Egr参与调控皮质酮处理的睾丸Leydig细胞中FasL启动子的活性

目的观察转录因子Egr(early growth response factor,早期生长反应因子)是否参与调控皮质酮处理的Leydig细胞中FasL启动子的活性。方法经RT-PCR检测皮质酮处理的Leydig细胞中Egr-2及Egr-3的mRNA的水平,采用双荧光素酶报告基因系统评价Egr-2和Egr-3表达载体对Leydig细胞中FasL启动子活性的影响。结果Leydig细胞经皮质酮处理后,细胞内Egr-2及Egr-3在转录水平呈显著上调。双荧光素酶报告基因检测发现,Egr-2及Egr-3表达载体均可上调皮质酮处理的Leydig细胞中FasL启动子的活性,并以Egr-3的作用为显著。结论Egr-2及Egr-3参与调控皮质酮处理的Leydig细胞中FasL启动子的活性。     

Ca2+和CaN凋亡信号通路参与皮质酮诱导的Leydig细胞凋亡中FasL的表达

目的观察在皮质酮诱导的大鼠Leydig细胞凋亡中,Ca2 和钙调神经磷酸酶(CaN)依赖的信号通路是否参与FasL表达的调控。方法利用钙定性探针Fluo-3/AM检测皮质酮作用下的Leydig细胞中Ca2 浓度变化。通过酶底物法测定CaN活性。以Westernblot检测FasL表达。用Annexin-Ⅴ-FITC和PI双标评价Leydig细胞凋亡率。结果经超生理剂量皮质酮处理的Leydig细胞中出现Ca2 浓度升高,CaN活性增加及FasL表达增加。环孢菌素A可抑制CaN活性,使FasL表达下调,细胞凋亡率下降。结论Ca2 和CaN依赖的信号通路参与了皮质酮诱导的大鼠Leydig细胞凋亡;CaN介导了由Ca2 引发的FasL表达,Ca2 和CaN在大鼠Leydig细胞凋亡过程中起重要作用。     

PrLZ基因3′-UTR区荧光素酶报告基因载体的构建及活性检测

目的分析前列腺亮氨酸拉链(PrLZ)基因3′非编码区(3′-UTR)可能的微小RNA(miRNA)调控位点,构建PrLZ基因3′-UTR荧光素酶报告载体,为研究PrLZ基因转录后的miRNA调控提供有效的工具。方法使用PCR方法从PrLZ阳性的C4-2细胞中扩增含PrLZ基因3′-UTR区序列,插入到T-Vector载体上,提高PCR产物的连接、克隆效率,通过Xbal和BamHI限制性内切酶双酶切后将其连入经相同内切酶双酶切后的荧光素酶报告基因载体pLUC中,构建pLUC-PrLZ 3′-UTR载体,使用生物信息学方法预测PrLZ基因3′-UTR可能是miR-34a的作用靶点,使用慢病毒载体构建对照载体(质粒名称-NC)和过表达miR-34a(质粒名称-miR-34a),包装成病毒颗粒后分别感染293-T细胞,然后通过X-tremeGENE HP DNA Transfection Reagent转染试剂将pLUC空质粒和pLUC-PrLZ 3′-UTR重组质粒分别转染293-T细胞,Promega试剂盒测定荧光素酶的活性。结果得到含PrLZ基因3′-UTR序列的荧光素酶报告重组质粒,并用凝胶电泳和基因测序的方法验证了其序列的正确性。在miR-34a高表达组的293-T细胞中,重组质粒组荧光素酶活性比空质粒组低40%,差异具有统计学意义(P0.05)。结论成功构建PrLZ基因3′-UTR荧光素酶报告载体,miR-34a可以显著降低荧光素酶的活性,PrLZ基因3′-UTR上可能有miR-34a的调控作用靶点。     

