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Objective To investigate the expression of B7-H3 in muscle infiltration bladder transitional cell carcinoma and its clinical significance.Methods The clinical and pathological data of 115 patients with muscle infiltration bladder transitional cell carcinoma who underwent radical surgery were retrospectively analyzed.The expression of B7-H3 in bladder cancer tissues and corresponding paracancerous tissues was detected by immunohistochemical staining,and the relationship between B7-H3 and clinicopathological characteristics was analyzed.Results The positive expression of B7-H3 was mainly in the cytoplasm,presenting brownish yellow granules.B7-H3 had positive expression in all bladder cancer tissues but no expression in paracancerous tissues.The high positive rate of B7-H3 expression in bladder cancer was 68.7％.The abnormal expression of B7-H3 was correlated with the distant metastasis and intravascular tumor thrombi (all P＜0.05),but not with gender,age,tumor stage and grade,lymph node metastasis and recurrence (all P＞0.05).The survival analysis result showed that the survival time of the patients with high expression of B7-H3 in the bladder cancer tissues was significantly lower than that of patients with low expression of B7-H3,and the difference was statistically significant (P＜0.05).Conclusion The expression of B7-H3 in muscle infiltration bladder cancer tissues is up-regulated and is positively associated with the distant metastasis of bladder cancer and intravascular tumor thrombus.The expression of B7-H3 may indicate poor prognosis of the patients with bladder cancer.
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Objective To investigate the expression of adenylate kinase 4 (AK4) in prostate cancer and its clinical significance.Methods The clinical data of 85 patients with prostate cancer who have underwent radical surgery were retrospectively analyzed.The expression of AK4 in prostate cancer tissues and corresponding paracancerous tissues was detected by immunohistochemistry to analyze the relationship with clinicopathological features.Results The positive rates of AK4 in prostate cancer and paracancerous tissues were 85.9％ and 38.8％ respectively,and the difference was statistically significant (P＜0.05).The abnormal expression of AK4 was correlated with recurrence and distant metastasis (all P＜0.05).The survival time of AK4-overexpressing patients with prostate cancer was significantly lower than that of AK4 low-expression patients,and the difference was statistically significant (P＜0.05).Conclusions The expression of AK4 in prostate cancer tissues is up-regulated,which is related to the distant metastasis of carcinoma and vascular tumor thrombus,which suggests that the expression of AK4 may indicate the poor prognosis of patients with prostate cancer.
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Objective To investigate the histopathologic characteristics of bladder tumor and provide theoretical basis for the reasonable selection of treatment modality.Methods This retrospective study collected the pathological data of 4 200 bladder tumor from May 2001 to October 2014.There were 3 443 male and 757 female, and the average diameter of these tumors was (1.8 ± 0.6) cm (ranged 0.2 to 6.5 cm).Among all cases, 3 214 (76.5％) cases were solitary tumor while 986 (23.5％) were multiple tumors.The histologic subtype, pathological grade and stage, the existence of vascular and lymphovascular invasion, tumor in situ, abnormal variants and rare subtypes were recorded and analyzed.Results 162 cases (3.9％)were benign tumors and 4 038 cases (96.1％)were malignant tumors including 4 008 cases of urothelial cancer (UC), 18 cases of primary adenocarcinoma and 12 cases of primary bladder squamous carcinoma.Furthermore, 2 460 (61.4％)cases were high grade UC while 1 548(38.6％)cases were low grade.320 cases were found intravascular tumor embolus or lymphovascular tumor thrombus and 391 (9.3％)cases were found metaplasia of squamous epithelium.Moreover, there were 230 cases of squamous differentiation, 120 cases of glandular differentiation, 110 cases of both squamous and glandular differentiation, and 39 cases (0.9％)of other rare subtypes or variations.On pathological stage, 112 (2.8 ％) cases were carcinoma in situ, 548 (13.7％)cases were Ta, 2 599(65.1％)cases were T1, 480(12％)cases were T2, 92 cases(2.3％)were T3 and 23 cases(0.6％)were T4 stage, with the rest cases being unable to be accurate staging.Multiple Logistic regression analysis revealed that lymphovascular invasion was related to tumor grade , pathological stage and abnormal differentiation (P ＜ 0.02).Moreover, UC with squamous and glandular differentiation were related with tumor recurrence and progression (P =0.02).Conclusions Most bladder tumors were high grade and low stage urothelial cancer with various forms of differentiation.Squamous and glandular differentiation were most common variation which should be avoided to diagnosed as hybrid carcinoma.Lymphovascular tumor thrombus and abnormal differentiation were correlated with tumor stage and grade.
