【作者】 乔跃兵；

【导师】 崔慧先；

【作者基本信息】 河北医科大学 ， 人体解剖与组织胚胎学， 2009， 博士

【摘要】 1994年,Zhang等学者利用定位克隆技术成功克隆了小鼠的肥胖基因(obese gene,ob)及人类的同源序列,ob基因编码的蛋白质产物被称为leptin,译为瘦素。leptin的发现使脂肪量和某些中枢部位可能存在着一定信号联系参与调节能量平衡和生殖这一存在了40多年的假说得以证实。ob/ob鼠和db/db(diabete gene,db)鼠分别由于leptin基因突变和leptin受体的基因突变而具有相似的表现:严重的早发肥胖,严重的胰岛素抵抗,饮食过多,能耗降低,不孕不育。但是,给予ob/ob鼠腹腔注射leptin后,不仅体重和摄食恢复至正常水平,生育能力也得以纠正;在雌性ob/ob鼠卵巢和子宫的重量增加,血清黄体生成素(luteinizing hormone,LH)浓度增加,原始卵泡和囊状卵泡数量增多;在雄性ob/ob鼠睾丸和精囊腺重量增加,生成精子数量增多。这些实验结果说明leptin在调节摄食与能量消耗的同时也参与了对生殖功能的调控。尽管有大量的证据支持leptin调节生殖功能的角色,然而leptin究竟是直接还是间接调节下丘脑-垂体-性腺轴,仍旧需要更多的工作去阐明。因此,本实验通过观察leptin对下丘脑POMC(Proopiomelanocortin)神经元表达的影响以及对下丘脑-垂体轴激素分泌的调节作用,研究leptin对下丘脑-垂体轴的可能影响,初步探讨leptin调控生殖的可能作用机理,为leptin的临床应用提供实验依据。研究内容与结果如下:一、Leptin对POMC基因及蛋白水平的调节目的:观察侧脑室微量注射leptin对去卵巢给予一定量雌激素补充的雌性大鼠下丘脑POMC基因及转录后水平的影响。方法:选用成年雌性Wistar大鼠制备OEP模型(ovariectomized, estrogen primed)。在无菌条件下行双侧卵巢切除术,术后从第8天开始每天皮下注射苯甲雌二醇25μg/只,注射5天后根据George Paxinos和Charles Watson编著的《The Rat Brain in stereotaxic coordinates》定位侧脑室:以Bregma点后1.0 mm,旁开1.5 mm,进针深4.0 mm,行侧脑室微量注射。实验组注射leptin 5μl,对照组注射生理盐水5μl;分别于微量注射后1h、2h、4h、6h以视交叉、乳头体为前后界,下丘脑沟为侧界,深约3mm左右为标准取下丘脑。迅速取出大鼠的下丘脑组织,称重,在低温下研磨制成匀浆,4℃3000rpm离心15min,取上清于-80℃保存。按照提取RNA的要求严格取下丘脑,置冻存管中保存于液氮中,依照Trizol的说明提取下丘脑RNA。经过RNA的完整性检测和定量,用RT-PCR的方法检测微量注射后大鼠下丘脑组织POMC mRNA表达的变化。根据Western blot的实验步骤,检测大鼠下丘脑组织POMC蛋白表达的变化,并且对实验结果进行统计分析。结果:1)大鼠下丘脑组织POMC mRNA的表达在侧脑室微量注射leptin 1h后有所增高,但与对照组相比没有显著性差异(P>0.05);在注射2h后POMC mRNA表达增高明显,4h后POMC mRNA表达量达到高峰,与对照组相比有统计学差异(P<0.05);6h后POMC mRNA表达显著降低,与对照组比较没有统计学差异(P>0.05)。2)侧脑室微量注射leptin 1h后,大鼠下丘脑组织POMC蛋白含量没有变化,与对照组相比在统计学上没有显著性差异(P>0.05);leptin注射2h后POMC蛋白含量开始增高,4h后POMC蛋白含量达到高峰,6h后POMC蛋白含量明显降低,三组与对照组比较均具有明显的统计学差异(P<0.05)。结论:1)侧脑室微量注射leptin后,对下丘脑组织中的POMC mRNA表达的调节具有正向调节作用。2)侧脑室微量注射leptin后,对下丘脑组织中的POMC蛋白表达具有正向调节作用。3)leptin可能通过POMC神经元对机体能量平衡与营养状态起着调节作用。二、侧脑室注射Leptin、α-MSH对下丘脑—垂体轴激素分泌的影响目的:采用侧脑室微量注射leptin、α-MSH,观察在注射后不同时间段对大鼠血浆GnRH、LH、FSH的影响,初步探讨POMC神经元对下丘脑—垂体—生殖轴的调节作用。方法:选用成年清洁级雌性Wistar大鼠,随机分为对照组、Leptin组、α-MSH组。在无菌条件下行双侧卵巢切除术,制作OEP大鼠模型。