Assessment of released acrosin activity as a measurement of the sperm acrosome reaction

Aim： To develop a method for assessing sperm function by measuring released acrosin activity during the acrosome reaction （AR）. Methods： Human semen samples were obtained from 24 healthy donors with proven fertility after 3-7 days of sexual abstinence. After collection, samples were liquefied for 30 min at room temperature. Standard semen parameters were evaluated according to World Health Organization （WHO） criteria. Calcium ionophore A23187 and progesterone （P4） were used to stimulate the sperm to undergo AR. After treatment, sperm were incubated with the supravital dye Hoechst33258, fixed in a glutaraldehyde-phosphate-buffered saline solution, and the acrosomal status was determined by fluorescence microscopy with fluorescein isothiocyanate-labeled Pisum sativum agglutinin （FITC-PSA）. The percentage of sperm undergoing AR （AR%） was compared to sperm acrosin activities as assessed by spectrocolorimetry. The correlation between AR% and acrosin activity was determined by statistical analysis. Results： The AR% and released acrosin activity were both markedly increased with A23187 and P4 stimulation. Sperm motility and viability were significantly higher after stimulation with P4 versus stimulation with A23187 （P 〈 0.001）. There was a significant positive correlation between released acrosin activity and AR% determined by FITC-PSA staining （r= 0.916, P 〈 0.001）. Conclusion： Spectrocolorimetric measurement of released acrosin activity might serve as a reasonable alternative method to evaluate AR. （Asian J Androl 2008 Mar, 10： 236-242）     

Relations of transcription expression of IL—2 with nuclear factor of activated T cells as well as changes of C—Fos and C—Jun after trauma

Objective:To observe the relations among expression of interleukin-2(IL-2)in spleen lymphocytes,DNA binding activity of nuclear factor of activated T cells(NFAT)and expression of the partly family members C-Fos,C-Jun after trauma.Methods:A murine closed trauma model was used,animals were sacrificed6,12hours and 1,4,7,10,14days,respectively after injury,Spleen lymplocytes were isolated from injured mice and stimulated with concanavalin-A,The culture supernatants were harvested and assayed for IL-2activity,Total RNA was extracted from spleen lymphocytes and assayed for IL-2mRNA.Nuclear protein was extracted,and the DNA binding activity of NFAT was measured using an electrophoretic mobility shift assay(EMSA),the expressions of C-Fos,C-Jun protein determined by Western blot analysis.Results:The expressions of IL-2 activity and IL-2mRNA in spleen lymphocytes were decreased in injured mice compared with those in control mice,and the most obvious decrease appeared on the 4th day after injury,The DNA binding activity of NFAT decreased gradually and reached the minimum that was only41%of the control on the 4th day after injury,which was cloely associated with the decline of IL-2activity and IL-2mRNA.An decrease in the expression ofC-Fos on the lst and 4th day after injury,trauma had no significant effect on the C-Jun expression.Conclusions:These results suggest that the inhibition of IL-2 expression is partly due to the impairment in the activation of NFAT in injured mice;and the decline in the DNA binding activity of NFAT is partly due to trauma block in the C-Fos expression.     

表达端粒酶逆转录酶siRNA的溶瘤腺病毒对裸鼠肾癌移植瘤的治疗作用

Objective To investigate the antitumor effect of oncolytic adenovirus armed with small interference RNA targeting hTERT gene for renal cancer therapy. Methods Nude mice were divid-ed randomly into 4 groups (8 mice/group),and were treated by intratumoral injections of ZD55-hTERT ( an oncolytic adenovirus armed with small interference RNA targeting hTERT gene) ,ZD55-EGFP ( an on-colytic adenovirus) and Ad-hTERT (replication-defective adenovirus armed with small interference RNA targeting hTERT gene) with three consecutive daily at 7 × 108 pfu/day or treated with PBS as a control. The expression of E1A and hTERT, and apoptosis of tumor xenografts were assessed by immunohistochemi-cal technique at the 7th day after injections. The tumor volume was measured at the 50th day after injec-tions. Results The tumor volume in ZD55-hTERT treatment group ( 124.1±27.5) was significantly less than that in ZD-EGFP (499.8±77.1 ) and Ad-hTERT ( 609.0±102.5 ) treatment groups. The E 1A pos-itive expression in ZD55-hTERT treatment group was significantly higher than that in Ad-hTERT treatment group. The hTERT positive expression in ZD55-hTERT treatment group was significantly lower than that in Ad-hTERT treatment group. ZD55-hTERT treatment of tumor xenografts resulted in an increased apoptotie cell death as compared with ZD55-EGFP and Ad-hTERT treatment. Conclusion The antitumor effect of ZD55-hTERT was more potent than oneolytie adenovirus ZD55-EGFP and Ad-hTERT.     

