Studies on Androgen Receptor mRNA expression in Pancreas,Hypothalamus and Ovary of Androgen Sterilized Rats

Objective To investigate the androgen receptor （AR） mRNA expression in pancreas, hypothalamus and ovary of androgen sterilized rats （ASR）.Methods ASR model was established by subcutaneous injection of testosterone propionate to SD female rats at the age of 9 days. Around the age of 106 days （proestrus）, all rats were killed, serum Δ4-andronestedione （Δ 4-A）, total testosterone （TT）, free testosterone （FT）, insulin （Ins） and C-peptide （C-P） were measured by radioimmunoassay （RIA）. Total RNA in pancreas, hypothalamus and ovary were extracted and the amount of AR mRNA was quantitatedly analyzed by RT-PCR with single base mutant template as inner standard. Results Serum concentrations of Δ4-A, TT, FT, Ins and C-P in ASR model rats were significantly higher than those in control group （P〈0.05, P〈0.01）. The expression of AR mRNA in pancreas, hypothalamus and ovary increased significantly （P〈0. 05, P〈0.01） of model rats as compared with control group. Conclusion The elevated serum androgen levels in ASR model could enhance the expression of AR mRNA levels in pancreas, hypothalamus and ovary, which further induce hyperinsulinemia and anovulation.

The effect of traditional Chinese medicine──Tian Gui Recipeon obesity and anovulation in androgen-sterilized rats

Objective: A traditional Chinese medicine Tian Gui Recipe (TGR) has been used to effectively treat clomiphene resistant anovulatory disease and obesity, especially in polycystic ovary syndrome (PCOS) cases with hyperinsulinemia. The effect of TGR on obesity and anovulation was investigated in androgen-sterilized rats (ASR). Methods: Female SD rats at the age of 9 days were divided into 3 groups. Group ASR (n=15): anovulation was demonstrated at the age of 70 days by vaginal smear in rats while 1. 25 mg of testosterone propionate was injected subcutaneously on its 9th neonatal day. Group A+H (n = 25): TGR were administered to ASR at the age of 80 days for 3 weeks. Rats with regular estrous cycle and ovulation after herbal treatment were included in this group. Group C (n = 15): normal ovulated rats were recruited. Around the age of 112 days or on proestrous day, all rats were sacrificed under anesthesia. Serum leptin, testosterone (T), estrogen (E2), follicular stimulating hormone (FSH), luteinizing hormone (LH) levels were measured with radioimmunoassay (RIA). Double immunofluorescent staining combined with confocal laser scanning microscope and dual in situ hybridization were carried out on sections through areas of arcuate nucleus (ARC) to determine whether estrogen receptor (ER) immunoreactivity (IR) or long form of leptin receptor (OB-Rb) mRNA were expressed in neuropeptide Y (NPY) immuno-and mRNA-containing neurons, and its relationship to proopiomelanocortin (POMC) mRNA-containing neurons. Immunohistochemistry and in situ hybridization were used to observe the levels of ER, NPY, gonadotrophin releasing hormone (GnRH) and gene expression levels on NPY, OB-Rb, POMC. Meanwhile, the criteria of energy state, including daily food intake, retroperitoneal fat depot pad and body weight, were measured and evaluated. Values of immunohistochemistry and in situ hybridization were expressed in mean optic density (MOD). Results: Seventeen out of the 25 rats (68%) in Group A+H were ovulated after herbal treatment. ASR characterized with significantly high metabolic rate, energy imbalance and obesity (P<0. 01, P<0. 01, P<0. 01). The mean serum leptin, T, E2 levels of group ASR were significantly higher than those of Group C and Group A+H (P< 0. 01,P<0. 01 ), while FSH, LH levels were lower (P<0. 01, P<0. 01 ). ERs were proved to be distributing in NPY-containing neurons, OB-Rb coexpressed in NPY-syn- thesizing neurons, and both NPY and POMC mRNA expressing neurons overlapped in ARC. Compared to Group C, levels of NPY mRNA and NPY, POMC mRNA expressions were increased (P<0. 01, P<0. 01, P<0. 01 ), OB-Rb mRNA, ER and GnRH immunoreactive expression were decreased significantly in the hypothalamus of ASR (P<0. 01, P<0. 01, P<0. 01 ). These dramatic neuroendocrine changes became normal in ovulated ASR after the herbal treatment (all P>0. 05). Conclusion: The elevated peripheral E2 caused by high T and leptin levels in ASR may down-regulate their corresponding receptors oriented on hypothalamic NPY- containing neurons respectively, therefore challenge the NPY mRNA and NPY, POMC mRNA overexpressions. The overexpression, of NPY, POMC gene transcription and translation stimulate food intake, promote the deposit of adipocyte tissue, and inhibit GnRH expression and GnRH/FSH, LH release, which contribute to the occurrence of obesity and anovulation in ASR. TGR may reverse these abnormal neuroendocrine cascades by lowering the levels of T, E2 and leptin.

