BACKGROUND: Prostate cancer stem cell is an important reason for the invasion and recurrence of prostatic carcinoma. However, separation efficiency of prostate cancer stem cells is very low.OBJECTIVE: To explore the efficient method for isolating and identifying the prostate cancer stem cells from human prostatic carcinoma cell lines PC-3 and LNCap. METHODS: Prostate cancer cell lines PC-3 and LNCap were cultured in serum free medium (SFM) and serum supplemented medium (SSM), respectively. The percentage of prostate cancer stem cells from different medium was detected by flow cytometry through markers CD133 and CD44, and the properties of prostate cancer stem cells were preliminarily identified using inducing differentiation experiments.RESULTS AND CONCLUSION: PC-3 and LNCap formed sphere cells in SFM, which can be induced into adherent cells after culture in SSM. Higher percentage of CD44+/CD133+cells was obtained from LNCap cells (1.71%; 0.73%) than PC-3 cells (0.59%; 0.32%) in both SFM and SSM. The number of CD44+/CD133+ LNCap cells was more than PC-3 using both methods (P < 0.05), but the efficiency of SFM and SSM had no statistical significance (P > 0.05). However, the culture cycle was longer and number of obtained cells was less by SFM culture, directly influencing functional determination of prostate cancer stem cells. Compared with suspension culture method with SFM, SSM is more convenient and effective in isolating prostate cancer stem cells from LNCap cells.

BACKGROUND: Construction of recombinant adenovirus plasmid plays a central role in preparation of recombinant adenovirus.However, conventionally intracellular homologous recombination method is limited by complex procedures, low successful ratio,and long experimental cycle.OBJECTIVE: To construct the recombinant replication-defective adenovirus vector containing interleukin-10 (Ad.dL-10) in a SD rat, and to provide experimental evidences for eukaryotic expression and animat model studying.DESIGN, TIME AND SEI-FING: Opening study was conducted in the First Affiliated Hospital of Sun Yat-sen University from July 2005 to April 2006.MATERIALS: A SD rat, AdEasy system (USA), ThermoscdptTMRT kit & Tdzol (USA), and HEK-293 (Guangzhou, China) were used in this study. The primer of rlL-10 gene of clone rat was synthesized and sequenced in Shanghai, China.METHODS: rlL-10 gene was cloned from total RNA of healthy SD rat spleen tissue with RT-PCR, and then recombinant adenovirus plasmid named pAd.rlL-10 was obtained by homologous recombination within E.ColiBJ5183 carded with AdEasy-1 system. Last, the recombinant adenovirus was packaged, proliferated by HEK-293 cells and purified.MAIN OUTCOME MEASURES: The Ad.rlL-10 was identified using Western blot and RT-PCR methods.RESULTS: rlL-10 gene was successfully cloned from fresh spleen tissue of a SD rat and incorporated into recombinant adenovirus plasmid pad in order to obtain the Ad.rlL-10. Western blot and RT-PCR showed the rlL-10 gene and protein expressions in the cells. Finally, the rlL-10 recombinant adenovirus was obtained with the titers of 1.0x1014 pfu/mL after amplification and purification.CONCLUSION: AdEasy-1 system characterizing by simple operation and reliable results is commonly used to obtain enough quantity and quality adenovirus after homologous recombination, and HEK-293-induced package, amplification, and purification.

