Objective To evaluate the different methods of continent urinary diversion and the ileal orthotopic neobladder after total cystectomy. Methods 4 different kinds of continent urinary diversion were undertaken after total cystectomy for 68 cases.Continence and catheterization, volume and pressure of the reservoirs, image and hydroelectrolyte condition were investigated. Results Of the 3 cases with intussusceptive efferent tract,2 had partial deintussusception resulting in incontinence and had to be reoperated;44 cases with tapered terminal ileal efferent tract were continent and could be catheterized easily with 16～20F catheters, whereas only one case had difficulty in catheterization. 39 cases with detubularized and reshaped intestinal segment reservoirs, including 3 ileal,22 colonic and 14 ileocolonic, all achieved the demand of low intrareservoir pressure, whereas 8 cases operated in the early period had dilated reservoirs with a volume as large as 1 470～1 650 ml;8 cases with detenia cecocolonic reservoir had the volume of 430～600 ml and as intrareservoir pressure of 30～45 cmH 2O with peristalsis waves,2 of them had urine leakage in the early stage after operation.21 cases with ileal orthotopic neobladder had a volume of 350～480 ml and an intrareservoir pressure of 12～20 cm H 2O.1 being incontinent at daytime and 2 at night whereas all the others were continent. Conclusions Continent urinary reservoirs constructed by 30 cm detubularlized cecocolon can achieve the demand of low intrareservoir pressure.Detenial colonic reservoirs had more chance of urine leakage and adhesion, and higher intrareservoir pressure. Tapered terminal ileal efferent tract is better than ileal intussusception efferent tract for its excellent continence, large caliber, easy catheterization and fewer complications. Ileal orthotopic neobladder has the advantages of excellent continence, higher quality of life, but has limited indications.

Objective The aim of this study was to investigate the distribution and clinical significance of CD8+ T cells in bladder cancer tissues in situ.Methods Immunohistochemistry were used to examine the distribution of CD8+ T cells in bladder cancer tissues,which were obtained from January 2003 to December 2009 from 302 patients.Among all the patients,262 were male while 40 were female;mean age is 60 years;tumor size ≤ 3 cm was in 235 and tumor size ＞ 3 cm was in 67;Unifocal tumor was in 214 and multifocal tumors were in 88.Amount of tumor stage Ta-T1 was 212 and T2-T4 was 90.Sixteen patients have lymph node metastasis.Histological low grade was diagnosed in 175 and histological high grade was diagnosed in 127.According to the differences between anatomic structure and cellular composition,bladder tumor tissues can be classified to two localization patterns:(1) intratumoral regions,defined as tumor cell nests;(2) stromal regions,defined as stromal areas that lack direct contact with tumor cells.Therefore,we divided 302 bladder cancer patients into two groups based on the median frequency of intratumoral CD8+ T cells (median,3/× 400 high resolution) and stromal CD8+ T cells (median,37/× 400 high resolution),respectively.x2 analysis was used to evaluated the correlation between CD8+ T cell density and clinicalpathological variables.Kaplan-Meier analysis and Cox proportional hazards regression models were applied to estimate overall survival (OS).Results CD8+ T cells were predominantly located in the intratumoral regions (mean,14 ± 2/× 400 high resolution) rather than in associated stromal regions (mean,50 ± 3/× 400 high resolution,P ＜ 0.05).The density of intratumoral CD8+ T cells was inversely associated with age (P =0.026),tumor size (P ＜ 0.05) and tumor stage (P ＜ 0.05),and could represent a favorable prognostic predictor of OS (HR =0.427,P =0.003).However,the density of stromal CD8+ T cells was positively associated with age (P =0.004) and histological grade (P ＜ 0.01),and could represent an adverse prognostic predictor of OS (HR =2.206,P =0.009).Conclusions Our findings suggest that intratumoral/ stromal CD8+ T cells could potentially serve as favorable/ adverse prognostic markers for bladder cancer patients,respectively.

