Biological effects of eukaryotic recombinant plasmid pReceiver-M61-BAI-1 transfection on T24 cells and HUVECs.

The aim of the current study was to investigate the biological effect on T24 cells and human umbilical vein endothelial cells (HUVECs) of transfection with brain-specific angiogenesis inhibitor-1 (BAI-1). The recombinant plasmid pReceiver-M61-BAI-1 was transfected into human superficial bladder tumor cells (T24) and HUVECs, in parallel with the vector control. mRNA and protein expression levels of BAI­1 were then detected by quantitative polymerase chain reaction (qPCR) and western blotting, respectively. Cell apoptosis of T24 cells and HUVECs prior and subsequent to transfection with BAI­1 was analyzed by flow cytometric analysis. Proliferation of T24 cells and HUVECs prior and subsequent to transfection of BAI-1 was assessed by the MTT method. T24 cells and HUVECs transfected with pReceiver­M61­BA1­1 were classed as the experimental group; T24 cells and HUVECs transfected with p­Receiver­M61 were the control group. qPCR and western blotting methods confirmed that there was positive expression of BAI­1 in T24 cells and HUVECs transfected with pReceiver­M61­BAI­1, however BAI­1 was not expressed in T24 cells and HUVECs transfected with pReceiver­M61. The results of the MTT assay demonstrated that absorbance was markedly reduced in HUVECs at 12, 48 and 72 h subsequent to transfection with pReceiver-M61-BAI-1 when compared with that of the control group and in T24 cells transfected with p­Receiver-M61-BAI-1. Furthermore, flow cytometry results also indicated that the apoptotic rate of HUVECs transfected with p­Receiver­M61­BAI­1 was significantly increased compared with that of the control group and T24 cells transfected with p­Receiver­M61­BAI­1. BAI­1 was observed to markedly inhibit the proliferation of vascular endothelial cells in vitro, however, no direct inhibition by BAI­1 was observed in T24 cells. In conclusion, BAI-1 is suggested to be a potential novel therapautic target for the inhibition of tumor neovascularization.

Predictive value of high residual platelet reactivity by flow cytometry for outcomes of ischemic stroke patients on clopidogrel therapy.

High residual platelet reactivity (HRPR) assessed by multiple tests has been associated with worse clinical outcomes. However, the clinical impact of HRPR assessed by flow cytometry is unknown. The aim of this study was to validate the predictive value of HRPR measured by flow cytometry for clinical outcomes in ischemic stroke patients during clopidogrel therapy. Overall, 198 consecutive patients with ischemic stroke taking clopidogrel underwent platelet function testing on flow cytometer including adenosine diphosphate (ADP)-induced platelet aggregation (PAg) and platelet activation markers (CD62P, CD63, and PAC-1). Poor outcome was defined as poor prognosis and ischemic events during 12-month follow-up. By receiver operating characteristic curve analysis, residual platelet reactivity assessed by flow cytometry was able to distinguish between patients with and without poor outcomes, when platelet inhibition was evaluated with ADP-PAg (area under the curve [AUC], .77; 95% confidence interval [CI], .69-.84; P < .001), CD62P (AUC, .73; 95% CI, .64-.81; P < .001), CD63 (AUC, .72; 95% CI, .64-.80; P < .001), and PAC-1 (AUC, .70; 95% CI, .62-.78; P < .001). The prevalence of HRPR was 25.8% for ADP-PAg, 32.8% for CD62P, 41.4% for CD63, and 56.1% for PAC-1. The multiple logical regression analysis demonstrated that HRPR was an independent predictor of poor outcomes (ADP-PAg: odds ratio [OR] 13.03, 95% CI 5.66-29.98, P < .001; CD62P: OR 8.55, 95% CI 3.94-18.57, P < .001; CD63: OR 8.74, 95% CI 3.89-19.64, P < .001; PAC-1: OR 4.23, 95% CI 1.98-9.08). In conclusion, HRPR, assessed by flow cytometry, is able to detect ischemic stroke patients at increased risk of 12-month poor outcomes on clopidogrel treatment.

Influence of CYP2C19 polymorphisms on platelet reactivity and clinical outcomes in ischemic stroke patients treated with clopidogrel.

