[Expression of COX10 in human non-obstructive azoospermia testes].

OBJECTIVE: To evaluate the expression of COX10 mRNA in the testes of non-obstructive azoospermia patients and normal men. METHODS: A cDNA microarray containing COX10 and some other genes as RBM and EIF1AY was used to identify the differential gene expression profiles in the normal and azoospermic testes. The cDNA probes were prepared by labeling mRNA from azoospermic and normal testis tissues with Cy5-dUTP and Cy3-dUTP respectively through reverse transcription. The mixed cDNA probes were then hybridized with cDNA microarray. Later the fluorescent signals were scanned and the values of Cy5-dUTP and Cy3-dUTP on each spot were calculated and analyzed. After that an ISH was employed to detect the expression of COX10 mRNA in 10 fertile and 39 non-obstructive azoospermic testes, and the expression levels were compared to evaluate the significance. RESULTS: We obtained 128 differentially expressed genes that might be related with azoospermia, among which 56 were up-regulated and 72 down-regulated, with the expression of COX10 significantly decreased. In situ hybridization confirmed that the mRNA expression of COX10 was stronger in the spermatogenic cells of the normal fertile than the azoospermic testes. CONCLUSION: COX10 may play a certain role in the development and progression of azoospermia. The technique of cDNA microarray can be applied to further studies of screening non-obstructive azoospermia associated genes.

[A multi-center clinical trial of Qianlieantong tablets for chronic prostatitis].

OBJECTIVE: To observe the efficacy and safety of Qianlieantong Tablets in the treatment of chronic prostatitis. METHODS: A multi-center, self-controlled open clinical trial was conducted. A total of 280 subjects with chronic prostatitis were enrolled and treated by Qianlieantong Tablets, 3 times a day, 5 tablets each time. Before and after 2 and 4 weeks after the administration, NIH-CPSI scores and white blood cell counts in the prostate secretion were recorded. RESULTS: Of the 273 subjects evaluated, the rates of excellence, effectiveness and ineffectiveness were 35.2% (n = 96), 47.6% (n = 130) and 17.2% (n = 47), respectively, with a total effectiveness rate of 82.8%. After 4 weeks'medication, the scores of the subjects on NIH-CPSI pain, voiding and quality of life and white blood cell counts in prostate secretion were significantly decreased compared with pre-treatment (P < 0.01). No adverse events or laboratory abnormality related to the medication were observed. CONCLUSION: Qianlieantong Tablets has a significant effect on chronic prostatitis with high safety, particularly indicated in chronic prostatitis with pelvic pain.

Biphasic effect of androgens on prostate cancer cells and its correlation with androgen receptor coactivator dopa decarboxylase.

The aim of this study was to explore the mechanism underlying the dual effect of androgen on prostate cancer cells and further explore its correlation with dopa decarboxylase (DDC), an androgen receptor (AR) coactivator and a traditional neuroendocrine differentiation (NED) marker. Cell proliferation and cycling after treatment with synthetic nonmetabolizable androgen R1881 was determined by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) method and flow cytometry. Differential gene expression was analyzed by cDNA microarrays. DDC expression during the dual effect of R1881 was further explored with microarray, quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot, and enzyme activity assays. Proliferation of LNCaP cells was inhibited by 1 nM R1881 but stimulated by 0.1 nM R1881. Compared with the untreated cells, 320 (2.26%; 170 up-regulated, 150 down-regulated) and 4608 (32.65%; 2046 up-regulated, 2562 down-regulated) genes were found to be expressed differentially in the 1 nM and 0.1 nM R1881-treated cells, respectively. The results were partially confirmed by RT-PCR and Western blot. The DDC gene was down-regulated in the 1 nM R1881-treated cells and up-regulated in 0.1 nM R1881- and 30 nM hydroxyflutamide-treated cells. The enzymatic activity of DDC in the latter 2 groups was also strengthened. Meanwhile, the NED markers CgA and synaptophysin were not affected by these AR activators. R1881 had a dose-dependent biphasic effect on LNCaP cell proliferation. AR coactivator DDC was respectively down- and up-regulated in high and low concentrations of R1881. DDC up-regulation by exogenous AR activators is not accompanied by up-regulation of definitive NED markers.

Change of the cell cycle after flutamide treatment in prostate cancer cells and its molecular mechanism.