表达端粒酶逆转录酶siRNA的溶瘤腺病毒对裸鼠肾癌移植瘤的治疗作用

Objective To investigate the antitumor effect of oncolytic adenovirus armed with small interference RNA targeting hTERT gene for renal cancer therapy. Methods Nude mice were divid-ed randomly into 4 groups (8 mice/group),and were treated by intratumoral injections of ZD55-hTERT ( an oncolytic adenovirus armed with small interference RNA targeting hTERT gene) ,ZD55-EGFP ( an on-colytic adenovirus) and Ad-hTERT (replication-defective adenovirus armed with small interference RNA targeting hTERT gene) with three consecutive daily at 7 × 108 pfu/day or treated with PBS as a control. The expression of E1A and hTERT, and apoptosis of tumor xenografts were assessed by immunohistochemi-cal technique at the 7th day after injections. The tumor volume was measured at the 50th day after injec-tions. Results The tumor volume in ZD55-hTERT treatment group ( 124.1±27.5) was significantly less than that in ZD-EGFP (499.8±77.1 ) and Ad-hTERT ( 609.0±102.5 ) treatment groups. The E 1A pos-itive expression in ZD55-hTERT treatment group was significantly higher than that in Ad-hTERT treatment group. The hTERT positive expression in ZD55-hTERT treatment group was significantly lower than that in Ad-hTERT treatment group. ZD55-hTERT treatment of tumor xenografts resulted in an increased apoptotie cell death as compared with ZD55-EGFP and Ad-hTERT treatment. Conclusion The antitumor effect of ZD55-hTERT was more potent than oneolytie adenovirus ZD55-EGFP and Ad-hTERT.     

表达端粒酶逆转录酶siRNA的溶瘤腺病毒对裸鼠肾癌移植瘤的治疗作用

Objective To investigate the antitumor effect of oncolytic adenovirus armed with small interference RNA targeting hTERT gene for renal cancer therapy. Methods Nude mice were divid-ed randomly into 4 groups (8 mice/group),and were treated by intratumoral injections of ZD55-hTERT ( an oncolytic adenovirus armed with small interference RNA targeting hTERT gene) ,ZD55-EGFP ( an on-colytic adenovirus) and Ad-hTERT (replication-defective adenovirus armed with small interference RNA targeting hTERT gene) with three consecutive daily at 7 × 108 pfu/day or treated with PBS as a control. The expression of E1A and hTERT, and apoptosis of tumor xenografts were assessed by immunohistochemi-cal technique at the 7th day after injections. The tumor volume was measured at the 50th day after injec-tions. Results The tumor volume in ZD55-hTERT treatment group ( 124.1±27.5) was significantly less than that in ZD-EGFP (499.8±77.1 ) and Ad-hTERT ( 609.0±102.5 ) treatment groups. The E 1A pos-itive expression in ZD55-hTERT treatment group was significantly higher than that in Ad-hTERT treatment group. The hTERT positive expression in ZD55-hTERT treatment group was significantly lower than that in Ad-hTERT treatment group. ZD55-hTERT treatment of tumor xenografts resulted in an increased apoptotie cell death as compared with ZD55-EGFP and Ad-hTERT treatment. Conclusion The antitumor effect of ZD55-hTERT was more potent than oneolytie adenovirus ZD55-EGFP and Ad-hTERT.     

