Enhanced recovery after surgery for radical cystectomy with ileal urinary diversion: a multi-institutional, randomized, controlled trial from the Chinese bladder cancer consortium.

PURPOSE: Enhanced recovery after surgery (ERAS) has played an important role in recovery management for radical cystectomy with ileal urinary diversion (RC-IUD). This study is to evaluate ERAS compared with the conventional recovery after surgery (CRAS) for RC-IUD. METHODS: From October 2014 and July 2016, bladder cancer patients scheduled for curative treatment from 25 centers of Chinese Bladder Cancer Consortium were randomly assigned to either ERAS or CRAS group. Primary endpoint was the 30-day complication rate. Secondary endpoints included recovery of fluid and regular diet, flatus, bowel movement, ambulation, and length of stay (LOS) postoperatively. Follow-up period was 30-day postoperatively. RESULTS: There were 144 ERAS and 145 CRAS patients. Postoperative complications occurred in 25.7 and 30.3% of the ERAS and CRAS patients with 55 complications in each group, respectively (p = 0.40). There was no significant difference between groups in major complications (p = 0.82), or type of complications (p = 0.99). The ERAS group had faster recovery of bowel movements (median 88 versus 100 h, p = 0.01), fluid diet tolerance (68 versus 96 h, p < 0.001), regular diet tolerance (125 versus 168 h, p = 0.004), and ambulation (64 versus 72 h, p = 0.047) than the CRAS group, but similar time to flatus and LOS. CONCLUSIONS: ERAS did not increase 30-day complications compared with CRAS after RC. ERAS may be better than CRAS in terms of bowel movement, tolerance of fluid and regular diet, and ambulation.

Triptorelin relieves lower urinary tract symptoms in Chinese advanced prostate cancer patients: a multicenter, non-interventional, prospective study.

BACKGROUND: Although triptorelin is increasingly used in China for biochemical castration, its effects on primary prostate cancer symptoms remain unclear. This study aimed to assess the prevalence of lower urinary tract symptoms (LUTS) in Chinese prostate cancer patients and the effectiveness of triptorelin on LUTS. METHODS: In this 48-week multicenter, non-interventional, prospective study, we enrolled patients with locally advanced or metastatic prostate cancer. Patients received triptorelin (15 mg) intramuscularly at baseline and at weeks 12, 24, and 36 with symptom assessment using the International Prostate Symptoms Score (IPSS). The primary endpoints were the prevalence of LUTS at baseline per IPSS categories and the percentage of patients with moderate to severe LUTS (IPSS > 7) at baseline, having at least a 3-point reduction of IPSS score at week 48. RESULTS: A total of 398 patients were included; 211 (53.0%) and 160 (40.2%) among them had severe and moderate LUTS, respectively. Of the patients with IPSS scores available at baseline and at week 48 (n = 213), 81.2% achieved a reduction in IPSS of at least 3 points. Of the patients with moderate to severe LUTS at baseline and IPSS scores available at baseline and at week 48 (n = 194), 86.6% achieved a total IPSS reduction of at least 3 points. CONCLUSIONS: The vast majority of Chinese patients with locally advanced or metastatic prostate cancer scheduled to receive triptorelin as part of their standard treatment have severe or moderate LUTS. Triptorelin therapy resulted in sustained improvement of LUTS in these patients.

[Gujing Maisiha Tablets combined with natural vitamin E for the treatment of idiopathic asthenospermia].