CYP2C19 genetic polymorphisms influence clopidogrel response and clinical outcomes of cardiovascular disease. However, data on their relationship in stroke patients are scarce. We aimed to investigate the influence of CYP2C19 polymorphisms on platelet reactivity and clinical outcomes in ischemic stroke patients treated with clopidogrel. A total of 211 patients were enrolled. All patients were given clopidogrel treatment and underwent CYP2C19 genotyping and platelet function testing by flow cytometry including adenosine diphosphate-induced platelet aggregation (ADP-PAg) and platelet activation markers (PAC-1, CD62P and CD63). The modified Rankin Scale (mRS) was used and ischemic events were evaluated. A total of 129 (61.1%) of the 211 enrolled patients were carriers of CYP2C19 loss-of-function (LOF) alleles (*2, *3). After clopidogrel therapy for 7 days, the levels of ADP-PAg, PAC-1, CD62P and CD63 were higher in carriers than noncarriers. CYP2C19 carriage was associated with more frequent high residual platelet reactivity. CYP2C19 polymorphisms alone could explain 12.9%, 4.3%, 8.9% and 5.5% of the inter-individual variability of ADP-PAg, PAC-1, CD62P and CD63 after clopidogrel treatment, respectively. At 6-month follow-up, 38 (19%) patients were scored poor prognosis and 15 (7.6%) ischemic events were observed. Carriers had poorer prognosis than noncarriers (P=0.025). No significant association of CYP2C19 carriage with ischemic events was found. Multiple regression analysis showed that CYP2C19 carriage was an independent predictor of poor prognosis (odds ratio, 3.01; 95% confidence interval, 1.23-7.38; P=0.016). In conclusion, carriage of the CYP2C19 LOF allele has significant influence on clopidogrel response and prognosis in patients with ischemic stroke.

Suppression of bladder cancer growth by adeno-associated virus vector-mediated combination of HSV-TK and endostatin in vitro.

BACKGROUND: Gene therapy may offer a new tool for the treatment of bladder cancer. Previously, we have shown a significant antitumor effect in bladder cancer xenografts in a nude mouse model using intratumoral herpes simplex virus thymidine (HSV-TK) and endostatin gene monotherapy. OBJECTIVES: Given the high vascularity of human bladder cancer and the ability of HSV-TK or endostatin monotherapy to eradicate the tumors, we decided to test a novel combination of cytotoxic and antiangiogenic gene therapy using HSV-TK and endostatin adeno-associated viruses (AAV) in vitro. METHODS: We constructed the plasmid AAV-TK-IRES-Endostatin (pAAV-TIE) and packaged the AAV particles containing gene fragments of HSV-TK and endostatin. The combination anticancer effect of recombinant AAV-TIE (rAAV-TIE) was measured in vitro while rAAV-HSV-TK and rAAV-Endostatin were used as control groups. RESULTS: The inverted terminal repeat sequences were amplified using only one primer and the fragment between two ITRs of pAAV-TIE measuring about 4 kb, which indicated a stable sequence of pAAV-TIE. Three clear bands representing the AAV capsid proteins VP1, VP2, and VP3 could be seen on both lanes against a very low background, which demonstrated that chloroform extraction could effectively extract contaminants from rAAV stock without significant loss of the rAAV. In vitro, our results found that the transduction efficiency, measured from GFP-transduced tumors, was about 62%. The combination therapy led to an obvious apoptosis of bladder tumor cells compared with single HSV-TK or endostatin treatment. CONCLUSIONS: We concluded that the inhibition of angiogenesis using endostatin gene transfer, together with the cytotoxic HSV-TK gene therapy, resulted in a significant antitumor effect in vitro compared to the single gene based therapy in BTCC.

Potent antitumor activity of the combination of HSV-TK and endostatin by adeno-associated virus vector for bladder cancer in vivo.

