Objective To investigate the effect of N-myc downstream regulated gene 2 (NDRG2) gene on the renal cancer 786-O cells' proliferation and expressions of Cyclin D1 and p21.Methods The lentiviral vector were infected into renal cancer cell line 786-O in vitro.The up-regulation of NDRG2 protein was determined by indirect immunofluorescence assay.The protein expressions of Cyclin D1 and p21 in the cells infected with Lenti-NDRG2 and Lenti-mCherry were determined by Westernblot respectively.Methyl thiazol tetrazolium (MTT) assay and plate colony formation were used to determine the effect of proliferation of cells.The variation of cell apoptosis was detected by flow cytometry.Results After infected by lentivirus,it had been verified that the protein expression of NDRG2 in the cells by indirect immunofluorescence assay.The expression of Cyclin D1 became low,moreover the expression of p21 became high.With the MTT assay(inhibition ratio 12 h 3.96％,24 h 4.78％,36 h 9.32％,48 h 20.27％,60 h 22.85％ and 72 h 43.7％) and plate colony formation (56.3％,55.2％ and 36.7％ respectively),NDRG2 exhibited significant inhibiting the growth and proliferation of 786-O cells.The apoptosis rate of cells transfected with Lenti-NDRG2 (17.20-1.44) ％ was significantly higher than control group (6.30 ±0.46)％,(t=12.49,P＜0.01) and Lenti-mCherry group (6.00±0.31)％,(t=13.17,P＜0.01).Conclusion The lentiviral vector expressing NDRG2 could inhibit the proliferation of 786-O,furthermore it could down regulate the expression of Cyclin D1 and up-regulate the expression of p21.

HLA antibodies and sCD30 levels were detected in the serum sampled from 620 renal graft recipients at 1 year post-transplantation, which were followed up for 5 years. Six-year graft and patient survivals were 81.6% and 91.0%. HLA antibodies were detected in 45 recipients (7.3%), of whom there were 14 cases with class I antibodies, 26 cases with class II, and 5 cases with both class I and II. Much more graft loss was record in recipients with HLA antibodies than those without antibodies (60% vs. 15.1%, p < 0.001). Significantly higher sCD30 levels were recorded in recipients suffering graft loss than the others (73.9 ± 48.8 U/mL vs. 37.3 ± 14.6 U/mL, p < 0.001). Compared with those with high sCD30 levels, recipients with low sCD30 levels (< 50 U/mL) had much better 6-year graft survival (92.4% vs. 46.6%, p < 0.001). Further statistical analysis showed that detrimental effect of de novo HLA antibodies and high sCD30 on graft survival was not only independent but also additive. Therefore, post-transplantation monitoring of HLA antibodies and sCD30 levels is necessary and recipients with elevated sCD30 level and/or de novo HLA antibody should be paid more attention in order to achieve better graft survival.

Objective: To evaluate the safety and merits of 80 W potassium titanyl phosphate (KTP) laser prostatectomy in men with high cardiovascular or pulmonary risk, receiving oral anticoagulant medication, or with bleeding disorders. Methods: A prospective clinical trial was performed in 128 high-risk men with benign prostatic hyperplasia who underwent photoselective vaporisation of the prostate (PVP). The operation time, postoperative serum sodium and packed cell volume were recorded. The International Prostate Symptom Score (IPSS), maximal flow rate (Qmax) and post-void residual urine volume (PVR) were measured at every follow-up visit. Results: In all patients, KTP laser prostatectomy was performed successfully. The mean (SD) operation time was 51.6 (22.8) min. No blood transfusions were required. No major complications occurred during surgery, and there was no mortality. The mean (SD) catheterisation time was 2.8 (1.6) days. IPSS decreased from 19.2 (6.1) before surgery to 11.5 (8.2), 7.8 (6.4), 6.7 (5.3) and 6.1 (4.6), respectively, at 1, 3, 6 and 12 months after. Qmax increased from 7.0 (2.8) ml/s to 14.5 (9.3), 20.2 (11.5), 21.3 (8.6) and 24.8 (8.0) ml/s, respectively. PVR decreased from 168 (89) ml to 35 (29), 30 (26), 27 (21) and 23 (34) ml, respectively. Conclusions: 80 W KTP laser prostatectomy is a virtually bloodless, safe and effective procedure for men with high cardiovascular or pulmonary risk or bleeding disorders or taking oral anticoagulant medication.