Research on 30 days Feeding Test of Rats with Irradiated Pet Food

[ Object] The research is aimed to provide scientific basis for irradiated pet food＇s standardized production, supervision and commercial- ized application. E Method] Take broiler breast meat with high-dose irradiation as testing material, then evaluate its safety after 30 days feeding test of rats [ Result] Compared with control group （Pet food＇s irradiation dose is 0 kGy）, the general situation, weight, food-intake, food utilization rate, hematological examinations, bio-chemical analysis of blood, viscera weight, ratio of viscera and body weight, histopathological examinations, physi- ological feature, life＇s basic characteristics （including height, tail length, body temperature, heart rate and blood pressure ） and other indexes of rats, which were fed respectively with 10, 15, 25 kGy irradiation dose pet food for 30 days, were not found abnormal. [ Conclusion] Pet food with 10, 15, 25kGy irradiation dose is safe.

Effect of Tiangui Recipe (天癸方) on Serum Leptin and Pituitary Gonadotro pin in Androgen-Sterilized Rats

Objective: To explore the effect of Tiangui Recipe (TGR) on obesity and reproductive function in androgen-sterilized rats (ASR).Methods: ASR model was established by subcutaneous injection of testosterone propionate to SD female rats at the age of 9 days. Change of serum leptin, follicular stimulating hormone (FSH) and luteinizing hormone (LH) in model rats aged 115 days before and a fter feeding them with TGR was measured by radioimmunoassay (RIA). Meanwhile, criter ia of energy state, including diurnal food intake, retroperitoneal fat depot weight and body weight, were measured and evaluated.Results: ASR had the characteristics of high metabolic rate, energy imbalance and obesity. Serum level of leptin increased and FSH, LH decreased significantly (P<0.01) in model rats as compared with the normal rats. Significant positive correlation between leptin level and body weight (r=0.8977, P<0.001) was observed, and negative correlation was seen between FSH, LH and leptin (r=-0.7517, P<0.001; r=-0.8444, P<0.001, respectively). The endocrine changes of ASR all returned to normal range after the TGR treatment.Conclusion: Obesity and concerned hypogonadotrophic hypogonadal anovulation were expressed in ASR. TGR may play an important role in reducing weight and inducing ovulation.

Experimental Study on Effect of Tonifying Kidney Herbs on Pituitary, Ovary, and Adrenal Gland in Androgen Sterilized Rats

Objective: To observe the effect of tonifying Kidney herbs (TKH) on pituitary gland, ovary, and adrenal gland of androgen sterilized rats (ASR).Methods: ASR model was established by injecting testosterone propionate subcutaneously to Sprague-Dawley (SD) female rats of 9 days age. Morphological and endocrinological change of pituitary gland, ovary and adrenal gland in rats of 100–107 days old before and after feeding TKH extract were observed by light and electron microscope, cell cultures, immunohistochemical studies, and radioimmunoassay.Results: In the ASR groups, there were intracytoplasmic lipid drops, crinophagy, vacuole, and autophagy in pituitary gland, anovulation in ovary, and hyperplasia of reticular zone in adrenal gland. At the same time, serum follicular stimulating hormone and luteinizing hormone decreased (P < 0.05-0.005) and serum testosterone and dehydroepiandrosterone levels increased significantly (P < 0.01-0.001). These morphologiacl and endocrinological changes were all returned to normal range after the TKH treatment.

