Abstract:Objective: To determine the adjuvant potential of artemisinin with a soluble leishmanial antigen in vaccinating BALB/c mice.Methods: Seventy two female BALB/c mice were randomly assigned into six groups. The mice were vaccinated with soluble Leishmania antigens (SLA) alone, artemisinin co-administered with SLA, SLA and Bacille Calmette Guérin (BCG) vaccine, and artemisinin and BCG alone. Unvaccinated mice formed the control group. The induction of cell-mediated immunity following vaccination was determined by measuring in vitro lymphocyte proliferation and the production of interleukin (IL)-4, IL-5 and gamma interferon (IFN-γ) determined by flow cytometry. Protection against L. major was determined by quantifying parasite burdens in L. major infected footpads using a limiting dilution assay and by measuring lesion sizes of the infected footpad compared to the contralateral uninfected footpad. Results: Mice receiving SLA plus artemisinin produced significantly high levels of IL-4 and IL-5 (P < 0.05) and low levels of IFN-γ, resulting in exacerbated disease. In addition, subcutaneous administration of SLA + artemisinin, artemisinin alone or SLA alone resulted in the development of large footpad swellings and high parasite loads that were comparable to those of the control unvaccinated mice (P > 0.05), resulting in exacerbated disease. Conclusion: These data suggest that artemisinin is not a suitable adjuvant for Leishmania vaccines. However, since artemisinin has been shown to be effective against Leishmania parasites in vitro and in vivo, further studies ought to be conducted to determine its immunochemotherapeutic potential when co-administered with Leishmania antigens.

Abstract:Objective: To investigate the effects of Panax notoginseng saponins (PNS) on hydrogen peroxide (H2O2)-induced apoptosis in cultured rabbit bone marrow stromal cells (BMSCs). Methods: The effects of different concentrations of PNS on proliferation and early osteoblast differentiation of BMSCs were determined by the MTT assay and an alkaline phosphatase (ALP) assay. An optimal effective concentration of PNS was determined and used in subsequent experiments. The cultured BMSCs were divided into three groups: untreated control, H2O2 treated, and PNS pretreatment of H2O2 treated. The oxidative stress level was assessed by superoxide dismutase (SOD) and malondialdehyde (MDA) assays. Flow cytometry was used to determine BMSC apoptosis by staining with annexinV-FITC/propidium iodide (PI). The activity of caspase-3 enzyme was measured by spectrofluorometry. Results: PNS (0.1g/L) significantly increased both BMSC proliferation rate and ALP activity, while it decreased the indicators of oxidative stress, caspase-3 activity, and the apoptosis rate of BMSCs induced by H2O2.. Conclusion: PNS, acting as a biological antioxidant, had a protective effect on H2O2-induced apoptosis in cultured rabbit BMSCs by decreasing oxidative stress and down-regulating caspase-3.

Abstract:Objective: To study the effect of interferon-alpha IFN-α in the serum of SLE patients on the differentiation and maturation of dendritic cells (DCs) derived from CD34+ hematopoietic precursor cells (HPCs). Methods: Serum samples from SLE patients and normal controls were collected and the concentration of IFN-α detected by ELISA. CD34+HPCs were purified from cord blood by a magnetic cell sorting system (MACS), and cultured to differentiate to DCs. Normal serum, normal serum with exogenous IFN-α, SLE serum with raised levels of IFN-α, or SLE serum with anti-IFN-α neutralizing antibody was added to the culture medium. The phenotype of DCs was analyzed by flow cytometry (FCM) and the capacity of DCs to stimulate allogenic T lymphocyte proliferation was evaluated in a mixed lymphocyte reaction by the Cell Counting Kit-8. Cytokine production was assessed by ELISA. Results: Serum levels of IFN-α were significantly higher in SLE patients than in normal controls and this correlated positively with disease activity. Cultured in SLE serum with raised levels of IFN-α, CD34+ HPCs could differentiate into DCs that expressed higher levels of HLA-DR, CD80 and CD86, and showed an enhanced allogenic T-cell stimulatory capacity, while producing lower levels of IL-12 and higher amounts of IL-10 compared with those DCs cultured in normal serum. Conclusion: Increased levels of IFN-α in SLE serum promotes the differentiation and maturation of DCs derived from CD34+ HPCs and could contribute to the pathogenesis of SLE.

Abstract:Objective: To explore the in vitro effects of curcumin on the proliferation and apoptosis of the human renal cell carcinoma cell line ACHN, and to investigate its mechanisms of action. Methods: The human renal cell carcinoma cell line ACHN was treated with different concentrations of curcumin for 24 h. The MTT assay was used to evaluate the cytotoxic effects of curcumin and flow cytometry was utilized to observe and detect the apoptosis of ACHN cells induced by curcumin. The expression levels of Bcl-2, Bax and NF-κBP65 mRNA were evaluated by Reverse Transcription-Polymerase Chain Reaction (RT-PCR), while the expression of Bcl-2, Bax, NF-κBP65 and IκB proteins was evaluated by Western blot. Results: The concentrations of curcumin used significantly inhibited the proliferation of ACHN human renal cell carcinoma cells in vitro in a dose and time-dependent manner (Ftime=5.55, P < 0.05; Fdose=110.05, P < 0.05). Obvious apoptosis of cells treated with different concentrations of curcumin could be observed by FCM. Compared with the control group, the apoptosis rates of curcumin-treated cells were markedly increased (F=96.35, P < 0.05). Lower dose of curcumin significantly induced the apoptosis of ACHN cells. With intervention of different concentrations of curcumin (0, 10, 20 and 40 μmol/L) for 24 h, the expression levels of Bcl-2 and NF-κBP65 mRNA in ACHN cells were decreased while the expression level of Bax mRNA was increased (P < 0.05), and Bcl-2, and NF-κBP65 protein decreased, while Bax and IκB protein increased compared with those in the untreated group. Conclusion: Curcumin inhibited proliferation and increased apoptosis of the human renal cell carcinoma cell line ACHN. These curcumin effects appear to involve up-regulating IκB, down-regulating NF-κB, and regulating the expression of the apoptosis genes Bcl-2/Bax.

