c-Fos对大鼠间质细胞睾酮生成的影响及其作用机理

目的　本研究拟通过反义技术观察c fosASODNs对hCG诱导大鼠睾丸间质细胞睾酮分泌及睾酮合成过 程中限速酶表达水平的变化,并探讨其作用机制。方法　(1)放射免疫方法研究c fosASODNs对hCG诱导大鼠睾 丸间质细胞睾酮分泌的影响。(2)用免疫组织化学方法观察离体大鼠间质细胞Fos蛋白的变化。(3)用RT PCR 法观察在睾酮合成过程中大鼠间质细胞P450C17mRNA表达水平的变化。结果　(1)1.2×10-2IU/mlhCG可显著 性地增加间质细胞睾酮的分泌(P<0.01),并使c Fos蛋白染色阳性的间质细胞百分率和P450C17mRNA表达水 平显著性地升高(P<0.01)。(2)c fosASODNs呈剂量依赖性地抑制hCG诱导的离体间质细胞的睾酮分泌,同时 使c Fos蛋白染色阳性的间质细胞百分率下降,而无义tatODN没有相应作用。(3)当二丁酰cAMP与c fosA SODNs作用时可明显逆转c fosASODNs对hCG诱导间质细胞睾酮分泌的抑制作用,同时c Fos蛋白染色阳性的 间质细胞百分率和P450C17mRNA表达又恢复至接近hCG刺激水平。(4)维拉帕米(10-6mol/L)对hCG诱导的 大鼠间质细胞睾酮的分泌及c Fos蛋白染色阳性的间质细胞百分率均未见明显改变。结论　c fos参与hCG诱导 大鼠间质细胞睾酮的分泌,其机制很可能是通过cAMP PKA依赖性途径使c fos癌基因表达增     

重组人绒毛膜促性腺激素β-亚基(hCGβ)抗血清对hCG生物活性能力的影响

该文将重组hCGβ免疫C57BL小鼠，观察了抗hCG抗血清对hCG诱发的睾丸间质细胞分泌睾酮和雌幼SD大鼠子宫增重的影响。结果表明：重组hCGβ抗血清可不同程度抑制hCG诱发的睾丸间质细胞分泌睾酮和雌幼sn大鼠子宫增重，提示重组hCGβ抗血清与hCG形成的复合物缺乏生物活性，重组hCGβ抗血清具有中和hCG的生物活性能力。     

酪氨酸抗hCG诱导的大鼠离体Leydig细胞睾酮生成与蛋白质合成的关系

近年来我室的实验结果证明,酪氨酸除具有抗hCG 致孕酮生成作用外,对LH/hCG 诱导的雄激素和cAMP 生成也有一定的抑制作用,而且,酪氨酸还能拮抗外源cAMP 诱导大鼠离体Leydig 细胞的睾酮生成。据文献报道,hCG 能促进大鼠离体Leydig 细胞蛋白质合成,并认为新合成的蛋白质在hCG 诱导睾酮生成的过程中起重要作用。为了探讨酪氨酸抗hCG 致睾酮生成的作用机理,本文观察     

ATP50对生长激素诱导大鼠睾丸问质细胞睾酮分泌的影响

目的：研究ATP50在SD大鼠睾丸Leydig细胞中对生长激素（Growth hormone,GH）诱导睾酮合成及生长激素受体功能的影响。方法：采用离体细胞体外孵育法,观察反义ATP50寡脱氧核苷酸（Oligo deoxy nucletides,ODN）对GH诱导大鼠睾丸Leydig细胞分泌睾酮的影响及GH受体功能的影响,以睾酮放射免疫试剂盒测定睾酮合成的情况,cAMP放免试剂盒检测cAMP含量来评定GH受体的功能,免疫组织化学检测ATP50蛋白的表达。结果：反义ATP50ODN呈剂量依赖性抑制GH诱导睾丸Leydig细胞睾酮的分泌（P〈0．05）,同时使Leydig细胞中ATP50蛋白表达下降,而无义tat ODN则没有相应的作用,免疫组织化学显示ATP50蛋白阳性颗粒在Leydig细胞中中呈密集性分布,表达强度明显高于GH组（P〈0．05）。结论：在SD大鼠睾丸Leydig细胞中,ATP50可能通过增强GH受体的功能,进而刺激GH诱导睾酮的合成。提示ATP50参与GH诱导大鼠睾丸Leydig细胞睾酮的分泌过程。     