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Objective To analyze the features of onset, clinical pathological characteristics about the primary bladder mucinous adenocarcinoma.Methods From January 1990 to June 2015, we retrospectively reviewed the data from 15 patients diagnosed as primary bladder mucinous adenocarcinoma, including 10 male patients and 5 female patients.Their mean age was 58 years old, ranged from 41 to 78 years old.Among the fifteen patients, the initial symptoms included hematuria in 13 cases, lower abdominal pain in 1 case and urinary irritation symptom in 1 case.The ultrasound and CT scan revealed bladder tumors, which the size ranged from 2 to 6 cm.The location of bladder tumors included front wall in 12 cases, trigone zone in 2 cases and top wall in 1 case.Nine cases was suspected as tumor from urachal remnants.Eleven patients underwent partial cystectomy, three patients accepted the radical cystectomy and one case accepted transurethral resection of bladder tumor (TURBT).Result Pathological diagnosis was bladder mucinous adenocarcinoma in all patients, including nine from urachal remnant and the others from urothelimn.The tumor exhibited the mushroom liked prominence, which was associated with surface ulceration and infiltrated into the depth of bladder.Meanwhile, it was covered with thick mucinous substances.The histologic examination revealed the presence of andenoid structure, composed by various degree of diferenfiated mucinous cells.In cases with adenocarcinomas from urachus, the residue of urachal tissue could be noticed.The bladder mucous was intact or ulcerated.No sign of metaplasia was observed.In the pathological diagnosis, the classification included grade Ⅲ in 3 cases, grade Ⅱ in 7 cases and grade Ⅰ in 5 cases.Ten persons reported the information of the follow up.Eight of them, whose tumor originated from urachus, accepted bladder-sparing surgery.One died from acute myocardial infarction after 23 months postoperatively.And one died from cerebral hemorrhage 45 months postoperatively.The others have been followed up from 8 to 65 months with no sign of recurrence.In two cases with urothelial carcinoma, one was found the new urothelial carcinoma 50 months after TURBT and one died from cancer metastasis 29 months after partial cystectomy.Conclusions Primary mucinous adenocarcinoma of the bladder possess the relatively high malignant tendency.The hematuria is the main initial symptoms.The histologic examination revealed the presence of different differentiated mucinous cells and formed the andenoid structure.The case with urachal remnant adenocarcinoma has the better prognosis than other types.
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Micro-ribonucleic acids (miRNAs) are endogenous single-stranded small non-coding RNAs. miRNAs bind to a com-plementary site in the 3' untranslated region of their target mRNAs through canonical base pairing, which can direct the degradation or translational repression of these transcripts. Thus, miRNAs can effectively silence the protein expression of target genes post-transcrip-tionally. miRNAs may also regulate the expression of oncogenes and tumor suppressor genes and could be involved in almost all known hallmarks of cancinogenesis. In this paper, we discuss the following in detail:(1) biogenesis and main functions of cellular miR-NAs, (2) stability and detectability of exosomal miRNAs in biological fluids;and (3) feasibility of miRNAs as a potential new class of biomarkers derived from urinary exosome in the malignancy of urinary system. Finally, we summarize studies on urinary exosomal miRNAs as potential biomarkers of prostate, bladder, and kidney cancers.
