【作者】 张汝仙；

【导师】 杜惠兰；

【作者基本信息】 河北医科大学 ， 中西医结合临床， 2008， 硕士

【摘要】 目的:虚寒证与能量代谢密切相关。本实验旨在通过研究虚寒证模型大鼠下丘脑、垂体、卵巢组织线粒体功能(SDH和ATP酶含量)的改变,生殖激素水平的变化,探讨这些改变是否与虚寒证生殖功能损伤有关,金匮温经汤是否通过调节性腺轴能量的储存和分解利用两个环节发挥作用,从而揭示妇科虚寒证的本质,为中医证的研究提供理论和实验依据。方法:选用一级SD雌性成年健康未孕大鼠50只,体重226±16克,3月龄,由河北医科大学实验中心提供,清洁级动物。动物适应性饲养一周后,按随机数字表法将其分为5组,每组10只。即正常对照组(简称“正常组”),模型对照组(简称“模型组”),虚热对照组(简称“对照组”),模型+温经汤低剂量组(简称“低剂量组”),模型+温经汤高剂量组(简称“高剂量组”)。金匮温经汤组成:吴茱萸9g,当归6g,赤芍6g,川芎6g,人参6g,桂枝6g,阿胶6g,丹皮6g,生姜6g,甘草6g,半夏6g,麦冬9g。由石家庄乐仁堂药店购入。采用传统煎煮方法,每天一剂煎煮,浓缩制成水煎剂。高剂量组每毫升药液含生药1.3克(相当于临床用量的10倍),低剂量组每毫升药液含生药0.65克(相当于临床用量的5倍)。置于4℃冰箱密闭保存备用。正常组常规饲养,模型组、低剂量组、高剂量组大鼠采用冰水冷冻的方法,将大鼠置于0℃～1℃冰水中20min/12h,连续2周。对照组大鼠用附子、干姜(1:1)制成100%水煎剂, 4ml/24h灌胃,2周即可[1]。低剂量组、高剂量组于造模的第一天开始,分别按6.5g/kg/d、13g/kg/d(分别相当于临床等效剂量的5倍、10倍)给予温经汤灌胃,每日1次,连用2周,正常组给予同体积的蒸馏水。自造模第一天起,每日专人定时进行阴道涂片,观察大鼠动情周期的变化。末次造模给药后,禁食不禁水lOh,每只动物游泳5min作为负荷,每两小时进行阴道细胞涂片,选择动情间期的大鼠断头取血,采用放射免疫分析法测定血清孕酮(P)、雌二醇(E2)、睾酮(T)、卵泡刺激素(FSH)、黄体生成素(LH)的含量。无菌操作取下头颅,马上沿正中线开颅,充分暴露大脑并仔细翻开,断开视交叉,以神经分离棒分离并取出下丘脑,以眼科钳仔细夹取垂体的包膜,轻轻取出,随后马上将下丘脑、垂体经双蒸水反复冲洗吸干后称重。打开腹腔,摘取卵巢,称重,4%戊二醛和1%锇酸双固定,取下丘脑、垂体和卵巢用冰冷的分离介质制成10%匀浆,3000～4000rpm低温离心10min,取上清以12000rpm低温高速离心10min,沉淀即为线粒体。按照试剂盒说明书测定琥珀酸脱氢酶(SDH)含量。将分离的线粒体用冰冷匀浆介质制成混悬液,用超声粉碎机使线粒体破碎。按照试剂盒(南京建成生物工程研究所)说明书测定Na十-K十-ATP酶和Ca2+-Mg2+-ATP酶的活性。结果:1各组大鼠一般状况正常组大鼠毛色白而光泽,精神佳,活泼,反应灵敏,饮食、二便正常。模型组大鼠受冻后开始出现寒战,继而表现蜷缩少动,反应迟钝,爪尾部苍白,耳色苍白,喜扎堆,呼吸微弱,被毛无光泽,饮水量少,小便色清,大便湿烂等症状。高、低剂量组大鼠仅表现有轻微的畏寒喜暖,少动等症状。2各组大鼠体重、饮水量、饮食量的变化模型组、对照组大鼠体重减轻,与正常组比较有显著性差异(P<0.01),经温经汤治疗,低剂量组、高剂量组体重皆增加,与模型组比较差异均有统计学意义(P <0.01,P<0.05),各治疗组与正常组之间比较无统计学意义。对照组与模型组比较体重减轻但其差异无统计学意义。模型组大鼠饮水量减少,对照组大鼠饮水量增加,与正常组比较均有统计学意义(P<0.01),经温经汤治疗,低剂量组、高剂量组饮水量增加,与模型组比较差异均有统计学意义(P<0.01,P<0.05),与正常组之间比较无统计学意义。模型组、对照组大鼠饮食量减少,与正常组比较有显著性差异(P<0.01),经温经汤治疗,低剂量组、高剂量组皆增加,与模型组比较差异均有统计学意义(P<0.01,P<0.05),与正常组之间比较无统计学意义。3各组大鼠游泳时间的变化模型组、对照组大鼠游泳时间减少,与正常组比较有显著性差异(P<0.01),经温经汤治疗,低剂量组、高剂量组游泳时间皆增加,与模型组比较差异均有统计学意义(P<0.01,P<0.05),各治疗组与正常组之间比较无统计学意义。对照组与模型组比较其差异无统计学意义。4各组大鼠动情周期的变化模型组大鼠动情周期延长、间期延长,与正常组比较有显著性差异(P<0.01),经温经汤治疗,低剂量组、高剂量组动情周期和动情间期皆缩短,高剂量组与模型组比较差异有统计学意义(P<0.01,P<0.05),低剂量组动情周期和动情间期皆有缩短趋势,但与模型组比较无统计学意义。各治疗组与正常组之间比较无统计学意义。对照组与模型组比较动情周期、动情间期缩短,但其差异无统计学意义。5各组大鼠卵巢重量指数的变化模型组大鼠卵巢重量指数与正常组比较有降低趋势,但无统计学意义(P>0.05),经温经汤治疗,高剂量组卵巢重量指数与模型组比较有升高趋势,其差异无统计学意义(P>0.05)。6各组大鼠血清生殖激素变化与正常组相比,模型组血清E2、P、T、FSH、LH显著降低(P<0.01,P<0.05),经温经汤治疗后,低剂量组、高剂量组E2、P、LH均升高,与模型组比较差异有统计学意义(P<0.01,P<0.05),高剂量组T、FSH升高,与模型组比较差异有统计学意义(P<0.01,P<0.05),对照组E2、P、LH、FSH升高,与模型组比较差异有统计学意义(P<0.01,P<0.05),高剂量组与对照组比较E2、P升高差异有统计学意义(P <0.05)。7各组大鼠下丘脑、垂体、卵巢组织Na+-K+-ATP酶的变化与正常组相比,模型组垂体、卵巢Na+-K+-ATP酶含量显著降低(P<0.01,P<0.05),经温经汤治疗后,低剂量组、高剂量组垂体、卵巢Na+-K+-ATP酶均升高,与模型组比较差异有统计学意义(P<0.01,P<0.05),与正常组比较无统计学意义(P>0.05)。对照组下丘脑、垂体、卵巢Na+-K+-ATP酶升高,与模型组比较差异有统计学意义(P<0.01,P<0.05),与正常组相比,模型组下丘脑Na+-K+-ATP酶有降低趋势,但无统计学意义(P>0.05)。8各组大鼠下丘脑、垂体、卵巢组织Ca2+-Mg2+-ATP酶的变化与正常组相比,模型组垂体、卵巢Ca2+-Mg2+-ATP酶含量显著降低(P<0.01,P<0.05),经温经汤治疗后,低剂量组、高剂量组垂体、卵巢Ca2+-Mg2+-ATP酶均升高,与模型组比较差异有统计学意义(P<0.01,P<0.05),与正常组比较无统计学意义(P>0.05)。对照组下丘脑、垂体、卵巢Ca2+-Mg2+-ATP酶升高,与模型组比较差异有统计学意义(P<0.01,P<0.05),与正常组相比,模型组下丘脑Ca2+-Mg2+-ATP酶有降低趋势,但无统计学意义(P>0.05)。