Abstract:Objective: To assess the efficacy and toxicity of gefitinib as a single agent treatment in Chinese patients with advanced non-small cell lung cancer (NSCLC). Methods: Forty-five patients with advanced NSCLC were treated with gefitinib at 250 mg daily until the disease progressed or the patient could not tolerate the toxicity. Results: None of the patients achieved a complete response (CR), while 15 patients achieved a partial remission (PR) and 17 experienced a stable disease (SD). Thirteen patients continued to have a progressive disease (PD). The response rate and the disease control rate were 33.3% and 71.1%, respectively. The symptom remission rate was 72.5%, and the median remission time was 8 days. The median survival time was 15.3 months. The median progression-free survival time was 6.0 months. The most common toxicities included rash (53.3%) and diarrhea (33.3%). Dehydration and pruritus of the skin developed in 26.7% and 22.2% of the patients, respectively. Hepatic toxicity occurred in 6.7% of patients and oral ulceration occurred in 4.4% of patients. Conclusion: Single agent treatment with gefitinib is effective against advanced NSCLC, and is well tolerated in Chinese patients.

Abstract:Objective: To investigate the antiproliferative and apoptogenic activities of polysaccharides extracted from Dioscorea bulbifera L (DBP) and Chinese Angelica (CAP) and a 3:2 mixture of these compounds (DCCP) on two esophageal carcinoma cell lines, EC9706 and Eca109. Methods: A MTT assay was used to detect the effects of DBP (10 μg/ml and 100 μg/ml), DCCP(17 μg/ml and 170 μg/ml) and CAP (10 μg/ml and 100 μg/ml) on the proliferation of EC9706 and Eca109 cells. DNA content analysis by flow cytometry was used to determine the cell cycle distribution, and Annexin V-FITC/PI stained fluorescence-activated cell sorter (FACS) was used to detect the apotosis rate of treated cells. Western blots were used to examine protein levels. Results: DBP and DCCP strongly inhibited the proliferation and viability of both the EC9706 and Eca109 cells. CAP enhanced the effects of DBP. DCCP primarily arrested the EC9706 and Eca109 cells at the G1 phase of the cell cycle. DCCP induced apoptosis in both esophageal carcinoma cell lines, and reduced the expression of pIκBα and Bcl-2 proteins. Conclusion: DCCP triggered apoptosis in esophageal carcinoma cells by inhibiting the NF-κB signaling pathway.

Abstract:Objective: To investigate the effect of the calcium channel blocker verapamil on adipocyte differentiation and its mechanism of action. Methods: Preadipocytes from 3T3-L1 strain mouse embryos were cultured and differentiated into matured adipocytes in vitro. Verapamil was added to the culture medium in the concentration of 30 μmol/L on Day 0. Cell differentiation was determined by Oil Red O staining and marker gene mRNA expression was evaluated and compared by RT-PCR. The fluo-3/AM probe and laser scanning confocal microscopy were used to measure intracellular calcium concentrations. Results: ①The differentiation rate of 3T3-L1 preadipocytes exposed to verapamil was lower than that of untreated cells. ②Verapamil promoted the retention of pref-1 gene expression. Lipoprotein lipase expression in the verapamil group was significantly lower than that in the control group on Day 4, Day 6 and Day 8 (P < 0.05) and resistin expression was significantly lower than that in the control group on Day 6, Day 8 and Day 10 (P < 0.05). Fatty acid synthase expression in the verapamil group was significantly lower than that in the control group from Day 2 (P < 0.05). ③ Intracellular concentrations of calcium [Ca2+]i in the verapamil group were significantly decreased compared with those in the control group on Day 2, Day 4 and Day 6 (P < 0.05), while there was no obvious difference between the two groups on Day 0 (P > 0.05). Conclusion: In 3T3-L1 preadipocytes verapamil significantly reduced adipocyte differentiation, down-regulated the mRNA expression of three marker genes for adipocytes differentiation, and prolonged the mRNA expression of an inhibitor of differentiation. The inhibitory effect of verapamil on differentiation may involve its role as a blocker of calcium influx in adipocytes.

Abstract:Objective: To investigate the effect of activation of angiotensin II (AngII) type 1 (AT1) receptors in the median preoptic nucleus (MnPO) of rats on renal sodium excretion. Methods: After anesthesia, the rats were injected into the MnPO via an implanted cannula. Urine samples were collected via a bladder cannula, and the urine sodium concentration was assayed with flame spectrophotometry. The serum level of endogenous digitalis-like factor (EDLF) and Na+,K+-ATPase activity in the renal cortex tissue were assayed respectively with a radioimmunoassay and with an ammonium molybdophosphate-based kit. Results: Both the urinary volume and the sodium excretion peaked 60 min after AngII was administered into the MnPO. The responses were accompanied by an increase in serum EDLF and a decrease in Na+,K+-ATPase activity in the renal cortex. The responses of diuresis and natriuresis, as well as an increase in serum EDLF and a decrease in Na+,K+-ATPase activity in the renal cortex induced by MnPO adminstration with AngII were inhibited by pior treatment with the AngII receptor blocking agent losartan into the MnPO. Conclusion: These results suggest that activation of AT1 receptors in the MnPO of rat induces diuretic and natriuretic responses. The responses are associated with an increase release of EDLF and with the inhibition of Na+,K+-ATPase activity in renal cortex tissue.