OBJECTIVE: To investigate the therapeutic efficacy and safety of Gujing Maisiha Tablets combined with natural vitamin E in the treatment of idiopathic asthenospermia. METHODS: This study included 135 outpatients with idiopathic asthenospermia received in our hospital from February 2015 to January 2016. We randomly divided them into a treatment group (n = 65, aged 22－44 ï¼»mean 32.8ï¼½ yr) and a control group (n = 55, aged 21－43 ï¼»mean 33.7ï¼½ yr) to be treated with Gujing Maisiha Tablets combined with natural vitamin E or natural vitamin E only, both for 90 days. We obtained total sperm motility and the percentage of progressively motile sperm (PMS) from the patients before and after medication and evaluated the clinical effects by comparing the collected parameters and pregnancy rates between the two groups. RESULTS: The baseline total sperm motility and PMS were (25.23 ± 5.57)% and (17.53 ± 5.78)% in the treatment group, with no statistically significant differences from (26.05 ± 6.77)% and (15.11 ± 6.55)% in the control (P >0.05). After 90 days of medication, both the treatment and the control groups showed remarkable increases in total sperm motility (ï¼»48.73 ± 8.66ï¼½% and ï¼»36.54 ± 8.09ï¼½%, P <0.05) and PMS (ï¼»32.77 ± 6.04ï¼½% and ï¼»26.99 ± 6.87ï¼½%, P <0.05). However, both total sperm motility and PMS were significantly higher in the treatment than in the control group after medication (P <0.05), and so was the total rate of clinical effectiveness (73.85% vs 54.55%, P <0.05). No adverse reactions were observed in either of the two groups during the treatment. CONCLUSIONS: Gujing Maisiha Tablets combined with natural vitamin E is safe and effective for the treatment of idiopathic asthenospermia.

Reduction of fertility in male mice immunised with pSG.SS.C3d3.YL.Bin1b recombinant vaccine.

OBJECTIVES: To study the anti-fertility effect of a DNA vaccine using Bin1b as the target antigen in male mice. METHODS: A novel recombinant eukaryotic vector containing a fusion gene sequence of mouse Bin1b in tandem with three copies of C3d fragment (C3d3) was used to construct pSG.SS.C3d3.YL.Bin1b. The correct expression of the Bin1b-C3d3 protein was confirmed in transfected HEK293 cells by indirect immunofluorescence and western blot analysis. The fertility of immunised mice was determined by a mating experiment and sperm motility test. Anti-Bin1b antibody titres in sera were examined by ELISA assays. Binding activity of C3d3 fragment of the fusion protein was verified in C3d receptor-expressing Raji cells and flow cytometric analysis. RESULTS: Immunisation of pSG.SS.C3d3.YL.Bin1b recombinant DNA vaccine significantly decreased sperm motility and compromised fertility in male mice. ELISA results showed that the titres of anti-Bin1b IgG in sera of immunised mice increased markedly with the immunisation process. Further, the anti-fertility effect of pSG.SS.C3d3.YL.Bin1b was significantly better than that of pSG.SS.YL.Bin1b DNA vaccine and generated higher titres of anti-Bin1b antibody. CONCLUSIONS: Our results show that recombinant DNA vaccine targeting Bin1b can markedly reduce fertility in male mice, providing an alternative approach for birth control.

Detection and mechanism of action of ESM-1 in rat kidney transplantation under various immune states.

OBJECTIVE: To investigate whether ESM-1 expression change reflects the impairment of endothelial cells and rejection after kidney transplantation, ESM-1 expression was detected under various immune states in this study. METHODS: Kidney transplantations were performed from BN to LEW rats. Syngenic LEW-LEW grafts were used as controls. The LEW recipient rats were divided into acute rejection (AR) group, ciclosporin A (CsA) group and control group. In each group, 10 rats were sacrificed at 1, 5, and 7d after operation, respectively, and blood and kidney samples were collected. In the rat model of kidney transplantation, ESM-1 mRNA and ESM-1 protein expression were detected in various immune states to verify if ESM-1 can reflect endothelial cell impairment sensitively. RESULTS: ESM-1 mRNA (1d vs. 3d, P<0.01;3d vs. 7d, P=0.018) and ESM-1 protein expression was upregulated significantly in the AR group (P<0.01, 5 and 7d), when compared to CsA group and control group. In CsA group, the cell apoptosis rate decreased when compared to AR group (P<0.01). Pathological impairment was more serious in AR group than in CsA group (P<0.01). CONCLUSIONS: Peripheral blood ESM-1 mRNA and ESM-1 protein expression in kidney grafts can reflect the severity of endothelial cell impairment. Thus, ESM-1 may be used as a new indicator for AR prediction and diagnosis. Nevertheless, further investigation is required to test if it meets the criteria for clinical utility.

Surgical management of intractable chyluria: a comparison of retroperitoneoscopy with open surgery.

