeks serum ALT, TG, IHCL were decreased significantly, while CT value ratio of liver to spleen increased significantly in NAFLD group. It was suggested that IHCL could be measured precisely by ¹HMRS. NAFLD was treated effectively by Ganzhixiao capsule.

The effects of synthetic Smac peptide (SmacN7) on chemotherapeutic sensitivity of bladder cancer cells were investigated. SmacN7 penetratin peptide was synthesized and delivered into T24 cells. MTT assay was used to evaluate the viability of T24 cells induced by low-dosage of MMC. Flow cytometry was used to analyze the proportions of apoptosis. Western blot was used to detect the expression of XIAP and Caspase-3. The activity of Caspase-3 was measured and the effect of SmacN7 combined with MMC on T24 cell lines was also determined. The results showed that SmacN7 penetratin peptide could successfully interact with endogenous XIAP, increase the proportions of apoptosis of T24 cell lines induced by low-dosage of MMC in a dose-and time-dependent manner. An obvious down-regulation of XIAP expression and up-regulation of Caspase-3 was identified by Western blot. The activity of Caspase-3 in experimental group was significantly increased as compared with that in the control group. As compared with MMC group, the viability of T24 cells in SmacN7 penetratin peptide+MMC group was markedly decreased to 2.22 and 3.61 folds at 24 h and 48 h respectively. It was concluded that SmacN7 penetratin peptide could act as a cell-permeable IAP inhibitor, inhibit the proliferation, induce apoptosis and enhance the chemo-sensitivity of bladder cancer cells to MMC. These findings indicate that SmacN7 penetratin peptide may be a very promising ageut for bladder cancer treatment when used in combination with chemotherapy.

The effect of cyclin-dependent kinase inhibitors Cip1/Waf1 (p21) on regulatory expression of survivin transcription in human hepatocellular carcinoma cell HepG2 was observed and the related mechanisms explored. Doxorubicin (DOX) was used to treat HepG2. Eukaryotic vector pEGFP-C2-p21 was transfected into HepG2 by lipofectamine and positive clones were screened out by G418. The mRNA expression of p21 and survivin was detected by real-time fluorescent quantitative polymerase chain reaction (RQ-PCR). Flow cytometry was used to examine the cell cycle, and reverse transcription polymerase chain reaction (RT-PCR) was used to measure the levels of E2F-1 and p300. The results showed that: (1) After treatment with DOX, the expression of p21 was increased, whereas that of survivin was reduced during 24 h of treatment; (2) After transfection of pEGFP-C2-p21 into HepG2, p21 level was significantly enhanced to 2100.11-folds or 980.89-folds in comparison to HepG2 or HepG2-C2 group, and survivin level was markedly down-regulated to 0.54% or 0.59% relative to the control groups; (3) Overexpressed p21 resulted in G1/G0 phase arrest (F=31.59, P<0.01), meanwhile E2F-1 mRNA and p300 mRNA were reduced as compared with those of controls (F_E2F-1=125.28, P<0.05; F_p300=46.01, P<0.01). It was suggested that p21 could be a potential mediator of survivin suppression at transcription level in HepG2 cell, which might be through the block at G1/G0 phase and down-regulation of transcription factors E2F-1 and p300.

The effects of Oxymatrine (Oxy) on the proliferation and apoptosis of human esophageal carcinoma Eca109 cell line and the mechanism were investigated. The human esophageal carcinoma Eca109 cells were cultured in vitro. The Oxy-induced apoptosis of Eca 109 cells was assayed by using flow cytometry. The expressions of p-ERK_1/2, Cyclin D1, p21^waf/cip1, Bax and Bcl-2 were detected by Western blot. Flow cytometry revealed that Oxy could induce the apoptosis of Eca109 cells. Western blot showed that Oxy of different concentrations suppressed the expressions of p-ERK_1/2, Cyclin D1 and Bcl-2, but up-regulated the expression of p21^waf/cip1 and Bax, and the ratio of Bax/Bcl-2 was increased. It was suggested the Oxy could induce the apoptosis of Eca109 cells, which might be related to the upregulation of p21^waf/cip1 and the downregulation of p-ERK_1/2, Cyclin D1 and p21^waf/cip1. The possible pathway may be related to Bcl-2/Bax.