BACKGROUND: Gene therapy may offer a new tool for the treatment of bladder cancer. Previously, we have shown a significant antitumor effect in bladder cancer xenografts in a nude mouse model using intratumoral herpes simplex virus thymidine (HSV-TK) and endostatin gene monotherapy. METHODS: Given the high vascularity of human bladder cancer and the ability of HSV-TK or endostatin monotherapy to eradicate the tumors, we decided to test a novel combination of cytotoxic and antiangiogenic gene therapy using intratumorally delivered HSV-TK and endostatin adeno-associated viruses (AAV). We constructed plasmid AAV-TK-IRES-Endostatin (pAAV-TIE) and packaged the AAV particles containing gene fragments of HSV-TK and endostatin. The combined anticancer effect of recombinant AAV-TIE (rAAV-TIE) was measured in vivo with rAAV-HSV-TK and rAAV-Endostatin as the control groups. RESULTS: The inverted terminal repeat sequence was amplified using only one primer and the fragment between two ITRs of pAAV-TIE measuring about 4 kb, which indicated a stable sequence of pAAV-TIE. Three clear bands representing the AAV capsid proteins VP1, VP2, and VP3 could be seen on both lanes against a very low background, which demonstrated that chloroform extraction could effectively extract contaminants from rAAV stock without significant loss of the rAAV. In vivo, our results showed that the tumors in mice injected with the rAAV-TIE not only took significantly longer to emerge but also that their growth, once established, was significant slower than that of tumors grown with single HSV-TK or endostatin treated animals. CONCLUSIONS: We concluded that the inhibition of angiogenesis using endostatin gene transfer, together with the cytotoxic HSV-TK gene therapy, resulted in a significant antitumor effect compared to the single gene based therapy in BTCC.

Diabetes mellitus increases the risk of bladder cancer: an updated meta-analysis.

PURPOSE: Studies have indicated that diabetes mellitus (DM) is a risk factor for bladder cancer; however, not all evidence supports this conclusion. The aim of this meta-analysis was to collate and evaluate all primary observational studies investigating the risk of bladder cancer associated with DM. METHODS: The PubMed and Google Scholar databases were searched to identify studies that estimated the association of DM and bladder cancer. Summary effect estimates were derived using a random-effects meta-analysis model. RESULTS: A total of 23 studies (8 case-control studies, 15 cohort studies) including 643,683 DM and 4,819,656 non-DM cases were identified. Analysis of all studies showed that DM was associated with an increased risk of bladder cancer compared with non-DM overall (OR=1.68, 95% CI 1.32-2.13). Analysis of subgroups demonstrated this to be the case in both case-control studies (OR=1.59, 95% CI 1.28-1.97, I2=58%) and cohort studies (RR=1.70, 95% CI 1.23-2.33, I2=96%). There was no gender difference in DM-associated bladder cancer risk. Bladder cancer risk was increased in Asia and the North America region, but not in Europe. Furthermore, DM-associated bladder cancer risk was obviously higher in Asia than North America and Europe or in those with Caucasian ethnicity. With extension of follow-up time, the bladder cancer risk was not increased for the patients with DM. CONCLUSIONS: This meta-analysis provided further evidence supporting the DM association with a significantly higher risk of bladder cancer obtained from observational studies.

[Toll-like receptor 4 expression of human peripheral blood mononuclear cells treated by recombinant hIFN-α-2b-BCG and its role of immune activation].