BACKGROUND: Retroperitoneoscopic renal pedicle lymphatic disconnection has been described in the management of intractable chyluria. METHODS: We retrospectively reviewed the clinical outcomes of 76 patients with intractable chyluria undergoing renal pedicle lymphatic disconnection via a retroperitoneoscopic (n = 59) or conventional open approach (n = 17). Operative time, intraoperative blood loss, volume of postoperative drainage, postoperative draining time, postoperative intestinal recovery, intraoperative and postoperative complications, and postoperative hospital stay were evaluated. RESULTS: Compared with open surgery, retroperitoneoscopy was superior in terms of operative time, intraoperative blood loss, postoperative drainage, postoperative draining time, postoperative intestinal recovery time, and postoperative hospital stay. Recurrence developed in 2 patients due to the contralateral chylous efflux confirmed by cystoscopy during the period of 9-85 months of follow-up. CONCLUSIONS: With minimal invasion, sparing operative time, less blood loss, and rapid recovery, retroperitoneoscopic renal pedicle lymphatic disconnection can achieve at least the same clinical efficacy as open surgery.

Resveratrol induces apoptosis associated with mitochondrial dysfunction in bladder carcinoma cells.

OBJECTIVE: Resveratrol shows chemopreventive activity in a variety of human cancers by targeting mitochondria and triggering apoptosis. The purpose of this study was to investigate the antitumor action of resveratrol in bladder cancer and its underlying mechanism. METHODS: Using two different bladder cell lines, BTT739 and T24, the cytotoxicity of resveratrol were determined by MTT assay. The apoptosis induced by resveratrol was assayed by transferase dUTP nick end labeling staining. To show whether the mitochondrial dysfunction involved in the effects of resveratrol, mitochondrial function was detected by mitochondrial membrane potential, reactive oxygen species production and adenosine 5'-triphosphate content. In addition, the markers of apoptosis in the intrinsic mitochondrial-dependent pathway were analyzed by the release of cytochrome c and the activities of caspase-9 and caspase-3. RESULTS: Resveratrol effectively decreased cell viability and induced apoptosis in a concentration- and time-dependent manner. In addition, resveratrol significantly disrupted the mitochondrial membrane potential in both intact cells and isolated mitochondria. Resveratrol also increased reactive oxygen species production and reduced adenosine 5'-triphosphate concentrations. Western blot analysis showed that resveratrol provoked the release of cytochrome c from mitochondria to the cytosol. Furthermore, resveratrol significantly promoted the activation of caspase-9 and caspase-3. CONCLUSIONS: These findings suggest that resveratrol efficiently triggers apoptosis in bladder cancer cells through the intrinsic mitochondrial-dependent pathway, which is associated with mitochondrial dysfunction. Resveratrol might have great pharmacological promise in the treatment of bladder cancer.

Construction of a DNA vaccine encoding Flk-1 extracellular domain and C3d fusion gene and investigation of its suppressing effect on tumor growth.

Although the critical role of complement component C3d as a molecular adjuvant in preventing virus infection is well established, its role in cancer prophylaxis and treatment is unclear. In this study, we constructed a recombinant plasmid encoding Flk-1 and C3d3 fusion proteins and investigated its transient expression in vitro in transfected eukaryotic cells and its antibody response in immunized mice. Subsequently, we investigated the vaccine's ability to elicit an immune response leading to suppression of angiogenesis and tumor growth in mice bearing bladder transitional cell carcinoma. Using Western blotting, immunocytochemistry, and flow cytometry, we detected the expression of Flk-1 and C3d3 fusion proteins in COS-7 cells transfected with these recombinant plasmids. Further binding experiment using CR2 (C3d receptor) positive Raji cells that were incubated with transfected COS-7 supernatant indicated that C3d was successfully fused to Flk-1. Although both vaccines elicited peak antibody levels at 5 weeks, Flk-1-specific antibody titer in pSG.SS.Flk-1(ECD).C3d3.YL-immunized mice was significantly higher when compared to pSG.SS.Flk-1(ECD).YL-immunized mice. The results of experiments with bladder tumor-bearing mice showed that the vaccine inhibited tumor growth significantly. These results suggest that C3d plays a critical role in tumor immunotherapy by promoting antibody response in Flk-1-based DNA vaccines. This approach may provide a new strategy for the rational design of anti-angiogenic therapies for the treatment of solid tumors and provide a basis for the further exploitation and application of the anti-angiogenesis DNA vaccines.

