Protective effect of Rhodiola rosea polysaccharides on cryopreserved boar sperm.

Cryopreservation brings sublethal damage to sperm, resulting in reduced fertile life of sperm. Rhodiola rosea polysaccharides (RPs) have antiviral, antioxidant and antitumor activities. In the present study, the cryoprotective effect of RPs on boar sperm quality parameters after frozen-thawed process was investigated. Boar sperm was cryopreserved in the extender with RPs added at concentrations of 0 (used as control), 2, 4, 6, 8 and 10mg/L and their effects on the quality of frozen-thawed boar sperm were assessed. Addition of RPs significantly improved sperm motility, mitochondrial activity, acrosomal integrity, plasma membrane integrity, superoxide dismutase and glutathione peroxidase activity and decreased sperm malonaldehyde level (p<0.05). The results indicated that the addition of RPs to the freezing extender decreased the cryodamage to the boar sperm.

Alloantigen-specific T-cell hyporesponsiveness induced by dnIKK2 gene-transfected recipient immature dendritic cells.

Immature dendritic cells (iDCs) have been shown to be able to induce peripheral T-cell tolerance through distinct pathways. Here, we investigated the tolerogenic property of recipient iDCs whose maturation was arrested by a dominant negative mutant of inhibitor of nuclear factor kappa-B kinase 2 (dnIKK2) gene. We found that dnIKK2-iDCs presented a typical semi-mature morphology and expressed lower levels of CD80 and CD86, slightly higher MHC-II than untransfected iDCs. The expression of these molecules had no significant change even dnIKK2-iDCs were pulsed by donor antigen. In primary mixed leukocyte reaction (MLR), dnIKK2-iDCs exhibited impaired ability to stimulate allogeneic T-cells, but induced CD4(+)CD25(-) T-cell formation. In co-culture MLR, these CD4(+)CD25(-) T-cells suppressed T-cell alloreaction in an antigen-specific manner. Besides, CD4(+)CD25(-) T-cells inhibited IL-2 and IFN-γ release, whereas promoted IL-10 and TGF-ß secretion. These data suggested recipient dnIKK2-iDCs could maintain peripheral tolerance through down-regulating costimulatory molecule expressions and inducing CD4(+)CD25(-) T-cell formation.

[The value of histopathologic examination of testis tissue from testicular sperm aspiration].

OBJECTIVE: To evaluate histopathologic examination of the testis tissue from testicular sperm aspiration (TESA). METHODS: We analyzed the results of inverted microscopy and histopathologic examination of 96 samples of testis tissue from TESA, and compared the accuracy of the two methods in detecting sperm in the testis tissue. RESULTS: Among the 11 cases in which sperm was found by inverted microscopy, 9 were confirmed by histopathologic examination, and among the 57 cases in which sperm was not detected by inverted microscopy, 11 (19.3%) were found with sperm by histopathologic examination. Histopathologically, the cases in which sperm was not found by inverted microscopy included Sertoli-cell-only syndrome (n = 34), maturation arrest (n = 12) and hypospermatogenesis (n = 11). CONCLUSION: Histopathologic examination may reveal sperm in the TESA testis tissue proved to be sperm-absent by microscopy, and thus offer valuable information for a second testicular sperm retrieval.

Prolongation of rat renal allograft survival by CD4⁺CD25⁻ T cells induced by recipient dendritic cells transfected with IKK2dn.