BACKGROUND: The nutritional support, as well as the water and electrolyte balance during the perioperative period in the renal transplantation recipients at diuresis stage are important to the functional restoration of transplanted kidneys.OBJECTIVE: To explore the method and opportunity of the nutritional support and the handling of the water and electrolyte balance in perioperative period of renal transplantation.DESIGN, TIME AND SETTING: A retrospective clinical analysis was performed in the Department of Urology, Xijing Hospital from June 2003 to June 2007.PARTICIPANTS: Ninety-six patients of chronic renal failure underwent allograft renal transplantation. They comprised 59 males and 37 females, aged 17-67 years, with a mean of 35.7 years.METHODS: The perioperative physiological features of the renal transplantation recipients were summarized retrospectively. The recipients' condition during the perioperative period was divided into two stages at the opening point of allograft blood current. The vital signs of the patients maintained at a stable level before operation. All patients received blood transfusion since the operation began, and were supplemented with albumin before opening the vessels. Urinary production exceeding 100 mL per hour indicated the beginning of fluid replacement, which was a simplified transfusion for the patients at diuresis stage following renal transplantation.MAIN OUTCOME MEASURES: Blood inosine, urea nitrogen, electrolyte, blood sugar and urine of the patients were detected at one day postoperatively.RESULTS: During 12-16 hours postoperatively, the urinary production was 260-1 200 mL, average 520 mL per hour. Blood routine test showed 8 cases developed mild hyponatremia, accounting for 8.3%, 3 cases occurred high potassium and healed after renal functional recovery, 1 case presented low potassium and healed with supplement therapy. There were no abnormal changes of blood chlorine. The blood glucose among 21 cases (21.9%) was higher than the normal level, and recovered following hormone maneuver. The electrolytes and blood glucose were detected to be normal in other patients, without any case with low calcium or magnesium. The urine specific gravity arranged during 1.010-1.015.CONCLUSION: The colloid such as erythrocytes, blood plasma and albumin should be mainly infused before the opening of allograft blood current. And the water and electrolytes is recommended to administrate promptly and regularly during the diuresis stage. The healing of the stoma benefits from the adequate nutritional support. The metabolic acidosis still should be prevented when the urinary production returns normal.

Sensitization in kidney transplantation is associated with more acute rejections, inferior graft survival, and an increase in delayed graft function. This study was designed to evaluate the efficacy and safety of preoperative single bolus antithymocyte globulin (ATG) induction therapy in sensitized renal transplant recipients. Methods. Fifty-six cadaveric donor kidney transplant recipients were divided into two groups: Group I (nonsensitized group, n = 30) and group II (sensitized group, PRA > 10%, n = 26). ATG was given as a single preoperative bolus induction therapy to group 11 (ATG IV; 9 mg/kg). The group I patients were treated with mycophenolate mofetil preoperalively as induction therapy. The basic immunosuppressive regimen included tacrolimus (FK-506) or cyclosporine, mycophenolate mofetil, and prednisolone. After hospital discharge, patients were followed on a routine outpatient basis for 12 months. Results. Acute rejection episodes (ARE) occurred in 20% (6/30) of group I and 15.38% (4/26) of group II patients (P = NS). Infections occurred in eight patients (26.7%) as 11 episodes (36.7%), averaging 1.4 episodes per infected patient in group 1, and 6 patients (23.1 %) for a total of 10 episodes (38.5 %), averaging 1.7 episodes per infected patient, in group II (P = NS). Occurrence of side effects and hospital stay were almost comparable in the two groups. No delayed graft function was observed in either group. The 12-month actuarial patient and graft survival were 100% in Group I and II. Conclusion. A preoperative single bolus ATG induction therapy was an effective and safe therapeutic measure, yielding an acceptable acute rejection rate in presensitized renal transplant recipients.

Identification of renal graft candidates at high risk of impending acute rejection (AR) and graft loss may be helpful for patient-tailored immunosuppressive regimens and renal graft survival. To investigate the feasibility with soluble CD30 (sCD30) as predictor of AR, sCD30 levels of 70 patients were detected on day 0 pre-transplant and day 1, 3, 5, 7, 10, 14, 21, and 30 post-transplant. AR episodes in 6 months were recorded and then patients were divided into Group AR (n=11) and Group UC (n=59). Results showed that the patients had higher pre-transplant sCD30 levels than healthy people. A significant decrease of sCD30 was observed on the first day post-transplant and continued until day 14 post-transplant. Soluble CD30 presented a stable level from day 14 to 30 post-transplant. Pre-transplant sCD30 levels of Group AR were much higher than those of Group UC (P<0.001). Patients of Group AR also had higher sCD30 levels than those of Group UC on day 1, 3, 5, 7, 10 and 14 (P<0.001). The sCD30 level presented a significantly delayed decrease in the patients of Group AR. Statistical results showed that the highest value of area under ROC curve (0.95) was obtained on day 5 post-transplant, suggesting that sCD30 levels on day 5 are of high predictive value. Therefore, sCD30 level may be a good marker of increased alloreactivity and of significant predictive value. It's necessary to monitor the variation of sCD30 in the early period post-transplant.