含Survivin启动子条件复制型腺病毒的构建及其对前列腺癌抑瘤效应的体外研究

目的：构建携带有Survivin启动子和报告基因的条件复制型腺病毒并观察该病毒对前列腺癌细胞的特异性溶瘤作用。方法：以前列腺癌细胞系LNCaP细胞全基因组DAN为模板,PCR扩增Survivin启动子,构建pGL3BSurvivin质粒表达载体,荧光素酶检测系统观察前列腺癌细胞中Survivin启动子活性。将pGL3BSurvivin亚克隆至穿梭质粒pShuttle中,构建含有Survivin启动子的重组腺病毒reADGL3BSurvivin。转染HEK293细胞进行重组病毒的扩增、纯化和滴度检测。利用CCK-8法检测重组腺病毒对前列腺癌细胞的生长抑制作用,以正常前列腺细胞为对照。结果：经多种限制性内切酶酶切、PCR及测序鉴定,证实成功构建了含Survivin启动于报告基因质粒表达载体。荧光素酶报告基因检测结果表明前列腺癌细胞内Survivin启动子活性明显高于正常细胞组。同时也证实成功构建了含Survivin启动子和报告基因的腺病毒载体;CCK-8结果显示reADGL3BSurvivin可有效抑制前列腺癌细胞增殖而对正常细胞无增殖抑制作用。结论：成功构建含Survivin启动子的条件复制型腺病毒具有选择性杀伤前列腺癌细胞的能力,实验结果为前列腺癌靶向治疗提供了良好的条件复制型病毒载体及新的治疗策略。     

pCGN-HAM-N1-wcAPC质粒的构建及其功能

目的 构建带有HAM标签的真核表达重组质粒pCGN-HAM-N1-wcAPC及探讨其功能.方法 制备高效率感受态;用聚合酶链反应(PCR)扩增,将腺瘤息肉病(APC)基因截短的片段APC1克隆于T载体;同时将pCMV-neo-bam-APC酶切,得到APC基因的另一个8300 bp截短片段kAPC亚克隆于pCGN-HAM-N1真核表达载体,得重组质粒pCGN-HAM-N1-kAPC;再运用双酶Sal Ⅰ和Kpn Ⅰ同时酶切TA克隆、pCGN-HAM-N1-kAPC,将APC1片段插入载体pCGN-HAM-N1-kAPC,获含野生型全长APC基因重组质粒pCGN-HAM-N1-wcAPC.用酶切,测序鉴定.脂质体法分别将含全长,截短的重组质粒,空载体与WNT信号系统的β-连环蛋白(β-catenin)、topflash和荧光素酶共同转染293T细胞,利用双荧光报告测试系统,检测转染后荧光素酶活性.结果 重组质粒经酶切其大小与预期一致,经测序比对证实与GeneBank中给出的序列一致.转染pCGN-HAM-N1-wcAPC组的荧光素酶活性明显低于转染空载体组pCGN-HAM-N1(P＜0.05).结论 含野生型全长9000 bp大片段APC基因分段成功插入pCGN-HAM-N1载体,带有HAM标签的真核表达重组质粒pCGN-HAM-N1-wcAPC构建成功,pCGN-HAM-N1-wcAPC抑制T细胞因子/淋巴增强因子(Tcf/Lef)启动子的活性.     

养精胶囊通过调控StAR启动子促进睾酮合成的研究

目的探讨养精胶囊促进睾丸间质(Leydig)细胞合成睾酮(T)功能的具体作用机制。方法在Leydig细胞(MLTC-1细胞系)中加入不同剂量养精胶囊提取液24 h后,用化学发光法检测细胞上清T浓度;用免疫荧光显微镜分析类固醇快速调节蛋白(StAR)的表达情况;用RT-PCR及Western blot法测StAR mRNA及蛋白质的表达;用双荧光素酶报告基因实验检测StAR启动子的活性。结果低、中、高剂量养精胶囊提取液作用24 h后,均可以促进MLTC-1细胞合成睾酮的功能;养精胶囊提取液可以促进MLTC-1细胞StAR免疫荧光的表达;养精胶囊提取液可以促进MLTC-1细胞StAR mRNA和蛋白质的表达;养精胶囊提取液可以提高MLTC-1细胞中StAR启动子的活性。结论养精胶囊能通过调控Leydig细胞中StAR启动子的活性,促进StAR mRNA和蛋白的表达,进而提高睾酮合成的功能。     