Previous studies have demonstrated that recipient-derived immature dendritic cells transfected by recombinant adenovirus-mediated IKK2dn (Adv­IKK2dn) and loaded with donor splenocyte lysate generate CD4+CD25- T cells (Adv-IKK2dn-CD4+CD25- T cells). These cells may inhibit T cell responses in vitro. In the present study, Lewis (LW) rats were administered with an intravenous injection of naive CD4+ T cells, empty adenovirus (Adv-0)-dendritic cell-generated CD4+CD25- T cells (Adv-0-CD4+CD25- T cells), Adv-IKK2dn-CD4+CD25- T cells or an equal volume of normal saline, seven days prior to transplantation. The potency and the mechanism of action of Adv-IKK2dn-CD4+CD25- T cells was analyzed, as well as an investigation of their tolerogenic properties in vivo. Administration of Adv-IKK2dn-CD4+CD25- T cells in vivo to LW rats was observed to markedly prolong the survival of a kidney allograft from Brown Norway rats. Furthermore, the Adv-IKK2dn-CD4+CD25- T cell-treated group exhibited significantly reduced levels of interleukin (Il)-2 and interferon-γ production and increased Il-10 and transforming growth factor-ß (TGF-ß) secretion. The serum creatinine levels remained at low levels in the Adv-IKK2dn-CD4+CD25- T cell-treated group. Their ability to induce allogeneic T cell proliferation was markedly reduced compared with the other groups. These observations indicated that Adv-IKK2dn-CD4+CD25- T cells induce prolongation of kidney allograft survival in vivo, which is hypothesized to be due to the high expression levels of Il-10 and TGF-ß.

[Expressions of interleukin-15 and osteopontin mRNA in early stage of acute rejection of renal allograft in rats].

OBJECTIVE: To study the expressions of interleukin-15 (IL-15), osteopontin (OPN), granzyme B (GraB) and perforin (PFP) mRNA in early stage of acute rejection (AR) of renal allograft in rats. METHODS: The rat renal transplantation model was established. Male Brown Norway and Lewis rats were used as donors and recipients. Four groups were designated: CsA group (BN-->LEW, n = 10, recipients were treated with CsA i.p.); AB group (BN-->LEW, n = 10, recipients were treated with anti-IL-15 neutralizing antibody i.p.); AR group (BN-->LEW, n = 10, recipients were treated with normal saline i.p.) and control group (LEW-->LEW, n = 6, recipients were treated with normal saline i.p.). The blood samples of recipients were drawn at Days 1, 3, 5 and 7 post-transplantation. The serum expressions of IL-15, OPN, PFP and GraB mRNA of recipients were detected by real-time PCR. Allograft tissues were analyzed by pathological assays. RESULTS: In comparison with other groups, the expressions of OPN, IL-15, PFP and GraB mRNA in AR group were gradually up-regulated and peaked at Day 5. The expressions of IL-15 mRNA in CsA and AB groups were 9685 +/- 1440 and 4346 +/- 741 respectively at Day 5 post-operation. It was significantly lower than that in AR group (17 022 +/- 2153, P < 0.01). The expression of IL-15 mRNA in AB group was significantly lower than that in CsA group (P < 0.01). The expressions of OPN mRNA in CsA and AB groups (13 226 +/- 1565 vs 19 112 +/- 2908) were both significantly lower than that in AR group (24 663 +/- 2449, P < 0.01). But the expression of OPN mRNA in AB group was higher than that in CsA group (P < 0.01). At Day 5 post-transplantation, both the expressions of PFP and GraB mRNA in AB and CsA groups was lower than that in AR group (P < 0.01). The pathological results showed that severe AR occurred at Day 7 post-transplantation in AR group and whereas the extent of rejection sign relieved in AB group. CONCLUSION: In early stage of AR of renal allograft in rats, the expressions of OPN, IL-15, PFP and GraB mRNA are up-regulated. Blocking IL-15/IL-15R pathway in early stage of AR can down-regulate the expressions of PFP and GraB mRNA.

[Expressions of survivin and the splice variants survivin-2B and survivin-DeltaEx3 in bladder cancer and their clinical significance].

BACKGROUND AND OBJECTIVE: Studies on survivin over the past 5 years have shown that survivin participates in the genesis of several human cancers, including bladder cancer. Recent studies have indicated that survivin splice variants appeared to have unique subcellular localizations and functions as well. This study was to explore the roles of survivin and its two splice variants survivin-2B and survivin-DeltaEx3 in transitional cell carcinoma of bladder (BTCC). METHODS: The relative amount of survivin, survivin-2B, and survivin-DeltaEx3 mRNA of fresh carcinoma tissues from 60 patients with BTCC and 12 non-cancerous bladder tissues were detected by real-time quantitative reverse transcription polymerase chain reaction (RT-PCR), and the relationships of their expression levels in different pathologic grades to clinical stages of bladder cancer were analyzed. The time of follow-up was 4-24 months. RESULTS: Survivin, survivin-2B, and survivin-DeltaEx3 mRNA were detected in all BTCC tissues, and their relative expressions were 0.333+/-0.163, 0.056+/-0.017, and 0.124+/-0.096, respectively. In the control group,three and four samples expressed survivin and survivin-DeltaEx3 mRNA respectively, and all samples expressed survivin-2B mRNA. The expressions of survivin and survivin-DeltaEx3 mRNA were positively correlated with the pathologic grades and clinical stages (0 < r 's < 1,P < 0.05), however, survivin-2B mRNA was negatively correlated with those (-1 < r 's < 0, P < 0.05). CONCLUSION: Detecting the expression levels of survivin and its two splice variants survivin-2B and survivin-DeltaEx3 mRNA in BTCC by real-time PCR could have potential values to evaluate tumor progression and recurrence rate.