The effects of nanometer realgar suspension on proliferation and apoptosis of human uterine cervix cancer cell line SiHa cells and oncogenic genes HPV16E6/E7 were investigated. A “micro-jet efflux” strategy was used for the preparation of nanometer realgar suspension. SiHa cells were treated with nanometer Realgar suspension in various concentrations (6.25, 12.5, 25 and 50 mg/L) for different durations (12, 24, 48 and 72 h). The inhibitive effect of nanometer realgar suspension on growth of SiHa cells was detected by MTT method. Special morphological changes of apoptosis were observed by transmission electron microscopy (TEM) and DNA fragments electrophoresis. The apoptotic rate was quantified by flow cytometry (FCM). The expression of HPV16E6/E7 mRNA and protein was assayed by RT-PCR and Western blot respectively. The results showed after being treated with 25 50 mg/L nanometer realgar suspension for 48 h, the survival rate of SiHa cells was decreased, and apoptotic rate markedly increased in a time-and concentration-dependent manner. TEM and DNA electrophoresis revealed the special morphological changes of apoptosis. The apoptotic rate of SiHa cells treated with nanometer realgar suspension was significantly higher than in the control group (P<0.01), and G₀/G₁ phase arrest appeared following treatment with nanometer realgar suspension in 25 and 50 mg/L for 48 h. RT-PCR and Western blot assay indicated that nanometer realgar suspension reduced the HPV16E6/E7 gene expression. Nanometer realgar suspension could inhibit the proliferation and induce apoptosis of SiHa cells. The mechanism may be related to the down-regulation of the HPV16E6/E7 gene expression.

To enhance the fusion of graft bone in thoracolumbar vertebrae and minimize the postoperative loss of correction, short-segment pedicle screw fixation was reinforced with posterior moselizee bone grafting in vertebrae for spinal fusion in patients with thoracrolumbar vertebrate fractures. Seventy patients with thoracrolumbar vertebrate fractures were treated by short-segment pedicle screw fixation and were randomly divided into two groups. Fractures in group A (n=20) were reinforced with posterior morselized bone grafting in vertebrae for spinal fusion, while patients group B (n=50) did not receive the morselized bone grafting for bone fusion. The two groups were compared in terms of kyphotic deformity, anterior vertebral height, instrument failure and neurological functions after the treatment. Frankel grading system was used for the evaluation of neurological evaluation and Denis scoring scale was employed for pain assessment. The results showed that the kyphosis correction was achieved in both group A and group B (group A: 6.4 degree; group B: 5.4 degree)/ At the end of follow-up, kyphosis correction was maintained in group A but lost in group B (P=0.0001). Postoperatively, greater anterior height was achieved in group A than in group B (P<0.01). During follow-up study, anterior vertebral height was maintained only in Group A (P<0.001). Both group A and group B showed good Denis pain scores (P1 and P2) but group A outdid group B in terms of control of severe and constant pain (P4 and P5). By Frankel criteria, the changes in neurological functions in group A was better than those of group B (P<0.001). It is concluded that reinforcement of short-segment pedicle fixation with morselized bone grafting for the treatment of patients with thoracolumbar vertebrae fracture could achieve and maintain kyphosis correction, and it may also increase and maintain anterior vertebral height. Morselized bone grafting in vertebrae offers immediate spinal stability in patients with thoracolumbar vertebrate fractures, decreases the instrument failure and provides better postoperative pain control than without the morselized bone grafting.

