OBJECTIVETo investigate the reproducibility of Gleason scores for prostate cancer.

METHODSBased on the revised Gleason Scoring System of the International Society of Urological Pathology ( ISUP) , we analyzed the reproducibility and difference of Gleason scores in 49 cases of prostate cancer using the methods of combination and grouping.

RESULTSThe total reproducibility of Gleason scores among 15 pathologists was good (κ = 0.642), 62.2% by the combination method, the highest in Gleason 5 + 5 (81.2%) and 5 +4 (73.3%), then in Gleason 4 + 4 (67.5%), 3 + 3 (64.0%), 4 +3 (61.3%), and 3 + 4 (44.0%), and the lowest in Gleason 4 + 5 (38.9%) and 3 + 5 (33.3%). The total reproducibility of Gleason scores by the grouping method was 71.4%, the highest in Gleason 9-10 (84.9%) , then in Gleason 7 (76.7%) and 6 (64.0%), and the lowest in Gleason 8 (60.7%).

CONCLUSIONThe reproducibility of Gleason scores remains to be further improved in prostate cancer, mainly concerning the understanding of Gleason 3 and 4 carcinoma.

Aim To investigate the molecular mechanisms of differentiation in human leukemia HL-60 cells induced by diallyl disulfide(DADS)using suppression subtractive hybridization(SSH).Methods In our privious study,the subtractive cDNA library was constructed successfully and efficiently. 30 clones were randomly analyed with restriction enzyme.The inserts of cDNAs were analyzed by restrictive enzyme EcoR I.Positive clones were sequenced and the homology of resulting cDNA sequences were analyzed through bioinformatics software Blastn.Results 18 clones contained 100～600 bp cDNA inserts.10 differantiation genes were obtained and involved in cell cycle,signal transduction,metabolism and RNA binding.And 3 of 10 genes,p21,STAT1 and CAMTA1 were up-regulated and detected by RT-PCR,the results matched with SSH.Conclusion sThere are tight correlation between the differentiation induced by DADS and three-upregulated gene:p21,STAT1 and CAMTA1.

Aim To investigate JAKs/STATs signal transduction change in HL-60 cells differentiation induced by diallyl disulfide(DADS)and molecular mechanism regulating the differentiation.Methods After incubation of HL-60 cells with DADS or AG490(50 ?mol?L -1),the cell differentiation indexes were observed by cytomorphology, NBT reduction ability assay,cell myeloid differentiation antigen CD11b by flow cytometry. Kinase activity of JAKs/STATs was tested by western-blotting and expressions of nucleus transcription genes stats,c-myc,c-fos,c-jun were detected by immumocyte chemistry method.Results Cell differentiation index changes indicated that HL-60 cells were induced differentiation toward granulocytic lineage by DADS, Western blot test demonstrated that constitive phosphorylation of Jak1,stat3 kinase was suppressed. Stat3,c-myc gene expression decreased and c-fos, c-jun gene expression increased in HL-60 cells treated with DADS through immunocyte chemistry.Conclusion Inhibition of phosphative Jak1, Stat3 was involved in HL-60 cells differentiation induced by DADS, its molecular mechanism might be related to modulation of gene expression associated proliferation and differentiation,and inhibition of DNA systhesis, induction differentiation.

Objective To investigate the expression of paired box2(Pax2),proliferation cell nuclear antigen(PCNA) and cell apoptosis in steroid-sensitive and steroid-resistant groups with primary nephrotic syndrome(PNS) and to find out the action of Pax2 expression in PNS with steroid-resistance.Method The expressions of Pax2,PCNA were evaluated by immunohistochemistry and cell apoptosis by fluorescence micoscope.Results Pax2 expression in renal tubule had a positive correlation with PCNA expression in steroid-sensitive group.In steroid-resistant group,Pax2 expression had no correlation with PCNA.Pax2 had a negative correlation with cell apoptosis.Conclusions Pax2 proper expression facilitate PCNA expression and repair tubulointerstitial lesions in steroid-sensitive group.Renal tubular epithelial cell proliferation coordinated with cell apoptosis.Pax2 overexpression in steroid-resistant group lead to the decrease of cell proliferation and cell apoptosis and lead to the severe tubule lesions,which made to glucocorticoid resistance.