OBJECTIVE: To examine the toll-like receptor 4 (TLR4) expression of human peripheral blood mononuclear cells (hPBMC) treated with recombinant bacillus Calmette-Guérin (rBCG) and its role of immune activation. METHODS: hPBMC was treated with recombinant human interferon (hIFN)-α-2b-BCG (rBCG) or wild BCG (wBCG) in vitro and the TLR4 expression detected by flow cytometry. The TLR4 functional blocking antibodies were applied for intervening the TLR4 signaling pathway of hPBMC. Then rBCG, wBCG, hIFN-α-2b and phosphate-buffered solution (PBS) were used to stimulate the hPBMC of blocking and non-blocking groups. The changes of human tumor necrosis factor-alpha (hTNF-α), human interleukin-12 (hIL-12) and hIFN-γ between the blocking and non-blocking groups by ELISA. RESULTS: The expression of TLR4 in hPBMC treated with rBCG or wBCG were stronger than that treat with PBS (all P < 0.05). In TLR4 non-blocking groups the expressions of hTNF-α and hIFN-γ were rBCG group > wBCG group > hIFN-α-2b group > PBS group, the expressions of hIL-12 was hIFN-α-2b group > PBS group > rBCG group > wBCG group (all P < 0.05). Application of TLR4 functional blocking antibodies to intervene hPBMC 48 h, comparing the changes in rBCG group, wBCG group, hIFN-α-2b group and PBS group, the expressions of hIFN-γ in TLR4 blocking groups (27.3 ± 1.2, 20.6 ± 0.9, 20.3 ± 0.8, 18.4 ± 0.7)were significantly inhibited than those in non-blocking groups (84.6 ± 1.3, 34.0 ± 1.0, 24.9 ± 0.9, 22.9 ± 0.7) (all P < 0.05). The expressions of hTNF-α in TLR4 blocking groups (1431 ± 28, 1032 ± 21, 104 ± 6, 109 ± 4) were significantly inhibited than those in non-blocking groups (1553 ± 28, 1065 ± 31, 343 ± 6, 299 ± 4) (all P < 0.05). The expressions of hIL-12 in TLR4 blocking groups (0.646 ± 0.005, 0.592 ± 0.015, 0.638 ± 0.008, 0.595 ± 0.019) were significantly inhibited than those in non-blocking groups (1.120 ± 0.012, 0.946 ± 0.015, 1.254 ± 0.011, 1.112 ± 0.024) (all P < 0.05). CONCLUSION: rBCG regulates the secretion of Th1 cytokines through the TLR4 signaling pathway.

The adeno-associated virus-mediated HSV-TK/GCV suicide system: a potential strategy for the treatment of bladder carcinoma.

Novel treatment strategies such as gene therapy are warranted in view of the failure of current treatment approaches to cure a high percentage of patients with advanced bladder cancers. The emergence of cancer gene therapy potentially offers a number of exciting treatments. The majority of approaches involve strategies to suppress the function of activated oncogenes to restore the expression of functional tumour suppressor genes or to initiate tumour self-destruction. One gene therapy approach against tumours that holds great promise is suicide gene therapy. Herpes simplex virus thymidine kinase (HSV-TK) phosphorylates ganciclovir (GCV), which in turn interacts with cellular DNA polymerase and interferes with DNA synthesis to cause death of rapidly dividing cells. The development of an effective delivery system is absolutely critical to the usefulness and safety of gene therapy. At present, the adeno-associated virus (AAV) vector has the most promising potential in view of its non-pathogenicity, wide tropisms and long-term transgene expression in vivo. Gene therapy studies using different serotypes of recombinant AAV (rAAV) as delivery vehicles have proved rAAVs to be an effective modality of cancer gene therapy. In the present study, we investigated the suppression effect of AAV-mediated HSV-TK/GCV system on the bladder cancer cells and in mice xenograft models of bladder cancer. Our data demonstrate that rAAV-HSV-TK system controlled tumour cell growth and achieves strong antitumour efficacy in vivo. These findings provide a foundation for the development of potential targeted clinical therapies for bladder cancer in humans.

Potent antitumour activity of the combination of HSV-TK and endostatin armed oncolytic adeno-associated virus for bladder cancer in vitro and in vivo.

