Long-term effect of transurethral partial cystectomy with a 2-micrometer continuous-wave laser for non-muscle-invasive bladder cancer.

Purpose: We have reported the efficacy and safety of 2-micrometer continuous-wave laser cystectomy of non-muscle invasive bladder tumor (NMIBC) (J Urol. 2009;182:66-9). In this study, we evaluated the long-term outcomes of patients with NMIBC who underwent transurethral partial cystectomy with a 2-micrometer continuous-wave laser, and explored the risk factors for tumor recurrence. Methods: This was a retrospective study of patients with NMIBC planned to undergo transurethral partial cystectomy with a 2-micrometer continuous-wave laser at the Fourth Medical Center of the PLA General Hospital between January 2012 and December 2014. The primary outcome was bladder cancer recurrence. Results: A total of 75 patients were enrolled. Sixty-two (82.7%) were male. The patients were 59.8 ± 12.9 years of age. The mean operation time was 38.7 ± 20.4 min. No Clavien grade >2 complications occurred. The duration of catheter indwelling was 3.6 ± 1.8 days. The hospital stay was 6.0 ± 2.3 days. The median follow-up was 80 months. A total of 17 patients had a recurrence during follow-up, and the recurrence-free survival (RFS) rate was 77.3%. In the multivariable analysis, the tumor risk group were independently associated with the recurrence of NMIBC (p = 0.026). Conclusions: After TURBT with a 2-micrometer continuous-wave laser, RFS was 77.3% at the median follow-up of 80 months. All complications were mild. Only tumor risk group was independently associated with the recurrence of NMIBC.

A novel convolutional neural network for kidney ultrasound images segmentation.

BACKGROUND AND OBJECTIVE: Ultrasound imaging has been widely used in the screening of kidney diseases. The localization and segmentation of the kidneys in ultrasound images are helpful for the clinical diagnosis of diseases. However, it is a challenging task to segment the kidney accurately from ultrasound images due to the interference of various factors. METHODS: In this paper, a novel multi-scale and deep-supervised CNN architecture is proposed to segment the kidney. The architecture consists of an encoder, a pyramid pooling module and a decoder. In the encoder, we design a multi-scale input pyramid with parallel branches to capture features at different scales. In the decoder, a multi-output supervision module is developed. The introduction of the multi-output supervision module enables the network to learn to predict more precise segmentation results scale-by-scale. In addition, we construct a kidney ultrasound dataset, which contains of 400 images and 400 labels. RESULTS: To highlight effectiveness of the proposed approach, we use six quantitative indicators to compare with several state-of-the-art methods on the same kidney ultrasound dataset. The results of our method on the six indicators of accuracy, dice, jaccard, precision, recall and ASSD are 98.86%, 95.86%, 92.18%, 96.38%, 95.47% and 0.3510, respectively. CONCLUSIONS: The analysis of evaluation indicators and segmentation results shows that our method achieves the best performance in kidney ultrasound image segmentation.

MBANet: Multi-branch aware network for kidney ultrasound images segmentation.

Due to the influence of kidney morphology, heterogeneous structure and image quality, segmenting kidney in ultrasound images is challenging. To alleviate this challenge, we proposed a novel deep neural network architecture, namely Multi-branch Aware Network (MBANet), to segment kidney accurately and robustly. MBANet mainly consists of multi-scale feature pyramid (MSFP), multi-branch encoders (MBE) and master decoder. The design of MSFP can make the network more accessible to different kinds of class details at different scales. The information exchange between MBE can reduce the loss of feature information and improve the segmentation accuracy of the network. In addition, we designed a multi-scale fusion block (MFBlock) in the MBE to further extract and fuse more refined multi-scale image information. In order to further improve the robustness of MBANet, this paper also designed a step-by-step training mechanism. We validated the proposed approach and compared to several state-of-the-art approaches on the same kidney ultrasound datasets using six quantitative metrics. The results of our method on the six indicators of pixel accuracy (PA), intersection over union (IoU), precision, recall, specificity and F1-score (F1) are 98.83%, 92.38%, 97.10%, 95.03%, 99.46% and 0.9601, respectively. Compared with the comparison method, the average values on the six indicators are improved by about 2%. The evaluation results and segmentation results demonstrate that the proposed approach achieves the best overall performance on kidney ultrasound images segmentation.

