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Objective To construct and identify the efficacy of a lentiviral vector harboring RNAi sequence targe-ting NSBP1 gene. Methods Three siRNA targeting the NSBP1 mRNA were designed, the pGCSIL-GFP-NSBP1 lentivirus vectors were constructed and confirmed by DNA sequencing. A total of 293T cells were co-transfected with pGCSIL-GFP-NSBP1, pHelper1.0 and pHelper2.0 for the virus stocks produced, the titer of the virus was test-ed. After lentivirus transfecting into DU145 ceils, Western-blot and MTT methods were used to determine the ex-pression and biological activity of NSBP1 gene, the cells were transplanted into nude mice, then inhibitive effect was observed. Results PCR analysis and DNA sequencing demonstrated that the RNAi sequence targeting the hu-man NSBP1 gene was successfully inserted into the lentiviral vector. The titer of the recombinant]entiviral vector was 2 × 10~8TU/mL. NSBP1 protein expression level in transfected cells was significantly decreased and growth rate of cells transfected with lentivirus was decreased by MTT assay, the downregulation of NSBP1 reduced growth rate of transplantated tumor, whereas tumorgenicity was not influenced. Conclusion The construction of the]entiviral vector of NSBP1 has been successfully prepared and NSBP1 plays an important regulatory role in androgen-inde-pendent prostate cancer cell proliferation.
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The number of application and granted funds for NSFC were analyzed statistically in Urology institute of Peking University from 2001 to 2007.The result showed the development of urological research projects and scientific research potential of institute of Urology in recent years.Meanwhile,some suggestions for existing problems of the scientific research were put forward in this paper.
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Objective To investigate the crosstalk between Wnt signaling pathway and TGF-? signaling pathway in prostate cancer cell line PC-3. Methods Luciferase report assay and RT-PCR methods were used to explore the crosstalk between two pathways. Results Wnt signaling pathway activated TGF-? responsive CAGA-luciferase reporter activity. ?-catenin(WT), TCF-4(WT), GSK3? (KM) activated while TCF-4(DN) inhibited CAGA reporter activity. ?-catenin(WT), TCF-4(WT) synergized to activate TGF-? signaling while TCF-4(DN) had converse effect.TGF-? signaling pathway activated LEF-1 luciferase activity through a Smad3-dependent manner.Smad7 activated LEF-1 luciferase activity,Smad3 and Smad4 also synergically activated LEF-1 reporter activity.?-catenin(WT),TCF-4(WT) cooperated with each other to the activity of cyclinD1 promoter luciferase while Smad3 inhibited cyclinD1 promoter activity.The inhibitory effect of Smad3 on cyclinD1 promoter could be partially reversed by co-transfecting ?-catenin(WT),TCF-4(WT).TGF-? induced the expression of VEGF in PC-3 cells and this effect was enchanced by LiCl addition. Conclusions Wnt signaling pathway and TGF-? signaling pathway can activate each other in prostate cancer cell line PC-3.Two pathways have crosstalk and this might be important for the development and progression of prostate carcinoma.
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Objective: To evaluate the effect of clusterin with and without leader sequence on overexpression preventing apoptosis in human prostate LNCaP cells. Methods:The plasmid pIRES2 EGFP was used to generate the clusterin expression constructs with full length or without the leader sequence (designated as pIRES2 EGFP/cluac, pIRES2 EGFP/clubc, respectively). Western blot analysis was employed to compare clusterin expression levels in the lysis and supernatant fluid of clusterin transfected LNCaP cells in vitro . The distribution of different functional domains of clusterin in cells was detected with Immunocytochemical staining. The clusterin's protective role of Na 2SeO 3 induced apoptosis in LNCaP cells was examined by flow cytometry (FCM) and fluorescence microscope. Results: Clusterin expression was detected in the lysis and supernatant fluid of pIRES2 EGFP/cluac transfected LNCaP cells, while clusterin was found only in lysis liquid of pIRES2 EGFP/clubc transfected LNCaP cells, but not found in their supernatant fluid. The distribution of cluserin in the plasm of pIRES2 EGFP/cluac transfected cells was aggregative, and on the other hand, clusterin distributed dispersedly in pIRES2 EGFP/clubc transfected cells. Its anti apoptotic property in LNCaP cells was proved by FCM and fluorescence microscope.Conclusion: It is apparent that clusterin plays an important role in preventing apoptosis in prostate cancer, and the presence of the leader sequence is necessary for clusterin's anti apoptotic function.
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Objective : To investigate the mutation of VHL gene, an important tumor suppressor gene in primary sporadic human renal cell carcinoma (RCC),and analyse its relationships with pathological stage and grade of renal cell carcinoma. Methods: We analyzed 57 cases of primary sporadic Chinese renal clear carcinoma using the polymerase chain reaction (PCR) and denaturing high performance liquid chromatography(DHPLC).All positive cases in DHPLC analysis were further characterized by direct sequencing. Results: Somatic mutations were detected in 30 (53%) of 57 clear cell renal carcinomas including 13 deletions, 2 insertions, and 15 missense mutations. These mutations mainly occurred in the last one third region of exon 1, 2,and 3. Conclusion: VHL tumor suppressor gene is one of the major tumor suppressor genes in human renal cell carcinoma,and there are frequent mutations of VHL in primary sporadic Chinese renal clear cell carcinomas. The mutations of VHL gene were irrespective of the age and pathological grade and stage of patients.