Objective To evaluate the effectiveness and security of Botulinum toxin type A (BTX-A) in treating interstitial cystitis /bladder pain syndrome,and also the effect of different site injection.Methods Sixty-nine consecutive BPS/IC patients from October 2011 to February 2016 were divided into three groups randomly,including control group (group A) and treatment group (group B and group C).There were 5 males and 64 females,with age from 23 to 66 years old (average 44.5 years old).Twentythree patients (1 male and 22 females,aged from 23 to 69 years old,with mean age of 44.6 years old) in control group (group A) underwent hydrodistention.Twenty-four patients (2 males and 22 females,aged from 27 to 65 years old,with mean age of 42.8 years old) in group B underwent BTX-A detrusor combined triangle injection plus hydrodistention.Twenty-two patients (2 males and 20 females,aged from 30 to 68 years old,with mean age of 44.3 years old) in group C underwent BTX-A triangle injection alone plus hydrodistention.The parameters such as daytime frequency,nocturia,visual analogue scale/score (VAS),functional cystemetric volume (FCV),post-void residual volume (PVR),QOL score,maximal bladder capacity (MBC),interstitial cystitis symtom index (ICSI),interstitial cystitisproblem index (ICPI),Qmax,and Hamilton anxiey scale (HAMA) score between pre-treatment and 1,3,6 and 9 months after therapy in the three groups were compared.Results There were no serious complications observed in the three groups.All parameters 1 month after therapy were obviously superior to that of pretreatment.The efficacy in control group decreased significantly 3 months after treatment except nocturia (3.0 vs.5.0),daytime frequency(7.0 vs.14.0)and Q (14.0 ml/s vs.13.0 ml/s).However,all parameters in research groups except PVR were still obviously superior to pretherapy.The parameters except Q and nocturia in group B had statistically significant difference from contemporaneous group A (all P ＜ 0.05).However,the parameters except Q nocturia and somatic anxiety score in group C had statistically significant difference from contemporaneous group A (all P ＜ 0.05).VAS (2.0 vs.3.0) and somatic anxiety score (6.0 vs.10.0) in group B were superior to group C (all P ＜ 0.05).When it came to 6 months after therapy,urinary frequence and urgency and pain symptoms were the same to pretherapy and all parameters had no statistically significant difference between pretherapy and after therapy in group A(all P ＞ 0.05).But all parameters except PVR in research group was still superior to pretherapy.the parameters except PVR,Q and QOL in group B had statistically significant difference from contemporaneous group A (all P ＜ 0.05).However,The parameters except PVR,Q MBC,ICSI and QOL in group C had statistically significant difference from contemporaneous group A (all P ＜ 0.05).Meanwhile,efficacy in group B was better than group C in term of ICPI (6.0 vs.8.0) and somatic anxiety score (7.0 vs.10.0) (all P ＜ 0.05).The efficacy decreased significantly 9 months after treatment in both group B and C,with no statistically significant difference compared with that of pretreatment.ICSI(10.0 vs.13.0),ICPI(9.0 vs.13.0),QOL(5.0 vs.6.0)in group B,and QOL(5.0 vs.6.0)in group C had statistically significant difference compared with the contemporary parameters in group A.ICSI(10.0 vs.12.0),MBC(285.0 ml vs.237.5 ml) in group B was better than that in group C (P ＜ 0.05).Conclusions Symptoms in IC/BPS patients can be alleviated significantly by detrusor BTX-A injection plus hydrodistention.Quality of life can be improved remarkably and HAMA scores can be reduced significantly after treatment.Thus,it's an effective therapeutic mnethod for IC/BPS,and detrusor combined triangle injection can provide a better effect than single triangle injection.

Objective The aim of this study was to investigate the expressions of and clinical significance of F-box/WD-40 domain protein 10(FBXW10) as well as the expression of cell cycle protein cyclin E( cyclin E) in renal clear cell carcinoma. Methods Immunohistochemistry SP method was used to detect the expressions of FBXW10 and cyclin E protein in 60 cases of renal clear cell carcinoma and 20 cases of adjacent normal tissues. The relationship between the expressions of FBXW10 and cyclin E,and the clinical pathological characteristics was analyzed. Results The expression rates of FBXW10 and cyclin E protein in renal clear cell carcinoma were 40. 0% ,70. 0% ,respectively and adjacent normal tissues were 55. 0% and 25. 0% ( P<0. 05). The expression of FBXW10 was correlated with the histologic grade of renal clear cell carcinoma(P=0. 041),histologic grade( P=0. 030);the ex-pression of cyclin E was correlated with the pathological tumor stage of clear cell renal cell carcinoma(P=0. 005),degree of differen-tiation(P=0. 035),and distant metastasis(P=0. 011). There was a significant correlation between the expressions of FBXW10 and cyclin E in renal clear cell carcinoma(r=0. 533,P<0. 001). Conclusion FBXW10 and cyclin E may play important roles in the development of renal clear cell carcinoma.