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Objective To study the clinical and histopathologic features of small renal carcinoma (diameter≤4 cm)and provide theoretical basis for evaluating the safety,efficacy and prognosis of nephron sparing surgery.Methods This retrospective study collected the pathological data of 490 patients with small renal cell carcinoma,who were treated in our hospital,from May 2000 to October 2014.We recorded and analyzed the tumor size,histological subtype,Fuhrman grading,pathological stage,the existence of mulifocality,vascular invasion,tumor psuedocapsule,hemorrhage or necrosis and distant metastasis.Results The median diameter of tumor was (3.2 ± 0.6) cm,ranged 0.6 to 4.0 cm.Of all the subjects,422 (86.1％) were clear cell carcinoma,32 (6.5％) were chromophobe cell carcinoma,23 (4.7％) were papillary carcinoma and 13 (2.7％) were other rare types.Among the 422 clear cell carcinoma cases,27 were Fuhrman grade Ⅰ,157 were Ⅰ-Ⅱ grade,210 were grade Ⅱ,21 were Ⅱ-Ⅲ grade,7 were grade Ⅲ and no one was grade Ⅳ.Multifocal tumors were found in 18 cases (3.7％) and tumor embolus of renal vein was found in 6 cases (1.2％).Intact psuedocapsule were found in 326 (66.5％) tumors with the thickness ranged from 0.2 to 1.0 mm.Tumor infiltration without the psuedocapsule penetration were found in 82 cases (16.7％),penetrated into the psuedocapsule were found in 11 cases (2.2％),infringement of perirenal fat were found in 9 cases (1.8％).Hemorrhage and necrosis were found in 240 cases (48.9％),synchronous lung metastases occurred in 3 patients (0.6％).Logistic regression analysis revealed that tumor invasion and pseudocapsule penetration were related to Fuhrman Ⅱ-Ⅲ,Ⅲ and tumor diameter (P =0.04).Moreover,tumor size was related with histological grade and renal capsule invasion (P =0.02).Nevertheless,there was no relationship among tumor size,renal vein embolus or mulifocality (P =0.35).Conclusions Although most small renal tumors are high differentiation and low grade,but rare cases are aggressive with infringement of perirenal fat or early distant metastasis,suggesting heterogeneity in its biological behavior.Most small renal tumors have obvious psuedocapsule.When the tumor size is greater than 3.0 cm and its Fuhrman classification was high,the psuedocapsule and perirenal fat are more likely to be infiltrated.Nephron sparing surgery should remove the tumor and its surface adipose tissue entirely.
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Tumor-derived heat shock protein-peptide complex 96 (HSPPC-96) containing tumor antigenic peptides can elicit po-tent tumor-specific and protective immunity. Autologous HSPPC-96 vaccine has been shown to effectively prolong recurrence-free sur-vival and increase the overall survival of many tumors, thereby suggesting extensive future applications. However, as an autologous tu-mor-derived individual vaccine, the development of HSPPC-96 vaccine is challenged by the lack of an adequate autologous tumor, lim-ited efficacy for advanced-stage cancer, etc. This paper summarized the progress, future perspectives, and challenges in the clinical de-velopment of HSPPC-96 vaccine immunotherapy.
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Objective: To investigate the molecular changes in bladder urothelial carcinoma via different pathways. Methods:Polymerase-chain reaction (PCR) or coamplification at low denaturation temperature-PCR and Sanger direct sequencing were per-formed to detect the status of fgfr3, p53, and h-ras gene mutations in 88 tissue samples of human bladder cancer and 10 normal control tissues. The relative mRNA expression levels of motility-related protein-1 (MRP-1)/CD9 and the relationship between genes and tumor recurrence were also determined. Logistic regression and relative analyses were conducted to compare the significance and interrelation of genes among tumor recurrences. Results:The mutation rate of p53 increased as pathological grades and stages increased. Recurrence rate was higher in patients with MT-p53 genotype than in patients with WT-p53 genotype. Conversely, the mutation rate of fgfr3 gene decreased as pathological grades and stages increased. Recurrence rate was also higher in patients with WT-fgfr3 genotype than in pa-tients with MT-fgfr3 genotype. In low-grade and early stage tumors, MT-fgfr3/WT-p53 was the most prevalent genotype;in high-grade and late stage tumors, WT-fgfr3/MT-p53 was the most prevalent genotype. The mutations of h-ras were mainly observed in low-grade tumors in early stages. Moreover, the relative mRNA levels of MRP-1/CD9 decreased as pathological grades and stages increased. The mRNA levels of MRP-1/CD9 were negatively correlated with p53 mutations and positively correlated with fgfr3 mutations. Logistic re-gression analysis results showed that patients with WT-fgfr3 genotypes exhibited 3.88 times higher relative risk of tumor recurrence than those with MT-fgfr3 genotypes;by contrast, patients with MT-p53 genotypes exhibited 4.53 times higher relative risk of tumor re-currence than those with WT-p53 genotypes. Conclusion:Fgfr3 and h-ras gene mutations may play important roles in tumorigenesis of low-grade and early stage bladder cancer. p53 gene mutation and mRNA levels of MRP-1/CD9 may be implicated in the tumorigenesis of high-grade tumors in late stage of bladder cancer. In general, the two variants of urothelial carcinoma exhibit distinct genetic defects. fgfr3 gene mutation revealed a pathway of favorable prognosis, and p53 gene mutation demonstrated a pathway associated with poor prognosis.
