植物雌激素对体外培养的大鼠睾丸间质细胞分泌睾酮的作用

目的:研究植物雌激素(大豆苷元、染料木素)对睾丸间质细胞分泌睾酮的刺激作用,并初步研究其可能的分子作用机制。方法:采用Percoll不连续密度梯度离心,分离获得3月龄Sprague-Dawley(SD)大鼠的睾丸间质细胞。以人绒毛膜促性腺激素(hCG)为阳性对照药物,测定不同浓度(0、0.02、0.1、0.5、1、5μmol/L)大豆苷元、染料木素对睾丸间质细胞分泌睾酮的影响。RT-PCR检测睾酮生物合成过程中胆固醇侧链裂解酶细胞色素P450(P450scc)的表达。结果:0.1μmol/L的染料木素能显著刺激原代大鼠睾丸间质细胞的睾酮分泌,RT-PCR结果显示染料木素能显著上调睾酮合成过程中P450sccmRNA的表达。在较高浓度下(5μmol/L),大豆苷元和染料木素均能抑制睾丸间质细胞分泌睾酮。结论:染料木素在低浓度下(0.1μmol/L)显著促进睾丸间质细胞分泌睾酮,但随着浓度的增加(5μmol/L),大豆苷元和染料木素均显著抑制Leydig细胞的睾酮分泌。     

c-jun在人绒毛膜促性腺激素诱导睾丸间质细胞睾酮分泌中的作用

目的 :本研究拟通过c jun反义寡脱氧核苷酸 (ASODNs)观察c jun在调节hCG诱导的睾丸间质细胞 (LC)睾酮分泌中的作用。　方法 :用c junASODNs拮抗c jun ,用hCG诱导体外培养大鼠LC的睾酮分泌 ,用放射免疫法检测睾酮水平。　结果 :hCG可刺激LC分泌睾酮 ,是LC功能研究的有用模型。c junASODNs呈剂量依赖性地抑制hCG诱导下的离体LC的睾酮分泌 (P <0 .0 5 )。　结论 :c jun促进hCG诱导的大鼠LC的睾酮分泌。     

维拉帕米对c-jun调节睾丸间质细胞睾酮分泌的影响

目的通过c-jun反义寡脱氧核苷酸(ASODNs)观察c-jun在调节人绒毛膜促性腺激素(hCG)诱导的睾丸间质细胞(LC)睾酮(T)分泌的作用。方法采用c-jun ASODNs拮抗c-jun,维拉帕米阻断钙通道,hCG诱导体外培养大鼠LC的T分泌,放射免疫方法检测T水平。结果c-jun ASODNs(0~2μmol/L)呈剂量依赖性抑制hCG诱导的离体LC的T分泌(P<0.01)。维拉帕米(10-5mol/L)可加强c-jun ASODNs抑制T分泌。结论c-jun促进hCG诱导大鼠LC的T分泌,c-jun表达可能与钙离子相关。     

壬基酚对体外培养大鼠睾丸间质细胞分泌雄激素影响的研究

目的:探讨壬基酚(NP)对大鼠睾丸间质细胞分泌雄激素的影响。方法:建立体外培养大鼠睾丸间质细胞原代培养体系,并采用3β-HSD染色对间质细胞进行鉴定;以不同浓度的NP(分别为较低浓度0.005、0.015、0.025、0.05、0.1μmol/L及稍高浓度0.5、5.0、10.0、15.0、25.0μmol/L)作用于间质细胞,采用3β-HSD染色观察间质细胞形态的变化,并检测间质细胞分泌睾酮含量的变化。结果:间质细胞经NP处理后,细胞形态发生变化,细胞密度降低,NP作用浓度达到25μmol/L时,细胞发生裂解。0.005及0.015μmol/L NP处理后,间质细胞睾酮分泌量增加,与对照组比较差异有显著性(P<0.05)。NP浓度达到0.5μmol/L后,睾酮分泌量与对照组相比显著下降(P<0.05),且当NP浓度为5,10,15,25μmol/L时睾酮分泌量下降极为明显(P<0.01)。结论:低浓度NP能促进间质细胞分泌睾酮,而高浓度NP抑制睾酮分泌,且高浓度NP能诱导间质细胞坏死。     