BACKGROUND: Gene therapy may offer a new tool for the treatment of bladder cancer. Previously, we have shown a significant antitumour effect in bladder cancer xenografts in a nude mouse model using intratumoural herpes simple virus thymidine (HSV-TK) and Endostatin gene therapy. OBJECTIVES: Given the high vascularity of human bladder cancer and the ability of HSV-TK or Endostatin monotherapy to eradicate the tumours, we decided to test a novel combination of cytotoxicity and antiangiogenisis gene therapy using intratumourally delivered HSV-TK and Endostatin adeno-associated viruses (AAVs). METHODS: We constructed plasmid AAV-TK-IRES-Endostatin (pAAV-TIE) and packaged the AAV particles contain gene fragments of HSV-TK and Endostatin. The function of HSV-TK and Endostatin was evaluated separately in vitro via T24 bladder tumour cells and human umbilical vein endothelial (HUVEC) cells. The combination anticancer effect of recombinant AAV-TIE (rAAV-TIE) was measured in vivo while rAAV-HSV-TK and rAAV-Endostatin as control groups. RESULTS: In vitro, rAAV-TIE was found to induce a significant increase in apoptosis in HUVEC cells equally as rAAV-Endostatin and confirmed that the inhibition of endothelial cells mediated by rAAV-TIE was associated with the apoptotic process. rAAV-TIE was found to induce a significant increase in apoptosis in T24 cells equally as rAAV-HSV-TK and confirmed that the inhibition of T24 cells mediated by rAAV-TIE was associated with the apoptotic process too. In vivo, our results showed that the tumours in mice injected with the rAAV-TIE not only took significantly longer to emerge but also that their growth, once established, was significant slower than that of tumours grown, compared with single HSV-TK or Endostatin treated animals. CONCLUSIONS: We concluded that the inhibition of angiogenesis using Endostatin gene transfer, together with the cytotoxicity HSV-TK gene therapy, resulted in a significant antitumour effect compared to the single gene based therapy in BTCC. The results warrant further development of the combination gene therapy, and suggest that this approach, directed towards systemic efficiency, could be used as an additional treatment for human BTCC.

[Stage I testis teratoma in adults: treatment options and recurrence factors].

OBJECTIVE: To study the clinical outcomes of stage I testis teratoma, including pure teratoma, and to provide information on the treatment options for this disease. METHODS: We retrospectively analyzed 27 cases of orchiectomy for stage I testis teratoma, excluding epidermoid cyst, and investigated its recurrence associated with treatment methods and clinicopathological factors. RESULTS: Four of the 27 cases relapsed, all in the orchiectomy group and confined to the retroperitoneal region, 3 with and the other 1 without risk factors, but with no death. No recurrence was found in those treated by orchiectomy followed by chemotherapy with bleomycin, etoposide and platinum (BEP). The total rate of recurrence was 15.8%. No severe side effects were observed in the 9 patients undergoing adjuvant BEP chemotherapy. CONCLUSION: Risk factors may increase the recurrence rate of stage I testis teratoma, while postoperative adjuvant chemotherapy can reduce it, including that of pure teratoma, though surveillance policy remains the most popular option after orchiectomy.

[TLR4 expression of human PBMC treated by BCG and its role of immune activation].

AIM: To investigate TLR4 expression of human peripheral blood mononuclear cells (hPBMC) treated with BCG and its role of immune activation. METHODS: hPBMC were treated with BCG in vitro. TLR4 expression were detected by flow cytometry, IFN-γ and TNF-α expression of hPBMC in both BCG stimulated group and the control group were detected by ELISA. RESULTS: The expression of TLR4 in hPBMC treated with BCG was stronger than the control group significantly (P<0.01) and increased with the time. In 72 h the TLR4 expression of BCG group was (44.73 ± 0.0066)%, while the control group was (1.02 ± 0.0024)%. BCG can promote hPBMC proliferation, and this enhancement was time-dependent. In 24 h, 48 h and 72 h IFN-γ and TNF-α expression of BCG group were significantly higher than the control group(P<0.05), and this enhancement was time-dependent. CONCLUSION: BCG can on enhance TLR4 expression and promote immune activation of hPBMC.

Suppression of bladder cancer growth in mice by adeno-associated virus vector-mediated endostatin expression.

Novel treatment strategies such as gene therapy are warranted in view of the failure of current treatment approaches to cure a high percentage of patients with advanced bladder cancers. Testing of the hypothesis that blocking the angiogenic switch may keep tumour growth in check has been facilitated by the discovery of endogenous inhibitors of angiogenesis and has also added another research dimension to the field of cancer gene therapy. Consequently, the concept of targeting the tumour vasculature with anti-angiogenic agents has emerged as an attractive new strategy in the treatment of cancer. Targeted biological therapies that selectively interfere with tumour angiogenesis could improve survival among patients with bladder cancer. Endostatin is a tumour-derived angiogenesis inhibitor and is the first endogenous inhibitor of angiogenesis to be indentified in a matrix protein. Gene therapy represents an attractive approach to treat cancers and other chronic diseases. The development of an effective delivery system is absolutely critical to the usefulness and safety of gene therapy. At present, the adeno-associated virus (AAV) vector has the most promising potential in view of its non-pathogenicity, wide tropisms and long-term transgene expression in vivo. Gene therapy studies using different serotypes of recombinant AAV (rAAV) as delivery vehicles have proved rAAVs to be an effective modality of cancer gene therapy. In the present study, an IgG fragment was inserted at the start of the sequence coding for endostatin with the aim of enabling continuous secretion of endostatin the serum. We also investigated the suppression effect of AAV-mediated endostatin expression on endothelial cells and in mice xenograft models of bladder cancer. Our data demonstrates that rAAV-endostatin controlled tumour cell growth and achieves strong anti-tumour efficacy in vivo.