[Expression of survivin mRNA in urine exfoliated cells of patients with bladder transitional cell carcinoma detected by real-time PCR].

BACKGROUND AND OBJECTIVE: Bladder cancer is the most malignant cancer of the urinary system. However a noninvasive and sensitive method of the early diagnosis for bladder cancer has not been developed. This study was to explore the expression of survivin mRNA in urine exfoliated cells of preoperative and postoperative patients with bladder transitional cell carcinoma (BTCC), and to analyze its value in early diagnosis and postoperative monitoring. METHODS: Urine of 30 patients with initially diagnosed BTCC was collected before operation and one week, one month, six months and 15 months after operation. Urine of 10 healthy volunteers and 15 patients with cystitis was used as control. Expression of survivin mRNA in urine exfoliated cells was detected by real-time fluorescent quantitative polymerase chain reaction (real-time PCR). RESULTS: The relative copy number of survivin mRNA in the patients was (96.01+/-42.33) before operation, which was significantly higher than that of healthy volunteers and cystitis patients (P <0.05). The level of survivin mRNA was apparently declined one week after operation (25.30+/-1.51) compared with its preoperative level (P <0.05); and the level became as low as the control group after one month (13.20+/-1.49) and six months (13.90+/-1.36) (P>0.05). Patients were followed up for 15 months, and three patients recurred, whose survivin mRNA level (97.83+/-27.47) was significantly higher than that at six months after operation (P <0.05). CONCLUSION: Detecting survivin mRNA in urine exfoliated cells is sensitive for the diagnosis of BTCC. Detection survivin mRNA after operation can monitor recurrence.

[All-trans retinoic acid enhances bystander effect of suicide-gene therapy against androgen-unresponsive prostate cancer].

OBJECTIVE: To investigate the enhancing effect of all-trans retinoic acid (ATRA) on the bystander effect of the herpes simplex virus thymidine kinase(HSV-TK)/ganciclovir (GCV) against androgen unresponsive prostate cancer. METHODS: The bystander effect of the HSV-TK/GCV system was measured by methyl thiazolyl tetrazolium (MTT) assay on PC-3 cells before and after ATRA treatment. The growth and the histopathology of transplant tumors were observed in 4 groups of nude mice with prostate cancer. RESULTS: ATRA augmented significantly the bystander effect of the HSV-TK/GCV system by reducing TK positive PC-3 cells from 50% to 30% (P < 0.05). HSV-TK showed an inhibiting effect, while ATRA with the HSV-TK/GCV system produced significant effect on prostate cancer 1 week earlier than the former (P < 0.05). CONCLUSION: ATRA can argument the in vivo and in vitro bystander effect of the HSV-TK/GCV system in the treatment of androgen unresponsive prostate cancer.

[Effects of survivin small interfering RNA on biological behaviors of bladder cancer T24 cells].