9各组大鼠下丘脑、垂体、卵巢组织SDH的变化与正常组相比,模型组垂体、卵巢SDH含量显著降低(P<0.01,P<0.05),经温经汤治疗后,低剂量组、高剂量组垂体、卵巢SDH均升高,与模型组比较差异有统计学意义(P<0.01,P<0.05),与正常组比较无统计学意义(P>0.05)。对照组下丘脑、垂体、卵巢SDH升高,与模型组比较差异有统计学意义(P<0.01,P<0.05),与正常组相比,模型组下丘脑SDH有降低趋势,但无统计学意义(P>0.05)。结论:1.下丘脑、垂体、卵巢组织的线粒体SDH和ATP酶含量变化与虚寒证的发生存在密切关系;2.虚寒证模型大鼠下丘脑、垂体、卵巢线粒体功能紊乱,能量代谢障碍;垂体及卵巢生殖内分泌功能低下;降低3.温经汤能显著改善虚寒证模型大鼠的症状,调节大鼠动情周期、下丘脑、垂体、卵巢的线粒体功能,改善垂体及卵巢的生殖内分泌功能,使之趋于正常。从治疗角度反证了虚寒证的发生与下丘脑、垂体、卵巢组织线粒体SDH和ATP酶含量的变化密切相关。更多还原

【Abstract】 Objective: Asthenia cold syndrome have closed correlation with energy metabolish. This experiment means approaching the changes if together with damage of genitor-function of Asthenia cold syndrome , through research the changes of hypothalamus, master gland, ovarian chondriosome function (content of SDH and ATPase) in Model Rats of Asthenia cold syndrome, and its relationship with gonadalhormone. Jinkiu Decoction for warming meridian if effect by adjusting store and dilapsus exploitation two link of sex gland axle energy, to reveal essence of department of gynecology Asthenia cold syndrome, provided theory and experiment evidence to investigation of syndrome of traditional Chinese medicine.Method: Adoption 50 depuratory SD female rats, which first class, healthy and unpregnancy, 226±16g in body weight,3-month in age. Provided by experiment center of Hebei Medical University .After raised for 1 week, these rats were divided into 5 groups randomly, there were 10 rats in each group : Normal Control ,Model Group, Control Group, Low-dose Group and High-dose Group.Composition of decoction for warming meridian: Evodiae,Fructus 9g, Angelica 6g, Paeoniae radix 6g, Chuanxiong 6g, Ginseng 6g, Ramulns cinnamomi 6g, Donkey-hide glue 6g, Cortex moutan 6g, Zingiberis recens,rhizome 6g, Licorice root 6g, Pinelliae tuber 6g, Ophiopogonis tuber 9g. Buy from Shijiazhuang Rerentang. Use traditional decoction method, one dose par day, condense liquid apozem. High-dose group containing crude drug 1.3g each milliliter physic liquor (correspond 10times of clinical dosage), low-dose group containing crude drug 0.65g each milliliter physic liquor(correspond 5times of clinical dosage), maintain to reserve by placing 4℃Refrigerator .Normal control breed routinely, model group, low-dose group and high-dose group adopt method of freezing by ice water ,which putting rats in 0℃-1℃ice water 20 minutes, twice everyday, continuous two weeks. Control group lavages 100% liquid apozem 4ml per day, what fainting by aconite root and zingiberis,rhizome(1:1), continuous two weeks. Low-dose group and high-dose group lavages decoction for warming meridian , differencely to give 6.5g/kg/d、13g/kg/d(correspond 5 times and 10 times of clinical dosage)from the first day of making model ,once daily,2 weeks. Normal control lavages same voluminal distilled water. From the first day of making model, special messenger undertake vaginal smear oriented to time everyday, to observe the changes of