正常及去势大鼠组织内雌激素受体mRNA表达水平的研究

通过切除大鼠卵巢复制实验性骨质疏松动物模型，从小块胫骨组织内提取ＲＮＡ，对正常对照、切除卵巢雌激素治疗组的标本进行逆转录－聚合酶链反应（ＰＴ－ＰＣＲ）检测，直接从生理状态下的成熟骨经检测出雌激素受体（ＥＲ）ｍＲＮＡ的表达，并经测序证实。实验结果还显示切旋卵巢大鼠的ＥＲ表达水平明显降低，而予以雌激素治疗组的ＥＲ表达接近正常。提示ＥＲ的表达依赖于雌激素的存在，而雌激素通过ＥＲ在骨代谢中起作用。     

大豆异黄酮的选择性雌激素受体调节剂作用

近年来 ,选择性雌激素受体调节剂和大豆异黄酮的研究不断深入 ,逐步证实大豆异黄酮在不同组织可以表现为雌激素激动剂和 (或 )拮抗剂的作用 ,本文将对大豆异黄酮的选择性雌激素受体调节剂的作用、作用机理以及应用和发展前景等进行概述。     

不同剂量大豆异黄酮对去卵巢大鼠骨密度和血脂影响的研究

目的探讨不同剂量的大豆异黄酮对去卵巢大鼠骨密度和血脂水平的影响.方法选用43只10w龄性成熟SD雌性大鼠行去势手术,2w后采用完全随机的方法分为5组:阴性对照组(溶媒花生油灌胃)、阳性对照组(戊酸雌二醇500ng/g·BW·d)、大剂量组(大豆异黄酮2500μg/g·BW·d)、中剂量组(大豆异黄酮500μg/g·BW·d)和小剂量组(大豆异黄酮50μg/g·BW·d).分别于给药前和给药6周后测量总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白(HDC)、低密度脂蛋白(LDL),并测量腰椎和股骨的骨密度.结果给大豆异黄酮6周后,大、中、小剂量组和阳性对照组腰椎骨密度均高于阴性对照组;大剂量纽骨密度的增加,有显著性差异(P＜0.05);中剂量组骨密度增加的程度和阳性对照组(戊酸雌二醇组)相似,但与阴性对照组比较无显著性差异(P＞0.05);大豆异黄酮对股骨骨密度无明显影响.大鼠去卵巢后TC和LDL下降,给大豆异黄酮后使TC和LDL进一步下降,以大剂量组最为显著,中剂量组的作用与阳性时照组相似,小剂量组无作用.结论大豆异黄酮具有雌激素样作用,能增加去卵巢大鼠的腰椎骨密度,进一步降低由于去卵巢引起的血清TC和LDL下降,它的作用强度与剂量呈正比.因此大豆异黄酮的作用值得进一步研究,以确定它是否具有临床价值.     

宁夏枸杞叶对去势模型大鼠雌激素受体表达的影响

BACKGROUND: Lycium barbarum polysaccharide, the main active component of leaves of lycium barbarum, has a remarkable therapeutic effect on osteoporosis in adult ovariectomized female rats. OBJECTIVE: To explore effects of leaves of Lycium barbarum on serum estradiol and bone estrogen receptor expressions in adult oxariectomized female rats． METHODS: Thirty-two 6-month-od female rats were randomized into sham-surgery, model, and treatment groups, respectively. Rat models of postmenopausal osteoporosis were induced by ovariotomy followed by orally administration of water solution of leaves of Lycium barbarum (500 or 1 000 mg/kg) or distilled water once daily for consecutive 12 weeks in treatment and model groups, respectively. Serum estradiol level was detected by radiation immunoassay method. Estrogen receptor α, β immunoreactivities in bone tissue were determined by immunohistochemical staining． RESULTS AND CONCLUSION: Serum estradiol level in the model group was significantly decreased compared with the sham-surgery group (P < 0.01), but that was significantly increased in the treatment groups, particularly at high dose, compared with the model group (P < 0.01). Estrogen receptor α, β immunoreactivities were weaker in the model group than the sham-surgery group (P < 0.01), and those were stronger in the treatment groups than the model group (P < 0.01). Our results suggest that leaves of Lycium barbarum treat osteoporosis through enhancing serum estradiol and estrogen receptor expressions in adult ovariectomized rats． 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