BACKGROUND & OBJECTIVE: Bladder cancer, a common tumor of urinary system, is related to genetic mutations that deregulate cell cycle and render tumor cells resistant to apoptosis. Survivin, a new member of the inhibitor-of-apoptosis (IAP) family, participates in the genesis of several human cancers, including bladder cancer, through inducing cell proliferation and inhibiting apoptosis. This study was to explore the effects of small interfering RNA (siRNA) targeting survivin on the biological behaviors of bladder cancer T24 cells. METHODS: A pair of survivin siRNA were designed and synthesized, and transfected into T24 cells with increasing concentrations (50-200 nmol/L) and injected in tumor-bearing mice at different doses (5 and 50 microg). The effects of survivin siRNA on the biological behaviors of T24 cells were assessed. RESULTS: Survivin siRNA efficiently down-regulated the expression of survivin mRNA. Its maximal effect was achieved at the concentration of 100 nmol/L, at which survivin expression level was down-regulated by 75.91%, the inhibition rate of cell proliferation was 55.29% (P<0.01), and the apoptosis rate was significantly higher in siRNA-transfected cells than in control cells (45.70% vs. 2.8%, P<0.01). Intratumoral injection of 50 microg survivin siRNA significantly inhibited the growth of bladder cancer in mice (P<0.01). CONCLUSIONS: Survivin siRNA could markedly inhibit survivin expression in T24 cells, induce cell apoptosis and inhibit the growth of transplanted tumor in mice. It might be a new gene therapy tool for bladder cancer.

[Experimental study on bladder cancer by the small interfering RNA targeting survivin].

OBJECTIVE: To study the influence of small interfering RNA (siRNA) targeting survivin on the biological behavior of bladder cancer. METHODS: One pair of survivin target sequence-specific siRNA was designed, then siRNA/liposome complex was used to transfect bladder cancer cell line-T24. The efficiency of transfection and the apoptosis were detected by flow cytometry. The transcriptional level of survivin was analyzed using real time PCR. DNA sequence corresponding to siRNA targeting survivin was cloned into pRNAT-U6.1/Neo to produce plasmid targeting surviving. RESULTS: The ratio of T24 cells releasing fluorescence in total cells were 92.3%; siRNA-survivin efficiently down-regulated survivin expression (mRNA) in a dose-and time-dependent manner. Its maximal effect was achieved at the concentration of 100 nmol/L, at which survivin expression level was down regulated by 75.91%. Similar results were found in the inhibition ratio of cell growth, which was 55.29%(P< 0.01). Simultaneously the apoptotic rate was markedly increased, which was 45.70%(P< 0.01). After cutting the vector with Bam H I and Hin d III and ligating the vector with the insert by using T4 ligase, the recombinant vector was confirmed by restriction digestion and DNA sequencing. CONCLUSION: The application of siRNA-survivin can markedly inhibit survivin expression in bladder cancer cell line, induce apoptosis and inhibit the growth of the tumor. It may be a new gene therapy tool for bladder cancer. The successful construction of the siRNA expressing plasmid will facilitate the application of RNA interference technique, and lay a foundation for further studies on the function of surviving.

[Establishment and application of an orthotopic murine bladder cancer model].

BACKGROUND & OBJECTIVE: The recurrence rate of superficial bladder cancer is still high even the patients received postoperative intravesical infusion of chemotherapeutic drugs, such as mitomycin C (MMC). Some studies showed that intravesical infusion of small interfering RNA (siRNA) could suppress the growth of bladder cancer in nude mice. This study was to establish an orthotopic animal model bearing human bladder cancer, monitor tumor progression by magnetic resonance imaging (MRI), and observe the synergistic effect of survivin short hairpin RNA (shRNA) in combination with MMC for intravesical treatment using this animal model. METHODS: Human bladder cancer cell line T24 was inoculated into the bladders of 25 BALB/c nude mice to establish orthotopic bladder cancer model. MRI was performed to monitor tumor progression, using Gd-DTPA as contrast agent. The pathologic morphology of the bladders was observed. Eighteen mice bearing bladder cancer were randomized into 3 groups: untreated group, MMC group, and combination group. The bladders were weighed after 6 intravesical infusions. RESULTS: All the 25 mice developed bladder cancer after T24 cell inoculation. On MRI, no change in the bladders was observed at 7 days after inoculation, filling defect in the bladders, accordant to actual tumor size, was detected at 14, 21, and 28 days after inoculation. Pathologic examination showed that tumors grew in mucosa of the bladders at 7 days after inoculation, infiltrated into muscle layer at 14-28 days after inoculation, and invaded serosa at 35 days after inoculation. The inhibition rate of tumor growth was significantly higher in combination group than in MMC group (56.34% vs. 33.45%, P<0.05). CONCLUSION: We successfully established an orthotopic bladder cancer model, which could simulate the progression of human bladder cancer approximately. MRI is a reliable way for dynamic detection of murine orthotopic bladder tumor. Down-regulating survivin expression by RNA interference could enhance the antitumor effect of MMC.