Abstract:Objective: To assess the relationship between left ventricular hypertrophy (LVH) or left ventricular geometry (LVG) and endothelial function in patients with essential hypertension (EH). Methods: Seventy-six patients and 30 normal subjects were first examined by echocardiography. Brachial artery dilatation induced by reactive hyperemia (DIRH) or nitroglycerin (DING) was detected using high-resolution ultrasonography. Results: DIRH was lower in patients with hypertension than in the controls, and the decrease in DIRH was greater in the patients with LVH than that in patients without LVH (4.36±2.54% vs 8.56±1.87 %; P < 0.0001). There were no significant differences in age, serum concentrations of total cholesterol, triglycerides or sugar, blood pressure and the brachial artery dilatation induced by nitroglycerin between the two groups (P > 0.05). While there was no significant difference in DIRH between the patients with normal left ventricular geometry or cardiac remodeling, the patients showing either eccentric or concentric left ventricular hypertrophy had lower DIRH than the patients with normal left ventricular geometry or cardiac remodeling. The DIRH was the lowest in patients with concentric hypertrophy. Although bivariate analysis showed that the left ventricular mass index (LVMI) correlated well with the brachial artery dilatation induced by reactive hyperemia, diastolic blood pressure and mean blood pressure (r=-0.61, P < 0.0001; r=0.27, P < 0.05; r=0.31, P < 0.05, respectively), a multivariate stepwise regression demonstrated that LVMI correlated only with the brachial artery dilatation induced by reactive hyperemia. Conclusion: Left ventricular hypertrophy was related to endothelial dysfunction in essential hypertension. The endothelial dysfunction might be basic and important in the progression of left ventricular hypertrophy.

Abstract:Objective: To investigated the apical constriction morphology of maxillary first premolars in the Chinese population. Methods: Eighty recently extracted human maxillary first premolars from a native Chinese population were used. The number and shape of apical constrictions were recorded under a dental operating microscope (DOM) at 12.5×2.5 magnification. After access preparation, a new K-file was inserted into the canal until the tip of the file was just seen at the apical constriction under the DOM. The teeth with files in the canals were X-rayed from a mesiodistal direction using a direct digital radiography (DDR) system, and the distance between the file tip and the center of radiographic apex was directly measured from the computer screen using DDR measurement software. Results: The percentage of teeth with an apical constriction was 78.5% (102/130). The most common apical constriction shapes were oval (55.9%) and round (35.3%). The mean distance between the apical constriction and the anatomical tip of the root was 0.61 mm, and 84.3% (86/102) were within 1 mm. Conclusion: The most common shape of an apical constriction was oval or round, and the distance to the apex was mostly within 1 mm, indicating that root canal therapy should stop 1 mm from the radiographic apex.

Abstract:A case is described of multi-recurrent cervical chordoma in a man over a 5 year period. The clinical features were of progressive spinal cord compression. The authors report a chordoma at C4 that recurred 3 times in five years. The patient underwent four operations and suffered distant metastases. This case confirms that thorough resection of the tumor during the first surgery and post-operative adjuvant treatment are the best assurance of a good prognosis with a chordoma. Multiple surgeries can stimulate biological activity of a chordoma and make its recurrence and distant metastases much more likely. The authors discuss the diagnosis, surgical treatment and the relationship between the histopathological changes and malignancy of a spinal chordoma after four operations. To our knowledge, this represents the first report of a 4th surgery for cervical chordoma.

Abstract:Hepatic venous stenosis may be a cause of graft failure in living donor liver transplantation (LDLT). Balloon dilation and metallic frame approaches have been used successfully to treat hepatic venous stenosis. Here, we report the effect of transfemoral venous balloon dilation for treating a child with hepatic venous stenosis after LDLT.

Abstract:Objective:To establish a stable GFP-LC3-expressed RAW264.7 cell line. Methods:The pcDNA3.1-GFP-LC3 plasmid was constructed and transfected into RAW264.7 cell with transfection reagent. The stable transfectants were screened by G418. The GFP-LC3 protein expression was analyzed by Western blot. The fluorescent signals were detected by inverted fluorescence microscope. ER stress-induced autophagy was detected by confocal microscope and Western blot. Results:Selected by G418,2 transfected cell lines showed high expression level of GFP-LC3,as demonstrated by Western blot analysis. More than 95% cells showed positive fluorescent signals under inverted fluorescence microscope. The formation of autophagosomes and the increases in the conversion of LC3-Ⅰ to LC3-Ⅱ was observed in the constructed cells when treated with the ER stress inducer,thapsigargin. Conclusion:A RAW264.7 cell line stably expressing GFP-LC3 was constructed successfully in the study.

Abstract:Objective:To investigate the effect of a common polymorphism T/C(rs11614913) in mature miR-196a2 on the expression of LSP1 gene. Method:The pCDNA3.1-miR196a2-C and pCDNA3.1-miR196a2-T expression plasmids were created containing T or C allele of pre-miR196a2. Meanwhile,mature miR-196a2-C and miR-196a2-U probes were chemically synthesized for the investigation. The 3’UTR from LSP1 gene was cloned downstream of the firefly luciferase gene in pMIR-REPORTTM Luciferase Vector. Two sets of cotransfection were performed with CHO,HEK293 and A549 cells,and the luciferase activity with the Dual-Luciferase Reporter Assay System were analyzed. Results:pCDNA3.1-miR196a2-C construct group showed significantly lower levels of luciferase expression compared with pCDNA3.1-miR196a2-T construct group(P < 0.05),when LSP1 3’UTR luciferase reporter plasmids were cotransfected with miR-196a2 expression plasmids in CHO,HEK293 and A549 cells. Mature miR-196a2-C probe group showed significantly lower luciferase activity compared with miR-196a2-T probe group(P < 0.05),while LSP1 3’UTR luciferase reporter plasmids were cotransfected with mature miR-196a2 probes in CHO,A549 and HEK293 cells. Conclusion: The miR-196a2 could effectively bind the predicted target gene of LSP1 and influence the expression on the level of transcription,while miR-196a2 carrying the rs11614913 C allele.