表达端粒酶逆转录酶siRNA的溶瘤腺病毒对裸鼠肾癌移植瘤的治疗作用

Objective To investigate the antitumor effect of oncolytic adenovirus armed with small interference RNA targeting hTERT gene for renal cancer therapy. Methods Nude mice were divid-ed randomly into 4 groups (8 mice/group),and were treated by intratumoral injections of ZD55-hTERT ( an oncolytic adenovirus armed with small interference RNA targeting hTERT gene) ,ZD55-EGFP ( an on-colytic adenovirus) and Ad-hTERT (replication-defective adenovirus armed with small interference RNA targeting hTERT gene) with three consecutive daily at 7 × 108 pfu/day or treated with PBS as a control. The expression of E1A and hTERT, and apoptosis of tumor xenografts were assessed by immunohistochemi-cal technique at the 7th day after injections. The tumor volume was measured at the 50th day after injec-tions. Results The tumor volume in ZD55-hTERT treatment group ( 124.1±27.5) was significantly less than that in ZD-EGFP (499.8±77.1 ) and Ad-hTERT ( 609.0±102.5 ) treatment groups. The E 1A pos-itive expression in ZD55-hTERT treatment group was significantly higher than that in Ad-hTERT treatment group. The hTERT positive expression in ZD55-hTERT treatment group was significantly lower than that in Ad-hTERT treatment group. ZD55-hTERT treatment of tumor xenografts resulted in an increased apoptotie cell death as compared with ZD55-EGFP and Ad-hTERT treatment. Conclusion The antitumor effect of ZD55-hTERT was more potent than oneolytie adenovirus ZD55-EGFP and Ad-hTERT.     

表达端粒酶逆转录酶siRNA的溶瘤腺病毒对裸鼠肾癌移植瘤的治疗作用

Objective To investigate the antitumor effect of oncolytic adenovirus armed with small interference RNA targeting hTERT gene for renal cancer therapy. Methods Nude mice were divid-ed randomly into 4 groups (8 mice/group),and were treated by intratumoral injections of ZD55-hTERT ( an oncolytic adenovirus armed with small interference RNA targeting hTERT gene) ,ZD55-EGFP ( an on-colytic adenovirus) and Ad-hTERT (replication-defective adenovirus armed with small interference RNA targeting hTERT gene) with three consecutive daily at 7 × 108 pfu/day or treated with PBS as a control. The expression of E1A and hTERT, and apoptosis of tumor xenografts were assessed by immunohistochemi-cal technique at the 7th day after injections. The tumor volume was measured at the 50th day after injec-tions. Results The tumor volume in ZD55-hTERT treatment group ( 124.1±27.5) was significantly less than that in ZD-EGFP (499.8±77.1 ) and Ad-hTERT ( 609.0±102.5 ) treatment groups. The E 1A pos-itive expression in ZD55-hTERT treatment group was significantly higher than that in Ad-hTERT treatment group. The hTERT positive expression in ZD55-hTERT treatment group was significantly lower than that in Ad-hTERT treatment group. ZD55-hTERT treatment of tumor xenografts resulted in an increased apoptotie cell death as compared with ZD55-EGFP and Ad-hTERT treatment. Conclusion The antitumor effect of ZD55-hTERT was more potent than oneolytie adenovirus ZD55-EGFP and Ad-hTERT.     

表达端粒酶逆转录酶siRNA的溶瘤腺病毒对裸鼠肾癌移植瘤的治疗作用

Objective To investigate the antitumor effect of oncolytic adenovirus armed with small interference RNA targeting hTERT gene for renal cancer therapy. Methods Nude mice were divid-ed randomly into 4 groups (8 mice/group),and were treated by intratumoral injections of ZD55-hTERT ( an oncolytic adenovirus armed with small interference RNA targeting hTERT gene) ,ZD55-EGFP ( an on-colytic adenovirus) and Ad-hTERT (replication-defective adenovirus armed with small interference RNA targeting hTERT gene) with three consecutive daily at 7 × 108 pfu/day or treated with PBS as a control. The expression of E1A and hTERT, and apoptosis of tumor xenografts were assessed by immunohistochemi-cal technique at the 7th day after injections. The tumor volume was measured at the 50th day after injec-tions. Results The tumor volume in ZD55-hTERT treatment group ( 124.1±27.5) was significantly less than that in ZD-EGFP (499.8±77.1 ) and Ad-hTERT ( 609.0±102.5 ) treatment groups. The E 1A pos-itive expression in ZD55-hTERT treatment group was significantly higher than that in Ad-hTERT treatment group. The hTERT positive expression in ZD55-hTERT treatment group was significantly lower than that in Ad-hTERT treatment group. ZD55-hTERT treatment of tumor xenografts resulted in an increased apoptotie cell death as compared with ZD55-EGFP and Ad-hTERT treatment. Conclusion The antitumor effect of ZD55-hTERT was more potent than oneolytie adenovirus ZD55-EGFP and Ad-hTERT.     