Effect of small interfering RNA targeting survivin gene on biological behaviour of bladder cancer.

BACKGROUND: Bladder cancer is the most common type of urinary system tumours. It is frequently associated with genetic mutations that deregulate the cell cycle and render these tumours resistant to apoptosis. Survivin, a newly discovered member inhibitor of apoptosis protein (IAP) family in several human cancers, by inducing cell proliferation and inhibiting apoptosis is frequently activated in bladder cancer. We studied the influence of small interfering RNA (siRNA) targeting survivin on the biological behaviour of bladder cancer cells. METHODS: A double strand survivin target sequence specific siRNA was designed and synthesized. After transfection of bladder cancer cell line T24 by siRNA/liposome complex with increasing concentrations (50200 nmol/L), the transfectant cells were intratumourally injected at different doses (5 microg or 50 microg). The effects were measured in vitro and in vivo. RESULTS: The selected siRNA efficiently down-regulated survivin mRNA expression in a dose and time dependent manner. The maximal effect was achieved at the concentration of 100 nmol/L, at which survivin expression level was down-regulated by 75.91%. The inhibition rate of cell growth was 55.29% (P < 0.01) and the markedly increased apoptotic rate was 45.70% (P < 0.01). In vivo intratumoural injection of 50 microg siRNA-survivin could notably prevent the growth of bladder cancer (P < 0.01) in xenografted animals. CONCLUSION: The application of siRNA-survivin could markedly inhibit survivin expression in bladder cancer cell line by inducing apoptosis and inhibiting the growth of the tumour. It may become a new gene therapy tool for bladder cancer.

[Expression of nerve growth factor in cavernous tissue and its effects on the treatment of rats with diabetic erectile dysfunction].

OBJECTIVE: To investigate the content of nerve growth factor (NGF) in penis of rats with diabetic erectile dysfunction (DED) and apply rhNGF to treat the DED rat model to study the pathogenesis mechanism of DED and treatment effects by NGF for DED rat model. METHODS: Fifty adult male SD rats were randomly selected to make up diabetic models. After 8 weeks, the mRNA and protein levels of NGF in rat penis were detected. Then the rest of rats were divided into 5 groups: normal control group, non-treated diabetes group, NGF treatment group, insulin treatment group, NGF plus insulin treatment group. After 8 weeks of treatment, intracavernous pressure (ICP) was measured. The content of neuronal nitric oxide synthase (nNOS) in the erectile tissue was also value- ated by immunohistochemistry staining. RESULTS: Compared with those of normal control group, the mRNA content of NGF and protein in the penis of non-treated diabetic rats is increased significantly. Compared with non-treated diabetic group, ICP was much higher in the treated groups which received NGF or/and insulin therapy, and the changes in nNOS staining of those groups were the same. CONCLUSION: The injury of pelvic splanchnic nerves in advanced stage of diabetes may lead to DED, which may be relevant to the abnormal of NGF level or the NGF receptors. The increased extent of NGF in penis of rat with DED suggests that erectile nerves be severely damaged by diabete and the increase of producing NGF couldn't compensate the needs for reproduction of the nerve fibers. The ability of combination of NGF with its receptor may also be damaged. It is helpful to use exogenous NGF to lessen the partly neuropathy and improve the erectile dysfunction of diabetic rats. The abnormal of NGF may play an important role in the pathogenesis of diabetic ED and its treatment.

[Enhancement effect of interferon gamma on the sensitivity of RT4 bladder cancer cells to 5'-deoxy-5-fluorouridine,and 5-fluorouracil through up-regulation of PD-ECGF/TP].