皮质酮诱导的大鼠Leydig细胞凋亡中caspase-3活化途径的研究

目的我们新近的研究表明，超生理剂量的皮质酮（大鼠体内的糖皮质激素）可通过激活caspase-3诱导大鼠Leydig细胞凋亡。本研究拟评价在皮质酮诱导的大鼠Leydig细胞凋亡过程中，caspase-3的激活是否有其上游的caspase-8平和 caspase-9的参与。方法采用荧光分光光度法榆测经皮质酮处理的大鼠Leydig细胞中caspase-8活性，以DNA梯状电泳条带作为评价细胞凋亡的指标，观察caspase-8抑制剂是否能够抑制细胞凋亡，采用RTPCR枪测皮质酮诱导的大鼠Leydig细胞中caspase-9的mRNA水平。结果在皮质酮诱导的大鼠Leydig细胞中出现caspase-8活性增高，以12h最为址著，升高的caspase-8的活性可被caspase-8抑制剂抑制，并导致Leydig细胞的凋亡过程被阻断。Leydig细胞中caspase-9的mRNA水平在皮质酮作用下上升，同样以12h最为显著。结论皮质酮诱导的大鼠Leydig细胞调亡与caspase-8和caspase-9有关。     

甾体类激素受体AR、ER对前列腺癌中L-plastin表达的调控

目的研究甾体类激素受体AR、ER对前列腺癌L-Plastin基因的调控作用，确定甾体类激素对前列腺癌的调节作用。方法构建L-plastin启动子于含荧光素酶的pGL3质粒，利用PCR定点突变法依次构建切除ARE1、ARE2、ARE3、ERE序列的pGL3重组子，将重组子转染到前列腺癌细胞系LNCaP，检测荧光素酶强度，以了解ARE1、ARE2、ARE3、ER调控L-plastin表达能力。结果成功构建了L-plastin启动子的荧光素酶pGL3重组子以及切除ARE1、ARE2、ARE3、ERE位点的重组子，将这些重组子转染细胞后其荧光素酶强度有明显改变，切除ARE1位点后，荧光素酶活性下降1／3；切除ARE2后，荧光素酶活性与单纯切除ARE1相比下降1／2；切除ARE3后荧光素酶活性升高1倍；切除ERE后。荧光素酶活性下降约4／5。结论在L-plastin启动子转录活性中ARE1、ARE2、ARE3、ERE在前列腺癌中对其下游基因L-plastin起到调控作用，ARE1、ARE2、ERE起促进作用，ARE3起抑制作用。而且，L-plastin启动子上存在其他受甾体类激素调控的位点。     

端粒酶逆转录酶启动子联合CD自杀基因靶向杀伤人肝癌细胞研究

目的 利用肿瘤特异性人端粒酶逆转录酶(hTERT)启动子介导自杀基因CD表达,检测其促进5-FC对肝癌细胞系的杀伤作用.方法 采用聚合酶链反应(PCR)技术获得hTERT基因的启动子片段,将其连至SV40增强子序列后,并克隆到表达荧光素酶基因的报告质粒上,检测hTERT基因启动子在肝癌细胞系Bel-7402和人成纤维细胞HDF中的转录活性,同时将CD自杀基因连至该调控序列后,转染肝癌细胞Bel-7402,通过逆转录(RT)-PCR、噻唑蓝(MTT)比色法检测其作为肝癌基因治疗中肿瘤特异性靶向杀伤载体的可行性.结果 成功构建pGL3-SV40-hTERT载体和SV40-hTERT-CD-GFP质粒载体,转染ST-CD载体后Bel-7402细胞表达CD基因,转染PGL3-SV40-hTERT质粒后Bel-7402细胞中hTERT启动子高表达,相对活性为354%.与未转染组比较,5-Fc对转染SV40-hTERT-CD-GFP质粒载体的肝癌细胞Bel-7402的杀伤效应明显增强(F=27.831,P<0.01).结论 hTERT启动子在肝癌细胞系中具有较强的转录活性,能有效地诱导CD自杀基因的表达.     