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Objective: To investigate the expression of peroxisome proliferator actived receptor ? (PPAR ?)and the inducement of apoptosis by PPAR ? ligand in renal cell carcinoma(RCC) derived cell lines.Methods:RT-PCR and Western blot analysis were performed to determined the expression of PPAR ? mRNA and protein in two RCC derived cell lines(786 O and A498) and two normal kidney(NK) derived cell lines(HK 2 and HMCC). Two RCC cell lines were treated with 50 ?mol/L troglitazoned for and evaluated for the effects of antidiabetic thiazolidinediones (TZDs) on the cells apoptosis by fluorescence microscopy and DNA ladder assay.The mutative expressions of Bcl 2 and Bax before and after TZDs treatment were also performed by western blot analysis. Results: The expression of PPAR ? was observed to be stronger in 786 O and A498 cells than in HK 2 and HMCC cells by RT-PCR and Western blot analysis. Treated with 50 ?mol/L troglitazone (for 48 h) it induced typical apoatosis in 786 O and A498 cells. After treatment, a decrease in Bcl 2 expression in RCC cells was observed by Western blot analysis,and the expression of Bax,however,was up regulated.Conclusion: The results reveal that troglitazone has the tumor suppressive effect on RCC cells. High affinity PPAR ? ligands (TZDs) may be the candidates for a novel approach to the treatment of this refractory neoplasm.
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Objective:To identify the androgen-responsive genes in prostate and screen the molecular targets for further studying human prostate cancer. Methods:The potential androgen-responsive gene pituitary tumor transforming gene 1 (PTTG1) was selected which had been previously screened by cDNA microarray in rat prostate and its mRNA level was detected by Northern blot in the castrated rat prostate with and without replacement of Mibolerone. Immunohistochemistry was performed to determine the expression and location of PTTG1 in human prostate tissues. Then human androgen-dependent prostate cancer cells LNCaP were used as a model to study the regulation of PTTG1 by Mibolerone. Results: PTTG1 mRNA was hardly detectable in the prostate of 7-day castrated rats, while it was up-regulated dramatically in the prostate of 7-day castrated rats treated with Mibolerone for 2 days. It was showed that high expression of PTTG1 was localized to the epithelial cells of human prostate cancer but not to the stromal cells with Immunohistochemistry. Northern blot analysis indicated that LNCaP cells treated with 0.1 nmol/L Mibolerone for 2 days led to the high PTTG1 mRNA expression. The basic expression of PTTG1 in human androgen-independent prostate cancer cell lines PC3 or DU145 was even higher than that in the human androgen-dependent prostate cancer cells LNCaP treated with Mibolerone. Conclusion: Androgen can up-regulate the PTTG1 expression in castrated rat prostate and human prostate cancer cell LNCaP. It suggests that PTTG1 is potential to play an important role in human prostate cancer progression.
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Objective To investigate the role of leader sequence in anti-apoptotic action of clusterin in LNCaP cell. Methods The wild type LNCaP cells(L), LNCaP cells transfected with the control vector(M),LNCaP cells transfected with clusterin expression vector with(A) and without(B) the leader sequence were cultured.RT-PCR was used to observe the expression of clusterin mRNA in group A,B,M and L.Cultured with TNF-?,the expression of clusterin mRNA in group L was measured,MTT and ELISA were used to determine the status of cell proliferation and apoptosis of the 4 groups. Results The expression of clusterin mRNA in group A and B was significantly higher than that in group L and M (all P 0.05).Clusterin mRNA of group L transiently elevated after treated with TNF-? for 2 h( A =15 642.0?64.3, t =-77.106, P
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Objective To evaluate urinary nuclear matrix protein(NMP 22) as an adjuvant diagnostic marker for transitional cell carcinoma of the upper urinary tract. Methods 24 patients with transitional cell carcinoma of the upper urinary tract and 20 patients with benign urinary diseass were evaluated with urinary NMP 22(cutoff level 10U/ml) and voided urinary cytology.NMP 22 was determined using a commercial test kit. Results The sensitivity and specificity of NMP 22 were 87.5% and 85.0% respectively whereas these of cytology were 58.3% and 95.0%. Conclusions Urinary NMP 22 might be an useful adjuvant diagnostic marker for transitional carcinoma of the upper urinary tract.
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Objective To study the mechanism of calcium oxalate monohydrate and calcium oxalate dihydrate stone formation. Methods 258 urinary stones were examined by infrared spectrophotometry, from which, 20 patients with calcium oxalate monohydrate stones (COM) and 10 patients with calcium oxlate dihydrate stones (COD) were selected for the study of some urinary parameters. The statistical study was carried out with SPSS t test . Results The urinary excretions of both calcium and phosphate varied obviously between the two groups of patients.In the COM group,the urinary calcium was (4.83?1.98)mmol/24h whereas in COD group it was (9.88?4.28)mmol/24h,( P