Comparison of urological complications with primary ureteroureterostomy versus conventional ureteroneocystostomy.

Urological complications after kidney transplantation may be serious and carry a high risk of graft loss. The purpose of this study was to compare urological complications with primary ureteroureterostomy versus conventional ureteroneocystostomy retrospectively. Between December 1993 and April 2008, 1287 kidney transplants were performed at our institution. Urological complications such as urine leakage, ureteral obstruction and vesicoureteral reflux (VUR) were summarized according to the different ways of urinary tract reconstruction, ureteroureterostomy (U-U) and ureteroneocystostomy (U-C). Overall, urological complications were encountered in 94 (7.3%) cases, including urine leakage (n = 44, 3.4%), ureteral obstruction (n = 36, 2.8%), VUR (n = 14, 1.1%). Seventy-six cases (7.8%) were in the U-C group and 18 cases (5.8%) were in the U-U group. Ninety-three recipients were successfully treated, and one lost the graft because of pelvis and ureteral necrosis. There was no recipient loss secondary to these complications. In conclusion, U-U does not change the overall incidence of urological complications comparing to U-C, but it can decrease the incidence of urine leakage. It is a good first option with a greater possibility of resolving a ureteral stenosis with endourology and no risk of reflux.

[One-hole high ligation of internal spermatic vein by embryonic natural orifice transumbilical laparoscopy: a recommendable treatment of varicocele].

OBJECTIVE: To evaluate the one-hole method for high ligation of the internal spermatic vein by embryonic natural orifice transumbilical laparoscopy. METHODS: We used the one-hole method for high ligation of the internal spermatic vein by embryonic natural orifice transumbilical laparoscopy in the treatment of 15 cases of varicocele, 13 in the left and 2 in the right side, and appraised the treatment results by follow-up 1 month after the surgery. RESULTS: All the operations succeeded and no complications developed. The average operation time was 28 minutes and the mean hospital stay was 4 days. Symptoms were significantly relieved in all the patients and the scars were inconspicuous at follow-up. CONCLUSION: The one-hole method is a novel option for high ligation of the internal spermatic vein by embryonic natural orifice transumbilical laparoscopy in the treatment of varicocele. It is recommendable for its advantages of simple procedure, less pain, few complications, quick recovery, mini-invasiveness and cosmetic acceptability.

[Urological complications in 1223 kidney transplants].

OBJECTIVE: To review retrospectively the urological complications in 1 223 kidney transplants. METHODS: A total of 1 223 kidney transplants were divided into ureteroneocystostomy group (n = 948) and ureteroureterostomy group (n = 275) according to the methods of urinary tract reconstruction. The incidence and management of urological complications such as urinary fistula, obstruction of ureter, vesicoureteral reflux (VUR) and urinary tract infection (UTI) were summarized respectively. RESULTS: Overall, urological complications were encountered in 217 (17.7%) cases, including 43 cases of urinary fistula (3.5%), 35 obstruction of ureter (2.9%), 14 VUR (1.1%) and 125 UTI (10.2%). Urinary fistula was 39 (4.1%) cases and 4 cases (1.5%) (P < 0.05), obstruction of ureter 22 (2.3%) & 13 (4.7%) (P < 0.05), VUR 14 (1.5%) & 0 (0%) (P < 0.05) and UTI 109 (11.5%) & 16 (5.8%) (P < 0.01) in the ureteroneocystostomy group and ureteroureterostomy group respectively. Seventy patients underwent surgical treatment. The 3-year survival rate of graft with urological complications and without urological complications were 82.3% and 84.7% respectively. CONCLUSIONS: Ureteroureterostomy can decrease the incidence of urological complications after kidney transplantation. Most of urological complications require surgical interventions. The long-term graft survival is not affected by a correctly treated urological complication.

Application of fluorescence in situ hybridization in the detection of bladder transitional-cell carcinoma: A multi-center clinical study based on Chinese population.