ATP50对生长激素诱导大鼠睾丸间质细胞睾酮分泌的影响

目的:研究ATP50在SD大鼠睾丸Leydig细胞中对生长激素(Growth hormone,GH)诱导睾酮合成及生长激素受体功能的影响.方法:采用离体细胞体外孵育法,观察反义ATP50寡脱氧核苷酸(Oligo deoxy nucletides,ODN)对GH诱导大鼠睾丸Leydig细胞分泌睾酮的影响及GH受体功能的影响,以睾酮放射免疫试剂盒测定睾酮合成的情况,cAMP放免试剂盒检测cAMP含量来评定GH受体的功能,免疫组织化学检测ATP50蛋白的表达.结果:反义ATP50 ODN呈剂量依赖性抑制GH诱导睾丸Leydig细胞睾酮的分泌(P＜0.05),同时使Leydig细胞中ATP50蛋白表达下降,而无义tat ODN则没有相应的作用,免疫组织化学显示ATP50蛋白阳性颗粒在Leydig细胞中中呈密集性分布,表达强度明显高于GH组(P＜0.05).结论:在SD大鼠睾丸Leydig细胞中,ATP50可能通过增强GH受体的功能,进而刺激GH诱导睾酮的合成.提示ATP50参与GH诱导大鼠睾丸Leydig细胞睾酮的分泌过程.     

地塞米松对原代培养胎盘绒毛滋养细胞11β-HSD2表达影响的研究

目的 探讨地塞米松(DEX)对原代培养的早产胎盘绒毛滋养细胞11β-羟基类固醇脱氢酶2(11β-HSD2)表达的影响.方法 采用酶消化法和组织块培养法进行早产胎盘原代绒毛滋养细胞培养、纯化和鉴定;用DEX(100 nmol/L)模拟两疗程糖皮质激素给药干预原代堵养的绒毛滋养细胞,荧光定量PCR和Western blot免疫印迹法检测滋养细胞11β-HSD2 mRNA及蛋白表达情况;此外在DEX干预的同时加入RU486(1 μmol/L),检测滋养细胞11μ-HSD2 mRNA和蛋白表达情况.结果 DEX干预原代培养滋养细胞,培养1 d后11β-HSD2 mRNA表达开始上升.第2 d和第3 d继续升高(P<0.05),第4 d改为无药物培养液后表达明显下降,第5 d～7 d重复给药后再次升高(P<0.05);在DEX干预的同时加入RU486(1μmol/L)共同作用,可见每天滋养细胞11β-HSD2蛋白和mRNA表达下降不显著(P>0.05).结论 重复给予DEX具有刺激原代培养的早产胎盘绒毛滋养细胞增加11β-HSD2蛋白和mRNA表达的作用}故早产胎盘滋养细胞可以通过词节11β-HSD2表达起到保护胎儿的屏障作用.     

膜联蛋白5对大鼠睾丸间质细胞睾酮分泌及3β-羟基类固醇脱氢酶表达的影响

目的先前研究发现促性腺激素释放激素(gonadotropin-releasing hormone,GnRH)刺激Leydig细胞后,膜联蛋白5(annexin 5)及睾酮水平同时增加,推测annexin 5和睾酮之间可能存在某种内在的联系,3β-羟基类固醇脱氢酶(3β-HSD)是间质细胞的标记酶。文中旨在研究annexin 5对大鼠睾丸间质细胞睾酮分泌及3β-HSD表达的影响。方法原代培养大鼠睾丸间质细胞,用不同浓度的annexin 5(0、10-10、10-9、10-8mol/L)处理间质细胞24h和用10-9mol/L的annexin 5处理细胞不同时间(6、12、24、36h),收集培养液,用化学发光法测定培养液中的睾酮含量;Western blotting方法分析睾丸间质细胞中3β-HSD的表达变化。结果不同浓度的annexin 5处理间质细胞24h后,与对照组比较,终浓度为10-10mol/L和10-9mol/L时,睾酮水平分别升高了18%[(14.38±0.30)nmol/Lvs(12.12±0.74)nmol/L](P<0.01)和26%[(15.25±0.47)nmol/Lvs(12.12±0...     