Egr在高浓度皮质酮诱导的大鼠Leydig细胞凋亡中的表达及作用

目的 观察在高浓度皮质酮处理的大鼠Leydig细胞中Egr mRNA的表达量,并评价Egr在高浓度皮质酮诱导的Leydig细胞凋亡的作用.方法 采用RT-PCR方法检测经高浓度皮质酮以及高浓度皮质酮加环孢菌素A (CsA)处理的Leydig细胞中早期生长反应因子(early growth response factor,Egr) Egr-2及Egr-3的mRNA表达水平:用Annexin-V -FITC和Pl双标法检测Egr-2和Egr-3表达载体对经高浓度皮质酮诱导的Leydig细胞凋亡率的影响.结果 经高浓度皮质酮处理的Leydig细胞其Egr-2及Egr-3mRNA的表达量均显著升高,而皮质酮的这一作用能被钙调神经磷酸酶(CaN)的抑制剂环孢菌素A(CsA)所抑制.Leydig细胞中过量表达的Egr-2及Egr-3均可促进皮质酮诱导细胞凋亡,并以Egr-3的作用较为显著.结论 高浓度的皮质酮可能通过活化CaN来诱导Leydig细胞中Egr-2和Egr-3的表达,由此促进了Leydig细胞的凋亡过程.     

NFAT2 is implicated in corticosterone-induced rat Leydig cell apoptosis

Aim： To investigate the activation of the nuclear factor of activated T cells （NFAT） and its function in the corticosterone （CORT）-induced apoptosis of rat Leydig cells. Methods： NFAT in rat Leydig cells was detected by Western blotting and immunohistochemical staining. Cyclosporin A （CsA） was used to evaluate potential involvement of NFAT in the CORT-induced apoptosis of Leydig cells. Intracellular Ca^2＋ was monitored in CORT-treated Leydig cells using Fluo-3/AM. After the Leydig cells were incubated with either CORT or CORT plus CsA for 12 h, the levels of NFAT2 in the nuclei and in the cytoplasm were measured by semi-quantitative Western blotting. The role of NFAT2 in CORT- induced Leydig cell apoptosis was further evaluated by observing the effects of NFAT2 overexpression and the inhibition of NFAT2 activation by CsA on FasL expression and apoptosis. Results： We found that NFAT2 was the predominant isoform in Leydig cells. CsA blocked the CORT-induced apoptosis of the Leydig cells. The intracellular Ca^2＋ level in the Leydig cells was significantly increased after the CORT treatment. The CORT increased the level of NFAT2 in the nuclei and decreased its level in the cytoplasm. CsA blocked the CORT-induced nuclear translocation of NFAT2 in the Leydig cells. Both CORT-induced apoptosis and FasL expression in the rat Leydig cells were enhanced by the overexpression of NFAT2 and antagonized by CsA. Conclusion： NFAT2 was activated in CORT-induced Leydig cell apoptosis. The effects of NFAT2 overexpression and the inhibition of NFAT2 activation suggest that NFAT2 may potentially play a pro-apoptotic role in CORT-induced Leydig cell apoptosis through the up-regulation of FasL.     

