Objective To investigate the possible injury mechanism of human kidney proximal tubular epithelial cell-2 (HK-2) induced by aristolochic acid (AA). Methods Cultured HK-2 cells were divided into 4 groups:normal control,treated by AA at the concentration of 30,60 and 120 ?mol/L for 48 h respectively. The morphological changes were observed by inverted phase contract microscopy. The cell viability was measured by the Cell Counting Kit-8 (CCK8) assay. Apoptotic cells were identified by flow cytometry. Expression of active Caspase-3 was measured by Western blot analysis. Automatic biochemical analyzer was used to detect the contents of LDH and ?-N-Acetylglucosaminidase (NAG) in the supernatant. The expression of E-cadherin and a-SMA was detected with laser scanning confocal microscope (LSCM). Enzyme linked immunosorbent assay (ELISA) was used to measure the levels of TGF-?1 and collagen Ⅲ in the supernatant quantitatively. Results AA inhibited HK-2 cells proliferation,induced cell apoptosis and activated Caspase-3 expression,and increased the LDH and NAG levels. All of these were in a concentration-dependent manner. AA at the concentration of 60 ?mol/L inhibited E-cadherin expression,increased ?-SMA expression and TGF-?1 and collagen Ⅲ secretion. Conclusion AA inhibits cell proliferation,induces apoptosis and epithelial-mesenchymal transition (EMT) in HK-2 cells. AA at relatively low concentration (≤60 ?mol/L) mainly induces EMT in HK-2 cells,while,that at high concentration (≥120 ?mol/L) causes apoptosis and cytotoxicity.

AIM:To approach the therapeutic effects of mycophenolate mofetil(MMF)in hormonal resistance nephrotic syndromes.METHODS:Patients were diagnosed as nephrotic syndrome and treated with prednisone at the dose of 1 mg?kg-1?d-1 for over 8 weeks,and 24 patients with unsatisfactory results or were palindromic were selected,and several patients in the 24 patients had been treated with cyclophosphamide or cyclosporine A.All patients were treated with MMF combined with low dose hormone.The initial dose of MMF was 1.0-1.5 g/d for 3 months,later the dose were reduced,and the maintenance dose of MMF was 0.5-1.0 g/d,the dose of prednisone was 5-20 mg/d,the follow-up visit period more than six months.The changes on urine protein,serum albumin,liver function,renal function were compared before and after treatment.RESULTS:Before and after treatment,urine protein decreased from(3.4?1.7)g/d to(0.9?0.2)g/d,serum albumin increased from(19.6?5.4)g/L to(36.1?7.7)g/L.serum creatinine level decreased from(105.7?6.4)?mol/L to(90.1?5.8)g/L.20 patients(83.3%)pathogenetic condition were relieved,15 patients(65.2%)were with complete remission.5 patients(20.8%)were partially recovered,and 4 patients(16.6%)had no response.The adverse effects were observed,including gastrointestinal events(n=8,33.3%),bacterial pneumonia(n=4,16.6%),herpes zoster(n=1,4.1%),hepatic function mild damage(n=3,12.5%).CONCLUSION:It is safe and effective to combine MMF with low dose hormone in treatment of hormonal resistance nephrotic syndrome,which could become a therapeutic option for refractory nephrotic syndrome(RNS).

To explore the relations of Morphology Immnuophe-notype Cytogenetics Molecular biology(MICM) detection on diagnosis andtreat,emt of leukemia． Methods: 68 cases of leukemia patients had beenanalyzed by morphology(FAB)． Immunohistochemistry(Flow Cytometry, FCM)． chromosome G banding technique and dual-color fluorescence insitu hybridization (D-FISH)．Technique:All patients were treated bychemotherapy． T test and X2 test of significance． Results: 7 cases have acute leukema aberration antigen expression． 5 out of 47 cases acutemyeliod leukemia patients accompany lymhocytic interrelated antigenexpression． 2/l5 cases acute lymphoid leukemia accompany myelocyteinterrelated antigen expression． 2 cases acute lmphoid leukemia are T cell and B cell interrelated antigen mingle expression． had been examined46,XY,t(8,2l) translocation of chromosome and bcr/abl fusion genes inthe acute leukemia patients． 16 out of 20 chronic myeloid leukemia patientshad philadelphia chromosome． 7 out of 20 patients had complicate karyotype． 5 out of 20 patients had bcr/abl fusion gene, l out of 4 patient had bcr/abl fusion gene that Ph chro mosome showed negative in CML． 3/4 cases patients had complicate chromosome． The ratio of CR use l time chemotherapy and the total ratio of CR using 2 times chemotherapywith aberration antigen expression in acute leukemia was significantly lessthan those of the acute leukemia patient had single system antigenexpression(P<0.05)． The time of CML-BC with complicate c hromosome karyotype was significantly short than those of Ph showed negative in CML(P<0.05)． Conclusion: The MICM classification is more acc urate for diagnosis of leukemia and more significant in guiding the leukemiatreamen．