Objective To investigate the expression profile and biological function of lncRNAs in urothelial cancer stem cells and explore whether they can be biomarkers for prediction of clinical characteristics for bladder cancer patients.Methods Microarray analysis was performed to identify differentially expressed lncRNAs in cancer stem cells and parental cancer cells.Expression profiles were validated by Coding Potential Caculator analysis,real-time polymerase chain reaction.By performing in vitro sphere formation assays,J82 cells with lentivirus-based knockdown of lncRNA-BCSC (bladder cancer stem cells) were used to confirmed its function.Samples were obtained from patients who underwent TURBT between January 2009 and December 2012.All tissues were initially confirmed by pathologists.The association of the clinicpathologic of bladder cancer and lncRNA-BCSC expression was analyzed by x2 analysis.Results 750 lncRNAs were highly expressed from microarray analysis in bladder cancer stem cells.Among these,lncRNA-BCSC was identified as potentially maintaining self-renewal of cancer stem cells.Knockdown of this transcript in J82 cells inhibited spheroid formation.lncRNA-BCSC expression were higher in 54.8％ (57/104) bladder cancer tissues.Moreover,using x2 analysis,lncRNA-BCSC expression in primary tumors was found to be a predictor for recurrence following transurethral resection in patients with nonmuscle-invasive bladder cancer (P =0.009).Conclusions Our study provides strong evidence that lncRNA-BCSC are indispensable modulators of self-renewal ability of bladder cancer stem cells.Overexpression of lncRNA-BCSC may have a predictive value for early recurrence in patients suffering from nonmuscle-invasive bladder cancer.

Objective To determine the efficacy of retrovirus-mediated HSV-TK gene transfer and GCV in a BALB/c mice model with urinary bladder transitional cell carcinoma. Methods A replication defective retroviral vector containing HSV-TK gene was used. In vivo experimental animals were divided into 3 groups.In group A,7 tumors were induced in BALB/c mice by subcutaneous injection of MBT-2 transitional cell carcinoma (TCC);the virus was directly injected into the tumor and the animals received intraperitoneal GCV.In group B, 6 tumors were induced in BALB/c mice by subcutaneous injection of transferred HSV-TK gene TCC and the animals received intraperitoneal GCV.In group C,6 tumors were induced in BALB/c mice by subcutaneous injection of MBT-2 TCC and the animals received intraperitoneal normal saline.Tumor-volume measurement was performed in these 3 groups 10 d and 20 d after treatment, and the volume changes were compared among the 3 groups. Results In vivo experiment indicated that the mean tumor volume of group A [MBT-2 group, (55.37?4.52) mm3] and group B [MBT-2/HSV-TK group,(49.77?4.15)mm3] was reduced significantly compared with that of group C [control group,(146.27?10.46)mm3](P0.05).At 20 d after treatment, the mean tumor volume of group A [(186.75?8.14)mm3] and group B [(72.50?6.70)mm3] was also reduced compared with that of group C [(441.76?41.80)mm3] (P

Objective To describe the cloning of cDNA of prostate specific membrane antigen (PSMA),construction of the recon of PSMA,induction of the expression of 6 ? His PSMA fusion protein from PSMS recon,and purification of the expression products. Methods The total RNA was extracted from prostate cancer tissues.The site of EcoR I lying in the middle of PSMA’s cDNA was used to perform the reverse transcriptase and polymerase chain reaction of PSMA in two different parts,which were then linked into the carrier of pEt 30(a),thus the full cDNA of PSMA and the pronucleus expression carrier pET 30(a) PSMA were obtained.The pET30a (+) PSMA recon was transformed into the Escherichia coli BL 21 and the engineering bacteria expressing protein PSMA was gotten. With the induction of IPTG, the recon was expressed. The expression products were purified with Ni NTA Agarose resin, then the purer protein PSMA was obtained. The antigenicity and specificity of the expressed PSMA were evaluated with Western blotting and SDS PAGE. Results The wholly right base sequence of PSMA’s cDNA was successfully cloned.PSMA protein was expressed and purified.This protein had better antigenicity and specificity. Conclusions This study provides experimental basis for the function study of PSMA and scanning of the phage antibody library.