Abstract:Objective: To study transcription level and catalytic activity of DNA methyltransferase (Dnmts) in lung cancer cell line A549 exposed in 5-Aza-CdR(5-Aza-2’-deoxycitydine),and the role of Dnmts in the process. Methods:A549 cells were exposed in the 5-Aza-CdR for demethylation. Dnmts mRNA levels were determined by semi-quantitative RT-PCR. The catalytic activity of Dnmts was detected by enzyme colorimetric determination kit. The apoposis and changes of cell cycles were observed by flow cytometry(FCM),and MTT assay was used to study the cell proliferation. Results:The transcription level of the drug group and control group were Dnmt1(1.23 ± 0.253;1.15 ± 0.166), Dnmt3b(0.760 ± 0.164;0.649 ± 0.181),No significant difference in quantity of Dnmts mRNA can be oberserved between the two groups (P > 0.05). Catalytic activity of the drug group and control group were Dnmt1 (0.195 ± 0.030;0.153±0.041),Dnmt3b(0.172 ± 0.029;0.116 ± 0.050). Catalytic activity decreased compared with the control group (P < 0.05). FCM recommended 5-Aza-CdR arrested cell cycle at G1/G0,the rates of apoptosis of the two groups were 0.62 ± 0.56;7.60 ± 1.92,and the rate of apoptosis of drug group was higher than the control group (P < 0.01). Cell proliferation of drug group was inhibited according to the results of MTT. Conclusion: 5-Aza-CdR has cytostatic agent in A549 cells cultured in vitro,and accelerates its apoptosis. The inhibition of Dnmts catalytic activity can account for demethylating agent of 5-Aza-CdR in A549 cells,not the Dnmts transcription level.

Abstract:Objective:To determine whether the non-adherent mesenchymal stem cells(MSCs) are present in rat peripheral blood and whether 1,25-(OH)2D3 can stimulate them to form colony forming unit-fibroblastic(CFU-f). Methods:Peripheral blood mononuclear and granular cells(PBMGC)fraction was isolated from the blood of six-week old male Wistar rat and were cultured in the absence or presence of 10-8 mol/L 1,25-(OH)2D3 for 16 days After 4 days and 8 days,the non-adherent cells were transferred into another fresh petri dishes to culture consensually until to day 16(“pour-off” culture). The resulting cells were stained with methylene blue and the number of colonies was counted. The resulting cells from 4-day “pour-off” cultures were strained immunocytochemically for vimentin,α-smooth muscle actin(α-SMA),type Ⅰ collagen and type Ⅲ collagen. Results:Not only total PBMGC,but also non-adherent PBMGC can give rise to CFU-fs and the treatment of 10-8 mol/L 1,25-(OH)2D3 increased their CFU-f forming efficacy significantly. The resulting cells from 4-day “pour-off” cultures expressed vimentin,α-SM actin and type Ⅲ collagen,but did not express type Ⅰ collagen. Conclusion:The non-adherent mesenchymal stem cells(MSCs) are present in rat peripheral blood and the treatment of 1,25-(OH)2D3 can stimulate them to form CFU-fs.

Abstract:Objective:To investigate effece of triptolide on the growth inbibitio of human endometrial cancer and to find out the role of triptolide on the expressions of Bcl-2 and VEGF. Methods:Human endometrial cancer cell line HEC-1B was seeded in the subcutaneous and a implant xenograft model was established by using nude mice. Mice were randomly divided into five groups,then treated with 0.9% NaCl solution,high,middle,and low dose triptolide(8,4,2 μg/d each one) and DDP 40 μg/d each one for up to 10~15 days respectively. Tumor size was measured in 2 dimensions at regular time-intervals and the expressions of Bcl-2 and VEGF in tumor tissues were detected by SABC. Results:After treatment, the size of the xenografts in high-dose triptolide group were 894.802+81.519 mm3,compared with1771.702+114 mm3 in the control group(P < 0.05,With inhibition rate of 50%). The expressions of Bcl-2 and VEGF in this group were significantly lower than that in the control group(P < 0.05). Conclusion:High dose triptolide can significantly inhibit the growth of the xenografts of endometrial cancer HEC-1B in nude mice. The mechanism may be closely related to down-regulating the expressions of Bcl-2 and VEGF in tumor cells by triptolide.

Abstract:Objective:To study the success rate improvement of porcine myocardial infarction model produced by ligating coronary artery. Methods:16 young farm pigs were devided into operated(n = 10) and sham-operated(n = 6) groups. Operated animals were anesthetized,incubated,mechanically ventilated and lidocaine injected intravenously. The heart was exposed by sterile median sternotomy and pericardiotomy,and the left anterior descending(LAD) was ligated from two third distal to its origin,lidocaine was intravenously injected before,during and after operation. Sham-Operated animals underwent a similar procedure except for the LAD ligation. The electrocardiogram(ECG) was monitored throught the entire operation,coronary angioplasty,positron emission tomography(PET),triphenyltetrazolium chloride(TTC) staining and histopathological examination were performed 3 weeks later. Results:The success rat of porcine myocardial infarction model in the operated group was 90%. S-T elevation to single-phase curve appeared on the ECG after operation;coronary angiography showed that Medion and distal LAD was totally;PET demonstrated that radioactivity was sparse or absent in the left ventricular free wall and apex;light microscopy examination showed symmetrical fibrous connective tissue proliferation in the infracted area where the tissue is not stained by the TTC method. There were no abnormal results from macroscopic,ECG,PET and histopathological examination in sam-operated rats. Conclution:LAD ligation by sterile median sternotomy is a available and feasible method to increase the success rate of porcine myocardial infarction model, lidocaine intravenously used.