实验性精索静脉曲张对大鼠睾丸生精细胞凋亡的影响

目的 :探讨大鼠精索静脉曲张对睾丸生精细胞凋亡的影响 ,阐明精索静脉曲张引起不育的病理机制。 方法 :选用雄性SD大鼠 32只 ,随机分为假手术组、4 5d模型组、6 0d模型组及 90d模型组。采用肾静脉缩窄法建立精索静脉曲张动物模型 ,流式细胞术分别检测各组大鼠睾丸凋亡细胞数及各级生精细胞数。 结果 :假手术组未出现明显的凋亡峰 ,各模型组均出现明显的凋亡峰 ,且随着造模时间的延长凋亡峰增高。 结论 :精索静脉曲张可引起睾丸生精细胞大量凋亡 ,各级生精细胞数减少 。

愈精颗粒抗大鼠雄性不育的药效学研究

目的 :观察愈精颗粒抗大鼠雄性不育的效应。方法 :用雷公藤多甙造成大鼠雄性不育模型 ,分别给予愈精颗粒高、低剂量及克罗米芬灌胃 ,观察给药 30d后睾丸、附睾系数 ;血浆性腺激素水平 ;附睾精子数量、活率、活力 ;生育力和睾丸形态学改变。结果 :愈精颗粒能增加不育大鼠睾丸、附睾重量 ;使睾酮含量增高 ,降低雌二醇水平 ;修复损伤的生精细胞和间质细胞 ;改善精子质量 ;明显提高雌鼠怀孕率及胎产数。结论 :愈精颗粒具有显著抗大鼠雄性不育作用 ,优于克罗米芬。

补肾中药对雄激素致不孕大鼠垂体、卵巢及肾上腺作用的实验研究

目的：探讨雄激素致不孕大鼠（ＡＳＲ）发病机理和中药治疗作用。方法：采用光镜、电镜、细胞培养、免疫组化、放免测定等方法，对补肾药治疗前后的ＡＳＲ的垂体、卵巢、肾上腺的形态学及内分泌功能进行同步观察。结果：ＡＳＲ的腺垂体细胞浆内有“脂滴”、“粒溶”、“空泡”、“自嗜”、卵巢呈无排卵；肾上腺皮质网状带增生，同时促卵泡激素（ＦＳＨ）、促黄体激素（ＬＨ）明显低下（Ｐ＜０．０５～０．００５）；睾酮（Ｔ）、脱氢表雄酮（ＤＨＡ）明显升高（Ｐ＜０．０１～０．００１）。治疗后ＡＳＲ的形态学及内分泌功能均恢复正常。结论：ＳＤ雌性幼鼠９日龄给予外源性雄激素后，影响性腺轴和肾上腺，在ＡＳＲ发病机理中起重要作用；补肾药是通过对性腺轴，肾上腺多水平，多靶器官的调节，起到降低雄激素、促排卵作用。

中药天癸方对雄激素致不孕大鼠血leptin及垂体促性腺激素的影响

目的：探讨中药天癸方对雄激素致不孕大鼠（ＡＳＲ）肥胖及生殖功能的影响。方法：运用放射免疫法测定天癸方治疗前后血ｌｅｐｔｉｎ、促卵泡激素（ＦＳＨ）、促黄体生成素（ＬＨ）的变化；同时对ＡＳＲ能量状态指标（每日进食量、腹膜后脂肪垫、体重）进行评估。结果：ＡＳＲ呈高代谢、能量失衡肥胖状态，其血ｌｅｐｔｉｎ水平明显增加（Ｐ＜００１），ＦＳＨ、ＬＨ明显低下（Ｐ＜００１）。ｌｅｐｔｉｎ与体重呈正相关（ｒ＝０８９７７，Ｐ＜０００１），与ＦＳＨ、ＬＨ呈负相关（ｒ＝－０７５１７，Ｐ＜０００１；ｒ＝－０８４４４，Ｐ＜０００１）。中药治疗后各内分泌指标均恢复正常。结论：ＡＳＲ有肥胖及低促性腺激素性性腺功能低下无排卵的特点。天癸方同时有减肥及促排卵作用。