To rapidly select potent anti-VSTM1-v2 scFv (single-chain antibody fragment) by construction and screening of a humanized scFv library in which a murine VH-CDR3 library was grafted onto a human scFv framework. A murine VH-CDR3 library was amplified from anti-VSTM1-v2 murine cDNA and grafted on human scFv (VH3-VK1) framework. Anti-VSTM1-v2 scFv templates were selected and enriched through ribosome display, TA-cloned into expression vector, and transformed into BL21 (DE3) for soluble expression of target scFv. A total of 1000 clones were randomly picked. Positive ones were first identified using colony PCR, indirect ELISA, Western blotting and then verified with sequencing and dose response ELISA. At last an anti-VSTM1-v2 humanized scFv with good binding affinity (EC50 = 21.35 nmol x L(-1)) was selected from the humanized library of 10(12) members generated in this study. This scFv antibody might have potential applications. This study provides a new approach for rapid screening of humanized antibodies.

Objective To explore the effect of Smad7 gene transfer on tubular cell cycle arrest, tubular apoptosis and fibronectin (FN) synthesis by transforming growth factor ? 1(TGF ? 1) induced. Methods Mouse Smad7 gene was transfected into renal tubular cells in primary cell culture by using Tfx 50 cationic liposome. Tubular cell proliferation was measured by MTT and cell cycle was observed by flow cytometry. The level of FN secretion in the supernatant was determined by ELISA. Results At 48 h after administration of 10 ng/ml TGF ? 1 to renal tubular cells, cell proliferation declined, G 0/G 1 arrested in cell cycle, and cell FN secretion increased significantly. These abnormalities were attenuated by liposome mediated Smad7 gene transfection. Conclusion Transfection and expression of Smad7 can markedly inhibit TGF ? 1 responses in renal tubular cells, which is helpful for further studies of Smad7 gene therapy in vivo .

Apoptosis of dopaminergic neurons in the nigrostriatal projection plays a crucial role in the pathogenesis of Parkinson's disease (PD). Although the detailed mechanisms responsible for dopaminergic neuron loss are still under investigation, oxidative stress is identified as a major contributor for neuronal apoptosis. In the current study, we studied the effects of MPP(+), a substrate that mimics oxidative stress, on neuron-like PC12 cells and the underlying mechanisms. PC12 cells were cultured and treated by 100 μmol/L MPP(+) for 4, 8, 16, 24 and 48 h, respectively. For drug pretreatment, the PC12 cells were incubated with N-acetyl-l-cysteine (NAC, 5 mmol/L), an antioxidant, SP600125 (20 μmol/L) or PD98059 (100 μmol/L), two pharmacological inhibitors of JNK and ERK1/2, for 1 h before addition of MPP(+). Cell apoptosis was measured by flow cytometry. The mRNA expression of Cu(2+)/Zn(2+)-SOD, GSH-Px, Bcl-2 and Bax was detected by RT-PCR. The protein expression of p-ERK1/2 and p-JNK was determined by Western blotting. Our results showed that MPP(+) exposure could induce substantial PC12 cell apoptosis. The pretreatment of SP600125 or PD98059 could effectively reduce the apoptosis rate by reducing the ratio of Bax/Bcl-2 mRNA levels. MPP(+) exposure also induced high level of reactive oxygen species (ROS), marked by dramatic increase of Cu(2+)/Zn(2+)-SOD and GSH-Px mRNA levels. The elevated ROS was strongly associated with the activation of JNK and ERK1/2 signal pathways after MPP(+) exposure, since the pretreatment of NAC significantly reduced the upregulation of p-JNK and p-ERK1/2. Finally, the pretreatment of SP600125, but not PD98059, alleviated the increase of Cu(2+)/Zn(2+)-SOD and GSH-Px mRNAs induced by MPP(+), suggesting that the activation of the JNK signal pathway, but not the ERK1/2 signal pathway, could, in some degree, antagonize the generation of ROS induced by oxidative stress. In conclusion, our results suggest that JNK and ERK1/2 signal pathways, which are activated via ROS, play a crucial role in neuronal apoptosis induced by oxidative stress.

Total mRNA was extracted from lymphocytes separated from the peripheral blood of allergic patients, and then variable region of heavy chain (VH) and variable region of light chain (VL) cDNA library were constructed by RT-PCR. Human scFv templates for rabbit reticulocyte lysate ribosome display were assembled by primers and linker peptide (Gly4Ser)3. mRNA bound in antibody-ribosome-mRNA complexes was recovered using in-situ single primer RT-PCR, and three rounds of anti-IgE scFv DNA were enriched. The target DNA fragments were double enzyme digested and ligated into plasmid pET22b (+), followed by transformation in E. coli Rosseta (DE3). Positive clones were screened using clone PCR, Dot blotting and antigen ELISA. The correct lengths of VH (400 bp) and VL (710 bp) PCR products were obtained. The expected 1,000 bp ribosome display templates were also observed in agarose gel electrophoresis. After three rounds of ribosome display target sequences were effectively enriched, leading to a library of 10(13) members. Antibodies with the highest ELISA value for IgE were generated in the strain pET-IgE-6. A human anti-IgE scFv library was successfully constructed as described herein. Ribosome display using single primer in-situ RT-PCR as the recovery procedure effectively enriched target sequences. Anti-IgE scFv with high affinity and specificity were identified. The prepared human anti-IgE scFv fragment might be self-developed to a lead drug for treating asthma. Our study provides an alternative method for rapid discovery of human antibodies of therapeutic importance.