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Objective To investigate the clinical and pathological characteristics of nephrogenic adenoma. Methods Eleven patients were diagnosed as nephrogenic adenoma including 5 men and 6 women, aged 37-78 years (56 on average). The pathological findings in all cases of nephrogenic adenoma were presented with a review of the literature. Results Eleven cases of nephrogenic adenomas were evaluated, 2 cases were in ureter and 9 cases were in the bladder. Eight of the 9 bladder cases underwent TUR-BT surgery in continuous epidural anesthesia, 1 case underwent partial cystectomy with general anesthesia. A right ureteroscopy and left ureterolithotomy were performed respectively in continuous epidural anesthesia for the 2 cases in ureter. The final diagnosis was based on histopathological findings. For all of cases, 8 cases were diagnosed as nephrogenic adenomas, 2 cases as atypical nephrogenic adenoma and 1 case as nephrogenic adenoma with malignant transformation. The microscopic appearance of nephrogenic adenoma demonstrated that morphology closely resembled aberrant tubules of the kidney. In addition, atypical nephrogenic adenomas appeared as the presence of cytologic atypia, including nuclear enlargement, nuclear hyperchromasia and prominent nucleoli. The morphologic changes of nephrogenic adenomas with malignant transformation were that tumor cells retained the basic structural characteristics of typical nephrogenic adenomas, and the similar morphological cells lost adhesion ability among cells and presented diffuse solid growth in the surrounding area.Intravesical perfusion was further performed for treating the patients with atypical nephrogenic adenomas or nephrogenic adenomas with malignant transformation. The mean patient follow up was 46 months (range, 24- 104 months), and there was only 1 case of recurrence. Conclusions Nephrogenic adenoma is an uncommon benign lesion of the urinary tract. The symptoms and cystoscopic manifestations are not unique. We reported one patient of nephrogenic adenomas with malignant transformation and provided some evidence for malignant alteration in morphology and invasive behavior. All patients underwent local excision of the lesions. Intravesical perfusion was further performed for treating the patients of atypical nephrogenic adenomas or nephrogenic adenomas with malignant transformation. Whether it is nephrogenic adenoma or atypical nephrogenic adenoma, long-term follow-up after treatment is necessary.