Effect of local testicular heating on spermatogenic cell apoptosis in rats

Aim: To study the effect of testicular local heating on spermatoge-nic cell apoptosis in rats. Methods: Seventy male SD rats were divided into the heat-treated and the control groups. The former was exposed to heat (43 ℃) for 12 hours. Each group was further divided into seven subgroups with respect to the time of observation after heat exposure, i.e., 12 h and 1 days, 3 days, 6 days, 10 days, 50 days and 80 days, respectively. In each subgroup, sper-matogenic cell apoptosis was examined by means of electron microscopy, flow cytometry and terminal deoxynucleotidyl trans-ferase-mediated dUDP-nick end labeling (TUNEL) methods. Results: The percentage of cells with sub-haploid and the percentage of positive TUNEL cells were significantly higher in the heat-treated groups than in the controls (P<0.01). The reaction of cell apoptosis to local heat was highly selective: spermatocytes were the most sensitive, followed by spermatids, spermatozoa and sper-matogonia in a decreasing order. Conclusion: Local testic     

端粒酶hTERT基因反义核酸对前列腺癌细胞PC3端粒酶活性的抑制作用

目的探讨hTERT基因的两端部分硫代修饰反义寡核苷酸（ASPS—ODN）对前列腺癌细胞PC3端粒酶活性的抑制作用。方法采用端粒重复序列扩增法及TRAP-PCR-ELISA法检测前列腺癌细胞的端粒酶活性；采用RT-PCR方法检测hTERT基因mRNA的表达水平；以免疫组化通过流式细胞仪检测hTERT基因蛋白水平的变化。结果hTERT基因的两端部分硫代修饰ASPS—ODN作用于PC3细胞48h，其端粒酶活性下降，作用72h，其端粒酶活性受到抑制。结论通过hTERT基因的两端部分硫代修饰ASPS—ODN特异性抑制hTERT基因mRNA的表达，可降低前列腺癌细胞PC3端粒酶活性。     

端粒酶hTERT基因反义核酸对TNF-α诱导前列腺癌PC3细胞凋亡的影响

目的:探讨hTERT基因的两端部分硫代修饰反义寡核苷酸(AS PS-ODN)抑制前列腺癌细胞PC3端粒酶活性后,肿瘤坏死因子α(TNF-α)对PC3凋亡的增敏作用。方法:采用端粒重复序列扩增法及TRAP-PCR-ELISA法检测前列腺癌细胞的端粒酶活性;采用MTT比色试验观察hTERTAS PS-ODN与TNF-α共同作用对前列腺癌细胞PC3生长活力的影响;倒置显微镜观察凋亡细胞的形态变化;通过流式细胞仪测定凋亡细胞的百分率。结果:hTERTAS PS-ODN作用于PC3细胞48h,其端粒酶活性下降,作用72h,其端粒酶活性受到抑制,与正义寡核苷酸组及空白对照组相比差异有显著性(P<0.05)。hTERTAS PS-ODN作用于PC3细胞后加入TNF-α作用48h,PC3细胞的抑制率与对照组、S PS-ODN组、AS PS-ODN组、TNF-α组及S PS-ODN+TNF-α组相比有统计学差异(P<0.01)。hTERTAS PS-ODN作用于PC3细胞后加入TNF-α作用48h,细胞出现典型的凋亡形态变化。hTERT ASPS-ODN作用于PC3细胞后加入4μg/ml TNF-α作用48h,凋亡细胞的百分率分别与对照组、S PS-ODN组、AS PS-ODN组、TNF-α组及S PS-ODN+TNF-α组相比差异有显著性(P<0.05)。结论:hTERTAS PS-ODN能降低前列腺癌细胞PC3端粒酶活性;hTERT基因反义核酸对TNF-α诱导前列腺癌细胞PC3凋亡有增敏作用。     