Abstract:Objective:To evaluate the effects of Formoterol(β2-adrenergic receptor-specific agonist) and ICI118551(β2-adrenergic receptor-specific antagonist) on rat osteoblast-like cells induced from marrow-derived mesenchymal stem cells(MMSC). Methods:MMSCs by whole bone marrow adherent culture derived from a three-week-old female SD rat are induced to osteoblast-like cells. After different concentrations of Formoterol and ICI118551 are added into the culture medium,thealkali phosphatase and osteocalcin is detected. Results:The alkali phosphatase,osteocalcin and proliferation of cells cultured with all different concentrations(10-5-10-9 mmol/L) of Formoterol are obviously low. As to ICI118551,high concentrations(10-5 and 10-6 mol/L) can inhibit the expression of the alkali phosphatase,osteocalcin and proliferation of cells while low concentrations(10-8 and 10-9 mol/L) can enhance the expression of the alkali phosphatase,osteocalcin and proliferation. Conclusion:The β2-adrenergic receptor-specific agonist can inhibit the activity of rat osteoblast-like cells in vitro while the effects of the β2-adrenergic receptor-specific antagonist are not single,depending on its concentration.

Abstract:Objective:To explore the antitumor effect of resveratrol against malignant melanoma in vitro and in vivo. Methods:Murine B16 and human A375 cells were cultured in vitro and the growth inhibition effects of resveratrol were detected by MTT method. After the pretreatment with different doses of resveratrol,cell protein was extracted and the expression levels of protein p-Akt were detected by Western blot. B16 cells transplant model was established in male ICR mice and the tumor growth inhibition effects induced by resveratrol were detected by measuring tumor volume every two days. Results:Cytotoxicity test showed that cell growth was significantly inhibited by resveratrol. Moreover,resveratrol effectively downregulated the expression of protein p-Akt. Animal experiments also demonstrated that resveratrol efficiently inhibited tumor growth without obvious bodyweight change. Conclusion:resveratrol was demonstrated as a potential anti-melanoma agent both in vitro and in vivo with the possible mechanism of downregulation of p-Akt expression. The current study provided the experimental basis for the clinical application of resveratrol against malignant melanoma and also offered a detailed strategy for developing the antitumon effect of Chinese herbal medicines.

Abstract:Objective:To investigate the effect of insulin-like-growth factor-1(IGF-1) on expression of pituitary tumor transforming gene(PTTG)in C6 cells of glioma. Methods:Glioma C6 cells were divided into four groups and treated with different concentration of IGF-1 for 24 h respectively(A:0 ng/ml;B:0.1 ng/ml;C:1.0 ng/ml;D:10.0 ng/ml). PTTG mRNA was detected by reverse transcription polymerase chain reaction(RT-PCR),Western blot was used to detecte the expression of PTTG protein. Results:The expressions of PTTG mRNA and protein increased dramaticly in the groups treated with IGF-1,and there was a significantly difference between each each groups(P < 0.01). Conclusion:IGF-1 can upregulate the expression of PTTG significantly in dosage-dependent manner.

Abstract:Objective:To establish the experimental rat model of type 2 diabetes by streptozotocin and high-glucose-high-fat diet and observe its stability. Methods:60 SD male rats were randomly divided into four groups:control group,streptozotocin group(STZ group),high-glucose-high-fat feeding group(H.E group)and high-glucose-high-fat feeding+STZ group(H.E+STZ group). STZ group and H.E + STZ group were intraperitoneal injected with STZ 35 mg/kg. 4 weeks later,we measured and statistically analyzed the changes of body weight,blood insulin level,fasting blood glucose level,blood lipid level,insulin sensitivity index(ISI)and area under curve and insulin sensitivity index(AUC)of hamsters of every group at different time points. Results:The fasting plasma insulin,Serum triglyceride(TG),total cholesterol(TC),ISI of H.E group and H.E+STZ group significantly increased(P < 0.01), the Fasting blood glucose and AUC of STZ group and H.E+STZ group increased obviously(P < 0.01). Compared with the control group and H.E group,the Fasting plasma insulin and TG,TC,ISI of H.E+STZ group injected with STZ for four weeks increased significantly(P < 0.01). Conclusion:High-glucose-high-fat diet significantly decreased insulin sensitivity index,and the type 2 diabetes rat models were established by high-glucose-high-fat diet combind with low dose injection of STZ with high success rate,effective stability and shorter model-making time.

Abstract:Objective:To assess the natural interaction between gambogic acid and oxaliplatin widely used in gastrointestinal cancer treatment,and to investigate the influence of gambogic acid on cell apoptosis and drug-associated gene expression. Methods:Synergistic interaction on human gastric cancer cell lines BGC-823 and MKN-28 was evaluated using the combination index(CI) method. The double staining with both Annexin-Ⅴ-FITC and PI was employed to distinguish the apoptotic cells from others. Expression of drug-associated genes,i.e.,excision repair cross-complementing(ERCC1),breast cancer susceptibility gene 1(BRCA1) of BGC-823 with or without gambogic acid treatment were measured by real-time quantitative RT-PCR. Results:Gambogic acid had a synergistic effect on the cytotoxicity of oxaliplatin in BGC-823 cells. The combination of gambogic acid and oxaliplatin could also induce apoptosis in a synergistic manner. Most prominently,ERCC1,BRCA1 mRNA levels of BGC-823 were markedly suppressed at 0.070-,and 0.140-fold respectively by the presentation of gambogic acid. Conclusion:Gambogic acid appears a promising candidate for combining with oxaliplatin in cancer chemotherapy treatment. The possible synergistic mechanisms maybe the synergistic apoptotic effect and the downregulation of drug-associated genes.