Leptin组注射leptin 5μl,α-MSH组注射α-MSH 5μl,对照组注射生理盐水5μl,于1min内注入侧脑室。分别于微量注射后1h、2h、4h和6h在麻醉状态下开胸,从右心房采集静脉血,离心后取上层血浆,在-20℃下储存备测。本实验采用ELISA酶免试剂盒进行血浆激素检测。结果:1)侧脑室注射Leptin后1h,大鼠血浆GnRH浓度与对照组相比明显增高,注射后2h,血浆GnRH浓度达到高峰。注射后4h,GnRH浓度迅速回落至1h时浓度以下,但仍较对照组浓度高。1h、2h、4h时GnRH浓度与对照组相比有统计学差异; Leptin注射后6h时,血浆GnRH浓度已恢复到对照组水平,与对照组之间没有统计学差异。2)侧脑室微量注射Leptin后1h,大鼠血浆LH浓度与对照组相比明显增高,注射Leptin后2h,血浆LH浓度继续增高并达到高峰。在4h时,LH浓度下降,但与对照组相比仍维持在较高水平。1h、2h、4h时的浓度与对照组相比具有统计学差异;在注射Leptin 6h后,LH浓度与对照组相比,没有明显的统计学差异。3)侧脑室注射Leptin后1h,大鼠血浆FSH分泌有所增多,但与对照组比较没有统计学差异;2h时FSH血浆浓度迅速升高并达到高峰,4h时FSH浓度明显降低。2h、4h时的FSH浓度与对照组相比具有统计学差异;在注射Leptin 6h后,FSH浓度与对照组相比,没有明显的统计学差异。4)侧脑室注射α-MSH后1h,大鼠血浆GnRH浓度降低,与对照组相比具有明显的统计学差异;注射2h后,血浆GnRH浓度继续降低,与对照组相比具有显著性统计学差异;在4h和6h时,GnRH浓度与对照组相比,没有明显的统计学差异。5)侧脑室注射α-MSH后1h,大鼠血浆LH浓度明显降低,2h时LH浓度继续降低,4h时LH浓度回升,较1h时浓度为高,6h时LH浓度恢复正常。注射α-MSH后LH浓度在1h、2h和4h时与对照组比较具有统计学差异;在6h时,LH浓度与对照组相比,没有明显的统计学差异。6)侧脑室注射α-MSH后1h,大鼠血浆FSH浓度略有降低,与对照组相比,没有明显的统计学差异;2h时FSH浓度明显降低,4h时FSH浓度回升,6h时FSH浓度恢复正常。注射α-MSH后FSH浓度在2h和4h时与对照组比较具有统计学差异;在6h时,FSH浓度与对照组相比,没有明显的统计学差异。结论:1)微量注射Leptin对去卵巢给予雌激素补充的雌性大鼠GnRH、LH、FSH分泌具有正向调节作用。2)微量注射α-MSH对去卵巢给予雌激素补充的雌性大鼠GnRH、LH、FSH分泌具有负向调节作用。三、Leptin、α-MSH对体外培养的大鼠垂体细胞LH、FSH分泌的影响目的:通过leptin、α-MSH对体外培养的大鼠垂体前叶(AP)细胞分泌LH、FSH的影响,初步探讨leptin、α-MSH在垂体水平对生殖功能的调节作用。方法:实验动物是清洁级成熟雌性Wistar大鼠,给予腹腔麻醉,酒精消毒大鼠头皮,用手术刀将头皮拨开,暴露颅骨,用止血钳将头盖骨剥离暴露大脑,快速取出大脑,并用眼科镊子取出靠近脑干部位的脑垂体,放入盛有D-Hanks平衡盐溶液的玻璃培养皿中。去除神经垂体,保留腺垂体。用眼科剪将漂洗好的腺垂体剪成<1mm3大小的腺垂体块,经过消化、离心、过滤、活细胞计数、培养,观察细胞生长情况。于培养的第4天,在培养液中分别加入不同浓度的leptin、α-MSH,继续培养24h后,取各组培养液,于-20℃储存,用酶免试剂盒对各组培养液中的LH、FSH含量作检测。结果:1)接种细胞最初为圆形,形态完整,6h内开始贴壁,24h内一些细胞开始长出突起,48h细胞贴壁良好,出现不规则的聚集生长。垂体前叶细胞呈现椭圆形、三角形或多角形,呈典型的上皮样细胞。在培养的7d内,腺细胞在形态上除了增大外,几乎没有明显的改变。从培养的第5d起,另一群细胞开始日渐明显,数量逐渐增多,并向成纤维细胞方向发展。7d成纤维细胞开始过度生长,而腺细胞开始失去了原有形态特点,到第10d时,基本被成纤维细胞取代。2)在leptin的刺激下,LH的释放量随leptin浓度的增高而增加,当leptin浓度达到10-9 mol/L时,LH释放量达到高峰,之后随leptin浓度的增加LH的释放量反而降低。除当leptin浓度为1×10-12mol/L时,LH的释放量与对照组比较,没有显著差异外,其他各组LH的释放量与对照组比较,均有显著差异。不同leptin浓度组之间比较,均有显著差异。3)在leptin的刺激下,FSH的释放量随leptin浓度的增高亦增高。当leptin浓度达到1×10-9 mol/L时,FSH释放量达到高峰,之后随leptin浓度的增加FSH的释放量反而减少。