AIM: To compare the effects of azathioprine (AZa) versus mycophenolate mofetil (MMF) based on corticosteroid combined with cyclosporine, which may induce hyperlipidemia, following renal transplantation. METHODS: Sixty-five patients underwent renal transplantation were selected from Department of Urinary Surgery, Xijing Hospital, Fourth Military Medical University of Chinese PLA between January 2003 and January 2006. The informed consent was obtained from the patients. ①All subjects were divided into AZa group (n =35) and MMF group (n =30), and they were treated by corticosteroid + AZa + cyclosporine and corticosteroid + MMF + cyclosporine, respectively within 1 year after renal transplantation. ②The lipid indexes including total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and very low-density lipoprotein cholesterol (VLDL-C) were measured before, 3, 6 and 12 months after transplantation, respectively. Meanwhile, serum creatinine level was evaluated. RESULTS: Totally 65 patients were involved in the result analysis, with no loss. ①For all patients, the prevalence of hyperlipemia was 35.2% during the pretransplant period, 62.7% at month 3, 52.3% at month 6, and 39.3% at month 12 after renal transplantation. ②TC and TG levels were significantly increased in both groups in the first year (P < 0.05). Three-month value for TC in MMF group was higher than that in AZa group (P < 0.05). No significant difference was observed in these two indexes between two groups 6 months after transplantation (P > 0.05). In both groups, HDL-C, LDL-C, and VLDL-C levels did not change during the one-year monitor (P > 0.05). CONCLUSION: Independent from hyperlipidemia risk factors, serum total cholesterol and triglyceride levels tend to increase during corticosteroid combined with cyclosporine therapy among patients undergoing renal transplantation, which has no relationship with MMF or AZa treatment for a long term.

Prostate cancer is relatively unique to man. There is no naturally occurring prostate cancer in the mouse. Pre-clinical studies involve the establishment of a genetically engineered mouse prostate cancer model with features close to those of the human situation. A new knock-in mouse adenocarcinoma prostate (KIMAP) model was established, which showed close-to-human kinetics of tumor development. In order to determine if the similar kinetics is associated with heterogeneous tumor architecture similar to the human situation, we utilized a new mouse histological grading system (Gleason analogous grading system) similar to the Gleason human grading system and flow cytometry DNA analysis to measure and compare the adenocarcinoma of the KIMAP model with human prostate cancer. Sixty KIMAP prostate cancer samples from 60 mice were measured and compared with human prostate cancer. Flow cytometry DNA analysis was performed on malignant prostate tissues obtained from KIMAP models. Mice with prostate cancer from KIMAP models showed a 53.3% compound histological score rate, which was close to the human clinical average (50%) and showed a significant correlation with age (P = 0.001). Flow cytometry analyses demonstrated that most KIMAP tumor tissues were diploid, analogous to the human situation. The similarities of the KIMAP mouse model with tumors of the human prostate suggest the use of this experimental model to complement studies of human prostate cancer.

BACKGROUND. Raising selectivity to tumor cells is a major challenge for most chemotherapy drugs. One of approaches to realizing this goal is antibody-directed enzyme prodrug therapy (ADEPT). This study was done to investigate the curative effect of a new ADEPT system for the treatment of prostate cancer.METHODS. Methotrexate (MTX) prodrugs were synthesized and anti-seminoprotein (SM) single-chain antibody/human carboxypeptidase-A fusion protein (scFv/hCPA) was prepared. Therapeutic effects of this ADEPT system were evaluated.RESULTS. The synthesis of prodrugs was successful and the prodrugs were confirmed no cytotoxicity, but hydrolysis with tumor-targeted scFv/hCPA fusion protein gave 1,000-fold higher cytotoxicity than MTX-alpha-Phe only. Cell cycle assays showed that tumor cells were arrested in theS phase after ADEPT treatment; furthermore, tumors were inhibited significantly in scFv/hCPA and MTX-alpha-Phe treated mice.CONCLUSIONS. Our results suggest that targeted activation cytotoxicity against established prostate cancer by scFv/hCPA mediated ADEPT is tumor-specific and has no systemic toxicity in vitro and in vivo.