AIM: To explore the effect of androgen receptor (AR) on the expression of the cell cycle-related genes, such as CDKN1A and BTG1, in prostate cancer cell line LNCaP. METHODS: After AR antagonist flutamide treatment and confirmation of its effect by phase contrast microscope and flow cytometry, the differential expression of the cell cycle-related genes was analyzed by a cDNA microarray. The flutamide treated cells were set as the experimental group and the LNCaP cells as the control. We labeled cDNA probes of the experimental group and control group with Cy5 and Cy3 dyes, respectively, through reverse transcription. Then we hybridized the cDNA probes with cDNA microarrays, which contained 8 126 unique human cDNA sequences and the chip was scanned to get the fluorescent values of Cy5 and Cy3 on each spot. After primary analysis, reverse transcription polymerase chain reaction (RT-PCR) tests were carried out to confirm the results of the chips. RESULTS: After AR antagonist flutamide treatment, three hundred and twenty-six genes (3.93%) expressed differentially, 97 down-regulated and 219 up-regulated. Among them, eight up-regulated genes might be cell cycle-related, namely CDC10, NRAS, BTG1, Wee1, CLK3, DKFZP564A122, CDKN1A and BTG2. The CDKN1A and BTG1 gene mRNA expression was confirmed to be higher in the experimental group by RT-PCR, while p53 mRNA expression had no significant changes. CONCLUSION: Flutamide treatment might up-regulate CDKN1A and BTG1 expression in prostate cancer cells. The protein expressions of CDKN1A and BTG1 play an important role in inhibiting the proliferation of cancer cells. CDKN1A has a great impact on the cell cycle of prostate cancer cells and may play a role in the cancer cells in a p53-independent pathway. The prostate cancer cells might affect the cell cycle-related genes by activating AR and thus break the cell cycle control.

[Antisense oligonucleotide targeting survivin induces apoptosis of renal clear-cell carcinoma cells and enhances their sensitivity to epirubicin in vitro].

OBJECTIVE: To investigate the effect of antisense oligonucleotide (ASODN) targeting survivin on the apoptosis and proliferation of renal cancer cell line 786-O and enhancement of its sensitivity to epirubicin. METHODS: ASODN targeting survivin was designed and constructed. Cultured cells were divided into 6 groups: control group, liposome group, sense oligonucleotide (SODN) group, 600 nmol/L ASODN group, and 600 nmol/L ASODN combined with epirubicin group. After transfected for 24 h, cultured cells were harvested to carry on the next tests. Cell morphological changes were examined by transmission electron microscopy. Survivin protein was detected by immunohistochemical method. Apoptosis index (AI) and proliferation index (PI) were examined by flow cytometry. RESULTS: Morphological abnormalities of cells were observed in ASODN transfected groups. Expression of survivin in ASODN groups were significantly decreased compared with that in the control group, liposomes group and SODN group. AI of ASODN groups was significantly higher than that in other groups. PI of ASODN groups was significantly lower than that in other groups. The PI of ASODN combined with epirubicin group was (35.7 +/- 1.67)%, but (9.3 +/- 0.34)% or (8.5 +/- 0.21)% in liposomes group or SODN group that had combined with epirubicin. The ASODN group achieved the strongest effects to enhance apoptosis in comparison with control group (P < 0.05), while SODN did not cause statistically significant change (P > 0.05). CONCLUSION: The expression of survivin protein in the renal clear cell carcinoma cell line 786-O is downregulated by survivin ASODN. ASODN targeting survivin induces apoptosis and inhibits proliferation of 786-O cells. Inhibition of survivin enhances sensitivity of 786-O to epirubicin.

Expression and significance of Rap1A in testes of azoospermic subjects.

AIM: To evaluate the Rap1A mRNA expression and its significance in the testes of normal and azoospermic subjects. METHODS: A cDNA microarray that contained Rap1A and some other genes such as RBM, EIF1AY was used to identify the differential gene expression profiles between the normal and azoospermic testes. cDNA probes were prepared by labeling mRNA from azoospermic and normal testicular tissues through reverse transcription with Cy5-dUTP and Cy3-dUTP, respectively. The mixed cDNA probes were then hybridized with cDNA microarray (each containing 4096 unique human cDNA sequences). The fluorescent signals were scanned and the values of Cy5-dUTP and Cy3-dUTP on each spot were analyzed and calculated. In situ hybridization was employed to detect the expression of Rap1A in the testes of 10 fertile and 39 azoospermic subjects. RESULTS: One hundred and twenty-eight differentially expressed genes were found to be possibly related to azoospermia, of which 56 were up-regulated and 72, down-regulated genes. The mRNA expression of Rap1A in the spermatogenic cells of azoospermic was stronger than that in those of the fertile testes. CONCLUSION: Rap1A may play certain roles in the development of azoospermia.