BACKGROUND & OBJECTIVE: Platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP) is an essential enzyme in converting 5'-deoxy-5-fluorouridine (5'-DFUR), and 5-fluorouracil (5-FU) to their active metabolites in vivo, and can be up-regulated by some cytokines such as interleukin-1, and interferon gamma (INFgamma). This study was to observe the regulative effect of INFgamma on expression of PD-ECGF/TP, and its relation with the anti-cancer effect of 5'-DFUR, and 5-FU on RT4 bladder cancer cells. METHODS: PD-ECGF/TP mRNA, and protein expression were determined by reverse transcriptase polymerase chain reaction (RT-PCR), and Western blot analysis, respectively. Cytotoxicity of 5'-DFUR, and 5-FU against RT4 cells was evaluated by 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymetho-xyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay. RESULTS: Expression levels of PD-ECGF/TP mRNA and protein were elevated in RT4 cells after cultured with INFgamma. INFgamma reduced IC50s of 5'-DFUR [from (9.7+/-0.2) mmol/L to (3.7+/-0.9) mmol/L], and 5-FU [from (130.0+/-21.2) mmol/L to (49.3+/-18.4) mmol/L] in RT4 cells. CONCLUSIONS: INFgamma enhances cytotoxicity of 5'-DFUR, and 5-FU against RT4 cells through induction of PD-ECGF/TP. INFgamma/5'-DFUR or INFgamma/5-FU combination treatment may lead to better chemotherapeutic results in human bladder cancer.

[The significance of platelet-derived endothelial cell growth factor mRNA expression in superficial bladder cancer].

OBJECTIVE: To investigate the mRNA expression of platelet-derived endothelial cell growth factor (PD-ECGF) in superficial bladder cancer and its significance. METHODS: PD-ECGF mRNA expressions were determined by RT-PCR in 28 cases of superficial bladder cancers and 6 cases of normal bladder mucosa. The relation between PD-ECGF mRNA expression and tumor invasion to lamina propria or recurrence after transurethral resection was also analyzed. RESULTS: Some degree of PD-ECGF mRNA expression was present in all the samples. The PD-ECGF mRNA level was 3.1-fold higher in pT(1) tumors than in normal bladder mucosa (t = 2.13, P < 0.05) and 2.2-fold higher in pT(1) tumors than in pT(a) tumors (t = 2.66, P < 0.05); G(3) tumors expressed 3.3-fold higher PD-ECGF mRNA than normal bladder mucosa (t = 2.44, P < 0.05) and 2.5-fold higher than G(1 - 2) tumors (t = 3.36, P < 0.01). Eleven cases recurred during the mean follow-up period of 18 months. Three-fold higher PD-ECGF mRNA expression was showed in cases who recurred after transurethral resection than that in cases who did not recur (t = 4.49, P < 0.01). The specificity and sensitivity of predicting tumor recurrence were 82.4% and 81.8% respectively using 0.095 as a cutoff value of PD-ECGF mRNA level in this group of superficial bladder cancer. CONCLUSION: PD-ECGF mRNA expression correlates with tumor dedifferentiation and plays an important role in the early invasion in superficial bladder cancer. To analyze the PD-ECGF mRNA level contributes to the evaluations of tumor differentiation and invasion to lamina propria as well as recurrence prediction in superficial bladder cancer.

[The mRNA expression of platelet-derived endothelial cell growth factor of bladder transitional cell carcinoma and its relation to invasiveness].

OBJECTIVE: To investigate the relationship between mRNA expression of platelet-derived endothelial cell growth factor (PD-ECGF) and invasion of bladder transitional cell carcinoma (BTCC). METHODS: The mRNA expression of PD-ECGF in BTCC was detected by RT-PCR. The target PCR bands were analyzed by NIH Image 1.62 software. RESULTS: The mRNA level of PD-ECGF in BTCC was 3.86 times as high as that of normal bladder mucosa (t = 2.36, P < 0.05). The expression level of stage Ta, T1 and T2-4 tumor was 1.33, 4.02 and 7.59 times as high as that of normal bladder mucosa, respectively. That of Grade 3 tumor was 2.27 times as high as that of Grade 1 - 2 tumor (t = 3.52, P < 0.01). CONCLUSION: The mRNA expression of PD-ECGF was positively correlated with the invasiveness and grade of BTCC. The results suggest that the mRNA level of PD-ECGF might be used as an indicator of tumor progression and a guide for clinical treatment of bladder transitional cell carcinoma.