不同雌激素对大鼠海马和脑皮质区域雌激素受体α和β表达的差异性调节研究

目的:探讨不同种类雌激素药物对雌性去势大鼠海马和脑皮质区域雌激素受体(estrogenreceptor,ER)的调节差异。方法:雌性去势大鼠分别给予倍美力或补加乐持续灌胃3个生理周期,采用半定量RT-PCR法检测雌激素受体ERα、ERβ的mRNA表达,免疫组化法检测ERα、ERβ蛋白的分布及表达。结果:倍美力组海马和脑皮质ERαmRNA表达均显著低于去势对照组,ERα蛋白在海马中相对表达量也低于对照组,但在脑皮质中表达无改变;该组上述两个区域中ERβmRNA以及脑皮质ERβ蛋白的表达无显著改变,仅海马ERβ蛋白表达升高。补加乐组脑皮质和海马ERβmRNA和蛋白表达均显著高于去势对照组,海马ERαmRNA表达也高于对照组,但脑皮质ERαmRNA及脑皮质和海马ERα蛋白的表达水平均无显著改变。结论:两种不同种类雌激素药物对雌性去势大鼠认知区域中ERα、ERβ表达的调节水平存在显著差异,这可能是选用不同的雌激素药物进行激素替代治疗产生的保护认知疗效存在差别的机制之一。     

大豆异黄酮对去势雌大鼠缺血再灌注脑组织细胞凋亡及calpain表达的影响

目的: 研究大豆异黄酮对大鼠脑缺血再灌注后缺血脑组织神经细胞凋亡和calpain表达的影响,探讨大豆异黄酮的神经保护作用。方法: 成年雌性大鼠36只,随机分为药物组和对照组,药物组再随机分为小剂量和大剂量组。所有动物切除双侧卵巢,1周后各组每日给予大豆异黄酮和生理盐水,1个月后建立大脑缺血再灌注模型,原位末端标记检测神经细胞凋亡,采用原位杂交技术观察calpain mRNA表达情况,同时比较各组calpain活性变化。结果: 大豆异黄酮组凋亡细胞数明显减少(P<0.05),神经细胞凋亡指数降低,calpain mRNA表达明显减少,calpain活性下降。药物大剂量组与小剂量组比较无明显差异。结论: 大豆异黄酮使脑缺血再灌注后神经细胞凋亡减少,可能通过下调calpain mRNA表达,降低calpain的活性发挥神经保护作用。     

大豆异黄酮对去势大鼠脂质过氧化及肝超微结构的影响

目的 研究大豆异黄酮(soybean isoflavone,SI)对去势大鼠脂质过氧化及肝超微结构的影响.方法 70只雌性SD大鼠按血清总胆固醇水平随机分为7组:高脂、雌激素,低、中、高剂量SI干预、假手术及正常对照组.前5组摘除双侧卵巢,恢复1周后开始干预,实验周期为12周,灌胃给药,每周称重1次,分别在去卵巢、给药4、8周、处死时抽取尾静脉血,实验结束后取肝脏标本,分别测定胆固醇(TC)、甘油三酯(TG)、血清高、低密度脂蛋白胆固醇(HDL-C、LDL-C)、脂蛋白(a)及抗氧化酶活性等.结果 SI干预组LDL-C水平显著低于高脂组但仍高于正常对照组;干预可使脂蛋白(a)水平发生变化但对HDL-C影响不显著;高剂量SI能缓解因去卵巢造成的肝细胞超微结构破坏,维护其完整性,全视野下以高剂量干预组与正常组结构最为接近,高脂组镜像损坏明显.结论 SI对去卵巢大鼠的脂蛋白(a)、脂质过氧化及肝超微结构存在影响,高剂量持续干预能降低去卵巢大鼠的LDL-C,并能维持肝超微结构.     