In order to provide anatomical basis for transoral approach (TOA) in dealing with the ventro lesions of craniocervical junction, and the design and application of artificial atlanto-odontoid joint, microsurgical dissecting was performed on 8 fresh craniocervical specimens layer by layer through transoropharyngeal approach. The stratification of posterior pharyngeal wall, course of vertebral artery, adjacent relationship of atlas and axis and correlative anatomical parameters of replacement of artificial atlanto-odontoid joint were observed. Besides, 32 sets of atlanto-axial joint in adults’ fresh bony specimens were measured with a digital caliper and a goniometer, including the width of bony window of anterior arch of atlas, the width of bony window of axis vertebra, the distance between superior and inferior two atlas screw inserting points, the distance between two axis screw inserting points etc. It was found that the width of atlas and axis which could be exposed were 40.2±3.5 mm and 39.3±3.7 mm respectively. The width and height of posterior pharyngeal wall which could be exposed were 40.1±5.2 mm and 50.2±4.6 mm respectively. The distance between superior and inferior two atlas screw inserting points was 28.0±2.9 mm and 24.0±3.5 mm respectively, and the distance of bilateral axis screw inserting points was 18.0±1.2 mm. The operative exposure position through TOA ranged from inferior part of the clivus to the superior part of the C3 vertebral body. Posterior pharyngeal wall consisted of 5 layers and two interspaces: mucosa, submucosa, superficial muscular layer, anterior fascia of vertebrae, anterior muscular layer of vertebrae and posterior interspace of pharynx, anterior interspace of vertebrae. This study revealed that it had the advantages of short operative distance, good exposure and sufficient decompression in dealing with the ventro lesions from the upper cervical to the lower clivus through the TOA. The replacement of artificial atlanto-odontoid joint is suitable and feasible. The design of artificial atlanto-odontoid joint should be based on the above data.

The effect of astrocytes on the growth and differentiation of co-cultured mesenchymal stem cells (MSCs) was studied. MSCs of Wistar rats were isolated, cultured and labeled with Hoechst33342. The labeled MSCs were co-cultured with astrocytes in DMEM/F12/10%FBS. The growth status and morphologic change of MSCs were observed. The expression of specific protein NeuN, GFAP and CNP was detected by using immunofluorescence staining. The results showed that the two kinds of co-cultured cells grew well. Some labeled MSCs stretched out long processes with spin, triangle and star shapes. Immunofluorescence stain showed that these cells expressed NeuN, GFAP or CNP. It was suggested that astrocytes could promote the prolifevation of co-cultured MSCs and induce them to differentiate into neural like cells.

The expression of Aurora B in normal endometria and endometrial carcinomas and its relation with clinicopathologic parameters of endometrial carcinomas were investigated. Streptavidin-biotin peroxidase (SP) immunohistochemical technique was used to detect the expression of Aurora B in 10 cases of normal proliferative phase endometria, 10 cases of normal secretory phase endometria and 72 cases of endometrial carcinomas respectively. According to the 1988 International Federation of Gynecology and Obstetrics (FIGO) grade, there were 37 patients in grade 1, 23 in grade 2 and 12 in grade 3 respectively. According to the FIGO stage, there were 59 patients in stage I–II and 13 patients in stage III–IV. Aurora B was expressed in both normal proliferative phase endometria, secretory phase endometria and endometrial carcinomas, but its positive labeling index (PLI) in proliferative phase endometria was significantly higher than that in secretory phase endometria (P<0.01) and endometrial carcinomas (P<0.01). The PLI of Aurora B was lower in tumors with well differentiation (G₁), low surgical staging (I–II), and ≤1/2 myometrial invasion than that in tumors with moderate and low differentiation (G₂—G₃), higher surgical staging (III–IV), and >1/2 myometrial invasion (all P<0.01). Aurora B exerts its functions in the replication of normal endometrial glandular cells; Expression of Aurora B is significantly correlated with biologic behavior of endometrial carcinoma, indicating that Aurora B may be a promising prognostic factor in endometrial carcinoma.