AIM:To investigate the production and activation of caspase-3 in primary rat renal proximal tubule cells in response to tumor necrosis factor-α(TNF-α) and the implication of nuclear factor-κB (NF-κB) in the process. METHODS:Isolated rat renal proximal tubule cells (PTCs) from male adult Sprague Dawley rats were treated with TNF-α according to the indicated time courses. A specific NF-κB inhibitor,Bay11-7082,was used alone or as a pretreatment for 1 h followed by exposure to TNF-α for 24 h.The protein levels of cleaved caspase-3,caspase-3,I-κBα,phosphorylated I-κBα,and GAPDH were detected by Western blotting using specific antibodies. RESULTS:The protein level of cleaved caspase-3 relative to caspase-3 was significantly increased in the presence of TNF-α for 6 h,12 h,and 24 h. Protein levels of caspase-3 were significantly decreased by 12 h and returned to baseline by 24 h in the presence of TNF-α. Treatment with Bay11-7082 for 25 h alone or pretreatment with Bay11-7082 for 1 h followed by addition of TNF-α for 24 h caused a remarkable reduction in both cleaved caspase-3 and caspase-3 as compared to control and TNF-α treated groups. An increase in phosphorylated I-κBα was observed from 15 min to 60 min after treatment with TNF-α at a dose of 10 μg/L in PTCs. CONCLUSION:NF-κB is not only associated with the activation of caspase-3 but also the production of caspase-3 in primary rat renal proximal tubule cells in response to TNF-α.

BACKGROUND:Smooth muscle cells and transitional epithelial cells were traditionally used to construct tissue-engineered bladder and to perform double-sided implantation of scaffold.However,double-sided implantation is difficult to perform,because smooth muscle cells are difficult to isolate or culture in vitro and passage is limited.OBJECTIVE:To verify the feasibility of tissue-engineered bladder reconstruction with bone marrow mesenchymal stem cells(BMSCs)and bladder acellular matrix(BAM).DESIGN:A basic empirical study.SETTING:Linbaixin Medical Research Center,Second Affiliated Hospital,Sun Yat-sen University.MATERIALS:Experiments were performed at the Linbaixin Medical Research Center,Second Affiliated Hospital,Sun Yat-sen University from March 2006 to Mav 2007.The laboratory was the Opening Laboratory of Hospital Affiliated to Health Department of China.One-month old SD rats of either sex,weighting 80-100 g were provided by Animal Experimental Center of Sun Yat-sen University.Fresh porcine bladders were offered by Animal Experimental Center of Southern Medical University.METHODS:Whole bone marrow culture and successive adherence method was used to culture rat BMSCs in vitro.Flow cytometry was employed to detect surface antigen.Eradicator washing method was applied to prepare porcine BAM and measure its purity and characteristies.Third passage of BMSCs were inoculated in BAM and cultured in a medium containing vascular endothelial growth factor(VEGF165)(25 ng/L)in vive and in vitro to test compatibility.Cells cultured alone were considered to be controls for the in vivo trial,and materials non-implanted with cells were considered to be controls for in vitro trial.Suitable microenvironment was simulated to induce the differentiation of BMSCs.Four weeks and eight weeks later,compound materials were respectively removed to perform tissue section test.Simultaneously,immunohistochemistry keratin staining was conducted to examine regeneration of epithelial cells.MAIN OUTCOME MEASURE:Biocompatibility of BMSCs and BAM.RESULTS:①BMSCs were cultured by whole bone marrow method.Flow cytometry demonstrated that third passage of cells were positive for CD29(99.43%).②BAM had good biological characteristics.Homogen matrix and byssoid collagen appeared under a microscope.Compatibility trials showed good compatibility of BMSCs and BAM and well-growth cells.③Four weeks later,histological section test confirmed inflammatory cell infiltration,closely-arranged collagen and elastic fiber.Immunohistochemistry keratin staining showed lamellar and discontinuous simple epithelium.Eight weeks later,no inflammatory cell infiltration was found,and closely-arranged collagen and elastic fiber were detected.Immunohistochemistry keratin staining showed lamellar and continuous multiple epitheliums.CONCLUSIoN:With good compatibility,BMSCs and BAM appear to be an ideal material for bladder tissue engineering.