Development and validation of a nomogram for predicting prostate cancer in patients with PSA ≤ 20 ng/mL at initial biopsy.

ABSTRACT: The aim of this study was to construct a nomogram for predicting prostate cancer (PCa) in patients with PSA ≤ 20 ng/mL at initial biopsy.The patients with PSA ≤ 20 ng/mL who underwent prostate biopsy were retrospectively included in this study. The nomogram was developed based on predictors for PCa, which were assessed by multivariable logistic regression analysis. The receiver operating characteristic curve, calibration plots and decision curve analysis (DCA) were used to evaluate the performance of the nomogram.This retrospective study included 691 patients, who were divided into training set (505 patients) and validation set (186 patients). The nomogram was developed based on the multivariable logistic regression model, including age, total PSA, free PSA, and prostate volume. It had a high area under the curve of 0.857, and was well verified in validation set. Calibration plots and DCA further validated its discrimination and potential clinical benefits. Applying the cut-off value of 15%, our nomogram would avoid 42.5% of unnecessary biopsies while miss only 4.4% of PCa patients.The nomogram provided high predictive accuracy for PCa in patients with PSA ≤ 20 ng/mL at initial biopsy, which could be used to avoid the unnecessary biopsies in clinical practice.

Kidney ventrally rotation technique in retroperitoneal robot-assisted partial nephrectomy for posterior hilar tumor: technical feasibility and preliminary results.

PURPOSE: The retroperitoneal robotic assisted partial nephrectomy (RAPN) is suitable for tumors locating on the posterior side of the kidney. However, the posterior hilar tumor poses an additional surgical challenge due to the special location and poor tumor exposure. We developed a novel kidney ventrally rotation technique to overcome this difficulty during retroperitoneal RAPN and evaluated its efficacy in a retrospective case-control comparative study. METHODS: From March 2016 to April 2019, a total of 39 patients with posterior renal hilar tumor underwent retroperitoneal RAPN. The kidney ventrally rotation technique, which improved the tumor exposure by opening the peritoneum and rotating the kidney ventrally, was applied in 24 cases, and the conventional RAPN was performed in the other 15 cases (control group). Perioperative data was analyzed to evaluate the efficacy of the kidney ventrally rotation technique. RESULTS: In kidney rotation group, the 24 patients underwent RAPN successfully without converting to open surgery or radical nephrectomy. The warm ischemia time was 17.4 ± 6.6 min, which was significantly shorter than 24.5 ± 8.3 min in control group. The mean operation time (80 ± 24 min) and estimated blood loss (104 ± 65 ml) were not different from the control group. No sever complications occurred, and no positive surgical margin was found in all the malignant cases. After 14 months follow-up, no recurrence or metastasis occurred in all cases. CONCLUSION: Kidney ventrally rotation technique is safe and feasible for improving the exposure of posterior renal hilar tumor during retroperitoneal RAPN. It could be regarded as an efficient option for the management of posterior hilar tumor.

Robot-assisted laparoscopic antegrade versus open inguinal lymphadenectomy: a retrospective controlled study.