The activation of mammalian target of rapamycin (mTOR) signaling pathway in endometrial carcinoma cells Ishikawa and HEC-1A was investigated. The expression of mTOR was detected by confocal fluorescence microscopy in Ishikawa and HEC-1A cells. The mRNA levels of PTEN and mTOR, the downstream substrate S6K1 and 4E-BP1 protein were assayed by RT-PCR and Western blot, respectively. The expression of PTEN in Ishikawa cells was deficient, but intact in HEC-1A cells respectively (P<0.01). There was mTOR expression in both Ishikawa and HEC-1A cells and the phosporylated substrate levels in Ishikawa cells were higher than those in HEC-1A cells (P<0.05). mTOR signaling pathway is activated in two endometrial carcinoma cell strains and the status of activation is related with PTEN expression of the cells. The activation level of mTOR is higher in PTEN-deficient endometrial carcinoma cells than that in PTEN-intact endometrial carcinoma cells.

The killing effects of lymphocytes on Hela cells expressing interleukin-12 (IL-12) in vitro were explored. By using gene transfection technique, full length IL-12 gene was transfected into Hela cells. The expression of IL-12 in Hela cells was detected quantitatively by ELISA; Changes in killing effects of lymphocytes on Hela cells expressing IL-12 were observed by MTT. It was found that Hela cells could express IL-12 between 24 h and 72 h after transfection. Killing activity of lymphocytes on Hela cells expressing IL-12 was significantly enhanced. It was concluded by cell transfection technique, Hela cells could express IL-12 and were more easily killed by lymphocytes.

The correlation between aquaporin 1 (AQP1) and hypoxia-inducible factor 1 (HIF 1) in breast cancer tissues was preliminarily studied. In 155 cases of breast cancer, the expression levels of AQP1 were detected by immunohistochemisty in HIF1-positive group or HIF1-negative group, and the correlation between AQP1 and HIF1 was analyzed. The overexpression of AQP1 and HIF1 were observed in 155 cases of breast cancer tissues. The expression level of AQP1 in HIF1-positive group was significantly higher than that in HIF1-negative group. The positive expression rate of AQP1 was 296.55±24.67 and 168.37±37.53 in HIF1-positive group and HIF1-negative group respectively with the difference being very significant between them (P<0.001). It was concluded that AQP1 was overexpressed in the HIF1-positive group and there were some correlations between AQP1 and HIF1, suggesting they interact each other and regulate the oncogenesis of breast cancer.

In order to investigate the role of Caspase-3 and Bax in the pathogenesis of lichen planus, immunohistochemistry was used to detect the expression of Caspase-3 and Bax in skin lesions of the patients with lichen planus and skin tissues of normal subjects. The results showed that positive rate of Caspase-3 and Bax expression in lichen planus were significantly higher than that in normal skins (both P<0.05). Meanwhile, there was a obvious correlation between the increase of Caspase-3 and that of Bax in lichen planus. The expression of Caspase-3 and Bax might play an important role in the development of lichen planus.

In order to investigate the expression of Th1 and Th2 cytokines in the vaginal candidiasis caused by Candida, the fungal vaginitis model was established in female ICR mice by intravaginal inoculation of suspension of C. albicans after the animals were pretreated with estradiol. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the expression of IL-2, IL-4, IL-10 and TGF-β1 in the vagina in the mice of different groups at different time points after the beginning of the experiment. The average expression level of IL-2 mRNA in group D (estrogen-treated mice) was significantly higher than that in groups H (estrogen-untreated mice) and I (control group) on the day 2. The average expression level of IL-4 mRNA in group D was significantly higher than that in groups I and H on the day 5. The average expression level of IL-10 mRNA in group D was significantly higher than that in groups H and I from day 7 to 11. The average expression level of TGF-β1 mRNA in group D was significantly higher than that in groups H and I at all time points. It was concludes that the high-level expression of IL-2 mRNA during early infection was associated with clearance of mucosal C. albicans, and the high-level expression of IL-10 mRNA during late stage of the infection was related to susceptibility to infection. TGF-β1 may play a predominant role when the virtual absence of changes in other Th-type cytokines during infection.