c-jun对离体睾丸间质细胞睾酮分泌作用的机理研究

目的本研究拟通过c-jun反义寡脱氧核苷酸(ASODNs)观察c-jun在调节hCG促进睾丸间质细胞(leydigcells,LC)睾酮分泌中的作用机制。方法用c-junASODNs拮抗c-jun,再加用cAMP观察其对睾酮分泌的影响,用放射免疫方法检测睾酮水平。结果hCG可刺激LC睾酮分泌,是LC功能研究的有用模型。c-junASODNs呈剂量依赖性地抑制hCG诱导下的离体LC的睾酮分泌(P<0.01)。加用cAMP后睾酮分泌增加。结论c-jun促进hCG诱导的大鼠LC的睾酮分泌,c-jun表达可能与cAMP相关。     

内皮素-1对大鼠睾丸间质细胞睾酮分泌影响的可能作用机理

目的 :了解内皮素 1(ET 1)影响大鼠睾丸离体间质细胞睾酮分泌的可能作用机理 ,为男性不育症治疗提供一定的理论基础。　方法 :采用大鼠睾丸间质细胞体外孵育技术以及12 5I 睾酮放射免疫法 ,观察ET 1对间质细胞睾酮分泌的影响及其可能作用机理。　结果 :内皮素受体A(ETA)、内皮素受体B(ETB)受体拮抗剂均可逆转ET 1对大鼠离体间质细胞睾酮分泌的抑制作用 ,但Ca2 +通道阻滞剂维拉帕米 (VER)未表现此作用。　结论 :ET 1通过与ETA及ETB结合而发挥作用。其Ca2 +的动员可能不是细胞外Ca2 +进入细胞内。     

大豆黄酮对小鼠精子质量、睾丸重量及睾酮水平的影响

目的 :探讨大豆黄酮对小鼠精子质量、睾丸生长和睾酮水平的影响。　方法 :给雄性小鼠饲喂 3个不同剂量的大豆黄酮 ( 5、10 0、10 0 0mg/kg日粮 ) ,2 1d后宰杀 ,睾丸称重 ,伊红丫染色确定精子存活率 ,顶体染色采用姬姆萨染色法 ,睾酮浓度采用放射免疫法测定。　结果 :5mg/kg日粮大豆黄酮能显著提高睾酮分泌水平 (P <0 .0 1) ,睾丸明显增重 (P <0 .0 5 ) ,提高了精液质量 ;10 0 0mg/kg日粮大豆黄酮抑制睾酮的分泌 (P <0 .0 1) ,精液质量变化不明显 ;10 0mg/kg日粮的大豆黄酮对精子质量等指标均无显著影响。　结论 :大豆黄酮能影响小鼠精子质量、睾丸生长及睾酮水平 ,并与剂量有关     

老年大鼠睾丸间质细胞结构和功能变化的实验研究

目的:研究老年SD大鼠(PADAM动物模型)睾丸间质细胞形态、分泌功能变化,探讨老年大鼠睾丸间质细胞的功能状态。方法:分别取青年SD大鼠和老年各20只,静脉血测定血清总睾酮和游离睾酮的浓度,并通过组织切片和透射电镜观察两个年龄组大鼠睾丸间质细胞形态学变化;此外,分别用hCG、Forskolin刺激体外培养的两个年龄组大鼠的睾丸间质细胞,比较培养基中睾酮和孕酮的浓度。结果:老年大鼠的血清总睾酮[(3.07±0.75)nmol/L]和游离睾酮[(0.71±0.65)nmol/L]均比青年大鼠[(10.89±6.11)nmol/L和(2.42±1.02)nmol/L]显著降低(P<0.05);细胞形态有较显著差异;体外培养的大鼠睾丸间质细胞分泌能力显著降低(P<0.05)。结论:老年SD大鼠血清睾酮和游离睾酮浓度显著低于青年SD大鼠,原因在于睾酮合成酶系统整体功能衰退。     