Objective A new knock-in mouse adenocarcinoma prostate model (KIMAP) was established, which showed a close to human kinetics of tumour development. This study used a new mouse histological grading system similar to the human Gleason grading system and flow cytometry DNA analysis to measure and compare the new KIMAP model with human CaP and transgenic mouse adenocarcinoma prostate (TGMAP) model. Methods According to heterogeneity of the clinical standard for prostate cancer diagnosis, a close to human mouse standard for histological grading and scoring system, Gleason analogous grading system, was established in this study. Sixty KIMAP and 48 TGMAP prostate cancer samples were measured and compared with human CaP. Flow cytometry DNA analysis was performed on malignant prostate tissues obtained from both TGMAP and KIMAP models. Results Mice with CaP from KIMAP (n = 60) and TGMAP (n = 48) models showed a different distribution of histological scores (p = 0.000). KIMAP mice showed higher percentage (53.3%) of compound histological score rate than TGMAP (25%), but closer to the human clinical average (50%), which showed significant correlation with age (p = 0.001), while TGMAP mice showed unbalanced and random score distribution in all age groups. Flow cytometry analyses showed that most tumour tissues in KIMAP were diploid, analogous to the human condition, while all the TGMAP mice showed aneuploid tumours. Conclusions Results of this study further show that KIMAP, a new generation of murine prostate cancer model, could be used as a supplementary model in addition to the currently widely used transgenic models.

Current prostate cancer research in both basic and preclinical trial studies employ genetically engineered mouse models. However, unlike in human prostate cancer patients, rodents have no counterpart of prostatic-specific antigen (PSA) for monitoring prostate cancer initiation and progression. In this study, we established a mouse serum tumor marker from a mouse homologue of human prostate secretory protein of 94 amino acids (PSP94). Immunohistochemistry studies on different histologic grades from both transgenic and knock-in mouse prostate cancer models showed the down-regulation of tissue PSP94 expression (P < 0.001), the same as for PSA and PSP94 in humans. The presence of mouse serum PSP94 was shown by affinity column and immunoprecipitation purification using a polyclonal mouse PSP94 antibody. A competitive ELISA protocol was established to quantify serum PSP94 levels with a sensitivity of 1 ng/mL. Quantified serum levels of mouse PSP94 ranged from 49.84 ng/mL in wild-type mice to 113.86, 400.45, and 930.90 ng/mL in mouse prostatic intraepithelial neoplasia with microinvasion, well differentiated, moderately differentiated, and poorly differentiated prostate cancer genetically engineered prostate cancer mice, respectively (P < 0.01, n = 68). This increase in serum PSP94 is also well correlated with age and tumor weight. Through longitudinal monitoring of serum PSP94 levels of castrated mice (androgen ablation therapy), we found a correlation between responsiveness/refractory prostate tissues and serum PSP94 levels. The utility of mouse serum PSP94 as a marker in hormone therapy was further confirmed by three-dimensional ultrasound imaging. The establishment of the first rodent prostate cancer serum biomarker will greatly facilitate both basic and preclinical research on human prostate cancer.

Longitudinal studies of mouse cancer models required large cohorts since autopsy was the only reliable method to evaluate treatment efficacy. This paper reports the use of high-resolution three-dimensional ultrasound micro-imaging to monitor prostate tumor development in genetically engineered mice. Twenty-nine genetically engineered prostate cancer mice, including castrated and uncastrated mice, were imaged by three-dimensional ultrasound. Qualitative comparisons of three-dimensional ultrasound images with histology sections of prostate tumors demonstrate the ability of ultrasound to accurately depict the size and shape of malignant masses in live mice. The correlation coefficient of tumor diameter measurements performed in vivo with three-dimensional ultrasound and at autopsy was 0.997. Prospective tumor detection sensitivity and specificity were 91.7% and 100%. Representative exponential growth curves constructed via longitudinal ultrasound imaging indicated diameter doubling times from 10 to 37 days for four prostate tumors during an initial period of rapid progression. Three-dimensional ultrasound will likely become the micro-imaging modality most readily adopted for mouse pre-clinical trial studies.

AIM:To construct anti-human CD3 single chain Fv antibody (scFv)/human p53 tetramerization domain fusion gene and express the fusion protein in Hela cells.METHODS:The anti-human CD3 scFv was cloned into the previously constructed plasmid pUC18/IgG3/p53 to construct anti-human CD3 scFv /human p53 tetramerization domain fusion gene. After enzyme digestion analysis and sequencing, the fusion gene was subcloned into the expression plasmid pSecTag2-B. Then the plasmid pSecTag2-B containing the fusion gene was transfected into Hela cells. The expressed products were analyzed by SDS-PAGE and Western blot. The binding of the purified fusion protein to human peripheral blood mononuclear cells (PBMCs) was detected by flow cytometry.RESULTS:DNA sequencing showed the fusion gene was constructed successfully. The expressed product of the fusion gene with a relative molecular mass (M(r)) about 35 000 was confirmed by SDS-PAGE and Western blot. The purified tetrameric anti-human CD3 scFv showed significantly stronger binding to PBMCs than scFv.CONCLUSION:The tetrameric anti-human CD3 scFv which can bind to PBMCs has been successfully expressed and purified for potential use in clinical studies.