[Effect of recombinant hIFN-alpha-2b-BCG on mouse bladder tumor MB49 cells in vitro].

OBJECTIVE: To investigate the antitumor effect of recombinant IFN-alpha-2b-BCG on mouse bladder cancer MB49 cells in vitro, and to explore its antitumor mechanisms. METHODS: MB49 cells were co-cultured with recombinant BCG or wild BCG, and than were examined by light and transmission electron microscopy. The cell growth was assessed by MTT assay, and apoptosis rate and MHC-I of the MB49 cells was detected by flow cytometry using AO and Hoechst33258 fluorescence immunostaining. RESULTS: The hIFN-alpha-2b-BCG-treated tumor cells showed slow growth, detachment of some cells, and various degree of degeneration. Light microscopy revealed organelle disorganization, chromatin aggregation, nuclear pyknosis, and cytolysis in some cells. Cellular membrane bulged and some bubbles were seen under fluorescence microscope using AO staining. Hoechst33258 assay also depicted frequent apoptosis in the tumor cells. The MTT assay showed that rBCG more actively than the wild BCG inhibited the proliferation of MB49 cells. The apoptosis rate of the recombinant BCG group was 19.7% and 46.6% at the time point of 24 h and 48 h, respectively, significantly higher than 10.8% and 20.9%, respectively, in the wild BCG group. The results of flow cytometry indicated that both types of BCG enhanced the expression of MHC-I in the MB49 cells, but more effective in the recombinant BCG group. CONCLUSION: The recombinant hIFN-alpha-2b-BCG has more strong immuno-modulatory properties, anti-tumor effect on MB49 cells and induces apparent cytotoxicity in the bladder cancer cells in vitro.

Synergistic effects of alpha-1,2-fucosyltransferase, DAF, and CD59 in suppression of xenogenic immunological responses.

BACKGROUND: Previous studies showed that alpha-1,2-fucosyltransferase (HT), decay accelerating factor (DAF), and CD59 have an inhibitory effect on the immunological rejection of xenogenic transplantation. METHODS: To investigate their possible synergistic effects in suppression of heterogeneic transplantation, we produced transgenic mouse lines expressing human HT, DAF, and/or CD59 by the standard pronuclear injection approach. PCR and Southern blot were used to identify the transgenic founder lines. Flow cytometry confirmed the high-level expression of HT, DAF, or CD59 in the transgenic mice. RESULTS: The deposition of IgM, C3c, or C9 in the cardiac vascular endothelial cells of the HT, HT/CD59, and/or DAF multiple positive transgenic mice was markedly decreased. The survival time and function of the hearts of the co-transgenic mice were significantly longer and higher than that of the single HT-positive transgenic mice (P < 0.05). CONCLUSION: The mice co-expressing HT/DAF or HT/CD59 could resist the hyperacute rejection better than those expressing HT alone. It is feasible to use HT and C-reactive proteins co-transgenic tissues to resist hyperacute rejection and xenograft rejection.

[Effects of double targeting gene therapy by suicide gene combined with endostatin gene in treatment of bladder cancer: an experimental study].