瘦素、神经肽Y对离体人卵巢颗粒细胞分泌功能的影响

目的　探讨瘦素、神经肽Y(NPY)对离体人卵巢颗粒细胞雌、孕激素生成的影响。　方法　将来自体外受精 胚胎移植的 2 5例离体人卵巢颗粒细胞纯化后 ,在不同浓度瘦素 (1、10、10 0 μg/L)、NPY(1× 10 -8、1× 10 -7、1× 10 -6mol/L)单独或与人绒毛膜促性腺激素 (hCG ,0、2 0IU/L)联合作用下体外培养 ,采用放射免疫方法测定培养液中雌二醇 (E2 )、孕酮(P)的水平。　结果　 (1)无论单独或与hCG联合不同浓度瘦素组E2 、P水平均无显著性差异 (P >0 .0 5 ) ;(2 )NPY浓度 1× 10 -7mol/L时 ,E2 水平显著低于空白对照组 (P <0 .0 5 ) ,但与hCG联合组显著高于单独组 (P <0 .0 5 ) ;(3)NPY浓度 1× 10 -7mol/L、瘦素浓度 10 μg/L组 ,E2 水平明显高于相应浓度NPY单独作用组 (P <0 .0 5 )。　结论　 (1)瘦素不直接影响体外培养颗粒细胞E2 、P的分泌 ,可能通过影响其它因子的作用而间接调节卵巢功能 ;(2 )NPY单独作用时 ,对颗粒细胞E2 的分泌有一定抑制作用 ,但当hCG存在时作用消失 ,说明在正常月经周期中NPY对黄体的生成和维持可能无明显不利影响 ;(3)瘦素可能阻断NPY对颗粒细胞分泌E2 的抑制作用 ,表明二者可能对卵巢功能有相互协调作用。     

卵泡抑制蛋白对大鼠睾丸间质细胞分泌睾酮的影响

目的 :探讨卵泡抑制蛋白 ( follistatin,FS)对离体大鼠睾丸间质细胞 ( L eydigcell)分泌睾酮的影响。方法 :用 Percoll梯度分离法分离和培养 Wistar雄鼠 ( 2 2 0～ 2 50 g)睾丸的 L eydig细胞。分别观察 FS( rh FS-2 88)、Ca2 +以及 FS加 Ca2 +在基础状态 (不加h CG)和刺激状态 ( h CG 1 .0 IU/ L )对 Leydig细胞分泌睾酮的影响。结果 :FS抑制基础和刺激状态的睾酮分泌 ,并与剂量相关。 Ca2 +亦有抑制作用 ,但在 1 0 0 mmol/ L时出现逸脱现象。FS加 Ca2 +表现为与剂量相关的抑制作用。结果 :FS呈剂量依赖性抑制睾酮分泌 ,Ca2 +可能是 FS的第二信使 ,单独高浓度 Ca2 +出现抑制逸脱的机制未明。     

睾酮对体外大鼠阴茎海绵体组织细胞增殖的影响

目的:探讨睾酮对雄性SD大鼠阴茎海绵体组织细胞增殖的影响。方法:无菌条件下获取大鼠阴茎海绵体组织,免疫组化法检测其雄激素受体的表达,再分别采用酶消化法培养平滑肌细胞和贴壁法培养成纤维细胞,用四氮唑蓝(MTT)还原法观察对照组及睾酮10-8mol/L、10-7mol/L、10-6mol/L、10-5mol/L、10-4mol/L、10-3mol/L浓度组对海绵体组织细胞增殖的影响。结果:雄激素受体在大鼠阴茎海绵体组织表达,10-5mol/L睾酮刺激可促进大鼠阴茎海绵体平滑肌细胞和成纤维细胞增殖,MTT还原法测定分别为68100±2200和70200±1300(P<0.05);10-4mol/L睾酮刺激可抑制平滑肌细胞和成纤维细胞增殖,MTT法测定A值为分别55000±1400和59100±1500(P<0.01);其他组作用不显著。结论:大鼠阴茎海绵体组织存在雄激素受体,睾酮可经雄激素受体途径对阴茎海绵体组织细胞增殖的产生影响,不同浓度睾酮可促进或抑制海绵体平滑肌细胞和成纤维细胞的增殖。     