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Objective Our hypothesis was if we rendered host' s cytotoxic tumor lymphocytes insensitive to TGF-β,these immune cells could be able to overcome the TGF-β mediated immunosuppression and reject the tumor.We aimed to develop a lentivirus mediated gene transfer program incorporating a herpes simplex virus thymidine kinase(HSV-tk)in our dominant negative TGF-β type Ⅱ receptor(TβRⅡDNglytk)expression vector.So we first need to construct the lentiviral pLenti6/V5-D-TOPO vector containing TβRⅡDNglytk and produce the recombinant lentivirus as the transfection vector.Methods PCR were used to amplify the genes TβRⅡDN and HSV-tk from the respective plasmids.Then the genes were linked by recombinant PCR technology to construct the fusion gene TβRⅡDNglytk and control vector TRANSglytk.According to the operation manual from the Invitrogen company,TOPO cloning technology was used to construct the plasmids of pLenti6/V5-D-TOPO(R)-TβRⅡDNglytk and pLenti6/V5-D-TOPO(R)-TRANSglytk.Both of the constructed plasmids were verified by sequencing.ViraPowerTM Lentiviral System and 293 FT cells provided by Invitrogen were used to produce the recombinant lentivirus vector,the tillers of the lentivirus were determined by 293 cells.Results The construction of the plasmids of pLenti6/V5-D-TOPO(R)-TβRⅡDNglytk and pLenti6/V5-D-TOPO(R)-TRANSglytk were completed succefully.The DNA sequencing results showed that both the plasmids were constructed correctly.Using them we successfully produced infectious lentivirus vectors with appropriate tilters.Conclusions Using TOPO cloning technology in the construction of the plasmids of pLenti6/V5-D-TOPO(R)-TβRⅡDNglytk and pLenti6/V5-D-TOPO(R)-TRANSglytk and recombinant PCR in the fusion genes is feasible.Recombinant PCR combine with TOPO cloning technology can be a simple,highly efficient and rapid way to construct lentiviral vector.The production of infectious lentivirus with appropriate tilters using 293FT is suitable and feasible.The construction of pLenti6/V5-D-TOPO(R)-TβRⅡDNglytk and production of the infectious lentivirus will lay a foundation for immunotherapy of prostate cancer.
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Objective: To construct the lentiviral plenti6/V5-D-TOPO vector containing TβR Ⅱ Dnglytk and control vector by developing a lentivirus mediated gene transfer program incorporating a herpes simplex virus thymidine kinase(HSVtk) in the dominant negative TGF-β type Ⅱ receptor (TβR Ⅱ Dnglytk) expression vector. Methods: The PCR methods were used to amplify the genes TβR Ⅱ DN and HSV-tk from the respective plasmids. Then the genes were Linked by recombinant PCR technology to construct the fusion gene TβR Ⅱ Dnglytk and control vector TRANSglytk. According to the operation manual (from the Invitrogen company), ToPe cloning technology were used to construct the plasmids of plenti6/VS-D-TOPO(R)-TβRⅡ Dnglytk and plenti6/VS-D-TOPO -TRANSglytk. Both of the constructed plasmids were verified by sequencing.Results: The constructions of the plasmids of plenti6/V5-D-TOPO(R)-TβR Ⅱ Dnglytk and plenti6/V5-D-TOPO(R)-TRANSglytk were completed smoothly. The DNA sequencing results showed that both the plasmids were constructed correcdy and can be used in the production of infectious lentivirus vectors. Conclusion: Using TOPO cloning technology in the construction of the plasmids of plenti6/VS-D-TOPO -TβR Ⅱ Dnglytk and plenti6/VS-D-TOPO(R)-TRANSglytk and recombinant PCR in the fusion genes are feasible. The recombinant PCR combined with ToPe cloning technology can be the simple, highly efficient and rapid way to construct lentiviral vector and construction of plenti6/V5-D-TOPO(R)-TβR Ⅱ Dnglytk, which will lay a foundation for tumor immunotherapy.