不同灭菌方法对实验动物配合颗粒饲料营养成分的影响

探讨不同的灭菌条件,对实验动物配合颗粒饲料(鼠料)中营养成分的影响。采用不同时间的高压蒸汽(121℃、0.14 MPa)(蒸汽灭菌)和不同剂量的60 Co-γ射线照射(照射灭菌)对鼠料灭菌,依据国标做细菌培养,观察灭菌条件及效果,并对鼠料进行营养成分的测定。结果表明,20 min蒸汽灭菌和20.0 kGy剂量60 Co-γ射线照射时细菌培养为阴性;蒸汽灭菌比照射灭菌更容易造成营养成分的破坏,两种方法差异显著(P<0.05),且不同时间下水分、粗蛋白、粗脂肪、赖氨酸、蛋氨酸、胱氨酸、苏氨酸、异亮氨酸及维生素的损失都有显著差异(P<0.05),矿物质除铁外损失无显著差异(P>0.05);不同剂量照射下,各种营养成分的损失差异不显著(P>0.05)。鼠料消毒灭菌的适宜条件为20 min蒸汽灭菌或20.0 kGy照射灭菌;蒸汽灭菌对鼠料的营养成分影响较大,而照射灭菌则影响较小。

补肾活络中药对雄激素致不孕大鼠卵泡数、子宫内膜面积和内分泌的影响

目的观察补肾活络中药用药前后雄激素致不孕大鼠(ASR)卵巢卵泡数、子宫内膜面积和内分泌的变化。方法建立雄激素致高胰岛素、高雄激素不孕大鼠模型,通过阴道涂片筛选各组大鼠;卵巢、子宫重量采用电子天平称重法,血清促卵泡刺激素(FSH)和促黄体生成素(LH)的测定采用放免法,卵泡数的测定采用免疫组化法。结果卵巢、子宫重量分析:正常组>中药组>阳性组>模型组;卵泡数、子宫内膜面积免疫组化定量分析:正常组>中药组>阳性组>模型组;血清FSH和LH水平:阳性组>正常组>中药组>模型组。结论中药补肾活络方能增加ASR卵巢重量,增加卵巢各级卵泡总数,使卵巢激素的靶器官子宫明显增重,内膜明显增厚,能增强垂体的内分泌功能,促进促性腺激素的合成和释放,提高垂体促性腺激素水平,以促进卵泡的发育和卵巢的排卵。

补肾中药对雄激素致不孕大鼠肾上腺皮质形态学观察

本实验选用ＳＤ雌性大鼠幼仔，出生第９日龄注射丙酸睾丸酮制作雄激素致不孕大鼠，第８０日龄灌服补肾中药水溶液２周，第１００日龄心脏灌注处死。采用光镜、透射电镜、银胶染色法和新生物素－生物复合法，分别测定核仁组成区嗜银蛋白、增殖前期细胞核仁抗原，观察肾上腺皮质的形态学变化。结果显示：在调节性腺轴功能过程中，包括了对肾上腺的调整。补肾中药水溶液能够使增生的肾上腺网状带恢复正常；细胞内增多的脂滴数和核仁组成区嗜银蛋白、增殖前期细胞核仁抗原阳性细胞计数达正常范围。

雄激素致不孕大鼠胰腺、下丘脑及卵巢雄激素受体mRNA表达

目的:研究雄激素致不孕大鼠(ASR)胰腺、下丘脑及卵巢组织中雄激素受体(AR)mRNA的含量变化。方法:9日龄SD雌性大鼠皮下注射丙酸睾丸酮制备ASR模型,于106日龄左右(动情前期)处死,取血放免法测定△4-雄烯二酮(△4-A)、总睾酮(TT)、游离睾酮(FT)、胰岛素(Ins)和C-肽(C-P),提取胰腺、下丘脑及卵巢总RNA,以单碱基突变模板为内对照的RT-PCR方法对ARmRNA进行定量分析。结果:ASR模型血△4-A、TT、FT、Ins、C-P均显著高于对照组(P<0.05,P<0.01),胰腺、下丘脑及卵巢的AR mRNA表达水平明显升高,与对照组相比差异有统计学意义(P<0.05,P<0.01)。结论:ASR模型血雄激素水平升高,上调胰腺、下丘脑及卵巢AR mRNA的表达,引起该模型的高胰岛素血症和无排卵。