4-羟基他莫昔芬对前列腺基质细胞增殖与凋亡的作用

目的:研究4-羟基他莫昔芬(OHT)对原代培养的前列腺基质细胞增殖与凋亡的影响。方法:以10-8~10-5mol/L的雌二醇(E2)、己烯雌酚(DES)、OHT以及10-8~10-6mol/L的E2与10-7mol/L的OHT混合物分别作用于原代培养的前列腺基质细胞,采用MTT法和TUNEL法分别检测细胞增殖和凋亡。结果:OHT对前列腺基质细胞增殖和凋亡的作用与E2和DES比较均有显著差异(P均<0.05),在10-7~10-5mol/L浓度下表现出与浓度相关(r=-0.383,P=0.005)的抑制增殖作用(P<0.05),10-7mol/L的OHT可以抑制相同甚至更高浓度(10-6mol/L)E2的促增殖作用(P<0.05);OHT在10-8~10-5mol/L浓度下显示与浓度相关(r=0.349,P=0.012)的促凋亡作用(P<0.05),且10-7mol/L OHT的促凋亡作用不能被相同甚至更高浓度(10-6mol/L)E2所逆转(P>0.05)。结论:OHT在一定浓度范围内对原代培养的前列腺基质细胞具有明显的抑制增殖和促凋亡作用,该作用可能不完全是通过竞争性抑制雌激素受体而实现的。     

Effects of leptin and neuropeptide Y on function of human ovarian granulosa cells in vitro

Objective: To investigate the effects of leptin and neuropeptide Y on steroidogenesis of human ovarian granulosa cells in vitro. Methods: Human ovarian granulosa cells were isolated from follicular fluid obtained during oocyte retrieval of in vitro fertilization-embryo transfer program and cultured for 2 days with various concentration of leptin(1,10,100 ng/ml) or neuropeptide Y (1×10-6, 1×10-7, 1×10-8 mol/L) alone or both,or with the combination of human chorionic gonadotropin (hCG 0, 20 IU/L). The medium was collected for estradiol (E2) and progesterone (P) measurements. Results: (1)Whether hCG existed or not, the adding of leptin did not alter estradiol and progesterone production by human granulosa cells (P>0.05).(2) Only when the concentration of neuropeptide Y was at 1×10-7mol/L,estradiol level was lower than that in the control (P<0.05).(3) The levels of estradiol in neuropeptide Y (1×10-7mol/L) plus hCG group were significantly higher than those with neuropeptide Y alone(P<0.05). (4) In the absence of hCG, the levels of estradiol in neuropeptide Y (1×10-7mol/L)plus leptin (10 ng/ml) group were significantly higher than those with neuropeptide Y(1×10-7mol/L)alone(P<0.05).Conclusions: (1)Leptin alone produced no direct effect on secretion of E2 and P from granulosa cells in vitro.(2)Neuropeptide Y alone may inhibit the secretion of E2, but the inhibition would probably be blocked with the presentation of hCG.(3)Leptin probably blocked the inhibition of neuropeptide Y on E2 secretion, and this may indicate that there were some coordination between leptin and neuropeptid Y on the level of ovarian function.     

GLP-1受体激动剂艾塞那肽对MG-63细胞增殖及成骨分化的影响

目的探讨GLP-1受体激动剂艾塞那肽(Exenatide)对人成骨肉瘤MG-63细胞株增殖及成骨分化的作用。方法将MG-63细胞制成单细胞悬液,接种于细胞培养板,分别用不同浓度的艾塞那肽(0 mol/L、10-9mol/L、10-8mol/L、10-7mol/L)处理细胞,24 h后采用CCK-8比色法检测MG-63细胞增殖率,并将艾塞那肽处理后的细胞裂解并取上清,使用碱性磷酸酶测定试剂盒测定AKP活力。结果 (1)艾塞那肽可促进MG-63细胞增殖(P0.05),但随着GLP-1受体激动剂浓度升高,细胞增殖程度降低(P0.05),低浓度的艾塞那肽(10-9mol/L)促增殖能力最强。(2)艾塞那肽不同浓度处理后各组AKP活力增加(P0.05),而且低浓度的艾塞那肽(10-9mol/L)升高AKP活力最明显(P0.05)。结论艾塞那肽能促进人成骨肉瘤MG-63细胞的增殖及成骨分化,且低浓度的艾塞那肽(10-9mol/L)促增殖分化能力最强。     