estrous cycle of rats. After the last administration of making model, ambrosia but no prohibit water 10h,each animal swimming 5min as load, undertake vaginal smear every tow hours, select rats of diestrus to decapitate and to obtain blood, then use radio immunoassay assaying the content of serum corporin, aquadiol, andrusol, thylakentrin and luteinizing hormone.sterile operation to take head, immediately open the skull along midline, exposure cerebrum sufficiently and carefully to turn open, cut off chiasm opticum, separate and take out of hypothalamus by using nerves separate stick , take hypophyseal amicula carefully by using ophthalmology clamp ,then immediately wash repeatedly ,blot and weigh the hypothalamus and pituitary gland use twin distilled water .Open the abdominal cavity and select ovaries,weighed,4% glutaral and 1% osmic acid fixed, make organism to 10% homogenate used icecold separating medium,3000-4000rpm centrifuge 10min in low temperature, take supernatant low temperature ,high speed centrifugation 10min,the sedimentum is chondriosome. According to kit directions to determine the content of SDH. Make the segregative chondriosome to suspl using icecold homogenate, quassate chondriosome by ultrasonic disintegrator . According to kit directions to determine Na十-K十-ATPase and Ca2+-Mg2+-ATPase content.Result:1 General state of animalThe normal rats have clear and luster color pattern, fine spirit, clever active and reaction, normal meat and drink, urine and stool. After being frozen, the model rats appeared such symptoms as wriggly and few move , dull reaction, pale ear, claw and tail, like to heap, weak breathe, bloom color pattern, few the quantity of drinking water, clear rine, wet and rotten stool. Low-dose group and high-dose group rats only appeared slightly symptoms as chilly, like warm, few move .2 The changes of weight, drinking and eating of ratsThe rats in model group and control group body weight all loss, compared with normal control ,these were significance difference ( P<0.01 ) ,treated by decoction for warming meridian ,the weight of low-dose group and high-dose group all increase, compared with model group , these were significance difference ( P<0.01 , P<0.05 ) , compared with normal control ,these were no significance difference. The rats in model group and control group body weight all loss, but no significance difference.The rats in model group drinking decreased, but in control group were increased, compared with normal control, these were significance difference(P<0.01), treated by decoction for warming meridian , the rats drinking in low-dose group and high-dose group all increase, compared with model group , these were significance difference(P<0.01,P<0.05), compared with normal control, these were no significance difference. The rats in model group and control group eating less, compared with normal control, these were significance difference(P<0.01). Treated by