OBJECTIVE: To evaluate the diagnostic value of fluorescence in situ hybridization (FISH) in bladder cancer. METHODS: We enrolled healthy volunteers and patients who were clinically suspected to have bladder cancer and conducted FISH tests and cytology examinations from August 2007 to December 2008. Receiver operating characteristic (ROC) curve analysis was performed and the area under curve (AUC) values were calculated for both the FISH and urine cytology tests. RESULTS: A cohort of 988 healthy volunteers was enrolled to establish a reference range for the normal population. A total of 4807 patients with hematuria were prospectively, randomly enrolled for the simultaneous analysis of urine cytology, FISH testing, and a final diagnosis as determined by the pathologic findings of a biopsy or a surgically-excised specimen. Overall, the sensitivity of FISH in detecting transitional-cell carcinoma was 82.7%, while that of cytology was 33.4% (p < 0.001). The sensitivity values of FISH for non-muscle invasive and muscle invasive bladder transitional-cell carcinoma were 81.7% and 89.6%, respectively (p = 0.004). The sensitivity values of FISH for low and high grade bladder cancer were 82.6% and 90.1%, respectively (p = 0.002). CONCLUSION: FISH is significantly more sensitive than voided urine cytology for detecting bladder cancer in patients evaluated for gross hematuria at all cancer grades and stages. Higher sensitivity using FISH was obtained in high grade and muscle invasive tumors.

Relationship between glucocorticoid receptor signal pathway and androgen-independent prostate cancer.

OBJECTIVE: To study the role of the glucocorticoid receptor (GR) signal pathway and downstream cytokine interleukin-6 (IL-6) in androgen-independent growth of prostate cancer (PC). METHODS: The human androgen-dependent PC (ADPC) cell line LNCaP and androgen-independent PC (AIPC) cell line DU145 were cultured in vitro. Immunocytochemistry was used to examine the expression of the androgen receptor (AR), GR, HSP90 and IL-6. The GR antagonist RU486 was used to treat cultured cells, and the effects of RU486 on the proliferation of both cell lines were analyzed by MTT assay. Expression of HSP90 and IL-6 mRNA and protein was assessed by RT-PCR and Western blots, respectively. RESULTS: LNCaP cells were AR-positive and GR-negative, whereas DU145 cells were GR-positive and AR-negative. The expression of HSP90 and IL-6 in DU145 cells were significantly stronger than that in LNCaP cells (p < 0.01). RU486 had no obvious effects on the growth of LNCaP cells, but exerted a significant time- and dose-dependent growth inhibition on DU145 cells at doses as low as 0.1 micromol/l. RU486 treatment of DU145 cells also resulted in a dose-dependent decrease in the expression of HSP90 and IL-6 mRNA and protein. CONCLUSIONS: The GR signal pathway may be the main survival pathway for DU145 cells. Abnormal hyperactivation of the GR signal pathway and its promoting the expression of HSP90 and IL-6 contribute to the progression of ADPC to AIPC after androgen ablation.

[Testis homotransplantation: a report of 12 cases].

OBJECTIVE: To evaluate the effect of testis homotransplantation in the treatment of androgen deficiency and infertility. METHODS: We retrospectively analyzed 12 cases of testis homotransplantation. RESULTS: Surgical success was achieved in 11 cases, all with a significantly increased level of serum testosterone, and markedly improved secondary sex characteristics and sexual function. CONCLUSION: Testis homotransplantation is highly effective for the treatment of androgen deficiency in males, but has little effect on spermatogenesis.

IL­23 concentration­dependently regulates T24 cell proliferation, migration and invasion and is associated with prognosis in patients with bladder cancer.

Interleukin­23 (IL­23, also known as IL23A), is an important proinflammatory cytokine whose role in the development and progression of tumors remains controversial. The present study on IL­23 focused on its impact on the tumor microenvironment. Existing studies on its direct role on tumor cells are limited. Previously, we reported that the expression level of IL­23 in human bladder urothelial carcinoma was significantly higher than that in adjacent tissues as determined by immunohistochemistry. In this study, we further validated the results of immunohistochemistry using the Oncomine database and we found that IL23A expression in non­muscle invasive bladder urothelial carcinoma (NMIBC) was significantly higher than that in muscle invasive bladder urothelial carcinoma (MIBC). Expression of IL23A was negatively correlated with the clinical stage of bladder urothelial carcinoma and had a positive correlation with prognosis. In vitro experiments revealed that different concentrations of IL­23 had different effects on T24 cells. A low concentration of 20 ng/ml IL­23 promoted T24 cell proliferation, migration, invasion and EMT transformation, while a high concentration of 40 ng/ml IL­23 inhibited these functions. These results indicated that IL­23 plays a dual role in the progression of bladder cancer. Low concentrations of IL­23 promote bladder tumor progression, while high concentrations of IL­23 have the opposite effect.