BACKGROUND: To investigate the surgical methods and clinical results of robot-assisted laparoscopic antegrade inguinal lymphadenectomy. METHODS: A retrospective study was performed on clinical data from 19 patients with penile cancer admitted from March 2013 to October 2017. Among them, nine patients underwent robot-assisted laparoscopic antegrade inguinal lymphadenectomy (robot-assisted group) and 10 patients underwent open inguinal lymphadenectomy (open group). In the robot-assisted group, preoperative preparation, patient position, robot placement, design of operating channel and establishment of operating space are described. Key surgical procedures and techniques are also summarized. In addition, the number of lymph nodes removed, postoperative complications and follow-up in both groups were statistically analyzed. RESULTS: For the 9 patients in the robot-assisted group, surgery was successfully accomplished at 17 sides without intraoperative conversion to open surgery. The surgery time for each side was 45~90 min using laparoscope with an average of 68.5 ± 13.69 min/side. The intraoperative blood loss was estimated to be < 10 ml/side, and the number of removed lymph nodes was not significantly different from that of the open group (12 ± 4.2/side vs.11 ± 5.8/side, P = 0.84). There were no postoperative complications such as skin necrosis, delayed wound healing and cellulitis in the robot-assisted group. Skin-related complications occurred in 9 (45%) of the 20 sides in the open group. During a median follow-up of 25 months in robot-assisted group and 52.5 mouths in open group, was not significantly different there were no statistical differences in recurrence-free survival between the groups (75% vs 60%, p = 0.536). CONCLUSION: Robot-assisted laparoscopic antegrade inguinal lymphadenectomy achieved the desired surgical outcomes with fewer intraoperative and postoperative complications. The robotic arms of the surgical system were placed between the lower limbs of each patient. There was no need to re-position the robotic arms during bilateral inguinal lymphadenectomy. This simplified the procedure and reduced the use of trocars. If necessary, pelvic lymphadenectomy could be performed simultaneously using the original trocar position.

Significant Diagnostic Value of Free-Serum PSA (FPSA)/Prostate-Specific Antigen Density (PSAD) and (F/T)/PSAD for Prostate Cancer of the Chinese Population in a Single Institution.

BACKGROUND The aim of this study was to investigate the diagnostic value of (F/T)/PSAD for prostate cancer detection in the Chinese population. MATERIAL AND METHODS Data were collected retrospectively from patients with prostate cancer or benign prostatic hyperplasia from July 2009 to September 2014. SPSS 19.0 software was used for the receiver operating characteristic curve (ROC), and calculating sensitivity, specificity, and positive predictive values (PPV) and negative predictive values (NPV), respectively. Comparison of the area under ROC (AUC) was performed using the MedCalc v. 10.4.7.0 software. RESULTS A total of 660 patients (including 251 patients with prostate cancer and 409 patients with prostatic hyperplasia) were included. Prostate volume (PV), prostate-specific antigen density (PSAD), free-serum PSA (FPSA)/PSAD, and free-to-total PSA (F/T)/PSAD had similar AUC (P>0.05), and had significantly higher AUC (P<0.001) than F/T, total-serum PSA (TPSA), and free-serum PSA (FPSA). Based on the optimal cutoff value, the sensitivity of (F/T)/PSAD and FPSA/PSAD was similar (P>0.05), and significantly higher than the PV and PSAD (P<0.05). The logistic regression model using a combination of age, FPSA, PV, PSAD, FPSA/PSAD, and (F/T)/PSAD showed higher AUC than each one alone (P<0.001). CONCLUSIONS (F/T)/PSAD can be used as a predictor for prostate cancer in the Chinese population aged >50 years and has a significantly lower false negative rate than PSAD and PV with a cutoff value of ≤0.731. A new parameter, FPSA/PSAD, has similar diagnostic accuracy comparable to (F/T)/PSAD. The diagnostic value of a combination of age, FPSA, PV, PSAD, FPSA/PSAD, and (F/T)/PSAD needs further investigation.

Identification of aberrantly methylated differentially expressed genes in prostate carcinoma using integrated bioinformatics.