雌激素及雌激素受体α减轻谷氨酸对神经细胞的损伤

 目的：建立谷氨酸诱导的神经细胞损伤模型,观察雌激素及雌激素受体α（estrogen receptor α, ERα）对谷氨酸诱导的神经细胞损伤的作用。方法：原代培养小鼠大脑皮层神经细胞,神经元特异性烯醇化酶（neuron-specific enolase, NSE）免疫组化染色鉴定神经元的纯度。建立谷氨酸诱导的神经细胞损伤模型。用前期实验中已构建成功的ERα重组慢病毒（V-ERα-RFP-flag）感染经谷氨酸诱导的神经细胞。实时荧光定量PCR和Western blotting检测ERα mRNA和蛋白表达水平。实验分为3组：（1）对照组：用空慢病毒（V-RFP-flag）感染经谷氨酸诱导的神经细胞;（2）雌激素组：用雌激素干预经谷氨酸诱导的神经细胞;（3）慢病毒组：用V-ERα-RFP-flag感染经谷氨酸诱导的神经细胞。流式细胞术检测各组神经细胞凋亡率;实时荧光定量PCR及免疫荧光染色检测各组神经细胞中N-甲基-D-天冬氨酸受体1（N-methyl-D-aspartate receptor 1, NMDAR1）及囊泡膜谷氨酸转运体蛋白1（vesicular glutamate transporter protein 1, VGLUT1）的变化。结果：成功原代培养神经细胞,经NSE免疫组化方法鉴定神经元纯度大于90%。成功建立经谷氨酸诱导的神经细胞损伤模型。MOI=7的V-ERα-RFP-flag感染神经细胞72 h后,在荧光显微镜观察下可见到红色荧光表达,与对照病毒相比,能增加神经细胞中ERα mRNA和蛋白表达水平（P<0.01）。与对照组相比,雌激素组和慢病毒组细胞凋亡率降低（P<0.05）,且实时荧光定量PCR结果显示,雌激素组和慢病毒组的NMDAR1和VGLUT1 mRNA表达降低（P<0.05）,免疫荧光实验结果提示NMDAR1和VGLUT1阳性的细胞数减少（P<0.01）。结论：雌激素和ERα能减轻谷氨酸对神经细胞的损伤效应,该保护作用可能通过抑制NMDAR1和VGLUT1的表达来实现。     

毛蕊异黄酮防治去卵巢大鼠骨质疏松的实验研究

目的复制去卵巢大鼠骨质疏松动物模型,观察不同剂量的毛蕊异黄酮对去卵巢大鼠骨质疏松的防治作用。方法 50只SD雌性大鼠,随机分成五组：设一假手术组;腹腔手术切除大鼠双侧卵巢,分为阴性对照组、毛蕊异黄酮低、高剂量组和雌激素对照组,分别给予标准饲料和不同剂量受试物,12周后进行骨密度、骨组织形态计量、生物力学测定,与雌激素对照,观察给与毛蕊异黄酮对绝经后骨质疏松的防治作用。结果大鼠去卵巢后骨密度显著下降,股骨的力学性能有较大变化,弯曲强度和弯曲弹性模量明显降低。给予毛蕊异黄酮后,可使骨密度显著提高（P〈0.01）,存在一定的剂量-效应关系,同时弯曲强度和弯曲弹性模量明显增加,但均低于雌激素对照组。结论通过去卵巢手术,成功建立绝经后骨质疏松动物模型;毛蕊异黄酮对去卵巢大鼠具有显著的骨保护效应,并存在一定的剂量效应关系,其作用弱于雌激素。     

雌性大鼠心内神经节中雌激素受体及其mRNA的表达

目的 在雌激素受体蛋白及ERmRNA水平提供雌激素对心内神经节中神经元作用的形态学依据。方法 采用免疫组织化学及原位杂交技术。结果 在心内神经节部分神经元中，雌激素受体免疫反应及其mRNA原位杂交反应阳性。雌激素受体免疫反应沉淀物呈棕黄色，定位于胞核，雌激素受体mRNA免疫反应沉淀物呈棕黄色，定位于胞浆。结论 大鼠心内神经节中，部分神经元能合成雌激素受体蛋白，说明ER阳性神经元可以为雌激素提供结合位点，因此，这些神经元可能受到雌激素的影响。     