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Objective To construct a naive human Fab fragment phage display library,from which the anti-gp96 antibodies may be panned by the gp96 purified from the tissue of urothelial carcinoma in the urinary bladder and provide a basis to new therapy for the malignant tumors.Methods Peripheral blood lymphocytes were isolated from 800 ml of blood,which was obtained from four healthy blood donors.The heavy chain Fd and light chain cDNA synthesized from the total RNA of lympbocytes were amplified by PCR with variable regions 5' and 3' primers of heavy and light chain, and the amplification products were ligated into the phagemid vector pComb3, then the ligated sample was transformed into competent E.coli XL1-Blue by electroperation.The transformed cells were infected with VCSM13 helper phage to yield recombinant phage antibody Fabs.The phagemids abstracted from amplified E.coli were cut with endonucleases such as Sac Ⅰ,Xba Ⅰ,Xho Ⅰ and Spe Ⅰ, and both the phage antibody Fabs and phage-raids abstracted from amplified E.coil were amplified by PCR to monitor the insertion of the genes of light chain or heavy chain Fd fragment.The gp96 purified from the urothelial carcinoma tissue of the bladder by affinity chromatog-raphy on eoncanavalin-A sephnrose and DEAE-sephnrose ion exchange chromatography were utilized as antigens to process three rounds of panning to the original Fab antibody library.Results The quantity of total RNA and cDNA were qualified.By combination of light chain and heavy chain genes, an antibody library containing 6.6×106 clones was obtained, and both the cutting of enzymes and PCR showed that there were the genes of light chain or heavy chain Fd fragment in the phagemids.The gp96 protein was obtained from urothelial carcinoma tissue in the urinary bladder.After having been panned by gp96, the original antibody library gained enrichment by 68 times.Conclusion Utilizing the technology of phage surface display, specific antibody can be gained from the human naive Fab phage display library,which can be used for immunological therapy for tumors.
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Objective:To obtain antibodies against amylin from a 'naive' human Fab fragment antibody phage diasplay library and to analyze the specificity of antigen binding activity.Methods:Panning and screening Fab antibody from the antibody library,the positive clones with well reactivity to amylin were selected after five times selection of 'adsorption-elution-enrichment'.Then the plasmid DNA which was extracted from the clones,was digested with Spe Ⅰ and Nhe Ⅰ to delete gⅢ (about 660 bp).The digested 47 000 bp DNA which was purified after separation of bands from agarose gel was ligated with T4-DNA ligase.The constructed expressing phagemids were transformed to the BL21(DE3)pLysS,soluble Fab was expressed in it by the induction of IPTG and its characteristics and specificity were determined by ELISA and Western blot.Results:Soluble Fab antibodies were expressed in E.coli.According with molecular weight of IgG Fab,protein band of about 47 kD was shown by SDS-PAGE.Western blot using the goat anti human IgG-HRP showed their binding activities.ELISA showed their specificity with amylin antigens and they did not react with bovine serum albumin.Conclusion:The high level expression and identification of the soluble human anti- amylin Fab fragment antibodies has been obtained successfully,which lays a solid foundation for further researching about the biological and pathological activities of amylin.
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Objective To study the gene mutation of fibroblast growth factor receptor 3 (FGFR3) and p53 in bladder cancer tissue and to explore their relationship with tumor recurrence. Methods DHPLC and PCR direct sequence were used to detect the mutation of FGFR3 and p53 in BTCC (n=98) and normal bladder mucosa (n=10). Genomic DNA of 98 BTCC was extracted. The exon 5-8 of P53 and the exon 7, 10, 15 were amplification by PCR. The products of PCR was screened by DHPLC to detect the mutation of the production. The results of the FGFR3 and p53 mutation were analyzed by Kaplan-Meier method and no recurrence survival rate was tested by log rank test. All the analysis were aim to explore the clinical biological value of the mutation of FGFR3 and p53. Results Mutation of FGFR3 in BTCC (44. 9%) was higher than normal bladder mucosa(0, P<0.01). Mutation in T_a-T_1 was 75. 6%(33/45) ,T_2 -T_4 was 26. 6%C10/53). Mutation in G_1 was84. 6%(11/13),inG_2 was 61. 4% (27/44), in G_3 was 14. 6% (6/41), (P<0. 05). The mutation rate was lower with the higher of stage and grade. Mutation of p53 in BTCC (34. 6%) was higher than normal bladder mucosa (0%) (P<0. 01). Mutation in T_a - T_1 was 20. 0% (9/45), T_2 - T_4 was 47. 2%(25/53). Mutation in G_1 was G_1 7. 7%(1/13), in G_2 18. 2%(8/44),in G_3 58. 1%(25/41) , (P<0. 05). The mutation rate was higher in the higher stage and grade. Kaplan-Meier method results revealed that mutation of FGFR3 indicating a favorable prognosis while mutation of p53 indicating a poor prognosis. As to the analysis of genotype, the type of FGFR3mut/p53wt had a relative longer recurrent interval (P<0. 01). Conclusions Mutation of FGFR3 indicated a relative longer recurrent interval, which revealed a favorable prognosis of BTCC. Mutation of p53 indicated a relative shorter recurrent interval, which revealed a poor prognosis.