Abstract:Objective:To explore the level of tryptase-positive mast cells in the bone marrow of MDS patients. Methods:Bone marrow biopsies from 10 healthy controls,8 IDA,20 myelodysplastic syndrome and 17 acute myeloid leukemias were embedded in cold plastic resin. Immunohistochemical method was used to show the number of tryptase-positive cells.The level of tryptase-positive cells were compared. Results:No difference was found between the level of tryptase-positive mast cells in IDA group(63.43 ± 44.29) and healthy group(61.43 ± 56.19)(P > 0.05);the level in MDS group(195.95 ± 117.39) was higher than that in control group(P < 0.05), high risk group(229.25 ± 139.68) was higher than low risk group(171.73 ± 98.07),but the difference was not significant(P > 0.05). AML group(608.44 ± 342.58) was higher than that of MDS group(P < 0.01). Conclusion:The number of tryptase-positive mast cells in the bone marrow of MDS patients was increased and may play a role in the pathogenesis and development of the disease.

Abstract:Objective:To investigate the expression of CD4+ Foxp3+ regulatory T cells and different cytokines in peripheral blood of patients with coronary heart disease(CHD),and to explore the roles of them in CHD pathogenesis. Methods:21 patients with acute myocardial infarction(AMI),16 patients with unstable angina(UA),21 patients with stable angina(SA)were recruitde. The percentage of CD4+ Foxp3+ Treg of patients was measured by flow cytometry,and ELISA was used to analyze the blood plasma levels of cytokines(TNF-α,IL-10,IFN-γ,IL-4,TGF-β). Results:①The percentage of CD4+ Foxp3+ Treg decreased significantly in patients with acute coronary syndrome(ACS)compared with normal group(P < 0.001). ②Compared with SA patients,the level of TNF-α in AMI patients significantly increased(P < 0.05). In addition,the percentage of CD4+ Foxp3+ Treg in AMI patients was positively correlated with level of IL-10(P < 0.05). ③No correlation was found between CD4+ Foxp3+ Treg percentage and other cytokines(TNF-α,IFN-γ,IL-4,TGF-β)in CHD patients. ④There was no correlation between CD4+ Foxp3+ Treg and levels of total cholesterol,triglyceride,high or low density lipoprotein. Conclusion:The decreased percentage of CD4+ Foxp3+ Treg and increased TNF-α in peripheral blood of ACS patients may participate in ACS development and progression.

Abstract:Objective:To study the effects of intranasal FP on the expression of AQP5 in epithelial cells of nasal mucosa and nasal polyps and further explore the possible mechanism of AQP5 in the onset of nasal polys. Methods:The primary culture of the epithelial cells of nasal mucosa was conducted for 14 days. After these cells were subcultured.The cells of FP group was treated with culture medium containing 0.2% FP.The cells of no FP group were treated with culture medium which was not containing FP. After 7 days,the expressions of AQP5 were studied in the cells with immunohistochemistric staining,by its integrated light intensity and its grey step. At the first day we obtain 12 nasal polyps samples from these inpatients who were trouble with chronic sinusitis and nasal polyps for the first time and were not treated with glucocorticoid partly or integreatly in the last three mouths. FP were intranasally used 7 days before operation.The other 12 nasal polyps were obtained and studied with immunohistochemical technique,by its light intensity and its grey step. Results:The grey step of AQP5 in the cells of FP-using group is lower obviously than using FP before(P < 0.05),the light intensity is higher obviously than using FP before(P < 0.05). In the nasal polyps of FP-using group,the size of nasal polyps become much smaller than using FP before.The grey step of AQP5 in both glandular cells and lymphocyte is higher obviously than using FP before(P < 0.05) but the light intensity is lower obviously than using FP before(P < 0.05). Conclusion:the expressions of AQP5 in cells which treated with FP became higher than cells which untreated,It indicated that when treated with FP,interstitial fluid can effuse easily,the concha nasalis media and the concha nasalis inferior became smaller and smaller. After nasal polyps samples treated with FP,the poloyps became smaller.At the same time,the expressions of AQP5 in glandular cells and lymphocyte became lower. There may be relationship between these. It can indicate that AQP5 contributed to edema of nasal polyps.

Abstract:Objective:To study the expression and diagnostic value of staining glial fibrillary acidic protein(GFAP) and neurofilament protein(NF) after primary brain-stem injury. Methods:The cases that definitely died of the primary brain-stem injury and other disease were selected randomly(65 cases). All cases were divided into experimental group(25 cases) and control group(20 cases died of cardiovascular disease and 20 cases died of other disease).The brain-stem tissues were cut and stained by hematoxylin-eosin and immunohistochemical GFAP and NF,the data were analyzed. Results:Compared with the control group,astrocyte appeared acute reaction and axon showed distinctive changes in brain-stem tissues. There were significant differences in astrocyte quantity and axonal diameter of brain-stem tissue between the experimental group and the control group(P < 0.05). Conclusion:The above histopathologic changes might be valuable for autopsy pathological diagnosis of primary brain-stem injury. With the measurement of astrocyte quantity and axonal diameter,we provide a referenced quantization index for diagnosis of primary brain-stem injury.