右美托咪定对脂多糖诱导RAW264.7细胞凋亡的影响

目的：探讨右美托咪定（ DEX）对脂多糖（ LPS）诱导的小鼠巨噬细胞RAW264.7凋亡的影响。方法：体外培养小鼠巨噬细胞系 RAW264.7,细胞随机分为正常对照组、LPS刺激组（10 mg/L）、DEX组（0.1、1、10、100μmol/L）和LPS＋DEX组（0.1、1、10、100μmol/L）;采用四甲基偶氮唑盐（ MTT）微量酶反应比色法检测细胞存活率,流式细胞术检测细胞凋亡,蛋白质免疫印迹法（ western blot）检测bax、bcl-2蛋白表达。结果：与正常对照组相比,DEX组（浓度为0.1、1、10μ mol/L ）对RAW 264.7细胞生存率、凋亡率以及bax、bcl-2的表达影响不明显（ P﹥0.05）;100μmol/L DEX对RAW264.7细胞生存率、凋亡率明显下降,bax的表达明显增加,bcl-2表达降低（P﹤0.05）;与 LPS刺激组相比,LPS＋DEX组（0.1,1,10μmol/L）可增加RAW264.7细胞的生存率,降低细胞凋亡率,bax表达降低,bcl-2表达增加（ P﹤0.05）,LPS＋100μmol/L DEX组细胞生存率显著降低,凋亡率显著增加,bax表达明显增加,bcl-2表达显著降低。结论：低浓度DEX（﹤10μmol/L）抑制LPS诱导RAW264.7细胞凋亡,高浓度则表现为增强作用。     

糖皮质激素对人卵巢癌细胞系HO-8910细胞周期的影响

目的:观察糖皮质激素对人卵巢癌细胞HO-8910细胞周期的影响,并初步探讨其作用机制。方法:将1×10-7mol/L地塞米松(dexamethasone,Dex)和1×10-6mol/L糖皮质激素受体阻断剂RU486分别单用或合用处理HO-8910细胞,采用流式细胞术测定细胞周期,核素掺入半定量RT-PCR检测细胞周期蛋白激酶抑制剂p21/WAF1的表达水平。结果:Dex诱导HO-8910细胞发生G0/G1期停滞,并使p21/WAF1 mRNA表达上调;RU486可逆转Dex引起的p21/WAF1表达的变化。结论:p21/WAF1的表达上调可能参与糖皮质激素引起的HO-8910细胞G0/G1期停滞作用。     

去势大鼠睾丸间质细胞移植的实验研究

目的：探讨对去势大鼠动物模型行睾丸间质细胞（Leydig细胞）移植的可行性。方法：随机选择SD大鼠离体睾丸进行Leydig细胞的体外培养及同种异体去势鼠大腿内侧肌群Leydig细胞移植，并进行绒毛膜促性腺激素（hCG)激发试验，监测其血清睾酮分泌情况的变化，结果：体外培养的Leydig细胞对hCG的刺激敏感，能分泌大量睾酮，而一旦进入机体后虽然短期内仍然成活，但分泌功能明显受阻，结论：通过施行同种异体睾丸Leydig细胞移植以提高血清睾酮含量的前景值得商榷。     

硫酸脱氢表雄酮对MIN6细胞葡萄糖刺激的胰岛素分泌的影响

目的探讨硫酸脱氢表雄酮(DHEAS)对胰岛β细胞株MIN6葡萄糖刺激的胰岛素分泌的影响。方法以葡萄糖刺激浓度为2.8mmol/L和16.7mmol/L的对数生长期MIN6细胞作为实验对象,分别以1、5、10μmol/LDHEAS干预10min和24h(不同浓度DHEAS组),以5μmol/LDHEAS或与10μmol/L糖皮质激素受体阻断剂RU486联合干预24h(DHEAS和DHEAS+RU486组),设立空白对照组。ELISA法测定细胞培养上清液中胰岛素分泌量,Real-TimePCR检测细胞胰岛素mRNA表达。结果干预后10min和24h时点,5μmol/L和10μmol/LDHEAS组MIN6细胞培养上清液中的胰岛素分泌量均显著高于空白对照组(P<0.05);干预后24h时点,DHEAS+RU486组与DHEAS组MIN6细胞培养上清液中胰岛素分泌量比较差异无统计学意义(P>0.05),但均显著高于空白对照组(P<0.05)。干预后24h时点,5μmol/L和10μmol/LDHEAS组MIN6细胞胰岛素mRNA表达均显著高于空白对照组(P<0.05)。结论 DHEAS对MIN6细胞葡萄糖刺激的胰岛素...     