OBJECTIVE: To investigate the effect of double targeting gene therapy by using recombinant adeno-associated virus-thymidine kinase (TK)-internal ribosome entry site (IRES)-endostatin (ES) (rAAV-TIE). METHODS: Bladder cancer cells of the line T24 were cultured and transfected with rAAV-ES, rAAV-MCS (blank virus), and rAAV-TIE respectively. 72 hours later the levels of ES in the supernatants were measured by ELISA and annexin V apoptosis test kit was used to examine the apoptosis. Human umbilical vein endothelial cells (HUVECs) were transfected with rAAV-ES, rAAV-TIE, and rAAV-MCS respectively. MTT method and flow cytometry were used to detect the apoptosis of the HUVECs. Balb/c nude rats were inoculated subcutaneously with T24 cells. Twenty rats with tumor were randomly divided into 4 equal groups to be treated by rAAV-MCS, rAAV-TK, rAAV-ES, or rAAV-TIE, and 5 rats were used as control group. Four weeks later, blood samples were collected to detect the ES level by ELISA. The tumors were taken out to undergo microscopy to calculate the microvessel density(MVD). RESULTS: 72 h after transfection, ES could be detected in the supernatants of the T24 cells transfected with rAAV-ES, and rAAV-TIE. The apoptotic rates of the T24 cells transfected with rAAV-TK and rAAV-TIE were 34.12% and 36.91% respectively, significantly higher than those of the T24 cells transfected with rAAV-MCS and of the control group (3.08% and 0.84%, all P < 0.05). Transfection of rAAV-ES and rAAV-TIE increased the apoptotic rate of the HUVECs time-dependently. Nine days after the transfection rAAV-ES, rAAV-TK, rAAV-TIE, the tumor volumes of the rAAV-ES, rAAV-TK, and rAAV-TIE groups were (0.75 +/- 0.08), (0.71 +/- 0.11), and (0.52 +/- 0.09) cm(3) respectively, all significantly lower than those of the rAAV-MCS group and control group [(1.27 +/- 0.13) and (1.24 +/- 0.17) cm(3) respectively, all P < 0.05]. CONCLUSION: rAAV-TIE effectively inhibits the tumorigenesis and angiogenesis in bladder cancer. Double targeting gene therapy against bladder cancer can be achieved by using rAAV.

[Acupuncture for chronic prostatitis: a meta-analysis].

OBJECTIVE: To determine the effect of acupuncture on chronic prostatitis. METHODS: We retrieved all the case-control studies on acupuncture for chronic prostatitis before August 2007 in MEDLINE and CNKI databases, screened the eligible literature according to the selection and exclusion criteria, and performed meta-analyses of the included studies with the software Revman 4. 2. RESULTS: Thirteen eligible reports were identified in this study, including 861 cases and 738 controls. The effectiveness and cure rates were significantly higher in the acupuncture therapy group than in the control, with pooled RR as 1.20 (95% CI, 1.14, 1.25; P < 0.01) and 1.85 (95% CI, 1.63, 2.11; P < 0.01), respectively. CONCLUSION: Acupuncture therapy exhibited a definite effect in the treatment of chronic prostatitis.

[Study on deproteinization and decoloration in extraction of polysaccharides from Rabdosia rubescens].

OBJECTIVE: To screen the method on deproteinization and decoloration in extraction of of polysaccharides from Rabdosia rubescens. METHODS: The effect of deproteinization with different methods was evaluated in terms of the ratio of protein removing and polysaccharide removing, the decoloration effect was also calculated. RESULTS: The deproteinization effect was optimal with TCA method and activated carbon also achieved a high decoloration rate. CONCLUSION: The result can establish foundation for the further study of the polysaccharides from Rabdosia rubescens.

[Intravesical perfusion of recombinant adeno-associated virus-endostatin in treatment of bladder cancer: experiments with mice].

OBJECTIVE: To study the anti-tumor effect of intravesical perfusion of recombinant adeno-associated virus-endostatin (rAAV-ES) in treatment of bladder cancer. METHODS: Forty-five C57BL/6 mice underwent intravesical perfusion of mouse bladder cancer cells of the line MB49 so as to establish orthotopic murine bladder cancer models and were divided into 3 equal groups, 3 days later to undergo intravesical perfusion of rAAV-ES, rAAV-EYFP, and PBS respectively once per week for 6 times. The anti-tumor effect of rAAV-ES on the tumor bearing mice was studied. RESULTS: The tumor weight of the rAAV-ES group was (145 +/- 30) mg, significantly lighter than those of the rAAV-EYFP and PBS groups [(250 +/- 32) mg and (250 +/- 30) mg respectively, both P < 0.05]. The survival time of the rAAV-ES-treated mice was (46 +/- 7) d, significantly longer than those of the rAAV-EYFP- and PBS-treated groups [(38 +/- 7) d and (38 +/- 6) d respectively, both P < 0.05]. CONCLUSION: An effective biologic agent in bladder cancer gene therapy, intravesical treatment with rAAV-ES inhibits the angiogenesis, thus inhibiting the tumor formation and progression.