BACKGROUND: Methylation plays a key role in the aetiology and pathogenesis of prostate cancer (PCa). This study aimed to identify aberrantly methylated differentially expressed genes (DEGs) and pathways in PCa and explore the underlying mechanisms of tumourigenesis. METHODS: Expression profile (GSE29079) and methylation profile (GSE76938) datasets were obtained from the Gene Expression Omnibus (GEO). We used R 3.4.4 software to assess aberrantly methylated DEGs. The Cancer Genome Atlas (TCGA) RNA sequencing and Illumina HumanMethylation450 DNA methylation data were utilized to validate screened genes. Functional enrichment analysis of the screened genes was performed, and a protein-protein interaction (PPI) network was constructed using the Search Tool for the Retrieval of Interacting Gens (STRING). The results were visualized in Cytoscape. After confirmation using TCGA, cBioPortal was used to examine alterations in genes of interest. Then, protein localization in PCa cells was observed using immunohistochemistry. RESULTS: Overall, 536 hypomethylated upregulated genes were identified that were enriched in biological processes such as negative regulation of transcription, osteoblast differentiation, intracellular signal transduction, and the Wnt signalling pathway. Pathway enrichment showed significant changes in factors involved in AMPK signalling, cancer, and adherens junction pathways. The hub oncogenes were AKT1, PRDM10, and FASN. Additionally, 322 hypermethylated downregulated genes were identified that demonstrated enrichment in biological processes including positive regulation of the MAPK cascade, muscle contraction, ageing, and signal transduction. Pathway analysis indicated enrichment in arrhythmogenic right ventricular cardiomyopathy (ARVC), focal adhesion, dilated cardiomyopathy, and PI3K-AKT signalling. The hub tumour suppressor gene was FLNA. Immunohistochemistry showed that AKT1, FASN, and FLNA were mainly expressed in PCa cell cytoplasm, while PRDM10 was mainly expressed in nuclei. CONCLUSIONS: Our results identify numerous novel genetic and epigenetic regulatory networks and offer molecular evidence crucial to understanding the pathogenesis of PCa. Aberrantly methylated hub genes, including AKT1, PRDM10, FASN, and FLNA, can be used as biomarkers for accurate PCa diagnosis and treatment. In conclusion, our study suggests that AKT1, PRDM10, and FASN may be tumour promoters and that FLNA may be a tumour suppressor in PCa. We hope these findings will draw more attention to these hub genes in future cancer studies.

[Establishment of a human bladder cancer cell line stably co-expressing hSPRY2 and luciferase genes and its subcutaneous tumor xenograft model in nude mice].

Objective To establish a human bladder cancer cell line stably co-expressing human sprouty2 (hSPRY2) and luciferase (Luc) genes simultaneously, and develop its subcutaneous tumor xenograft model in nude mice. Methods The hSPRY2 and Luc gene segments were amplified by PCR, and were cloned into lentiviral vector pCDH and pLVX respectively to produce corresponding lentivirus particles. The J82 human bladder cancer cells were infected with these two kinds of lentivirus particles, and then further screened by puromycin and G418. The expressions of hSPRY2 and Luc genes were detected by bioluminescence, immunofluorescence and Western blot analysis. The screened J82-hSPRY2/Luc cells were injected subcutaneously into BALB/c nude mice, and the growth of tumor was monitored dynamically using in vivo fluorescence imaging system. Results J82-hSPRY2/Luc cell line stably expressing hSPRY2 and Luc genes was established successfully. Bioluminescence, immunofluorescence and Western blot analysis validated the expressions of hSPRY2 and Luc genes. The in vivo fluorescence imaging system showed obvious fluorescence in subcutaneous tumor xenograft in nude mice. Conclusion The J82-hSPRY2/Luc bladder cancer cell line and its subcutaneous tumor xenograft model in nude mice have been established successfully.

MEIS1 inhibits clear cell renal cell carcinoma cells proliferation and in vitro invasion or migration.

BACKGROUND: Myeloid ecotropic viral integration site 1 (MEIS1) protein plays a synergistic causative role in acute myeloid leukemia (AML). However, MEIS1 has also shown to be a potential tumor suppressor in some other cancers, such as non-small-cell lung cancer (NSCLC) and prostate cancer. Although multiple roles of MEIS1 in cancer development and progression have been identified, there is an urgent demand to discover more functions of this molecule for further therapeutic design. METHODS: MEIS1 was overexpressed via adenovirus vector in clear cell renal cell carcinoma (ccRCC) cells. Western blot and real-time qPCR (quantitative Polymerase Chain Reaction) was performed to examine the protein and mRNA levels of MEIS1. Cell proliferation, survival, in vitro migration and invasion were tested by MTT, colony formation, soft-agar, transwell (in vitro invasion/migration) assays, and tumor in vivo growthwas measured on nude mice model. In addition, flow-cytometry analysis was used to detect cell cycle arrest or non-apoptotic cell death of ccRCC cells induced by MEIS1. RESULTS: MEIS1 exhibits a decreased expression in ccRCC cell lines than that in non-tumor cell lines. MEIS1 overexpression inhibits ccRCC cells proliferation and induces G1/S arrest concomitant with marked reduction of G1/S transition regulators, Cyclin D1 and Cyclin A. Moreover, MEIS1-1 overexpression also induces non-apoptotic cell death of ccRCC cells via decreasing the levels of pro-survival regulators Survivin and BCL-2. Transwell migration assay (TMA) shows that MEIS1 attenuates in vitro invasion and migration of ccRCC cells with down-regulated epithelial-mesenchymal transition (EMT) process. Further, in nude mice model, MEIS1 inhibits the in vivo growth of Caki-1 cells. CONCLUSIONS: By investigating the role of MEIS1 in ccRCC cells' survival, proliferation, anchorage-independent growth, cell cycle progress, apoptosis and metastasis, in the present work, we propose that MEIS1 may play an important role in clear cell renal cell carcinoma (ccRCC) development.