原癌基因C-jun对基础状态下大鼠睾丸间质细胞睾酮分泌的影响

目的　通过反义技术观察原癌基因c jun对基础状态下大鼠睾丸间质细胞 (Leydigcells ,LC)睾酮分泌的影响 ,探讨原癌基因c jun对基础状态下大鼠LC睾酮分泌的作用。 方法　用放射免疫方法研究c jun对基础状态下大鼠LC睾酮分泌的影响。结果　原癌基因c jun反义寡脱氧核苷酸 (c junASODNs)呈剂量依赖性地抑制基础状态的大鼠离体LC的睾酮分泌 (P <0 .0 5 )。结论　c jun能促进基础状态下LC的睾酮分泌。     

胰岛素样生长因子-I和人绒毛膜促性腺激素对成年大鼠Leydig细胞葡萄糖转运蛋白基因表达调控

目的:研究胰岛素样生长因子I(IGF I)和人绒毛膜促性腺激素(hCG)对成年大鼠睾丸Leydig细胞中葡萄糖转运蛋白(GLUT)基因表达的影响,为进一步探讨Leydig细胞中睾酮的合成、分泌与葡萄糖代谢的关系提供依据。方法:采用改良的Klinefelter方法从成年大鼠睾丸中分离获得Leydig细胞;用反转录聚合酶链技术检测IGF I和hCG对原代培养的Leydig细胞中GLUT基因表达的调控作用。结果:分离得到纯度为98%的大鼠Leydig细胞,并与对照组比较,hCG可显著增加Leydig细胞中GLUT8基因mRNA的表达水平(P<0.001),且此作用具有剂量依赖性与时效性。当在试验组细胞中单独加入IGF I或IGF I和hCG作用于细胞后,发现IGF I(100ng mL)可显著增加Leydig细胞中GLUT8基因mRNA的表达(P<0.01),也可与hCG协同作用显著提高GLUT8基因的mRNA表达,该结果与IGF I(100ng mL)和hCG(10ng mL)能协同作用极显著增加睾酮合成水平(P<0.001)的结果是相吻合的。在大鼠Leydig细胞中,无论10ng mL或100ng mL还是两者同时作用于细胞,都不能影响GLUT1和GLUT3基因的mRNA水平。结论:在成年大鼠Leydig细胞中,IGF I和hCG对细胞中的GLUT8基因表达的调节作用具有特异性,其协同作用能显著提高细胞中GLUT8基因mRNA水平,增强细胞摄取葡萄糖的能力,给细胞提供更多的代谢能源,最终增加Leydig细胞睾酮的合成与分泌。     

The effects of 2-Bromopropane on Viability and Testosterone Production Ability of Rat Leydig Cells in Primary Culture

Epidemiological surveys and animal experiments have shown that 2-bromopropane induces oligozoospermia in exposed workers and inhibits spermatogensis in laboratory animals. However, the mechanism by which 2-bromopropane exerts its effects is unknown. To this end, we examined the formation of testosterone by the Leydig cells and their survival of these cells in the presence of different concentrations of 2-bromopropane in vitro. Leydig cells were isolated following vascular perfusion, enzymatic dissociation and Percoll gradient centrifugation techniques. The cells were cultured in culture dishes. After 8 h, different cultures were exposed to 2-bromopropane at concentrations of 0.01 mmol/L, 0.10 mmol/L and 1.00 mmol/L. In order to stimulate Leydig cells to secrete testosterone, human chorionic gonadotropin (hCG) was also added. Cell viability was determined using the trypan blue dye exclusion test and cell numbers were counted by hemocytometer. Testosterone secretion was detected by radioimmunoassay. The cell viability decreased after exposure to 2-bromopropane in a dose-dependent way, but no morphological change was observed. The cell number decreased in the 2-bromopropane-treated cultures. The secretion of testosterone did not manifest detectable changes in the culture treated with 0.10 mmol/L and 0.01 mmol/L of 2-bromopropane; however, it decreased significantly (P<0.02) in the presence of 1.00 mmol/L. Therefore, our results strongly suggest that 2-bromopropane may exert its cytotoxic effects on Leydig cells in vitro. We speculate that the decrease in the numbers of Leydig cells caused by 2-bromopropane was mediated by a feedback mechanism resulting from a lower testosterone concentration.     