Abstract:Objective:To analyze the differential expression of proteome between metastatic lymph nodes in adenocarcinoma of lung and normal lymph nodes. Methods:The proteome techniques were used to separate the protein spots and analyze the differential expression of proteome in the metastatic lymph nodes and normal lymph nodes,and verification was performed at protein and mRNA level,Results:Compared with control,there were 23 protein spots significantly differentially expressed,of which 4 protein spots were newly found,1 protein spot disappeared,14 protein spots increased markedly,and 4 protein spots decreased significantly 11 kinds of the associated protein were identified,among them,ANX1,CK18,Rho-GDP,GDIR,TPMF,TGLC and IL-18 were down-regulated expression significantly in metastatic lymph nodes,but CLI1,ER60,CK,PDX and CH60 were up-regulated expression significantly. Conclusion:The proteomic expression in metastatic lymph nodes of adenocarcinoma of lung is significantly different compared with of the normal control,the 11 kinds of proteins may be associated with the mechanism of metastasis of adenocarcinoma of lung.

Abstract:Objective:To investigate the effects of 5-Aza-2’-deoxycytidine(5-Aza-CdR) a methylation inhibitor on the growth of human renal carcinoma cell line OS-RC-2,and the DNA CpG island demethylation of γ-catenin gene so as to provide insights into origin of renal carcinoma and the possibility of its application in clinical treatment. Methods:MTT method and flow cytometry were used to detect the growth and appoptosis of OS-RC-2 after 5-Aza-CdR treatment respctirely. The western blot was used to detevt γ-catenin protein levels in the cell line before and after treatment with 5-Aza-CdR.The methylation and demethylation status of γ-catenin gene were volume detected by methylation special PCR(MSP). Results:The morphological pattern changes were observed invert-microscope. OS-RC-2 cells treated with 5-Aza-CdR displayed a slowed growth in comparision with the control cells. The apoptotic rate was also increased after 5-Aza-CdR treatment. Western blot indicated that expression of γ-catenin protein was recovered by 5-Aza-CdR treatment. The high methylation status of γ-catenin gene promoter region was reversed with 10-5 mol/L 5-Aza-CdR treated for 72 h. Conclusion:5-Aza-CdR effectively causes the demethylation of γ-catenin gene CpG-rich promoter regions, and recover the γ-catenin protein expression, subequenely induce the appoptosis of OS-RC-2 cells.

Abstract:Objective:To study the biocompatibility and mechanical properties of PLGA membrane modified by Type-Ⅰcollagen and Chitosan,and to develop a novel tissue engineering materials as artificial spinal dura mater. Methods:PLGA membrane(membraneⅠ),PLGA/ Type-Ⅰcollagen composite membrane(membraneⅡ),PLGA/ Type-Ⅰcollagen/ Chitosan(9 ∶ 1) composite membrane(membrane Ⅲ) and PLGA/ Type-Ⅰcollagen/ Chitosan(5 ∶ 5) composite(membrane Ⅳ) were produced through a certain process. Contact angle,absorption rate,cytotoxicity study and determination of mechanical properties were used to research all type membranes. Results:Contact angle:membraneⅡ < membraneⅢ < membraneⅣ < membraneⅠ,P < 0.01; Absorption rate:membraneⅠ < membrane Ⅳ < membrane Ⅲ < membrane Ⅱ,P < 0.01;Cytotoxic experiment(MTT method):at the 1st day,the OD value between each group didn’t have significant difference,P > 0.05. At the 3rd and the 7th day,there was significant difference between membrane Ⅰand Ⅱor Ⅲ,membrane Ⅲ and Ⅳ,P < 0.05. After being modified by Type-Ⅰcollagen and Chitosan, PLGA membrane striking enhanced the adhesion and proliferation of L929 cell,and the comparison between all type membranes showed that there was significant difference between membrane Ⅰand other type membranes,P < 0.05,and there was no significant difference between membrane Ⅱ,Ⅲ and Ⅳ. Conclusion:With type-Ⅰcollagen and Chitosan(9 ∶ 1) appending to the exterior, PLGA can enhance the biocompatibility and Mechanical properties of membrane.

Abstract:Objective:To study the effect of the inhibitor of p38MAPK(SB202190)on focal cerebral ischemia/reperfusion in rats,and furth explore the nourprotective effect and mechanism of SB202190 on focal cerebral ischemia/reperfusion. Methods: The acute focal cerebral ischemia/reperfusion models were established by suture emboli. Healthy male Sprague-Dawley rats were randomly divided into five groups:normal control group,sham group,ischemia/reperfusion group,SB202190 and DMSO group. In SB202190 and DMSO group,SB202190(the specific inhibitor of p38MAPK) and 1 % dimetyl sulphoxide(DMSO) were injected into the lateral ventricle respectively. Behavior scores of rat neuralogical fanction were penformed for each group The rats were sacrificed at 24 h after ischemia/reperfusion. Nissl staining is rsed to observe morphologic changes of neuron cells in ischemia/reperfusion injury region;and immunohisochemistry is used to detect Bcl-2 and Bax protein positive cell. Results: Rats of ischemia/reperfusion group had a lower score of neurological function compared with sham group(P < 0.05). The score in SB202190 group was significantly higher than that in ischemia/reperfusion group(P < 0.05),but there was no difference between ischemia/reperfusion group and DMSO group(P > 0.05).There were massive cellular necrosis found in rats at 24 h after ischemia/reperfusion,the form of neuron cell were atrophica,cellular plasm dried up and nucleus were densely stained,intercellular space were more lager than normal neuron cell. SB202190 group:Compared with the ischemia/reperfusion group,the cellular swelling was more light at 24 h after ischemia/reperfusion,and the injury of cellular necrosis was mild after ischemia/reperfusion;Immunohisochemistry staining to detect Bcl-2 and Bax positive neurons:There were a few Bcl-2 and Bax positive cells,which distributed dispersively in ischemia/reperfusion injury region,and there was no obvious difference between normal control group and sham group(P > 0.05);For the Ischemia/reperfusion group:There were massive Bax positive cells distributed in ischemia/reperfusion injury region at 24 h after ischemia/reperfusion,but Bcl-2 positive cells were no obvious change(P > 0.05);For the SB202190 group:Bcl-2 positive cells obvious increased at 24 h after ischemia/reperfusion(P > 0.05),but Bax positive cells obvious decreased compared with the Ischemia/reperfusion group. Conclusion:The inhibitor of p38MAPK(SB202190) can protect neural function from ischemia/reperfusion injury after ischemia/reperfusion in rat. SB202190 can reduce the region of ischemia/reperfusion injury,and regulate the activation of Bcl-2 and Bax protein after ischemia/reperfusion in rat,and it may be one of the possible mechanisms of antiapoptotic effect of SB202190.