Prognostic Role of Neutrophil-to-Lymphocyte Ratio in Prostate Cancer: A Systematic Review and Meta-analysis.

Inflammation is increasingly reported to be associated with the prognosis of patients with cancers. And the prognostic role of neutrophil-to-lymphocyte ratio (NLR) in patients with prostate cancer (PCa) remains inconsistent. Therefore, we conducted this systematic review and meta-analysis to obtain a more reliable assessment of prognostic significance of NLR in PCa.A comprehensive literature research regarding the association of NLR and prognosis of PCa was performed through PubMed, Embase, Cochrane Central, and Web of Science. The hazard ratios (HRs) and its 95% confidence intervals (CIs) for overall survival (OS), progression-free survival, or recurrence-free survival were extracted and pooled using fix-effects model or random-effects model.A total of 14 studies that met our criterion were included in this meta-analysis. Our pooled results demonstrated that elevated NLR was not significantly associated with the poor OS (HR = 1.45; 95% CI 0.77-2.71; P = 0.248) or recurrence-free survival (HR = 1.34; 95% CI 0.89-2.02; P = 0.155) of patients with localized PCa. Although elevated NLR predicted poorer OS (HR = 1.57; 95% CI 1.41-1.74; P < 0.001) and progression-free survival (HR = 1.97; 95% CI 1.28-3.04; P = 0.002) of patients with metastatic castration resistant prostate cancer (mCRPC).Elevated NLR is a strong indicator of poorer prognosis of patients with mCRPC, whereas the NLR is not significantly associated with prognosis of patients with localized PCa. Therefore, NLR could be used in patients with mCRPC for risk stratification and decision making of individual treatment.

Lateroconal fascia suspension for management of peritoneal tear and "curtain" effect during retroperitoneal laparoscopic operations.

PURPOSE: To describe the method of lateroconal fascia suspension for the management of peritoneal tear and curtain effect during retroperitoneal laparoscopic operations. MATERIALS AND METHODS: Between May 2013 and October 2014, we performed lateroconal fascia suspension in 30 cases of retroperitoneal laparoscopic operations. Peritoneal tear occurred and retroperitoneal space collapsed in 18 cases of them during the operation, and free edge of the lateroconal fascia caused curtain effect and sheltered the field of view in another 12 cases after the lateroconal fascia was incised longitudinally. RESULTS: The curtain effect of lateroconal fascia was eliminated successfully, and the sheltered field of view got normal in all the 12 cases. The collapsed retroperitoneal space due to peritoneal tear got enlarged effectively and was sufficient for the following operations in 15 patients of the overall 18 cases, while the collapsed retroperitoneal space did not get enlarged significantly in the other three cases. After the insertion of an extra 5-mm trocar into peritoneal space, the collapsed retroperitoneal space got enlarged eventually. Finally, retroperitoneal laparoscopic operations were continued and completed successfully in all these 30 patients. It took 4 min to complete the suspension procedure, and no related complications occurred during the whole suspension process. CONCLUSION: Lateroconal fascia suspension method could manage most peritoneal tears and curtain effect effectively during retroperitoneal laparoscopic operations.