The effects of 2—Bromopropane on Viability and Testosterone Production Ability7 of Rat Leydig Cells in Primary Culture

Epidemiological surveys and animal experiments have shown that 2-bromopropane induces oligozoospermia in exposed workers and inhibits spermatogensis in laboratory animals. However, the mechanism by which 2-bromopropane exerts its effects is unknown. To this end, we examined the formation of testosterone by the Leydig cells and their survival of these cells in the presence of different concentrations of 2-bromopropane in vitro. Leydig cells were isolated following vascular perfusion, enzymatic dissociation and Percoll gradient centrifugation techniques. The cells were cultured in culture dishes. After 8 h, different cultures were exposed to 2-bromopropane at concentrations of 0.01 mmol/L, 0.10 mmol/L and 1.00 mmol/L. In order to stimulate Leydig cells to secrete testosterone, human chorionic gonadotropin (hCG) was also added. Cell viability was determined using the trypan blue dye exclusion test and cell numbers were counted by hemocytometer. Testosterone secretion was detected by radioimmunoassay. The cell viability decreased after exposure to 2-bromopropane in a dose-dependent way, but no morphological change was observed. The cell number decreased in the 2-bromopropane-treated cultures. The secretion of testosterone did not manifest defectable changes in the culture treated with 0.10 mmol/L and 0.01 mmol/L of 2-bromopropane; however, it decreased significantly (P < 0.02) in the presence of 1.00 mmol/L. Therefore, our results strongly suggest that 2-bromopropane may exert its cytotoxic effects on Leydig cells in vitro. We speculate that the decrease in the numbers of Leydig cells caused by 2-bromopropane was mediated by a feedback mechanism resulting from a lower testosterone concentration.     

Multifactor Regulation on Expressions of MMP-9 and TIMP-1 in Endometrial Stromal Cells

Recent evidence suggests that preparation of the endometrium for implantation is notmerely a question of adequate hormonal stimulation but that implantation also depends on theinteraction between the blastocyst and the endometrium and is mediated by cytokines,growthfactors,and adhesion molecules,which are produced and secreted by the endometrium andthe blastocyst[1].Implantation is a complex process that involves embryo apposition andattachment to the maternal endometrial epithelium,the extrace…     

Inhibitory Effect of Dexamethasone on TGF-β1 Expression of Rabbit Ciliary Pigment Epithelia Cultured in Vitro

In order to elucidate the effect of dexamethasone on the expression of transforming growth factor-beta1 (TGF-β1) in ciliary pigment epithelial (CPE) cells cultured in vitro, rabbit CPE cells were cultured in vitro, treated with DMEM medium containing 0,1×10-8 , 5×10-8 , 10 × 10-8 and 50×10-8 mol/L dexamethasone respectively for 5 days. The TGF-β1 expression was detected by immunohistochemistry Supervision methods and analyzed semi-quantitatively by HMIAS-2000 image system. As opposed to in vivo, rabbit CPE cells expressed TGF-β1 under cultured circumstance in vitro. The gray scales of the positive yellow staining in the groups of 1×10-8, 5×10-8, 10×10-8 and 50× 10-8 mol/L dexamethasone were 136. 57±4.43, 140. 20±6.10, 142.98±2. 99, 146. 80± 1.68 and 150. 05± 1.94 respectively. When the concentrations of dexamethasone were equal to or higher than 5×10-8 mol/L and, the expression of TGF-β1 was inhibited. 10-7 mol/L dexamethasone showed a significant inhibition. It was suggested that CPE cells possess the potential ability of synthesizing and expressing TGF-β1. The inhibition of TGF-β1 expression by dexamethasone may be beneficial to the treatment of proliferative vitroretinopathy, also exert some influence on the secretion of aqueous humor and ciliary inflammation.     

非酶糖基化终末产物介导的ERK1/2信号转导通路在大鼠睾丸间质细胞睾酮分泌中的作用

目的：探讨非酶糖基化终末产物(AGEs)对大鼠睾丸间质细胞睾酮分泌的影响及其潜在机制。方法首先,原代培养大鼠睾丸间质细胞,采用3β-HSD免疫细胞化学方法对间质细胞进行鉴定;其次,MTT法测定不同浓度的AGEs (25μg/mL、50μg/mL、100μg/mL、200μg/mL)及200μg/mL BSA作用Leydig细胞后的细胞活力;最后,用不同浓度的AGEs及200μg/mL BSA预处理Leydig细胞12 h,之后更换含相应浓度AGEs或BSA的培养基并加入终浓度为4 U/mL的hCG,其中对照组不含AGEs和BSA,ELISA法和Western blot分别检测睾酮分泌量和p-ERK1/2的表达量。结果 MTT法表明AGEs (≤200μg/mL)对Leydig细胞的活力在本实验所研究的时间内没有影响;ELISA法和Western blot结果显示,不同浓度AGEs处理后,hCG诱导的Leydig细胞睾酮合成量和ERK1/2蛋白的磷酸化都呈现浓度依赖性下降,与对照组相比,差异有统计学意义(P<0.01)。结论 AGEs通过抑制ERK1/2的磷酸化降低大鼠Leydig cells睾酮的分泌。