通络助孕方对慢性炎症大鼠输卵管的影响

目的 :观察中药通络助孕方对慢性炎症大鼠模型输卵管病理形态学和受孕率的影响 ,并探讨其疗效机理。方法 :用混合菌接种法制作大鼠慢性炎症动物模型 ,以不同剂量的通络助孕方和对照药妇炎康治疗后 ,观察用药前后光镜和电镜下输卵管病理形态学改变情况 ,并追踪大鼠受孕情况。结果 :通络助孕方和对照药能不同程度恢复模型大鼠输卵管病理形态学改变 ,并提高了受孕率。结论 :模型大鼠输卵管组织呈慢性炎症表现 ,混合菌接种法可以成功制作大鼠慢性炎症模型 ;通络助孕方能改善输卵管结构和功能 。

补肾调经方对雄激素致不孕大鼠子宫转化生长因子β_1及其受体的影响

目的:探讨补肾调经方对雄激素致不孕大鼠(androgen-induced sterile rats,ASR)子宫转化生长因子β1及其受体(TGFβ1/TGFβRⅠ)表达的影响。方法:给出生9日龄SD雌性大鼠一次性皮下注射丙酸睾丸酮(T),建立ASR模型。设乌鸡白凤口服液为阳性对照药。分为正常组、模型组、阳性药对照组和中药治疗组。ASR模型81日龄始灌服乌鸡白凤口服液、补肾调经方,连续7周,130、131日龄麻醉处死,采用UE染色及免疫组化技术分别观察子宫组织形态学及TGFβ1、TGFβRⅠ表达的变化。结果:补肾调经方可改善病理状态下子宫的形态结构,使模型大鼠子宫内膜、肌层TGFβ1以及肌层TGFβRⅠ的表达增强。结论:补肾调经方可通过提高ASR模型子宫内膜、肌层TGFβ1及肌层TGFβRⅠ的表达,促进子宫的发育,改善子宫内膜的分泌功能,有利于胚泡的植入。

补肾助孕汤对雄激素致不孕大鼠的促排卵及卵巢IGF-1表达的影响

目的探讨补肾助孕汤对雄激素致不孕大鼠(ASR)促排卵的作用机制。方法选择9日龄SD龄雌性大鼠随机分为4组,正常对照组:皮下注射中性茶油;ASR模型组:皮下注射丙酸睾丸酮;中药组及西药组:ASR模型自81 d龄起,每天灌胃给药;连续28 d。采用阴道涂片测定大鼠动情周期及排卵率,采用ELISA法测定促卵泡刺激素(FSH)、黄体生成素(LH)、雌二醇(E_2)及孕酮(P)水平;采用免疫组织化学sp检测卵巢细胞胰岛素样生长因子(IGF-1)表达。结果补肾助孕汤可明显增加大鼠卵巢组织各级卵泡及黄体数目,恢复排卵功能;提高血清中FSH、LH及P水平,并且降低卵巢细胞中IGF-1的表达。结论补肾助孕汤可通过调节卵巢局部因子IGF-1及相应激素,促进卵泡生长发育及排出。

