Objective To investigate the effect and mechanisms of recipient-derived immature dendritic cells(imDC) transfected with IKK2dn and loaded with donor antigen on renal allografts survival in the rats.Methods DC were cultured from recipient rats'(Lewis) bone marrow,transfected with IKK2dn and loaded with donor antigen.The expression of CD86 and MHC Ⅱ was detected,and the ability of DC stimulating lymphocyte proliferation in vitro was measured.Male Brown Norwav rats and Lewis rats were used as donors and recipients respectively.Four groups were set up(DC group,empty transfection group,transfection group and control group),receiving 1×10~7 DC,Adv-0-DC,Adv-IKK2dn-DC loaded with BN antigen,and equal volume of normal saline,respectivelv 7 davs before transplantation.In the third party donor-group,Wistar rats as donors were treated the same as DC;group before transplantation.After transplantation,the T lymphocyte proliferation in reciPients was measured and the expression of serum IL-2 and IFN-γ was detected.The survival time of recipients and the acute reiection were observed.Pathological changes were examined tO identify the grade of rejection.Results DC assessment in vivo revealed that the transfected DC could still express CD86 and MHC Ⅱ in a low level as compared with those not transfected with IKK2dn. After DC were loaded with donor's antigen,the expression of CD86 and MHC Ⅱ was up-regulated.After DC were transfected with IKK2dn before loaded with donor's antigen, the expression of CD86 and MHC Ⅱ had no significant change. When DC were loaded with donor's antigen, its allostimulatory activity of T lymphocyte proliferation was enhanced (P<0. 05). When DC were transfected with IKK2dn before loaded with donor's antigen, its allostimulatory activity of T lymphocyte proliferation was not enhanced. Compared with control groups, IKK2dn-transfected DC pulsed with BN splenocyte lysate markedly prolonged the survival of renal allografts (26. 8±1.76d, P<0.01), and elicited markedly lower proliferative responses and reduced IL-2 and IFN-γ production. The pathological grade of rejection was low in the transfection group. Conclusion Recipient-derived imDC transfected with IKK2dn and loaded with donor splenocyte lysate could prolong the renal allograft survival in rats probably by down-regulating the expression of DC costimulatory molecules and inhibiting the T_H 1 cytokine production.

OBJECTIVETo study the influence of small interfering RNA (siRNA) targeting survivin on the biological behavior of bladder cancer.

METHODSOne 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.

RESULTSThe 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.

CONCLUSIONThe 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.

Cell Line, Tumor ; Genetic Vectors ; genetics ; Humans ; Inhibitor of Apoptosis Proteins ; Microtubule-Associated Proteins ; genetics ; RNA Interference ; RNA, Small Interfering ; genetics ; Transfection ; Urinary Bladder Neoplasms ; genetics ; pathology

Aged ; Aged, 80 and over ; Biomarkers, Tumor ; urine ; Carcinoma, Transitional Cell ; urine ; Female ; Humans ; Inhibitor of Apoptosis Proteins ; Male ; Microtubule-Associated Proteins ; genetics ; Middle Aged ; Neoplasm Proteins ; genetics ; RNA, Messenger ; urine ; Reverse Transcriptase Polymerase Chain Reaction ; Urinary Bladder Neoplasms ; urine

OBJECTIVETo investigate the relationship between mRNA expression of platelet-derived endothelial cell growth factor (PD-ECGF) and invasion of bladder transitional cell carcinoma (BTCC).

METHODSThe mRNA expression of PD-ECGF in BTCC was detected by RT-PCR. The target PCR bands were analyzed by NIH Image 1.62 software.

RESULTSThe 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).

CONCLUSIONThe 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.

Adult ; Aged ; Carcinoma, Transitional Cell ; metabolism ; pathology ; Cell Differentiation ; Female ; Humans ; Male ; Middle Aged ; Neoplasm Invasiveness ; RNA, Messenger ; analysis ; Thymidine Phosphorylase ; genetics ; Urinary Bladder Neoplasms ; metabolism ; pathology

BACKGROUNDBladder 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.

METHODSA 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.

RESULTSThe 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.

CONCLUSIONThe 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.

Animals ; Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Female ; Humans ; Immunohistochemistry ; Inhibitor of Apoptosis Proteins ; Mice ; Mice, Inbred BALB C ; Microtubule-Associated Proteins ; analysis ; antagonists & inhibitors ; genetics ; Neoplasm Proteins ; analysis ; antagonists & inhibitors ; genetics ; Neoplasm Transplantation ; RNA, Small Interfering ; pharmacology ; therapeutic use ; Transfection ; Urinary Bladder Neoplasms ; pathology ; therapy

OBJECTIVETo investigate the mRNA expression of platelet-derived endothelial cell growth factor (PD-ECGF) in superficial bladder cancer and its significance.

METHODSPD-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.

RESULTSSome 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.

CONCLUSIONPD-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.

Carcinoma, Transitional Cell ; metabolism ; pathology ; Follow-Up Studies ; Humans ; RNA, Messenger ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Thymidine Phosphorylase ; genetics ; metabolism ; Urinary Bladder Neoplasms ; metabolism ; pathology

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-△Ex3 in transitional cell carcinoma of bladder (BTCC).Methods:The relative amount of survivin,survivin-2B, and survivin-△Ex3 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-△Ex3 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-△Ex3 mRNA respectively,and all samples expressed survivin-2B mRNA.The expressions of survivin and survivin-△Ex3 mRNA were positively correlated with the pathologic grades and clinical stages(0＜r'_s＜1,P＜0.05),however,survivin-2B mRNA was negativly 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-△Ex3 mRNA in BTCC by real-time PCR could have potential values to evaluate tumor progression and recurrence rate.

OBJECTIVETo evaluate histopathologic examination of the testis tissue from testicular sperm aspiration (TESA).

METHODSWe 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.

RESULTSAmong 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).

CONCLUSIONHistopathologic 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.

Adult ; Azoospermia ; pathology ; Humans ; Male ; Middle Aged ; Sperm Count ; Sperm Retrieval ; Testis ; pathology ; Young Adult

OBJECTIVETo 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.

METHODSFifty 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.

RESULTSCompared 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.

CONCLUSIONThe 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.

Animals ; Diabetes Mellitus, Experimental ; complications ; Erectile Dysfunction ; drug therapy ; etiology ; metabolism ; Male ; Nerve Growth Factor ; biosynthesis ; genetics ; therapeutic use ; Penis ; metabolism ; RNA, Messenger ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo 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.

METHODSThe 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.

RESULTSATRA 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).

CONCLUSIONATRA can argument the in vivo and in vitro bystander effect of the HSV-TK/GCV system in the treatment of androgen unresponsive prostate cancer.

Animals ; Antineoplastic Agents ; pharmacology ; Bystander Effect ; drug effects ; Cell Line, Tumor ; Cell Survival ; drug effects ; Ganciclovir ; pharmacology ; Genes, Transgenic, Suicide ; genetics ; Genetic Therapy ; methods ; Humans ; Male ; Mice ; Mice, Nude ; Prostatic Neoplasms ; genetics ; pathology ; therapy ; Reverse Transcriptase Polymerase Chain Reaction ; Simplexvirus ; enzymology ; Thymidine Kinase ; genetics ; metabolism ; Tretinoin ; pharmacology ; Xenograft Model Antitumor Assays ; methods