decoction for warming meridian , low-dose group and high-dose group all increase, compared with model group , these were significance difference(P<0.01,P<0.05), compared with normal control, these were no significance difference.3 The changes of swimming time of ratsThe rats in model group and control group wimming time all decreased, compared with normal control ,these were significance difference(P<0.01) ,treated by decoction for warming meridian ,the swimming time of low-dose group and high-dose group all increase, compared with model group , these were significance difference(P<0.01,P<0.05), compared with normal control ,these were no significance difference. The rats in model group and control group wimming time all decreased, but these were no significance difference.4 The changes of estrous cycle of ratsThe estrous cycle lengthened in model group, mainly appeared longer in duration of anestrum, compared with normal control , these were significance difference(P<0.01), treated by decoction for warming meridian ,the estrous cycle and anestrum of low-dose group and high-dose group all decurtated, compared with model group , high-dose group have significance difference(P<0.01,P<0.05), low-dose group have no significance difference . Compared with normal control ,these were no significance difference. The rats in model group and control group estrous cycle and anestrum all decurtated, but these were no significance difference.5 The changes in ponderal index of ovary Ponderal index of ovary of rats in model group have decreased tendency, compared with normal control ,these were no significance difference(P﹥0.05) ,treated by decoction for warming meridian , ponderal index of ovary of rats in high-dose group have increased tendency, compared with model group, these were no significance difference(P﹥0.05) .6 The changes in gonadal hormone of serumCompared with normal control, gonadal hormone of serum of rats in model group decreased(P<0.01,P<0.05), treated by decoction for warming meridian , the content of E2、P、LH of low-dose group and high-dose group all increase, compared with model group , these were significance difference(P<0.01,P<0.05),the content of T、FSH of high-dose group increased, compared with model group , these were significance difference(P<0.01,P<0.05), the content of E2、P、LH、FSH of control group increased, compared with model group , these were significance differenc(eP<0.01,P<0.05), the content of E2、P of control group increased, compared with high-dose group, these were significance difference(P<0.05). 