Activation of nuclear factor-kappaB contributes to induction of death receptors and apoptosis by the synthetic retinoid CD437 in DU145 human prostate cancer cells.

Activation of the transcription factor, nuclear factor-kappaB (NF-kappaB), results in up-regulation of not only antiapoptotic genes but also proapoptotic genes, including death receptor 4 (DR4) and death receptor 5 (DR5). Therefore, NF-kappaB activation either suppresses or promotes apoptosis depending on the type of stimulus or cell context. We showed previously that the synthetic retinoid, 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437), effectively induces apoptosis particularly in androgen-independent prostate carcinoma cells. This effect was associated with the ability of CD437 to induce the expression of DR4 and DR5. In the present study, we examined the hypothesis that NF-kappaB activation plays a role in CD437-induced death receptor expression and apoptosis. Treatment of DU145 cells with CD437 resulted in a rapid decrease (> or = 3 hours) of IkappaBalpha, which was accompanied by increased translocation of the NF-kappaB subunit p65 from the cytoplasm to the nucleus and increased NF-kappaB DNA-binding activity (> or = 4 hours). The NF-kappaB inhibitor, helenalin, inhibited CD437-induced IkappaBalpha reduction and p65 nuclear translocation. Accordingly, it also abrogated CD437-induced up-regulation of DR4, activation of caspase-8 and caspase-3, and increased DNA fragmentation. Overexpression of an IkappaBalpha dominant-negative mutant blocked not only CD437-induced p65 nuclear translocation but also DR4 up-regulation, caspase activation, and DNA fragmentation. CD437 was unable to decrease IkappaBalpha protein levels and up-regulate DR4 expression in CD437-resistant DU145 cells. Moreover, knockdown of Fas-associated death domain, caspase-8, and DR4, respectively, suppressed CD437-induced apoptosis. Collectively, these results indicate that CD437 activates NF-kappaB via decreasing IkappaBalpha protein and thereby induces DR4 expression and subsequent apoptosis in DU145 cells.

[Construction and expression of eukaryotic recombinant of mouse fertilin beta subunit in HEK293 cells].

OBJECTIVE: To construct the eukaryotic expression recombinant pSG. SS. C3d3. YL-Fbeta and analyze the expression of mouse fertilin beta subunit in HEK293 cells. METHODS: The cDNA fragment expressing the disintegrin domain of mouse fertilin beta was obtained by PCR, and then inserted into the eukaryotic plasmid pSG. SS. C3d3. YL to get recombinant plasmid pSG. SS. C3d3. YL-Fbeta, which was transfected into the HEK293 cell line to express the target protein Fbeta after identified by restriction enzyme digestion. And then Fbeta was detected by indirect immunofluorescence through confocal laser scanning microscopy, Western blot, immunohistochemistry staining and flow cytometry assay. RESULTS: The recombinant vector pSG.SS.C3d3. YL-Fbeta could express Fbeta in HEK293 cells. CONCLUSION: The expression of Fbeta in eukaryotic cells provides a foundation for further researches on the effect of high F, expression on fertilization process.

Cotargeting tumor and tumor endothelium effectively inhibits the growth of human prostate cancer in adenovirus-mediated antiangiogenesis and oncolysis combination therapy.