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Objective To study the biomarker panel of superficial bladder transitional cell carcinoma(SBTCC)and analyze the biological pathway in tumorigenesis by Shotgun proteomics strategy.Methods Normal urothelium cells and cancer cells were harvested by laser capture microdissection from clinical specimen and the proteomic expression profile was identified by two-dimensional liquid chromatography tandem mass spectrometry.The isoelectric point,molecular weight,grand average of hydropathicity,transmembrane helices were analyzed by using proteomics tools.Gene ontology was used to comment the identified proteins.The pathway analysis was performed by ArrayTrack software,and visualized by GenMAPP.Results There were 440 and 218 proteins expressed in cancer cells and normal cells respectively,among them 388 proteins were differerntially expressed.All the database about identified proteins was deposited in an accessible form to researchers at http://www.Proteome-SBTCC.org.cn and http://www.Proteome-NHTE.org.cn.There were 267(68.8%)differentially expressed proteins which had GO biological process comments.The biological pathwavs of these proteins included MAPK signaling pathway,focal adhesion,oxidative phosphorylation,ECMreceptor interaction,etc.Conclusion Shotgun strategy proteomies database of normal transitional epithelium and SBTCC is successfully constructed.And the basis for the understanding of cell biology and discovery of biomarker panel for SBTCC iS provided.
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BACKGROUND:Cancer stem cell (CSC) hypothesis suggests that tumorous clones are maintained by a rare fraction of cells with stem cell proprieties. Several kinds of CSCs of solid tumor have been isolated in recent years. However, there have been fewer studies on the objective existence of bladder cancer stem cells (BCSCs) and on the methods to effectively isolate and identify BCSCs. OBJECTIVE:To investigate possibilities of BCSC existence and of epithelial membrane antigen (EMA) used as a surface marker of BCSC. DESIGN:A control observation experiment. SETTING:Tianjin Institute of Urinary Surgery & Second Hospital of Tianjin Medical University. MATERIALS:This study was performed at the Room for Tumor Immunity of Tianjin Institute of Urinary Surgery (key laboratory for State "211 Project") from March 2006 to July 2007. Nine specimens of human bladder were obtained from patients who received treatment in the Second Hospital of Tianjin Medical University. These specimens corresponded to the diagnostic criteria of low malignant potential papillary urothelial neoplasm and low-grade papillary urothelial carcinoma. Additionally, 40 samples of human low malignant bladder transitional cell carcinomas (BTCC) and 10 samples of normal urothelium that were used for immunohistochemistry were obtained from the patients who received treatment in the Department of Urinary Surgery, Second Hospital of Tianjin Medical University. Written informed consent for the specimen providing was obtained from the patients, and the protocol was approved by the hospital’s Ethics Committee. METHODS:The genes that were differentially expressed between normal urothelium and BTCC were identified through a DNA array assay to preliminarily determine the existence of BTCC. Overpressed stem cell related genes, Bmi-1 and EZH2, were verified by immunohistochemistry. A total of 27 potential surface markers of BCSCs were assayed to determine the location of positive cells. EMA- subsets were obtained through an immunomagnetic bead cell sorting system to analyze their abilities for colony forming, self-renewal and extensive proliferation. MAIN OUTCOME MEASURES:Normal urothelium and BTCC gene expression difference, Bmi-1 and EZH2 protein expression difference between low malignant BTCC and normal urothelium; and experimental results of colony forming. RESULTS:A total of 268 genes (including Bmi-1 and EZH2) that were differentially expressed between normal urothelium and low malignant BTCC were identified through a DNA array assay. The Bmi-1 and EZH2 had been found overexpressed in the low malignant BTCC. Except for EMA, above-mentioned 26 out of 27 potential surface markers were null for isolating BCSCs. EMA- subsets were located in the basal layer and suprabasal epithelial layers of both normal and tumorous urothelium (potential location of BCSCs). EMA- subsets possesed the abilities for colony forming, self-renewal and extensive proliferation. CONCLUSION:The experiment confirms the existence of BCSCs. EMA- might be a surface marker of BCSCs.