7The changes of Na+-K+-ATPase of hypothalamus, pituitary gland and ovariesCompared with normal control, the content of Na+-K+-ATPase of pituitary gland and ovaries in model group decreased(P<0.01,P<0.05), treated by decoction for warming meridian , the content of Na+-K+-ATPase of pituitary gland and ovaries of low-dose group and high-dose group all increased, compared with model group , these were significance difference(P<0.01,P<0.05), compared with normal control, these were no significance difference(P>0.05). The content of Na+-K+-ATPase of hypothalamus,pituitary gland and ovaries in control group increased, compared with model group , these were significance difference(P<0.01,P<0.05), compared with normal control, the content of Na+-K+-ATPase of hypothalamus in model group have decreased tendency, but no significance difference(P>0.05).8 The changes of Ca2+-Mg2+-ATPase of hypothalamus, pituitary gland and ovariesCompared with normal control, the content of Ca2+-Mg2+-ATPase of pituitary gland and ovaries in model group decreased(P<0.01,P<0.05), treated by decoction for warming meridian , the content of Ca2+-Mg2+-ATPase of pituitary gland and ovaries of low-dose group and high-dose group all increased, compared with model group , these were significance difference(P<0.01,P<0.05), compared with normal control, these were no significance difference(P>0.05). The content of Ca2+-Mg2+-ATPase of hypothalamus, pituitary gland and ovaries in control group increased, compared with model group , these were significance difference(P<0.01,P<0.05 ) , compared with normal control, the content of Ca2+-Mg2+-ATPase of hypothalamus in model group have decreased tendency, but no significance difference(P>0.05). 9 The changes of SDH of hypothalamus, pituitary gland and ovariesCompared with normal control, the content of SDH of pituitary gland and ovaries in model group decreased(P<0.01,P<0.05), treated by decoction for warming meridian , the content of SDH of pituitary gland and ovaries of low-dose group and high-dose group all increased, compared with model group , these were significance difference(P<0.01,P<0.05), compared with normal control, these were no significance difference(P>0.05). The content of SDH of hypothalamus, pituitary gland and ovaries in control group increased, compared with model group , these were significance difference(P<0.01,P<0.05), compared with normal control, the content of SDH of hypothalamus in model group have decreased tendency, but no significance difference(P>0.05).Conclusions:1 The changes of SDH and ATPase of hypothalamus, pituitary gland and ovaries have closed relationship with the happen of asthenia cold syndrome.2 The content of E2、P、T、LH、FSH of serum in rats of asthenia cold syndrome decreased, the content of SDH, Ca2+-Mg2+-ATPase ,Na+-K+-ATPase of hypothalamus ,pituitary gland and ovaries all decreased.3 Decoction for warming meridian can strikingly improve symptom of the model of asthenia cold syndrome ,regulate the estrous cycle and the level of serum E2、P、T、LH、FSH, regulate the content of SDH, Ca2+-Mg2+-ATPase ,Na+-K+-ATPase of hypothalamus, pituitary gland and ovaries,to make them normal. disproofed the changes of SDH and ATPase of hypothalamus, pituitary gland and ovaries have closed relationship with the happen of asthenia cold syndrome from treated point.更多还原