反义人端粒酶逆转录酶基因转染对人胃癌细胞系的影响

目的探讨反义人端粒酶(hTERT)基因治疗的可行性。方法构建hTERT基因的反义表达载体,经脂质体介导转染人未分化胃癌细胞系HGC-27,通过Southernblot检测外源反义基因的整合;RT-PCR及DNA测序法检测反义基因的转录;RT-PCR半定量方法检测被封闭目的基因mRNA的转录水平;TRAP及PCRELISA方法检测细胞的端粒酶活性;流式细胞仪检测细胞周期变化。结果外源反义hTERT基因已整合入细胞并获稳定转录,且能显著封闭目的基因转录的mRNA,并显著抑制HGC-27细胞的端粒酶活性,抑制HGC-27细胞的增殖并促进其凋亡。结论端粒酶反义hTERT基因可有效地应用于胃癌的基因治疗。     

反义核酸对大鼠A型精原细胞端粒酶活性及其mTR亚基表达的影响

目的 :探讨小鼠端粒酶RNA(mTR)基因的反义寡核苷酸 (ASODN)对体外培养的大鼠A型精原细胞端粒酶活性及其mTR亚基表达的影响。　方法 :以脂质体LipofectAMINE 2 0 0 0 (LF 2 0 0 0 )介导将硫代磷酸修饰的端粒酶mTR的ASODN、正义寡核苷酸 (SODN)、随机寡核苷酸 (RODN)及单纯脂质体组分别转染体外分离纯化的SD大鼠A型精原细胞 ,采用生物发光技术和TRAP SYBR Green染色法检测精原细胞中端粒酶活性的改变 ,原位杂交检测mTR基因mRNA的表达。　结果 :端粒酶mTR的ASODN明显抑制精原细胞端粒酶活性 (P <0 .0 1) ;mTR ASODN作用于精原细胞 2 4h后 ,mTR基因mRNA的表达水平显著下调。单纯脂质体组及SODN、RODN对照组均无此抑制作用 (P >0 .0 5 )。　结论 :硫代修饰的端粒酶mTR ASODN可使精原细胞端粒酶活性明显受到抑制 ,其机制可能是在mTR基因转录水平影响精原细胞端粒酶活性。     

表没食子儿茶素没食子酸酯对肝癌细胞端粒酶逆转录酶和c-myc基因表达的调控作用

目的探讨表没食子儿茶素没食子酸酯(EGCG)作用肝癌细胞时端粒酶活性的变化、端粒酶逆转录酶(TERT)及cmyc基因表达的改变,以探讨EGCG对肝癌细胞端粒酶活性的调控机制。方法用聚合酶链反应免疫酶联吸附试验(PCR-ELISA)测定肝癌细胞端粒酶活性,逆转录聚合酶链反应(RTPCR)检测肝癌细胞中人端粒酶逆转录酶(hTERT)和c-mycmRNA表达。结果在一定时间、一定剂量范围内,EGCG能抑制肝癌细胞端粒酶活性,随着浓度的增加和时间的延长,端粒酶活性逐渐下调;尤其当EGCG浓度超过50mg/L或作用时间超过36h时,端粒酶活性下调更明显,与对照组比较差异用统计学意义(P<0.01);EGCG能抑制肝癌细胞hTRETmRNA的表达,其下降趋势与端粒酶活性下降趋势基本一致,且下调幅度较端粒酶活性下降更明显,两者呈正相关(r=0.931,P<0.01);而EGCG对肝癌细胞cmycmRNA表达的抑制率比hTRETmRNA表达的抑制率更高,并且c-mycmRNA表达先受到抑制,与hTRETmRNA表达的抑制相关明显(r=0.907,P<0.01)。结论EGCG对c-myc的抑制作用可能导致hTERTmRNA的抑制并进一步下调端粒酶活性。