Tumor-endothelial interaction contributes to local prostate tumor growth and distant metastasis. In this communication, we designed a novel approach to target both cancer cells and their "crosstalk" with surrounding microvascular endothelium in an experimental hormone refractory human prostate cancer model. We evaluated the in vitro and in vivo synergistic and/or additive effects of a combination of conditional oncolytic adenovirus plus an adenoviral-mediated antiangiogenic therapy. In the in vitro study, we demonstrated that human umbilical vein endothelial cells (HUVEC) and human C4-2 androgen-independent (AI) prostate cancer cells, when infected with an antiangiogenic adenoviral (Ad)-Flk1-Fc vector secreting a soluble form of Flk1, showed dramatically inhibited proliferation, migration and tubular formation of HUVEC endothelial cells. C4-2 cells showed maximal growth inhibition when coinfected with Ad-Flk1-Fc and Ad-hOC-E1, a conditional replication-competent Ad vector with viral replication driven by a human osteocalcin (hOC) promoter targeting both prostate cancer epithelial and stromal cells. Using a three-dimensional (3D) coculture model, we found that targeting C4-2 cells with Ad-hOC-E1 markedly decreased tubular formation in HUVEC, as visualized by confocal microscopy. In a subcutaneous C4-2 tumor xenograft model, tumor volume was decreased by 40-60% in animals treated with Ad-Flk1-Fc or Ad-hOC-E1 plus vitamin D3 alone and by 90% in a combined treatment group, compared to untreated animals in an 8-week treatment period. Moreover, three of 10 (30%) pre-established tumors completely regressed when animals received combination therapy. Cotargeting tumor and tumor endothelium could be a promising gene therapy strategy for the treatment of both localized and metastatic human prostate cancer.

[Long-term survival of human spermatogonial stem cells in vitro and its functional identification].

OBJECTIVE: The culture of human spermatogonial stem cells (SSC) has not been studied in detail yet. Here we tried to explore the optimized culture method of human SSC by using several different co-culture systems. METHODS: The alpha6 +Thy-1 +c-kit- cells acquired by the immunomagnetic beads sorting technique were cultured in different co-culture systems. Their morphological, biological characteristics and survival rates were intensively observed by microscopic or immunocytochemical assay. The long-term survival rate of human SSC during culture period was evaluated by germ cell transplantation technique. RESULTS: The alpha6 +Thy-1 +c-kit- cells could stably survive in the DMEM and DMEM/F12 mediums with fetal bovine serum (FBS) on feeder layer. The survival rates within 1 week were more than 90%. The long-time culture showed the cells were gradually attached on the surface of Sertoli cells by the manner of scattered single cell or accumulated masses. Part of the SSC became more tightly attachment with Sertoli cells or mounted among the Sertoli cells. They could survive or even proliferate for more than 3 months in vitro. Germ cells transplantation study showed that some alpha6 +Thy-1 +c-kit- cells labeled by PKH26 could resided on the basal membrane of seminiferous tubule of nude mice, appearing as single or coupled cells 2 months later after transplantation. The function evaluation of the cultured cells by counting the fluorescent cells in the seminiferous tubule showed 54.9% and 9.2% of SSC in the alpha6 +Thy-1 +c-kit- cells were still remained after cultured for 2 and 4 weeks, respectively. CONCLUSION: Human SSC could maintain survival in vitro for more than 3 months, but it was still need to seek for a more optimized and successful culture system for its efficient expansion and proliferation. Thus it will open up a wide prospect for the understanding of the biology of human SSC and the treatment of male sterility.

[Preliminary screening of specific surface markers of human spermatogonial stem cells].

OBJECTIVE: To explore the specific surface markers for the isolation and purification of human spermatogonial stem cells (SSC). METHODS: Specific markers of human SSC were screened and identified in fetal and adult testes by immunohistochemical assay, using HSC markers c-kit, Thy-1 and human ES integrins. RESULTS: In human adult testes, the alpha6 integrin extensively and significantly expressed on the surface of most of the germ cells in the seminiferous tubule, and beta1 integrin mainly expressed on the surface of the germ cells residing on or near the basal membrane in the seminiferous tubule. Thy-1 scattering expressed on the surface of some cells of the basal membrane, and on some Leydig cells as well. The three antigen markers expressed on the SSC of human adult testes specifically to some extent. SSEA-1 specifically expressed on the surface of the gonocytes in the fetal testes. CONCLUSION: The alpha6 and beta1 integrins and Thy-1 may be used for the SSC isolation as positive markers. SSEA-1 can be used as an identification marker for the fetus SSC.