Involvement of nuclear factor-kappa B on corticosterone- induced rat Leydig cell apoptosis

Aim:To investigate the activation of nuclear factor-kappa B(NF-kappa B)and its function in glucocorticoid-inducedLeydig cell apoptosis.Methods:The Leydig cells were isolated from male Sprague-Dawley rats(90 days of age)andwere incubated with corticosterone(CORT,glucocorticoid in rat)for 6 h,12 h and 24 h,respectively.The P65subunit of NF-kappa B(NF-kappa B/P65)in nuclei and the inhibitor of NF-kappa B(Ikappa B)in cytoplasm wereanalyzed by Western-blotting.The Leydig cells were treated with anti-Fas antibody for 3 h followed by Westernblotting to assay the changes of NF-kappa B/P65 in nuclei and in cytoplasm.The role of NF-kappa B in CORT-induced Leydig cell apoptosis was evaluated by observing the effects of NF-kappa B/P65 overexpression and inhibit-ing activation of NF-kappa B by 100μmol/L Pyrrolidine dithiocarbamate(PDTC)on this apoptosis.Results:Thetreatment of Leydig cells with CORT increased the levels of NF-kappa B/P65 in nuclei and decreased the levels ofIkappa B in cytoplasm.Following the Leydig cells were treated with anti-Fas antibody,the levels of NF-kappaB/P65was increased in nuclei and decreased in cytoplasm.The CORT-induced Leydig cell apoptosis was inhibited byoverexpressed NF-kappaB/P65 and was enhanced by incubation with PDTC.Conclusion:NF-kappa B is activatedby increased FasL/Fas in CORT-induced Leydig cell apoptosis.NF-kappa B may play an anti-apoptotic role in thisapoptosis.(Asian J Androl 2006 Nov;8:693-702)     

皮质酮诱导的Leydig细胞凋亡中Egr的表达及其作用

目的评价皮质酮是否通过活化钙调神经磷酸酶（CaN）来诱导Leydig细胞中转录因子Egr-2及Egr-3的表达及两种Egr对皮质酮诱导的Leydig细胞凋亡的影响。方法采用RT-PCR方法检测CaN的抑制剂环孢菌素A（CsA）对经皮质酮处理的Leydig细胞中Egr-2及Egr-3的mRNA表达水平；用Annexin-V-FITC和PI双标法评价Egr-2和Egr-3的表达载体对Leydig细胞凋亡率的影响。结果皮质酮能诱导Leydig细胞中Egr-2及Egr-3的表达，且该诱导作用可被CsA抑制；过量表达的Egr-2及Egr-3均可使经皮质酮诱导的Leydig细胞凋亡率增加，并以Egr-3的作用较为显著。结论高浓度的皮质酮可通过活化CaN诱导Leydig细胞中Egr．2和Egr．3的表达，两种Egr对经皮质酮诱导的Leydig细胞的凋亡均具有促进作用。     

皮质酮诱导大鼠Leydig细胞凋亡是一经caspase-3激活的过程

目的 超生理剂量的皮质酮(大鼠体内的糖皮质激素)能诱导大鼠Leydig细胞凋亡。但有关皮质酮诱导Leydig细胞凋亡的细胞内机制尚不清楚。本研究旨在观察皮质酮是否经caspase-3激活的途径诱导大鼠Leydig细胞凋亡。方法 采用Western Blot方法检测不同时间点上经皮质酮处理的大鼠Leydig细胞中caspase-3酶原及裂解的caspase-3酶表达情况。运用荧光发光法检测不同时间点上经皮质酮处理的人鼠Leydig细胞中caspase-3酶活性。结果 caspase-3酶原表达水平在皮质酮处理6h时开始上升，12h及24h时表达量下降，而具生物活性的、裂解的caspase-3酶于12h时开始出现，24h时的表达水平最为显著。caspase-3酶活性在皮质酮处理12h时明显升高，以24h时最为显著。Caspase-3抑制剂DEVD-CHO对经皮质酮处理12、24及48h的Leydig细胞中的caspase-3酶活性均具有明显的抑制作用，加caspase-3抑制剂的处理组其细胞基因组DNA电泳未见有凋亡特征性的梯状条带。结论 皮质酮诱导的大鼠Leydig细胞凋亡是一经caspase-3激活的过程。     

Erratum

To evaluate the effect of estrogen and androgen levels on erythrocyte deformability in endocrinological erectile dysfunction patients. Methods: The estrogen level, androgen level, IR of 30 psychogenic and 15 endocriological ED were studied and the correlation between the estrogen and androgen levels and RI were analyzed. Results: There is a negative correlation between the androgen and estrogen levels and IR;     