[The biological characteristics of beta glucuronidase cDNA transfected renal cancer cell line GRC 1/betaG].

AIM: To establish a renal carcinoma cell line which can highly express beta-glucuronidase(betaG), and to observe the biological characteristics of the transfected cells. METHODS: Recombinant eukaryotic expression vector pcDNA3.1-betaG was constructed. It was transfected into renal cancer cells GRC-1 via liposome. The transcription and expression of betaG gene were detected by dot blot and Western blot. The biological characteristics of the betaG gene transfected cells was observed under light microscope, transmission electron microscope and flow cytometry. RESULTS: Dot blot and Western blot detection confirmed that the betaG gene had been stably integrated into the genomic DNA of the GRC-1 cells and was highly expressed. Transmission electron microscope observation showed that the lysosomes and endoplasmic reticulum were abundant, the number of microvili and process was significantly increased in the transfected cells, but growth condition and cell cycle of GRC-1 cells had no notable difference before and after transfection. CONCLUSION: A renal carcinoma cell line that can highly express betaG gene was established, which lays the foundation for further study on gene therapy.

[A study of aspermia-related genes by genchips and analysis of the RAP1A gene].

OBJECTIVE: To study the differential gene expression profiles between the normal and aspermia human testes by genechips. METHODS: Probes were prepared from mRNA extracted from both normal and aspermia testes and employed on Biostar H-40s genechips to detect the differential gene expression profiles. A distinctly up-regulated gene RAP1A was analyzed by bibliogrphic retrieval. RESULTS: Six hundred and twenty-three differential expressed genes were found, among which the distinctly up-regulated gene RAP1A was closely related to human sperm regulation. CONCLUSIONS: Screening the differential gene expression profiles between the normal and aspermia human testes by genechips can be used in the study of aspermia-related genes.

[Protective effect of nitric oxide synthase inhibitor (L-NAME) on germ cell apoptosis in experimentally cryptorchid rats].

OBJECTIVE: To explore the protective effect of nitric oxide synthase inhibitor (L-NAME) on the germ cell apoptosis in the rat cryptorchid. METHODS: Immature rats (22 day-old Sprague Dawley) were subjected to unilateral cryptorchid. Thirty rats were divided into three groups: sham operation group (testes still in the scrotum after operation); operation group; operation + L-NAME group(given L-NAME 10 mg/kg after operation, dip). Seven days after operation germ cell apoptosis was detected by terminal-deoxynucleotidyl transferase mediated-dUTP nick end labeling(TUNEL). Biochemical parameters (NO, NOS) were evaluated with spectrophotometric determination. RESULTS: At the 7th day after the operation, compared with the control, the number of apoptotic germ cells in the cryptorchid testis was increased significantly, but the testis weight was decreased predominantly(P < 0.01). The levels of NO and NOS in the cryptorchid were significantly higher than the control. CONCLUSIONS: The levels of NO and NOS might be involved in the germ cell apoptosis in the cryptorchid; L-NAME could protect the germ cell from apoptosis in experimentally cryptorchid rats by reducing the activity of NOS and reducing the level of NO in the testis.

[The relationships of PSA, FPSAR, clinical and pathological stage in patients with prostate cancer].

OBJECTIVES: To investigate the relationship between clinical and pathological stage, serum prostate specific antigen (PSA) concentration and free-to-total PSA ratio (FPSAR) in patients with prostate cancer. METHODS: Clinical and pathological stage were determined on the basis of pathological examination and clinic material in 42 prostate cancer patients treated by prostatectomy. PSA and FPSAR were measured before the operation. Spearman rank correlation was applied to evaluate the relationship between clinical and pathological stage, serum PSA concentration and FPSAR. RESULTS: Serum PSA concentration was significantly positively correlated with pathological stage(P < 0.05) but not correlated with clinical stage (P > 0.05) in prostate cancer patients. FPSAR was significantly correlated with pathological stage and negatively correlated with clinical stage in prostate cancer patients (P < 0.05). CONCLUSIONS: FPSAR is a more powerful predictor of clinical stage, pathological stage and prognosis than PSA.