Combination of soy protein and high dietary calcium on bone biomechanics and bone mineral density in ovariectomized rats

OBJECTIVE: To determine if feeding soy in combination with a high-calcium diet would preserve bone mineral density and biomechanical bone strength to a greater extent than either soy or calcium alone. DESIGN: Rats were sham-operated (n = l0) and fed a control diet (AIN93G containing 0.2% calcium, 20% casein) or ovariectomized (n = 40) and randomized to one of the following diets (n = 10 per group): control, high calcium (2.5% calcium, 20% casein), soy (0.2% calcium, 20% soy protein), or soy plus high calcium (2.5% calcium, 20% soy protein) for 8 weeks. Bone mineral density of femurs and lumbar vertebrae 1 through 6 were measured by dual energy x-ray absorptiometry. Biomechanical strength properties of femurs and the fifth lumbar vertebrae were measured by three-point bending and compression, respectively. RESULTS: The dietary combination of soy and high calcium did not result in higher femur bone mineral density compared with other ovariectomized groups, and there were no differences in femur yield load or peak load among groups. In contrast, soy plus high calcium resulted in a higher (P < 0.05) vertebral bone mineral density compared with all other ovariectomized groups. Vertebral strength was preserved among rats fed either soy plus high calcium, soy, or high calcium whereas the ovariectomized group fed a control diet had lower (P < 0.05) vertebral strength than the sham-operated group. CONCLUSION: Bone mineral density of the lumbar spine was the only bone outcome that significantly benefited from the combination of soy and high calcium compared with soy or high calcium alone.     

Effect of estrogen therapy, soy isoflavones, and the combination therapy on the submandibular gland of ovariectomized rats

The objective of this study was to evaluate the effect of estrogen deficiency, estrogen therapy, and soy isoflavones on the salivary glands in female rats. Ninety-six animals were ovariectomized, and 24 were sham-operated. Among the ovariectomized rats, 24 received 17β-estradiol; 24 received isoflavone extract; 24 received a combination therapy of both; and 24 received water as placebo. The submandibular glands were histomorphometrically analyzed. As a result, the ANOVA test revealed that the hormonal deficiency affected the acini and the ducts of ovariectomized rats, reducing their percentage compared to the sham group. All treatments caused an increase in ducts and acini compared to the placebo group. It was concluded that the estrogen deficiency may be related to salivary gland function due to a reduction in the quantity of salivary acini and ducts secondary to ovariectomy. The estrogen therapy, soy isoflavone therapy, and the combination of both are effective in reducing the effects of ovariectomy on the salivary glands.     

辛伐他汀干预卵巢切除大鼠骨密度和生物力学性能的变化

BACKGROUND: Osteoporosis and its complications severely threaten the elder’s health. Simvastatin, widely accepted as a lipid-lowering drug, is reported to potentially promote bone formation, but it is in debate when orally administered, and there is no evidence to support whether this is due to the region difference. OBJECTIVE: To investigate the effect of orally administered simvastatin on bone mass and biomechanical properties of the femur and vertebrae in osteopenia rats induced by ovariectomy (OVX). METHODS: Twenty-four 6-month-old female Sprague-Dawley rats were subjected to OVX+orally administered saline vehicle (OVX group, n=8), OVX+orally administered simvastatin (5 mg/kg/d; intervention group, n=8) or sham surgery (sham group, n=8). After 8 weeks of treatment, all rats were sacrificed and the level of procollagen type I N-terminal propeptide in blood serum was assessed by ELISA. Bone mineral density was determined in the L5 vertebra and left femur using dual-energy X-rays. Furthermore, the biomechanical properties of the L4 vertebra and right femur, including maximum load and elastic modulus, were detected by compression testing and three-point bending test, respectively. RESULTS AND CONCLUSION: The serum level of procollagen type I N-terminal propeptide in the sham group was significantly lower than that in the other two groups. OVX rats showed significantly lower bone mineral density in both the L5 vertebra and left femur than sham rats (P < 0.05). Rats in the intervention group showed higher bone mineral density than those in the OVX group, with statistically significant difference in the L5 vertebra (P < 0.05), but insignificant difference in the femur. Maximum load and elastic modulus of the L4 vertebra in the OVX group were significantly lower than those in the sham and intervention group. Markedly lower elastic modulus of the femur was found in the OVX group than the sham and intervention groups. These findings demonstrate that simvastatin treatment can partially prevent bone loss in OVX rats with more notable effect on the vertebrae than the femur, and for this model, the vertebra is superior to the femur used in biomechanical test. 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