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Objective To screen the gene expression profiles of human bladder cancer cell line EJ,in which RNA interference was used to downregulate the EZH2 expression and to investigate the EZH2 molecular mechanism in cancer.Methods The vector expressing EZH2 shRNA was constructed and transfected into EJ cell line with Lipofectamine 2000.We used the gene chip to screen differential expression genes in EJ cells with transfection with EZH2 shRNA compared with untransfected EJ cells.The data were up-loaded to Passway miner serve((http://www.biorag.org/passway.htm))Results We found 436 differentiation genes,in which 257 genes upregulated and 179 genes downregulated.Bioinformation analysis showed that 115 genes locate in the known biological ways,including EGF,P53,proliferation,cell cycle,Notch and Wnt passway.EZH2 target genes such as WNT1,MYT1,CNR1,WT1 were upregulated,some of which were linked to tumor stem cell and cell differentiation.Conclusion The linkage between EZH2 expression and cell proliferation,which is induced by genes encoding cell differentiation.The research focusing on EZH2 regulating genes will explore its molecular mechanism in oncog-enesis.
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Objective To construct a new secreting recombinant hIFN-?-2B-BCG to provide a new tool for the treatment of bladder tumor. Methods BCG was genetically engineered to secrete recombinant human interferon-alpha 2B by transforming of shuttle plasmid phIFN-?-2B. Expression of hIFN-? was readily detectable by ELISA. Results The phIFN-?-2B was transformed in BCG correctly,and the value of hIFN-?-2B in supernatant of recombinant BCG culture was calculated to approximately 997.2 pg/ml. Conclusions This study demonstrates that the recombinant phIFN-?-2B can be expressed in BCG secretively. As the construction of the plasmid and its transformation and expression in BCG were accomplished successfully, a foundation of reformed BCG and new vaccine will be established.
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Objective To investigate the influence of endogenous transforming growth factor ? 1 (TGF? 1) on the cell cycle regulation and proliferation of bladder cancer. Methods A constructed replication defective retroviral vector pRevT? AS,which carried antisense RNA of TGF? 1,was transfected to a bladder cancer cell line EJ.The proliferation and clone formation of transferred cells were observed in vitro,and the alteration of cell cycle was also detected by flow cytometric analysis. Results TGF? 1 antisense RNA was transferred into EJ cell and expressed efficiently.After the inhibition of target gene expression in EJ cells,the reduced growth and clone formation rates were demonstrated,and the proliferative indexes were decreased by 12%.The ratios of G 0 and G 1 stage cells to the antisense RNA transfected EJ cells were increased,simultaneously,the ratio of S stage cells to the antisense RNA transfected EJ cells ratios were decreased,compared with the control group. Conclusions The proliferative mechanism of endogenous TGF? 1 in bladder cancer cells is to stimulate the G 1 to S stage transition.
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Objective To explore the effects of specific T-cell immunity against prostate cancer(PC) cells induced by dendritic cells(DCs) derived from fetal organs.Methods Mononuclear cells(MNCs) were obtained from the fetal bone marrow and liver.Then MNCs were cultured in medium with induction of rhGM-CSF,rhIL-4 and rhTNF-?to get DCs.Lysates of DU145 containing HSP-peptide complex were prepared by 50%-70%(NH4)2SO4 saturation.T lymphocytes from fetal spleen were co-cultured with DC loading DU145 antigen for 72 h,whereby CTL was obtained.The cytotoxicity of CTL against DU145,PC3 and EJ was detected by MTT assay.Results Mature DCs were induced from fetal organs,which expressed CD1a,CD_(86),HLA-DR and CD_(83) at high levels.DC stimulated with tumor lysates transformed T cells to specific CD~+_8 CTL.Phenotype of CD~+_8 cell was(14.09?(2.46))% before transformation,and(62.76?2.64)% after transformation,respectively(P