Abstract:Objective:To investigate the effect of basic fibroblast growth factor(FGF-2) on pim-3 in humanl lung cancer cell A459. Methods:Lung cancer cell A459 was treated by different concentrations(0-25-50-100 ng/mL) of FGF-2,pim-3 mRNA was measured by reverse transcription polymerase chain reaction(RT-PCR),and western blotting was used to detect the expression of PIM-3 protein. Results:The expressions of pim-3 mRNA and protein increased dramaticly in the groups treated with FGF-2 compared with conorol,and there was a significantly difference between each two experiment groups(P < 0.01). Conclusion:FGF-2 can significantly upregulate the expression of pim-3 in dosage-dependent manner.

Abstract:Objective:To explore the relative functional area of dealing classifiers and potential difference in various functional area by investigating the activation of brain when it deals with quantitative words. Methods:Eighteen healthy volunteers were asked to perform a nominal and verbal classifiers semantic decision tasks(to be wrong or to be right) during fMRI scanning in order to compare the difference in extent of different cerebal activation. Results:The major activating brain regions for nominal classifiers:left and right middle frontal gyrus,left inferior frontal gyrus,left and right cingulate gyrus,left and right cuneus,precuneus,left and right angular gyrus,left and right inferior temporal gyrus,left middle temporal gyral,lingual gyral,right cerebellar. The major activating brain regions for verbal classifiers:left and right middle temporal gyral,left and right inferior temporal,left and right cingulate gyrus,left and right basilar nucleus,left and right angular gyrus,left and right middle frontal gyrus,left inferior frontal,left insula,left and right cerebellar. Conclusion:The major activating brain regions for nominal and verbal classifiers are same. There is some difference in extent of cerebal activation. However,they all need co-ordinated operation of each functional regions on both cerebal hemisphere.

Abstract:Objective:To study the clinical features and management of Borderline OvarianTumors(BOT). Methods:39 cases with Borderline OvarianTumors were retrospectively analyzed. Results:One case(2.9%)died among 35 cases followed up to the end. Five cases(14.7%) relapsed including of 1 case of stage Ia,1 case of stage Ib,2 case of stage Ib and 1 case of stage Ⅲ. Three cases of recurrence were serious tumor,among that 1 case had micropapillary pattern associated with microinvasion. There were 3 recurrences among 6 patients treated by cystectomy,and 1 recurrence among 18 patients treated by oophorectomy. Conclusion:Surgery-pathology stage[FIGO,2000]is the main prognosis factor of BOT. Surgery is the key treatment method for BOT. In early stage disease,conservative surgery is indicated when fertility is desired by the patient. Oophorectomy.is better for conservative surgery. Radical surgery should be performed for advanced patients.

Abstract:Objective:To evaluate the significance of the retinal nerve fiber layer (RNFL) thickness measured by optical coherence tomography (OCT) in the diagnosis and prognosis of visual function of pituitary tumor. Methods:The thickness of RNFL was measured by OCT in 39 patients with pituitary tumor (39 eyes) and 76 normal subjects(76 eyes). Comparisons were made with regard to the RNFL thickness of quadrants and in means between normal and pituitary tumor group. The correlation between mean RNFL thickness and mean defect of visual field. Results:There was significant difference of RNFL thickness in various quadrants and in means between normal and pituitary tumor group (P < 0.000).The mean RNFL thickness was positively correlated with mean defect of visual field (R=0.41,P=0.000). Conclusion:RNFL thickness measured by OCT provides a new method for the diagnosis of pituitary tumor.

Abstract:Objective:To retrospectively analyze the effectiveness and safety of radiofrequency catheter ablation (RFCA) in elder patients with atrioventricular node reentrant tachycardia (AVNRT). Methods:From Jan 2005 to Jan 2008, 21 elder patients (9 males and 12 females) suffered from intermittent AVNRT and subjected to RFCA were enrolled in this study. The mean age was 68.2 ± 3.4 years (age range from 60 to 78 years). The major coincident disorders were hypertension, coronary heart disease and pulmonary emphysema. Results:First RFCA succeeded in 20 cases and failed in one case who refused second procedure (achievement ratio 95.2%). Post-operation follow-up for 9~36 months showed there was no relapse (relapse ratio 0%) or death. The side effects include mild pneumothorax in one patient with previous pulmonary emphysema and degree Ⅰ atrioventricular block (PR interval 0.28 s) during electric discharge in another patient (complication incidence 9.5%). Conclusion:RFCA is effective and safe for elder patients with AVNRT.