[Retroperitoneal laparoscopy combined with urethral resection for treatment of renal pelvic carcinoma].

OBJECTIVE: The purpose of this study is to explore the operation method and efficacy through retroperitoneal laparoscopy combined with urethral resection for treatment of renal pelvic carcinoma. METHODS: Total nephroureterectomy with excision of bladder cuff by retroperitoneal laparoscopy plus urethral resection was performed in 18 patients with pathologically confirmed pelvic transitional cell carcinoma (II-III, T1N0M0-T2N0M0). The operation was performed using Olympus celioscope (30 degrees or 0 degree) under general anesthesia. First, a 10 mm incision was made at the intersection of midaxillary line and superior border 2 cm from crista iliaca, then a self-made hyponome filled with 250-300 ml water was put through the small incision in order to open the retroperitoneal space, followed by getting the hyponome out and perfusing CO2 into the retroperitoneal space to make a pneumoretroperitoneum. Finally, the celioscope was put into the retroperitoneal space to operate. During the operation, electric coagulation was used to stop bleeding and the bladder was not irrigated. RESULTS: The operation was successfully performed in 18 patients without any complication. The operative time ranged from 150 to 190 min with a mean of 160 min. The hospital stay after operation was 7 to 10 days. There was no tumor recurrence or metastasis or implantation in all these patients after follow-up of 1-19 months. CONCLUSION: Compared with regular operation mode, retroperitoneal laparoscopy plus urethral resection for treatment of renal pelvic carcinoma is a minimally invasive treatment with less bleeding and quick recovery.

[Gene therapy of bladder cancer by using recombinant adeno-associated virus-endostatin: experiments in vitro and in vivo].

OBJECTIVE: To package recombinant adeno-associated virus-endostatin (rAAV-ES) and study its anti-tumor effect in vitro and in vivo. METHODS: rAAV-ES was packaged with co-transfection technique and transfected into the human bladder cancer cells of the line EJ. 24 h later ELISA was used to examine the concentration of ES in the supernatant. The inhibition of human umbilical veins endothelial cells (HUVECs) chemotactic movement were examined by Transwell system. Nude Balb/c mice were divided into 4 groups: (1) 5 mice were inoculated with the EJ cells transfected with rAAV-ES or rAAV-enhanced yellow fluorescence protein (rAAV-EYFP) for 3 days to the subcutaneous tissues of bilateral shoulders so as to observe the growth of tumor. (2) 24 mice were injected with rAAV-ES intramuscularly and then the serum ES was examined every 10 days since the 10 th day after the injection. (3) 36 mice were randomly subdivided into 3 equal subgroups to be injected with rAAV-ES, rAAV-EYFP, or RPMI medium, inoculated with EJ cells 2 weeks later, and then killed 50 days later to observe the size of tumor. (4) 4 healthy mice and 4 mice injected with rAAV-ES for 8 weeks were killed with their hearts and brains taken out to observe the side effects. RESULTS: rAAV-ES was packaged successfully. The ES concentration in the supernatant of culture fluid of the EJ cell transfected with rAAV-ES was 54.09 ng/ml. The inhibition rate of the HUVECs chemotactic movement was 37.45%. The xenograft formation rate was 2/5 for the EJ cells transfected with rAAV-ES. The serum ES levels of the mice injected with rAAV-ES remained high. The tumor size in the mice injected with rAAV-ES was significantly smaller than those of the other groups (both P < 0.01). No pathological changes was found in the hearts and brains in the mice injected with rAAV-ES. CONCLUSION: rAAV-ES inhibits tumor angiogenesis, and tumor formation and progression. Successful packaging of rAAV-ES has laid a foundation for gene therapy of bladder cancer.