FasL cDNA转染对直肠癌细胞顺铂耐药性的影响

目的:探讨FasLcDNA转染和表达对直肠癌细胞耐药性的影响。方法:用RT-PCR方法克隆人FasL全长cDNA,构建pcDNA3.1-FasL真核表达载体,用脂质体法转染HR-8348人直肠癌细胞,采用MTT法检测顺铂对转染和未转染直肠癌细胞的生长抑制率。结果:DNA测序证实克隆FasLcDNA898bp与GeneBank序列完全一致。构建真核表达载体转染HR-8348细胞后,FasLmRNA表达明显增强。在不同浓度顺铂(1、5、10、20、40mg/L)的作用下,FasL转染组直肠癌细胞抑制率分别为11.0%、25.4%、31.2%、37.8%、42.4%:对照组癌细胞抑制率分别为26.1%、34.4%、37.6%、42.9%、53.2%,其差异有显著性意义(t=4.43,P<0.05)。结论:FasL转染HR-8348细胞可增强癌细胞的耐药性,减弱顺铂对HR-8348细胞的杀伤作用。     

LIGHT／INF—γ基因配比转染HepG2细胞对其凋亡及Fas／FasL系统表达的影响

目的：观察脂质体介导的LIGHT和IFN—γ配比转染HepG2细胞后对其凋亡及Fas和FasL表达的影响。方法：将HepG2细胞分为LIGHT单独转染组、LIGHT和IFN—γ联合转染组和空白对照组，以脂质体为中介转染HepG2细胞；分别于转染后12、24和48h收集HepG2细胞，流式细胞术检测转染后HepG2细胞的凋亡率及Fas和FasL的表达。结果：LIGHT基因转染HepG2细胞能明显促进其凋亡，随时间延长凋亡率增加；Fas和FasL在HepG2细胞高表达，以Fas升高程度显著。结论：LIGHT和IFN—γ联合转染HepG2，其凋亡效果优于LIGHT单独转染，主要是通过上调Fas的表达来促进HepG2细胞凋亡。     

Inhibition of 11beta-hydroxysteroid dehydrogenase enzymatic activities by glycyrrhetinic acid in vivo supports direct glucocorticoid-mediated suppression of steroidogenesis in Leydig cells

Previous studies have suggested that glucocorticoid (GC) can directly affect testicular testosterone (T) biosynthesis by Leydig cells through a receptor-mediated mechanism. Interconversion of corticosterone (CORT), the active form in rodents, and 11-dehydroCORT, the biologically inert 11-keto form, is catalyzed by 11betaHSD1. This enzyme thus controls the intracellular concentration of active GC. We have postulated that elevated CORT levels resulting from stress exceed the Leydig cell's capacity for metabolic inactivation of CORT, resulting in suppressed T production. The present study tested whether inhibition of 11betaHSD1 in vivo, by the administration of glycyrrhetinic acid (GA), increases intracellular active GC concentration and thereby affects serum T concentration and Leydig cell T production. Adult Sprague-Dawley rats were treated with vehicle (corn oil), CORT, GA, or GA + CORT. Serum luteinizing hormone (LH), CORT, and T levels were measured, as were the steroidogenic capacities of purified Leydig cells. Twofold elevations of CORT were achieved by the administration of either CORT or GA alone, but in both cases there was no effect on serum T levels. However, when CORT and GA were administered in combination, serum CORT levels increased 3.5-fold (to 420 +/- 34 ng/mL) and serum T levels were reduced significantly (to 0.72 +/- 0.07 ng/mL; control, 2.12 +/- 0.23 ng/mL). Serum levels of LH were not affected by CORT, GA, or GA + CORT. Consistent with the reduced serum T levels following GA + CORT, steroidogenic enzyme expression and capacities were significantly reduced compared to control. These data support a role for 11betaHSD1 in modulating intracellular CORT concentrations and, in turn, for a direct effect of GC on Leydig cells in response to stress.