生物不育灭鼠饵剂现场鼠类控制效果研究

[目的]研究生物不育灭鼠饵剂在现场大规模应用时,对鼠类的控制效果。[方法]在不育剂试验区采用首次足量投放,第2次等量补充的方法投放生物不育灭鼠饵剂后,检查鼠对该饵剂的拖食情况,并在投放后第1、3、4、6、9、12个月测定鼠密度,分类后剖检成年鼠的生育能力,同时设抗凝血灭鼠剂对照区和自然对照区。[结果]现场23d内鼠类对不育剂保持较好的适口性,平均拖食率为14.37%。3个月后平均鼠密度与灭前比较下降75.76%,年平均下降率为41.41%,全年鼠密度处于较低水平。投饵后3、4、6个月的鼠密度与自然对照区比较,相关密度指数(RPI)值为23.52、19.27和48.51。不育剂试验区解剖鼠类的繁殖率为7.69%,没有发现具有繁殖能力的雌鼠;而抗凝血灭鼠剂对照区、自然对照区和投药前的鼠的繁殖率分别为44.44%、33.33%和31.57%。[结论]现场应用研究证明,该生物不育灭鼠饵剂具有良好的适口性,安全环保,与常规抗凝血灭鼠剂相比,在有效控制种群数量上具有重要价值。

中药天癸方对雄激素致不孕大鼠下丘脑Leptin受体及神经肽Y mRNA的影响

目的：从基因转录水平探讨中药天癸方对雄激素致不孕大鼠（ASR）肥胖及无排卵的影响。方法：采用α－32P标记瘦素（leptin）受体（OB－R）及神经肽Y（NPY）寡核苷酸探针原位杂交、放射自显影技术结合图像分析，观察中药治疗前后下丘脑弓状核（AR）两种物质mRNA的表达变化。结果：ASR中ARC的NPY基因表达水平明显增加（P＜0．01），OB－R基因表达水平明显降低（P＜0．01），ASR呈肥胖、无排卵状态。用中药天癸方治疗后，NPY基因表达下降，OB－R基因表达上升至正常大鼠水平。结论：ASR肥胖及无排卵可能与NPYmRNA过度表达、OB－RmRNA数目异常有关，中药可调节NPY和OB－RmRNA表达而发挥减肥及促排卵作用。

饮食和养阴药对雄激素致不孕大鼠排卵及能量状态、血脂的影响

目的探讨饮食和养阴药对雄激素致不孕大鼠(ASR)排卵及能量状态、血脂的影响及其相互关系。方法给出生9日龄SD雌性大鼠丙酸睾酮1.25mg皮下注射,建立ASR模型,将其随机分为①模型组;②中药组(80日龄起灌服滋肾阴药“天癸方”);③节食组(80日龄开始饲料减为自由进食时的70%);④中药+节食组;⑤设立对照组。每组14只。各组于110日龄称重,断头处死,测定各组血总胆固醇、低密度脂蛋白胆固醇、高密度脂蛋白胆固醇、三酰甘油、血糖的变化;同时对能量状态指标(每日摄食量、腹膜后脂肪垫、体重)进行评估;卵巢苏木精伊红染色作形态学观察。结果雄激素致不孕大鼠呈高代谢、能量失衡肥胖状态,其低密度脂蛋白胆固醇、葡萄糖较对照组增加(P<0.05),而高密度脂蛋白胆固醇、总胆固醇、三酰甘油无明显改变(P>0.05),镜下见卵巢卵泡囊性扩张;分别予滋肾阴中药、节食、滋肾阴+节食治疗后,能量失衡肥胖状态改善,低密度脂蛋白胆固醇、葡萄糖恢复正常,卵巢排卵功能不同程度地恢复,以中药+节食组治疗效果最佳(排卵率达71.43%),而中药组、节食组排卵率分别为42.86%,28.57%。结论雄激素致不孕大鼠有肥胖、脂质代谢紊乱及不排卵特点。养阴药+节食能改善能量代谢紊乱,促进排卵,且两者有协同作用。

无菌大鼠培育的技术创新及其繁育探讨

对清洁级SD大鼠进行剖腹取胎净化,经人工饲育建立无菌SD大鼠群,以满足科研单位对无菌大鼠的需求。在手术隔离器中剥离子宫,取35只仔鼠传入饲育隔离器,静置2 h后存活20只;用自行研制的灌胃注射器,和市售配方改良奶粉,对剖腹取胎仔鼠进行人工哺乳。每24 h人工哺乳5~6次。哺乳20只,离乳13只。粪便、垫料、棉拭子经无菌检查,均无细菌生长。通过优化人工乳配方,创新无菌鼠培育技术,成功建立无菌大鼠模型,并传代保种,解决了科学研究在国内买不到无菌大鼠的难题。