Objective To detect the differentially expressed microRNAs (miRNAs) in bladder cancer tissue and normal bladder tissue. Methods Total RNA was extracted from bladder cancer tissue and normal bladder tissue by Trizol, and then miRNAs were isolated and enriched from the total RNA. Mammalian miRNA microarrays were used to analyze the differentially expressed miRNAs between the bladder cancer tissue and normal tissue. Data analysis was performed by software of LuxS-can3. 0 and SAM version 2. 1. Choose let-7 gene which was interesting to us, and validation of mi-croarray results was carried out by northern blotting. Results Compared with normal bladder tissues, there were 71 differentially expressed miRNAs in bladder cancer tissue, of which there were 38 down-regulated ones and 33 up-regulated ones. Among these miRNAs, 26 miRNAs were the most significant with 12 up-regulated and 14 down-regulated. The expression of let-7 gene in bladder cancer was down-regulated to normal bladder tissue by northern blotting, which was in agreement with the results of the miRNA microarrays. Conclusion There are differentially expressed miRNAs between bladder cancer tissue and normal bladder tissue, and let-7 gene is probably as a tumor suppressor in bladder cancer.

AIM: To construct eukaryotic expression vector of small interfering RNA(siRNA) specific to bcl-2 and investigate the effect of recombinant plasmid on suppressing bladder cancer cell growth.METHODS: siRNA of bcl-2 gene was designed according to the principle of RNAi-based medicine, and was converted into cDNA coding expression of small hairpin RNAs(shRNA) of siRNA. The cDNA was synthesized and inserted into plasmid pGenesil-1. The recombinant eukaryotic expression vectors of pGenesil-1545 and pGenesil-1555 were controlled by the U6 promoter of RNA polymerase Ⅲ, identified by the restriction map and the sequence analysis, and transfected into T24 cells. After T24 cells were transfected for 72 h, expression of bcl-2 mRNA was assayed by RT-PCR; and MTT was used to observe the proliferation of T24 cells.RESULTS: The recombinant plasmids of pGenesil-1545 and pGenesil-1555 were identified by the restriction map and the sequence analysis. The sequences completely coincided with the designs. The expression of the bcl-2 mRNA in T24 cells transfected with recombinant plasmid decreased nearly 80%, and the growth of T24 cells was suppressed significantly.CONCLUSION: The siRNA eukaryotic expression vector against bcl-2 gene is successfully constructed. It effectively downregulates the expression of bcl-2 in T24 cells and suppresses the cell growth.

AIM: To identify the non-steroid transcription factors upregulating the expression of L-plastin in hormone-independent prostate cancer, and partly elucidate the mechanism of hormone-refractory prostate cancer. METHODS: TF SEARCH software was used to analysis the possible binding sites of transcription factors in the 3’ end of L-plastin promoter that had been identified as important part of regulation response elements. Gel shift assay and supershift assay were used to confirm the transcription factors binding the speculated response elements. PCR site-mutagenesis technique was performed to delete the binding site of transcription factor and luciferase activity assay was carried out after deletion of the binding site. RESULTS: SP-1 respond element GGTGGGGCGGGGA located at -54- -41 of L-plastin promoter was identified with the TF SEARCH software. Gel shift assay and supershift assay confirmed that SP-1 was the transcription factor binding to GGTGGGGCGGGGA. Mutant deleted the SP-1 binding-site had low-luciferase activity than that of the naive. CONCLUSION: SP-1 plays an important role in the up-regulation of L-plastin expression in hormone-independent prostate cancer.