Radiofrequency Ablation Versus Partial Nephrectomy in Treating Small Renal Tumors: A Systematic Review and Meta-Analysis.

Radiofrequency ablation (RFA) has emerged as an alternative treatment to surgical partial nephrectomy (PN) in the treatment of small renal tumors (SRTs). But its safety and oncological efficacy are still controversial. We conducted this systematic review and meta-analysis to compare the peritoperative and oncological outcomes of RFA and PN in the treatment of SRTs. Pubmed, EMBASE, Cochrane CENTRAL, and Web of Science were searched to identify eligible studies that compared the RFA and PN in the treatment of SRTs. Twelve retrospective studies that compared RFA with PN in the treatment of SRTs met our selection criterion and were included in this meta-analysis. The pooled results indicated that the local recurrence rate (4.14% vs 4.10%, RR: 1.18, 95% CI: 0.68, 2.07, P = 0.550) and distant metastases rate (2.76% vs 1.89%, RR: 1.31, 95% CI: 0.70, 2.46, P = 0.686) were not significantly different between the RFA group and the PN group. In terms of perioperative outcomes, RFA was associated with shorter length of stay (LOS) (WMD: -2.02 days, 95% CI: -2.77, -1.27, P < 0.001), lower eGFR decline after treatment (WMD: -3.90, 95% CI: -6.660, -1.140, P = 0.006). However, the overall perioperative complication rate (7.5% vs 6.2%, RR:1.10, 95% CI: 0.64, 1.87, P = 0.740) and the major complication rate (3.7% vs 4.4%, RR: 0.83, 95% CI: 0.43, 1.60, P = 0.579) were both similar between RFA and PN groups. Compared with PN, RFA achieves an equal oncological outcome for SRTs with similar local recurrence rate and distant metastases rate. Additionally, RFA is associated with a similar perioperative complication rate, lower decline of eGFR, and shorter LOS. Therefore, RFA is an effective option in the treatment of SRTs for selected patients.

[Association between obesity-related plasma hemodilution and the concentration of prostate specific antigen].

OBJECTIVE: To determine the effect of obesity on prostate specific antigen (PSA) in men with benign prostatic hyperplasia (BPH) and develop a PSA-related parameter that can eliminate the effect of obesity. METHODS: We reviewed the clinical data of 706 patients with BPH. Two PSA-related parameters, namely PSA mass (total circulating PSA protein) and PSA mass ratio (total circulation PSA protein per prostate volume), were calculated for all the patients and the association of BMI with PSA, PSA mass, and PSA mass ratio was assessed. RESULTS: A higher BMI was significantly associated with a greater plasma volume and prostate volume (P<0.05). Linear regression analysis revealed a greater adjusted R2 of BMI versus plasma volume than of BMI PSA (0.569 vs 0.027). PSA was positively associated with the prostate volume and negatively with BMI and plasma volume (P<0.05). PSA mass was positively associated with prostate volume (P<0.05) but was not associated with BMI or plasma volume (P>0.05). PSA mass ratio was not associated with prostate volume (P>0.05) but negatively associated with BMI and plasma volume. Plasma volume and prostate volume, PSA, and PSA mass ratio (P<0.05), but not PSA mass (P>0.05), differed significantly among normal-weight, overweight, and obese patients. CONCLUSION: A higher BMI is associated with a greater plasma volume in BPH patients. In obese patients with BPH, a lower PSA concentration may result from hemodilution caused by a greater plasma volume, and PSA mass can eliminate the effect of obesity on PSA.

Synergistic antitumor efficacy of combined DNA vaccines targeting tumor cells and angiogenesis.