Objective To explore the protective effect of Salvianolic acid B on isolated lung in rats.Method Twenty-four SD rats were randomly divided into 2 groups:control group and experimental group (n =12 each).The isolated lung in control group was perfused with raffinose-low potassium dextran (R-LPD),and that in experimental group was perfused with R-LPD and 800 mg/L of Salvianolic acid B.The model of isolated lung was established in all these rats.Wet/dry weight ratio (W/D),myeloperoxidase (MPO),malondialdehyde (MDA) and superoxide dismutase (SOD) were measured,and the pulmonary structures were observed by HE staining at different preservative periods of 6,9,12 and 24 h after infusion.Result Intragroup comparison was made in both groups:all parameters had no statistically significant difference before 12 h.However,W/D,MPO,MDA,and SOD at 24 h were higher than those at 12 h (P＜0.01).The result of interclass comparison showed that after 6,9 and 12 h preservation,all parameters of these two groups showed no significant difference.However,W/D,MDA and MPO were lower,and SOD was higher after 24 h preservation in control group (P＜0.05).Moreover,before 12 h preservation,the two groups did not show inflammatory injury,only manifested slight inflammatory reaction histologically.Destructive pulmonary structure,alveolar interstitial edema and inflammatory damage were seen more clearly in control group than in experimental group.Conclusion The perfusion with Salvianolic acid B had no apparent superiority before 12 h.However,after 12 h,it could ensure the quality and prolong the preservation time of the donor lungs more effectively.Salvianolic acid B might play an important role in donor lungs protection.

Objective To construct a MigR1-CD19 recombinant vector which contains CD19 gene, and to establish a CD19-K562 cell line over-expressing stably CD19 gene and its subcutaneous xenograft model in NOD-SCID mouse.Methods The CD19 gene was inserted into the retroviral vector (MigR1) through recombinant DNA technology after transfection into Plat-A packaging cells, and viral supernatant was collected to transduce K562 cell line repeatedly to obtain stable transduction CD19-K562 cell line.Flow cytometry was used to determine the transduction efficiency and RT-PCR was used to confirmed CD19 gene expression.Cell proliferation and apoptosis were detected by cell count and Annexin V/PI, respectively.Then the subcutaneous xenograft subtype of CD19-K562-a cell line was constructed through subcutaneous inoculation and was cultured in vitro and in vivo.Then its subcutaneous xenograft model in NOD-SCID mouse was established.The characteristics of CD19-K562-a cells were detected by RT-PCR, Wright staining and immunohistochemistry.Results MigR1-CD19 recombinant vector was successfully constructed, and the CD19 positive efficiency of K562 cell line was (99.80±0.17) ％ through retrovirus centrifugation transduction.The transduction and passage had no effects on proliferation and apoptosis of CD19-K562 cells.The CD19-K562-a cell line was constructed after CD19-K562 cells were injected subcutaneously and were passaged in vitro and in vivo.The CD19 positive efficiency of the xenograft subtype CD19-K562-a cell line was (99.78± 0.04) ％.CD19-K562-a and CD19-K562 cells were in an undifferentiated state.NOD-SCID subcutaneous xenografts were established through subcutaneous inoculation of CD19-K562-a cells.CD19 in the CD19-K562-a subcutaneous xenografts was positive, while it was negative in its counterparts K562 cells.Conclusion The CD19-K562 cell line over-expressing CD19 gene and its subcutaneous xenograft model in NOD-SCID mouse are successfully established.

Objective To establish a method for isolation of cynomolgus monkey umbilical cord mesenchymal stem cells.Methods Fresh cynomolgus monkey umbilical cord was directly minced into pasty fine pieces, and the pieces were cultured in tissue flask with DMEM/F12 medium supplemented with 10% fetal bovine serum.The morphological characteristics of the resulting cells were examined, and their expression of mesenchymal cell surface markers were analyzed by flow cytometry.The multidifferentiation potential was examined in vitro, too.Results The fibroblast-like cells were successfully isolated from the fresh umbilical cord by an adherent culture procedure.These adherent cells expressed mesenchymal markers including CD29, CD44, and CD90, and also could be induced to differentiate into adipocytes, osteoblasts and chondrocytes.Conclusion Mesenchymal stem cells can be isolated from fresh cynomolgus monkey umbilical cord by using an adherent culture procedure.