胰岛素和睾酮对Ishikawa细胞HOXAIO基因表达的影响

目的观察胰岛素（INS）和睾酮（T）对子宫内膜腺癌细胞HOXA10基因表达的影响。方法免疫细胞化学检测HOXA10蛋白在Ishikawa细胞定位表达;体外培养Ishikawa细胞,予不同浓度INS（90、60、30、3、0．3U／L）或T（10^-3、10^-4、10^-5、10^-5、10^-7mol／L）刺激Ishikawa细胞48h,MTT法检测INS、T对Ishikawa细胞生长的作用;最后分别以30U／LINS和10^-5mol／LT刺激Ishikawa细胞48h,逆转录聚合酶链反应（RT-PCR）方法测定INS和T对Ishikawa细胞HOXA10mRNA表达的影响。结果（1）HOXA10蛋白定位表达于Ishikawa细胞的细胞浆内。（2）不同浓度的INS均可促进Ishikawa细胞的生长,随着浓度的增加作用越强,INS浓度达60、90U／L时,细胞生长状况与浓度为30U／L相似。不同浓度的T均可抑制Ishikawa细胞的生长,随浓度增加抑制作用越明显,浓度达10^-4、10^-3mol／L时,细胞生长抑制状况与浓度为10^-3mol／L相似。（3）RT-PCR检测结果显示INS组和T组Ishikawa细胞中HOXA10mRNA表达均较对照组减弱（P〈0．01或P〈0．05）。结论不同浓度INS和T均可影响Ishikawa细胞生长,高浓度的INS和T降低细胞上HOXA10mRNA的表达。     

A study of the effect of B-EP and naloxone on the function of the hypothalamo-pituitary-testicular axis of the rat

To investigate whether endogenous opioid peptides (EOP) play an important role in intragonadal regulation of testicular function and regulation of the hypothalamic-pituitary-gonadal axis of the male rat, the authors employed two principal methods: culture of testicular Leydig cells and Sertoli cells, and in vitro perifusion of hypothalamo-pituitary Leydig cells of the adult rat. The results demonstrated that incubation of Leydig cells with B-endorphin (B-EP 10(-9) = 10(-6) mol/L) or naloxone (NAL 10(-5) = 10(-8) mol/L) manifested no significant changes of non-stimulated or hCG-stimulated testosterone secretion both in 20 and 60 day-old rats. Similar results were obtained when the cells were treated with B-EP (10(-10) = 10(-7) mol/L) for 48 h during culture. Pretreatment of incubated Leydig cells with B-EP in similar concentrations for 48 h showed no effect on the response to hCG stimulation. In addition, treatment with B-EP in vitro for 24 or 72 h manifested no effects on estradiol production by aromatization of cultured Sertoli cells. Neither NAL 10(-5) given in vitro nor NAL (5 mg/body weight) injected subcutaneously 1 h before decapitation affected LH and testosterone release from the perifused hypothalamo-pituitary Leydig cells system. These results could not support the hypothesis that B-EP is a local regulator of testicular function. The physiological significance of EOP in regulating the function of gonadal axis of adult male rat remains to be investigated further.     

Effects of uric acid on mitochondrial oxidative damage and apoptosis in human renal tubular epithelial cells

Objective To observe the effects of uric acid (UA) on mitochondrial oxidative damage and apoptosis in renal tubular epithelial cells (HK-2), and investigate the possible mechanism. Methods HK-2 cells were exposed to UA (480 μmol/L, 720 μmol/L) for different time (0 h, 24 h, 48 h) in vitro. The mitochondrial ROS production was detected by MitoSOX staining. The mitochondrial membrane potential was measured by JC - 1 staining. The expressions of prohibitin and AIF were examined by Western blotting and immunofluorescence cytochemistry. The cell apoptosis was measured by Annexin V-FITC/PI staining. Results The mitochondrial ROS production in HK-2 cells exposed to 480 μmol/L UA was increased than that of control group at 24 h (P﹤0.05), and increased gradually with UA concentration and incubation time increasing, while the mitochondrial membrane potential was reduced at the same time. There were no significant changes in AIF expression and apoptosis rate of HK -2 cells exposed to 480 μmol/L UA for 24 h compared with that of control group (P﹥0.05), while both of them were up-regulated when HK-2 cells were exposed to 480 μmol/L UA for 48 h and 720 μmol/L UA for 24 h and 48 h (P﹤0.05). The prohibitin expression in HK-2 cells exposed to 480 μmol/L UA was reduced than that of control group at 24 h (P ﹤ 0.05), and down - regulated gradually with UA concentration and incubation time increasing. Conclusion Uric acid can induce the mitochondrial ROS production increased, the mitochondrial membrane potential reduced, the prohibitin expression down-regulated and the mitochondrial apoptosis pathway activated in HK-2 cells.     