494 ng/ml. The test showed a sensitivity of 100%, a negative predictive vale of 100%. Conclusion VIDAS D dimer is well sited as diagnostic tool for the exclusion pulmonary embolism.

Objective To investigate the anti-tumor efficiency of cytotoxic T-lymphocyte (CTL) induced by activated B lymphocyte after hepatocellular carcinoma (HCC) alpha fetoprotein (AFP) mRNA transfection.Methods B lymphocytes were fractionated,purified and activated by recombinant human soluble sCD40L.PGEM4Z/AFP/A64-EGFP plasmid was established in vitro,mixed with polymerase T7RNA,and then transcribed into AFP mRNA with Poly (A) sequence.B lymphocytes electrotransfected with AFP mRNA were in the experimental group,B lymphocytes electrotransfected with GAPDH mRNA were in the negative control group,and untreated B lymphocytes were in the blank control group.The expressions of antigen-presenting cell (APC)markers (CD19,CD20,CD21,CD40,CD80,CD83) and major histocompatibility complex (MHC) of the 3 groups were detected.B lymphocytes of the 3 groups were cultured with T lymphocytes at ratios of 1∶40,1 ∶ 20,1∶10 and 1∶5 to induce and ampify CTL,and then the absorbance values were detected to evaluate the proliferation ability of T lymphocytes.The killing activity of CTL was investigated with HCC cell line SMMC7721 as the target cells.All data were analyzed using the paired t test,one-way analysis of variance or Tamhane's T2 test.Results The expressions of CD19,CD20,CD21,CD40,CD80 and CD83 of the experimental group were 74 ± 11,78 ±8,80 ± 10,90 ± 11,82 ± 6,56 ± 5,which were significantly higher than 51 ± 5,60 ± 7,53 ± 5,73 ± 8,50 ± 5 and 49 ± 6 of the negative control group,and 46 ± 3,54 ± 5,41 ± 3,56 ± 5,52 ± 6 and 21 ± 4 of the blank control group (t =5.302,4.812,7.627,5.932,9.142,7.813; 11.581,7.036,13.592,12.873,9.235,14.619,P ＜ 0.01).The proliferation of CTL of the experimental group was significantly higher than that in the negative control group and blank control group (t =18.203,23.714,15.062,9.417 ; 16.833,19.392,13.871,6.592,P ＜0.01).When the T lymphocytes were mixed with the HCC cell line SMMC7721 at the ratios of 40∶ 1,20∶1and 10∶1,the killing rates of HCC cells by CTL of the exprimental group were 43％ 4％,32％ ± 4％ and 22％ ±3％,which were significantly higher than 15％ ± 5％,7％ ± 3％ and 6％ ±2％ of the negative control group,and 7％±3％,8％±3％ and 9％±4％ of the blank control group (t =9.141,13.272,11.901; 14.372,12.835,9.507,P ＜ 0.01).Conclusion Activated B lymphocytes after HCC AFP mRNA transfection may effectively induce CTL to kill HCC cells.

AIM: The aim of this study was to determine the relationship between PAI-1,TIMP-1 gene expression and renal tubulointerstitial fibrosis(TIF) of ureteral obstruction ,and the interfering effects of hepatocyte growth factor (HGF) treatment. METHODS: Sixty rats were divided into normal control, sham operation,unilateral ureteral obstruction(UUO),and rhHGF treated groups (received 0 5 mg?kg -1 ?d -1 HGF for twenty-one days), and were sacrificed at postoperative day 3, 7, 14, 21. The levels of PAI-1, TIMP-1 mRNA were measured by RT-PCR. MMP2 and MMP9 activities were detected by substrate zymography, and renal fibrosis was assessed by measuring tissue hydroxyproline. RESULTS: Compared to controls, expression levels of PAI-1,TIMP-1 mRNA were significantly increased in UUO rats, and this was accompanied by decreased activities of MMP2 and MMP9 and increase in tissue hydroxyproline content. HGF treatment significantly decreased expressions of PAI-1, TIMP-1 mRNA, increased MMP2 and MMP9 activities,and decreased tissue hydroxyproline content in the obstructive kidney. CONCLUSIONS: These results indicate that the increases in PAI-1, TIMP-1 mRNA expression may be the major cause of sustained decreased matrix degradation during the development of tubulointerstitial fibrosis after unilateral ureteral obstruction. rhHGF efficiently ameliorates renal tubulointerstitial injury by the reduction of PAI-1,TIMP-1 mRNA expression, and increasing MMP2, MMP9 activities. [