To further enhance the antitumor efficacy of DNA vaccine, we proposed a synergistic strategy that targeted tumor cells and angiogenesis simultaneously. In this study, a Semliki Forest Virus (SFV) replicon DNA vaccine expressing 1-4 domains of murine VEGFR2 and IL12 was constructed, and was named pSVK-VEGFR2-GFc-IL12 (CAVE). The expression of VEGFR2 antigen and IL12 adjuvant molecule in 293T cells in vitro were verified by western blot and enzyme-linked immune sorbent assay (ELISA). Then CAVE was co-immunized with CAVA, a SFV replicon DNA vaccine targeting survivin and ß-hCG antigens constructed previously. The antitumor efficacy of our combined replicon vaccines was evaluated in mice model and the possible mechanism was further investigated. The combined vaccines could elicit efficient humoral and cellular immune responses against survivin, ß-hCG and VEGFR2 simultaneously. Compared with CAVE or CAVA vaccine alone, the combined vaccines inhibited the tumor growth and improved the survival rate in B16 melanoma mice model more effectively. Furthermore, the intratumoral microvessel density was lowest in combined vaccines group than CAVE or CAVA alone group. Therefore, this synergistic strategy of DNA vaccines for tumor treatment results in an increased antitumor efficacy, and may be more suitable for translation to future research and clinic.

[Effect of p53-regulated apoptosis-inducing protein 1 transfection on the biological characteristics of PC-3M human prostate cancer cells].

OBJECTIVE: To investigate the effect of p53-regulated apoptosis-inducing protein 1 (p53AIP1) gene on the proliferation, cell cycle, apoptosis, invasion and migration of PC-3M human prostate cancer cells in vitro. METHODS: The eukaryotic expression vector pDC316-p53AIP1 was constructed and confirmed by double enzyme digestion and PCR analysis. Then it was transfected into PC-3M human prostate cancer cells by Lipofectamine (TM) 2000. The expression of p53AIP1 protein was detected by Western blotting. The proliferation of PC-3M cells was tested by CCK-8 assay; the cell cycle and apoptosis rate were analyzed by flow cytometry combined with annexin V-FITC/PI staining; the effect of p53AIP1 on the cell invasion and migration was detected by Transwell(TM) assay. RESULTS The eukaryotic expression vector pDC316-p53AIP1 was constructed successfully. After transfected into PC-3M cells, Western blotting demonstrated that the protein p53AIP1 was effectively expressed. CCK-8 assay revealed that the proliferation of PC-3M cells was significantly inhibited by p53AIP1 (P<0.05). Flow cytometry indicated that the cells were arrested at S/G2-M phase (P<0.05) and cell apoptosis was evidently promoted (P<0.05). Transwell(TM) chamber experiments showed that p53AIP1 decreased the abilities of both invasion and migration of the cells (P<0.05). CONCLUSION: The p53AIP1 inhibits the proliferation of PC-3M cells, arrests cell cycle at S/G2-M phase, decreases the abilities of invasion and migration and promotes cell apoptosis.

[Expression and activity identification of a fusion protein for promoting adenovirus infection efficiency of dendritic cells].

OBJECTIVE: To construct a prokaryotic expression plasmid for CT40L, express the target protein in E. coli, purity the CT40L fusion protein and verify its antigenicity. METHODS: Gene sequences of Coxsackie and adenovirus receptor (CAR), bacteriophage T4 fibritin and mouse CD40L were found out in GenBank. Then functional domains of three molecules were linked to form a fusion sequence which was then optimized for prokaryotic expression. The optimized sequence was cloned into prokaryotic expression vector pET42a(+) to construct the recombinant expression vector pET42a-CT40L. The recombinant vector was transformed into BL21 (DE3) and the fusion protein CT40L/GST was induced by IPTG. The fusion protein was then subjected to purification using GST affinity chromatography and to identification of the immune activity using Western blotting and ELISA. RESULTS: The recombinant expression vector was verified correct by double digestion with Nco I and EcoR I. After IPTG induction, SDS-PAGE showed that the relative molecular mass of the fusion protein was about 78 kDa and that the purity of the purified protein reached 90%. Western blotting and ELISA demonstrated that the purified fusion protein had a valid antigenicity. CONCLUSION: The prokaryotic expression plasmid pET42a-Ct40L was successfully constructed and expressed in E. coli, and the purified fusion protein was proved to have a good antigenicity.

[Over-expression of human SPRY2 promotes the proliferation and survival of HEK293T cells].