Aim To study the effects of cycle arrest and molecular mechanism in human leukemia HL-60 cells induced by diallyl disulfide ( DADS ) . Methods Cell count, colony formation in soft agar experiments and flow cytometry analysis were employed to observe the DADS-induced cell growth inhibition and the effect of cycle arrest in HL-60 cells. The expressions of Chk1/2 and its downstream element in HL-60 cells were detected by Western blot. Results Cell count revealed that population doubling time increased to 35. 03 h and 71. 82 h, respectively, from 19. 14 h in HL-60 cells treated with 60 and 120 μmol·L-1 DADS ( P<0. 05 ) . Colony formation in soft agar experiments showed that colony formation inhibition rate of HL-60 cells exposed to 30, 60, 90 and 120μmol·L-1 DADS increased to 35. 06%, 62. 10%, 93. 79% and 99. 35%, respectively ( P<0. 05 ) . Flow cytometry a-nalysis exhibited that HL-60 cells treated with 60 and 120 μmol · L-1 DADS for 24 h and 48 h arrested in G2/M phase in a concentration-and time-dependent manner ( P <0. 05 ) . Western blot disclosed that the expression of p-Chk1 increased in a time-dependent manner ( P <0. 05 ); however, Chk1, Chk2 and p-Chk2 were not changed in HL-60 cells treated with 60μmol·L-1 DADS (P >0. 05). The expression of Cdc25C, CyclinB1 and CDK1 decreased after treated with 60 μmol·L-1 DADS in a time-dependent manner ( P<0. 05 ) , but the expression of 14-3-3 protein did not change ( P>0. 05 ) . Conclusion DADS can in-hibit the proliferation of HL-60 cells, and induce G2/M arrest through Chk1/Cdc25 C/CyclinB1/CDK1 path-way.

Aim Toresearchthemolecularmecha-nisms of DADS-induced apoptosis in human leukemia K562cells.Methods Cellviabilitywasmeasuredby MTT. Levels of DADS-induced ROS were measured by 2ˊ, 7ˊ-dichlorofluorescein diacetate ( DCFH-DA) fluo-rescence. DADS-induced mRNA levels of components of the NADPH oxidase were detected by Real-time PCR. The combination of protein Rac2 and p67phox was measured by immunoprecipitation assays. Flow cy-tometry methods were used to determine the percentage of apoptosis cells. DADS-induced Rac2 levels were measuredbyWesternblot.Results TheDADS-trea-ted K562 cells showed a dose-and time-dependent de-crease in cell viability and proliferation. There was sig-nificant up-regulation of the mRNA level of components of the NADPH oxidase complex in K562 cells after treatment with 6 mg·L-1 DADS for 6 h. Western blot results revealed that, compared with the control group, there was a significant up-regulation of Rac2 protein in K562 cells treated with 5. 0 and 10. 0 mg·L-1 DADS for 24h. And Rac2 combined with p67phox in DADS-induced apoptosis in K562 cells. PMA markedly in-creased the percentage of apoptotic cells, and DPI re-duced the percentage of apoptotic cells in DADS-in-duced K562 cells. Levels of DADS-induced ROS, also showed enhancement when exposed in PMA, but there was no DADS-induced ROS production evident when exposed in DPI in DADS induced K562 cells. Conclu-sions TheseresultsindicatethatNADPHoxidaseis the main source of DADS-induced ROS production. Diallyl disulfide induces apoptosis in human leukemia K562 cells through activation of NADPH oxidase.

The enzyme activity of ?- Acetolactate Decaroboxylases (ALDC) from different microbes was studied, the results demonstrated that it was quite different among them. There were diversities of their enzyme reaction velocities. It was clear that the enzyme activity was affected by the pH of the enzyme reaction system, for example, the optimum pH of ALDC from Lactococcus lactis was 6. 6, while for Aerobacter Aerogenes it was 5. 8. Addition leucine,valine and isoleucine into enzyme reaction system obviously affected the enzyme activity of ALDC from different microbes.