Isoflavone-supplemented soy yoghurt associated with resistive physical exercise increase bone mineral density of ovariectomized rats

OBJECTIVE: To determine the effects produced by the ingestion of a fermented soy product (soy yoghurt), supplemented with isoflavones and associated with a resistive exercise program, on the bone metabolism of mature ovariectomized (Ovx) and sham-ovariectomized (sham-Ovx) rats. METHODS: A total of 56 rats were used. They were divided into 2 sedentary control groups, the Ovx control group (C-Ovx) and the sham-Ovx control group (C-Sovx), each with 7 sedentary animals, and 2 treated groups, Ovx and sham-Ovx, with 21 animals each. These two treated groups were subdivided into three subgroups of seven animals each, which received the following treatments: consuming the soy yoghurt+sedentary, only subjected to resistive exercise, and consuming the soy yoghurt+resistive exercise. Both the program of resistive exercise and the consumption of soy yoghurt (at 3 mL/(kg body weight day)) continued for 12 weeks. The soy yoghurt was supplemented with isoflavones at 50mg/100g of product. The animals were sacrificed and their right-side femurs and tibias removed and assessed for bone mineral density (BMD). The alkaline phosphatase activity (AP) was determined in the blood serum. RESULTS: There was a significant increase in both femur and tibia BMD values and in serum alkaline phosphatase activity in all the treated subgroups, compared with the control groups (p<0.05). CONCLUSION: The ingestion of the soy yoghurt supplemented with isoflavones was capable of preventing a loss of bone mass in Ovx rats and of increasing bone mass in sham rats, whilst the resistive exercise program was effective in augmenting the bone mass in sham and Ovx rats.     

去卵巢大鼠骨质疏松对下丘脑雌激素α受体的影响

目的 观察去卵巢雌性大鼠骨质疏松对下丘脑雌激素α受体(estrogen receptor alpha,ERa)及基基因表达的影响。方法 将45只SD大鼠随机分为对照组、假手术对照组和去卵巢组,用免疫组织化学方法和原位杂交方法检测下丘脑ERa及其mRNA表达。结果 去卵巢组ERa受体及其mRNA阳性细胞与对照组比较显著减少(P<0.01)。结论 下丘脑ERa受体及其mRNA阳性细胞的减少可能在绝经后骨质疏松发生起重要作用。     

补肾壮骨方干预去卵巢骨质疏松模型大鼠骨代谢及骨密度的变化

背景：中医药以其良好的治疗效果及安全性越来越多的应用于骨质疏松症的治疗中,补肾壮骨方目前作为山东中医药大学附属医院协定方被广泛应用于临床中,获得了极好的治疗效果。目的：评价补肾壮骨方对去卵巢骨质疏松大鼠骨代谢及骨密度的影响。方法：60只SD大鼠随机分为6组,分别为假手术组、模型组、阿仑膦酸钠组、补肾壮骨方高、中、低剂量组,每组10只,后5组制备去卵巢骨质疏松症模型,假手术组只切除卵巢周围脂肪组织。造模8周后,补肾壮骨方高、中、低剂量组分别予以2.34,1.17,0.58 g/(kg·d)补肾壮骨方,阿仑膦酸钠组予阿仑膦酸钠1 mg/(kg·d),假手术组及模型组给予等体积的生理盐水,1次/d灌胃。连续灌胃12周后检测各组大鼠血清碱性磷酸酶、Runt相关转录因子2及核因子κB受体活化因子配体水平;取右侧股骨观察骨密度及骨微结构;苏木精-伊红染色观察组织病理改变;免疫组化法检测骨组织Wnt1、骨形态发生蛋白2蛋白表达。结果与结论：(1)造模8周后,与假手术组对比,模型组大鼠骨密度、骨小梁数明显降低(P <0.05),提示造模成功;(2)与模型组大鼠对比,补肾壮骨方低、中、高剂量组及阿...     