The inhibitory effects of (-)-epigallocatechin-3-gallate (EGCG) on the invasion of human malignant melanoma cell line A375 and the possible molecular mechanisms of this effect were investiaged. A375 cells were pretreated with 20 μg/mL EGCG for 24, 48 and 72 h respectively and the E-cadherin expression was detected by Western blot analysis. A375 cells were also pretreated with different concentrations of EGCG (1, 5, 10 and 20 μg/mL) for 72 h and the expression of E-cadherin was measured by RT-PCR. The adhesion and invasion of A375 cells were tested by cell-matrigel adhesion assay and matrigel invasion assay respectively. The results showed that EGCG could significantly up-regulate the expression of E-cadherin time-and concentration-dependently (both P<0.05). Statistical analysis showed that A375 cells invasion was inhibited by EGCG and correlated with the up-regulation of E-cadherin expression. It was suggested that EGCG strongly inhibited invasion of A375 cells, and the inhibition mechanism was possibly associated with the up-regulation of E-cadherin expression.

The effects of lutein on the growth and migration of bovine lens epithelial cells (BLECs) in vitro were observed in an attempt to find a drug that can prevent after-cataract. BLECs were cultured in vitro and different concentrations of lutein were added to the BLECs cultures of the second and third generations. The effects of lutein on the proliferation of BLECs in vitro were examined by the MTT method, and the migration of BLECs was evaluated by a scratch wound assay. The results showed that: (1) Lutein at concentrations of 1 to 16 μmol/L could inhibit the proliferation of BLECs in a dose-and time-dependent manner (P<0.01): (2) The migration of BLECs was evaluated by wound healing rate. As compared with the control group, the wound healing rate in the experimental groups was decreased from 0.672±0.164 to −0.234±0.144 and −0.597±0.063 (P<0.01) at 1 and 2 μmol/L lutein, respectively. It was concluded that lutein at concentration of ≥1 μmol/L inhibited the proliferation and migration of BLECs in vitro. Lutein may become an effective drug to prevent after-cataract.

The effects of A771726, the active metabolite of leflunomide, on experimental rat corneal neovascularization (NV) in vivo and on cultured human umbilical vein endothelial cells in vitro were studied. The corneal NV was induced by alkali burn in 40 SD rats. The rats were randomly divided into 4 groups with 10 rats in each group. Group A was treated with 0.9% sodium chloride (control group), and group B, group C and group D were given different concentrations of A771726 eye drops (0.5%, 1.0%, 2.0% respectively) 4 times daily during days 0–28. The occurrence and development of corneal NV were observed at 4, 7, 14, 21 and 28 day after alkali burn by a slit lamp microscope. The cultured human umbilical vein endothelial cells (ECV-304) were incubated with A771726 solution at different concentrations (20, 40, 80, 160, 320 mol/L) for 36 h. The proliferation of cells was assessed by methyl thiazolyl tetrazolium (MTT), and the expression of proliferating cell nuclear antigen (PCNA) in cells was detected by using immunofluorescence under the laser confocal microscope. The rat model showed that the onset of corneal NV was delayed and progression of corneal NV was inhibited in the groups C and D. The corneal NV areas in groups C and D were significantly smaller than in groups A and B (P<0.01). No significant difference was found in corneal NV areas between groups C and group D (P>0.05). A771726 solution (⩾40 μmol/L) could inhibit proliferation of human umbilical vein endothelial cells and decrease the expression of PCNA in cells significantly. A771726, as the active metabolite of leflunomide, strongly prevented corneal NV induced by alkali burn in the in vivo model, and inhibited proliferation of human umbilical vein endothelial cells in the in vitro model. Therefore, A771726 may serve as an angiogenic inhibitor in the treatment of corneal NV.