紫杉醇和吉西他滨对前列腺癌PC-3细胞的作用

目的 :观察紫杉醇协同吉西他滨对前列腺癌细胞系PC 3的体外作用 ,并探讨其可能的作用机制。　方法 :应用光镜形态学、噻唑蓝 (MTT)法、流式细胞仪和免疫细胞化学法观察 10 -6、10 -7、10 -8mol/L浓度紫杉醇和 10 -7、10 -8、10 -9mol/L浓度吉西他滨在体外单独或协同用药对前列腺癌细胞系PC 3的作用 ,对细胞DNA含量及CyclinD1表达的影响。　结果 :10 -8mol/L以上浓度吉西他滨作用 4 8h ,可增强 10 -7mol/L以上浓度紫杉醇对前列腺癌PC 3细胞系的生长抑制 [抑制率≥ (5 0 .8± 4 .2 ) % ,P <0 .0 5 ]及诱导凋亡作用 [凋亡率≥ (2 2 .9± 2 .3) % ,P <0 .0 5 ],下调CyclinD1的表达 [表达率≤ (9.6± 1.6 ) % ],与阳性对照组CyclinD1表达率 (2 5 .5± 4 .1) %相比差异有显著性 (P<0 .0 1)。吉西他滨使紫杉醇所致的G2 /M期细胞周期阻滞比例由 (70 .3± 9.7) %变为 (38.2± 4 .2 ) % ,部分地逆转了其G2 /M期细胞周期阻滞 (P <0 .0 1)。　结论 :紫杉醇和吉西他滨可以协同增强对前列腺癌细胞系PC 3的生长抑制和诱导凋亡作用 ,显示了紫杉醇和吉西他滨协同用于治疗激素非依赖性前列腺癌的可能性 ,并部分地说明了其作用机制。     

Does gonadotropin receptor complex have an amplifying role in cAMP/testosterone production in Leydig cells?

The dose-response relationship between luteinizing hormone/human chorionic gonadotropin (LH/hCG)-stimulated biological response and 125I-labeled hCG binding was studied in purified Leydig cells from adult rat testes. The concentration of hCG needed for one-half maximal stimulation of cyclic adenosine monophosphate (cAMP) and testosterone production (ED50) was 2.16 x 10(-11)mol/L and 5.6 x 10(-13)mol/L, respectively. This suggests that extremely low levels of hormone in the range of 10(-13)mol/L hCG are sufficient to generate enough cAMP (5.66 pmol; 2.83 x 10(-9)mol/L) for steroidogenesis, thereby preserving the catalytic potential of the receptor-cyclase system. Most of the cAMP formed at 10(-13)mol/L hCG was released into the medium, and the intracellular cAMP was much less and barely detectable (0.98 x 10(-9)mol/L; 1.96 pmol/2 x 10(6) cells). The specific binding of 125I-labeled hCG to purified Leydig cells at a correspondingly higher hCG concentration (3 x 10(-10)mol/L) was extremely low and did not display a dose-dependent increase in binding. Assuming the specific binding to represent 100% occupancy of high affinity receptors (14.2 fmol/2 x 10(6) cells per 2 ml), each mole of bound hCG generated 15,423 mol cAMP and 12,817 mol testosterone. The results show that the hormone interacts with cellular receptors as a catalyst to generate the biological response. Moreover, the true affinity of hormone-receptor interaction responsible for the physiologic action is possibly much greater than previously reported for this system. This information should prove useful for reconstitution studies using the hormone receptor/G-protein/adenylate cyclase system in vitro in soluble form.