AIM:To type the HLA-DR DNA for renal transplantation by PCR with sequence specific primers (PCR-SSP).METHODS:According to nucleotide sequences of HLA-DR, 16 pairs of specific primers and a pair of positive control primers were designed and synthesized for PCR-SSP. Then HLA-DR sites of 52 donors and recipients for renal transplantation were typed by the PCR-SSP.RESULTS:All the samples were successfully typed by PCR-SSP with the synthesized primers. The results were available within 3 hours after sampling and the accuracy and reproducibility were 100%.CONCLUSION:Genotyping for HLA-DR sites by PCR-SSP with primers reported herein was a simple and accurate technique suitable for clinical application.

OBJECTIVETo construct a recombinant vector that expresses anti-CD 3/anti-prostate-cancer bispecific single-chain antibody (BsAb), and study its biological activities and clinical significance.METHODSAn anti-CD 3/anti-prostate-cancer BsAb was constructed by PCR and molecular biological technique of DNA cloning. The fusion gene, confirmed by sequencing, was subcloned into the pSectag2-B plasmid from the pUC18 vector by digestion with EcoR I and Hind III restriction endonucleases, whose sites exist in both the vectors. Then the recombinant plasmid was transfected into HeLa cells. The expression products in the supernatant were analyzed by both SDS-PAGE and Western blot technique, then were purified with Ni(2+)-NTA superflow affinity chromatography. Its biological activities were examined by flow cytometry (FCM).RESULTSA fragment of 1.5 kb was inserted into the pUC18 vector, which was sequenced and verified to be identical with that designed. The expression of anti-CD 3 x anti-prostate-cancer BsAb yielded a soluble protein with an apparent molecular mass of 61 KD. The purification rate of the expressed BsAb was up to 90.537% and the yield of purified BsAb from this procedure was 0.09 mg/ml. The positive binding rates of BsAb to PBMC and to PC-3 cell were 54.1% and 53.7% respectively.CONCLUSIONThe anti-CD 3 x anti-prostate-cancer BsAb thus constructed exhibits beneficial biological activities and may play an important role in the treatment of prostate cancer.

AIM:To study the relationship between the expression of Fas/FasL protein and apoptosis in rats after renal ischemia/reperfusion. METHODS:Establish the models of renal ischemia/reperfusion injury in rat and SABC immunohistochemical methods were used to detect the changes of expression of Fas/FasL protein. Pathomorphological changes in renal ischemia/reperfusion injury were observed.RESULTS:The expression of Fas/FasL proteins was negative in the sham operated group.Fas/FasL proteins were increased in renal in ischemia/reperfusion group,and gradually upregulated with the duration of ischemia or reperfusion and peaked at 72 h of reperfusion.The expression of Fas/FasL proteins was stronger in 60 min ischemia group than 30 min ischemia group and they were mainly expressed in renal tubule.We observed local necrosis and inflammatory cells infiltration around infracted area in ischemia/reperfusion group by HE dyeing methods.And the necrosis area was mainly occurred around proximal convoluted tubule. CONCLUSIONS:These findings suggested that Fas/FasL proteins were over expressed after ischemia/reperfusion injury in rats renal.And Fas/FasL system was involved in the process of renal ischemia/reperfusion injury.

Objective:To investigate the effect of the diode laser therapy on serum total PSA,free PSA and free／total PSA ratio in pat ients with benign prostatic hyperplasia and to determine the reliability of fre e／total PSA ratio after such interventions.Methods:Using the Indigo 830 e diode laser system,72 cases of prostatic hyperplasia have been treated by transurethral prostatectemy with interstitial laser coagulation .Serum total PSA and free PSA levels were determined 1 hour before and 24 hours after laser therapy by using enzyme immunoassay.Preoperative and postoperativ e free and total PSA and free／total PSA ratio were statistically compared.Results:Although postoperative total PSA and free PSA in creased significantly compared with preoperative values (P＜0.01)，the diffe rence between preoperative and postoperative free／total PSA ratios was not stat istically significant (P＞0.05).Conclusion:Free ／total PSA ratio may be a reliable parameter in the early period after such int erventions as diode laser therapy.