The effect of quercetin (Que) on proliferation and apoptosis of human nasopharyngeal carcinoma HEN1 cells was investigated. Inhibition rate of quercetin on HEN1 was assayed by MTT method, apoptosis by flow cytometry (FCM), and the caspase-3 expression of each group by colorimetry set respectively. Quercetin inhibited HEN1 cells in in a dose-(r=0.709, P<0.01) and time-dependent manner (r=0.703, P<0.01). The ratio of apoptotic and necrosis cells was increased in the cells treated with quercetin. Cell cycle was specificly arrested in G₂/M phase. Apoptosis cusp was revealed by FCM. The activity of caspase-3 was significantly up-regulated in 5 groups treated with quecetin as compared with control group (P<0.05). It was concluded that the growth inhibition of quercetin was highly related to cell cycle arrest at the G₂/M phase and induction of caspase-dependent apoptosis in human nasopharyngeal carcinoma HEN1 cells.

The aim of this study was to evaluate the efficiency and safety of ultrasound-guided thrombin injection on femoral pseudoaneurysm (FPA) as compared to ultrasound-guided local oppression. Eleven cases of FPA were enrolled and 7 cases received ultrasound-guide thrombin injection (injection group), and the remaining 4 cases were treated with local oppression (oppression group). Efficiency and safety were analyzed by ultrasound and subsequent follow-up. The results showed that 1 case relapsed in oppression group while no relapse occurred in thrombin injection group. Ultrasound-guided thrombin injection is better for treatment of FPA in terms of effectiveness and safety.

The purpose of this study was to screen peptides that can specifically bind to human hepatocellular carcinoma (hHCC) cells using phage display of random peptide library in order to develope a peptide-based carrier for the diagnosis or therapy of hHCC. A peptide 12-mer phage display library was employed and 4 rounds of subtractive panning were performed using the hHCC cell line HepG2 as the target. After panning, the phages that specifically bound to and internalized in hHCC cells were selected. The selected phages demonstrated highly specific affinity to HepG2 cells analyzed by ELISA and immunofluorescence analysis. 57.3% of the selected phage clones displayed repeated sequence FLLEPHLMDTSM, and 4 amino acid residues, FLEP were extremely conservative. Based on the sequencing results, a 16-mer peptide (WH-16) was synthesized. The competitive ELISA showed that the binding of the phage clones displayed sequence FLLEPHLMDTSM to HepG2 cells was efficiently inhibited by WH-16. Our findings indicate that cellular binding of phage is mediated via its displayed peptide and the synthesized 16-mer peptide may have the potential to be a delivery carrier in target diagnosis or therapy for hHCC.

Whether inhibiting the activity of nuclear factor (NF)-κB potentiates cisplatin-induced apoptosis in non-small cell lung cell line A549 cells was investigated. The recombinant plasmid pcDNA3.1 (+)/IκBα expressing IκBα was constructed. The in vitro cultured A549 cells were transfected with pcDNA3.1 (+)/IκBα alone, or pcDNA3.1 (+)/IκBα combined with cisplatin. The mitochondrial membrane potential (Δψm) was determined by rhodamine 123, the activity of caspase-3 was tested by colorimetric assay, and cell apoptosis was detected by flow cytometry with the annexin V /propidium iodide assay. The results showed that the activity of NF-κB in A549 cells was inhibited by transfecting pcDNA3.1(+)/I. Transfection of pcDNA3.1(+)/I alone did not promote apoptosis. Treatment of cisplatin alone had a little effect on cell apoptosis. Transfection of pcDNA3.1(+)/I combined with cisplatin treatment significantly induced apoptosis of A549 cells. It was concluded that inhibiting the activity of NF-B potentiated cisplatin-induced apoptosis of A549 cells.