Objective:To explore the clinical feasibility of extra-peritoneal laparoscopic radical cystectomy based on the concept of 3D membrane anatomy.Methods:The clinical data of 10 male patients with bladder cancer who underwent 3D extra-peritoneal laparoscopic radical cystectomy + ileal-orthotopic-neobladder surgery from October 2020 to June 2021 were retrospectively analyzed. The median age was 67 years. The ASA score was 1-2 in 8 cases and 3 in 2 cases. There were 4 cases of hypertension, 2 cases of diabetes, 1 case of heart disease, no case of abdominal surgery history. During the operation, the concept of 3D membrane anatomy was used to identify the important fascia in the pelvic cavity and to find the key layers and structures in the pelvic cavity.It was separated from the prevesical fascia to the laterovesical space, and confluenced with Retzius space and Bogros space. It was dissected in the layer surrounded by the prevesical fascia, the vesicohypogastric fascia, and the urogenital fascia to complete the process of cystectomy.Results:The operations of 10 patients were completed successfully and there was no conversion to open operation. The median operation time was 276(237-325) minutes, and the median blood loss was 160(50-280)ml. The postoperative bowel recovery median time was 1.8(1-3)days, and the patients were out of bed about 1.3(1-2) days. The median postoperative hospital stay was 9(5-12) days. The number of median lymph node dissection in all patients was 10(6-20). Positive lymph nodes was found in 3 cases. Positive margin was found in no case. Postoperative tumor pathological stages were T 2 stage in 7 cases, T 3 stage in 3 cases. During the follow-up, all patients had no obvious complications. Conclusions:It is feasible to apply the concept of 3D membrane anatomy to identify and locate the key fascia structures and levels in extra-peritoneal laparoscopic radical cystectomy. The operative complications were less and the postoperative recovery was faster. The anatomy is clear during the operation, which has good safety and reduces the difficulty of the operation.

Exosomes are nanometer-sized vesicles that contain various types of biologically active components, including proteins, nucleic acids, carbohydrates, and lipids, which vary with the type and physiological state of the cell. In recent years, several studies have showed that exosomes can provide new non-invasive diagnostic and prognostic biomarkers in patients affected by cancers, including bladder cancer (BC), and the lipid bilayer membrane structure makes exosomes as promising delivery vehicles for therapeutic applications. Exosomes have the characteristics of high abundance, high stability, tissue specificity, and wide distribution in body fluids, and are secreted as various types by cells in different states, thereby possessing great potential as biomarkers for BC. Herein, we briefly summarize the functions and roles of exosomes in the occurrence and development of BC and the current progress of research on exosomes in BC, while focusing on potential clinical applications of the diagnosis, treatment, and prognosis of BC.

BACKGROUNDProstate specific membrane antigen (PSMA) can facilitate the growth, migration, and invasion of the LNCaP prostate cancer cell lines, but the underlying molecular mechanisms have not yet been clearly defined. Here, we investigated whether PSMA serves as a novel regulator of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling by employing PSMA knockdown model and PI3K pharmacological inhibitor (LY294002) in LNCaP prostate cancer cells.

METHODSPSMA knockdown had been stably established by transfecting with lentivirus-mediated siRNA in our previous study. Then, LNCaP cells were divided into interference, non-interference, and blank groups. We first testified the efficacy of PSMA knockdown in our LNCaP cell line. Then, we compared the expression of PSMA and total/activated Akt by Western blotting in the above three groups with or without LY294002 treatment. Furthermore, immunocytochemistry was performed to confirm the changes of activated Akt (p-Akt, Ser473) in groups. Besides, cell proliferation, migration, and cell cycle were measured by CCK-8 assay, Transwell analysis, and Flow cytometry respectively.

RESULTSAfter PSMA knockdown, the level of p-Akt (Ser473) but not of total-Akt (Akt1/2) was significantly decreased when compared with the non-interference and blank groups. However, LY294002 administration significantly reduced the expression of p-Akt (Ser473) in all the three groups. The results of immunocytochemistry further confirmed that PSMA knockdown or LY294002 treatment was associated with p-Akt (Ser473) down-regulation. Decrease of cell proliferation, migration, and survival were also observed upon PSMA knockdown and LY294002 treatment.

CONCLUSIONSTaken together, our results reveal that PI3K/Akt signaling pathway inhibition may serve as a novel molecular mechanism in LNCaP prostate cancer cells of PSMA knockdown and suggest that Akt (Ser473) may play a critical role as a downstream signaling target effector of PSMA in this cellular model.

Antigens, Surface ; genetics ; metabolism ; Cell Line, Tumor ; Glutamate Carboxypeptidase II ; genetics ; metabolism ; Humans ; Male ; Phosphatidylinositol 3-Kinases ; metabolism ; Prostatic Neoplasms ; enzymology ; genetics ; therapy ; Proto-Oncogene Proteins c-akt ; metabolism ; RNA Interference ; Signal Transduction ; genetics ; physiology