Association of estrogen receptor alpha gene polymorphisms with bone mineral density in postmenopausal Indian women

Bone mineral density (BMD) is the major determinant of osteoporotic fracture risk with a particular genetic background. However, consensus on the association of BMD with specific gene locus has not been reached. In the present study, we investigated the potential association of estrogen receptor alpha (ER alpha) gene intron I polymorphisms with BMD in 246 postmenopausal Indian women (average age 54.2+/-3.4 years). All the subjects were genotyped for XbaI and PvuII polymorphisms and underwent BMD measurements at spine and hip by dual energy X-ray absorptiometery. The average BMD of subjects with the genotypes XX and PP (absence of restriction sites for XbaI and PvuII, respectively) was 12.7 and 5.4% higher at the spine and 13.1 and 4.6% higher at the hip, respectively, than those with genotypes xx and pp. In age vs. BMD scatterplot, the intercept and slope of regression lines for genotypes xx and pp at spine and hip demonstrated comparatively rapid decrease in BMD across the age. The genotype XX was significantly prevalent (p<0.001) in women with normal bone mass (32%) and genotype xx in women with osteoporotic bone mass (35.3%), within the group. A significantly higher relative risk was associated with xx genotype. The study concludes that genetic variations at ER alpha gene locus, perhaps, are associated with BMD in Indian women and may influence some determinant of bone metabolism resulting in accelerated bone loss with age.     

同型半胱氨酸经甲基化修饰下调去卵巢大鼠主动脉雌激素受体α基因的表达

目的观察同型半胱氨酸(Hcy)对大鼠雌激素受体α(ERα)基因表达和其启动子CpG岛甲基化的影响。方法高效液相色谱仪检测血浆Hcy水平;RT-PCR和甲基化特异性PCR(MSP)分别检测ERαmRNA表达水平及基因启动子CpG岛的甲基化状态。结果去卵巢和给予甲硫氨酸饮食均可升高大鼠血浆Hcy水平。去卵巢和Hcy均可使大鼠ERαmRNA表达减少,但5′-Aza可使细胞ERαmRNA表达增多。假手术组大鼠主动脉ERα基因启动子有2例(20%)存在甲基化,去卵巢组有3例(30%)存在甲基化,而去卵巢 Hcy组有6例(60%)存在甲基化,对照组细胞ERα启动子CpG岛完全去甲基化,Hcy组细胞甲基化增强,5′-Aza使细胞去甲基化。结论Hcy可经甲基化修饰方式下调去卵巢大鼠主动脉ERαmRNA表达。     

去卵巢大鼠骨密度变化与骨髓组织血管生成的关系

目的： 探讨去卵巢大鼠骨密度变化与骨髓组织血管生成的关系。方法：30只SD雌性大鼠随机分为去卵巢组（OVX）和假手术组（sham）,分别在4周、8周和12周处死。取左侧股骨测量骨密度(BMD)。右侧远端股骨干骺端松质骨甲醛固定,EDTA-Na2脱钙。常规脱水、石蜡包埋、切片。行苏木素－伊红染色用于观察骨髓组织病理改变,用CD34标记血管内皮细胞观察微血管密度(MVD),采取大鼠腹主动脉血进行血管内皮生长因子(VEGF)测定。结果： 大鼠去卵巢8周后BMD明显低于假手术组, 提示骨质疏松模型建立,此时,骨髓造血组织容量减少,脂肪组织容量增高,与假手术组比较均有显著差异（P<0.01）,骨髓组织MVD与假手术组比略有减少。12周后,上述改变更为明显。同时,骨小梁容量减少,骨髓组织MVD明显减少（P<0.05）,且显示MVD与BMD、造血组织容量和骨小梁容量呈正相关,与脂肪组织容量呈负相关。但血浆VEGF含量测定OVX组与sham组间无明显差异,与骨髓组织各形态指标亦无相关性。结论：去卵巢大鼠在骨量丢失和造血组织容量减少的同时伴有骨髓组织MVD减少, 为骨质疏松症采用促微血管增生治疗提供了实验依据。