OBJECTIVE: To construct a recombinant expression vector pDC315/hSPRY2 carrying human SPRY2 (hSPRY2) gene, and transfect the recombinant plasmid into human embryonic kidney HEK293T cells to express the target protein, and thus explore preliminarily the effect of hSPRY2 on the proliferation and survival of HEK293T cells. METHODS: The recombinant adenoviral vector expressing hSPRY2 was constructed and transfected into HEK293T cells in vitro. The expression of hSPRY2 target protein in the transfected cells was identified by Western blot analysis. The effect of hSPRY2 on the proliferation and survival of HEK293T cells in a serum-free condition was tested using CCK-8 assay. RESULTS: The recombinant expression vector pDC315/hSPRY2 was constructed, and the target protein hSPRY2 was expressed in the transfected HEK293T cells. The proliferation and survival rates of HEK293T cells transfected with pDC315/hSPRY2 were significantly higher than those transfected with pDC315/EGFP (P<0.05). CONCLUSION: The recombinant expression vector for hSPRY2 was successfully constructed and its expression was demonstrated in the transfected HEK293T cells. Over-expression of SPRY2 promoted the proliferation and survival of HEK293T cells.

[Cloning, prokaryotic expression, and antigenicity identification of extracellular domain of mouse CD40].

OBJECTIVE: To construct a prokaryotic expression plasmid for extracellular domain of mouse CD40 (mCD40), express the mCD40/GST recombinant protein in E.coli, purify the mCD40/GST recombinant protein and characterize its antigenicity. METHODS: Extracellular domain of mouse CD40 was amplified by PCR from cell line DC2.4 and then was cloned into prokaryotic expression vector pGEX-6P-1 to construct the recombinant expression vector pGEX-6P-1-mCD40. The expression vector was transformed into E.coli BL21 (DE3) and the fusion protein mCD40/GST was induced by IPTG. The fusion protein was purified through sepharose 4B. Then antigenicity of the purified mCD40/GST protein was verified by Western blotting and ELISA. RESULTS: The PCR product was verified by DNA sequencing to be consistent with the sequence of mouse CD40 on GenBank. The recombinant plasmid was identified by double digestion successfully. SDS-PAGE analysis showed the relative molecular mass of the fusion protein induced by IPTG was 45 000. Western blotting and ELISA demonstrated that the purified mCD40/GST protein had a good antigenicity. CONCLUSION: The prokaryotic expression plasmid pGEX-6P-1-mCD40 was constructed successfully. In E.coli BL21 (DE3) transformed with the plasmid, the mCD40/GST fusion protein was expressed by IPTG induction. The purified mCD40/GST fusion protein had a high antigenicity, which provides a strong support for the future study of CD40.

[Construction and identification of a recombinant replication-defective adenovirus vector encoding p53AIP1 and its expression in human HeLa cells].

OBJECTIVE: To construct a recombinant replication-defective adenovirus vector carrying p53AIP1 (p53-regulated apoptosis-inducing protein 1) gene and observe its expression in human HeLa cells. METHODS: Specific primers were designed according to p53AIP1 gene sequence and inserted specific enzyme cutting sites. P53AIP1 gene was amplified by PCR and cloned into the adenovirus shuttle plasmid pDC316 to construct the recombinant vector pDC316-p53AIP1, which was co-transfected with helper plasmid pBHGloxδE1, 3Cre into HEK293 cells by Lipofectamine(TM); 2000. The recombinant replication-defective adenovirus (Ad-p53AIP1) was generated by means of homologous recombination of the two plasmids in HEK293 cells with the Cre-loxP recombinase system and harvested after 12 days. Ad-p53AIP1 and Ad-null were respectively transfected into HeLa cells at MOI=100. Then the expression of p53AIP1 gene was detected by Western blotting. RESULTS: pDC316-p53AIP1 was constructed successfully. The new constructed vector was confirmed by PCR analysis, double enzyme digestion and DNA sequencing. The results were in conformity with the expected. Western blotting demonstrated that the target p53AIP1 proteins were effectively expressed in the transfected HeLa cells. CONCLUSION: The recombinant adenovirus vector of Ad-p53AIP1 was successfully established, and it was proved to have a strong ability of infectivity.