除当leptin浓度为1×10-12和1×10-6 mol/L时,FSH的释放量与对照组比较,没有显著差异外,其他各组FSH的释放量与对照组比较,均有显著差异。不同leptin浓度组之间比较,除1×10-12mol/L组和1×10-6 mol/L组两组之间没有显著性差异外,各组之间均有显著差异。4)在α-MSH的刺激下,LH、FSH释放量与对照组比较没有显著性差异,不同α-MSH浓度组之间LH、FSH释放量比较也没有显著性差异。结论:1)体外培养的垂体细胞6h内开始贴壁,24h内一些细胞开始长出突起, 48h细胞贴壁良好,开始聚集成团。从第5d起,另一群细胞开始日渐增多,并向成纤维细胞方向发展。在培养的7d内,腺细胞在形态上除了增大外,几乎没有明显的改变。此时成纤维细胞开始过度生长,而腺细胞开始失去了原有形态特点。培养的垂体细胞最初为圆形,进而呈椭圆形、三角形或多角形。到第10d时,基本被成纤维细胞取代。2)Leptin对培养的腺垂体细胞LH、FSH的分泌均有影响,当Leptin浓度为10-9mol/L对LH、FSH的分泌可产生最大效应。Leptin浓度增高或降低作用均减弱。3)α-MSH对体外培养的大鼠垂体细胞LH、FSH的分泌没有影响。更多还原

【Abstract】 In 1994, Zhang et al. had successfully cloned the little mouse’s obese gene and human homology sequence using positional cloning technology. The protein of ob gene code is known as leptin. The discovery of leptin confirmed the hypothesis about forty years ago: energy stores represented by the adipose tissue might produce some signaling molecules able to modulate both energy balance and reproduction to CNS sites. Ob/ob mouse and db/db (diabetic gene, db) mouse have similar physical features because of the gene mutation of leptin and gene mutation of leptin receptor separately: the serious early obesity, the serious insulin resistance, hyperphagia, reduced energy expenditure and infertility in both sex. However, after injecting leptin to the abdominal cavity of ob/ob mouse, not only their weights and diets back to the normal level, but also retrieved reproduction ability. In female ob/ob mouse, ovary and uterine weight increased, serum LH raised, primordial follicles and vesicular follicles increased. weight gained in male ob/ob mouse’s testicle and seminal vesicle gland, sperm’s quantity increased. The observation suggested that leptin controlled reproduction as modulating energy expenditure and food intake.Though there are a large number of evidence supporting the role of leptin to regulate the reproduction function, whether leptin regulates the hypothalamus-pituitary-gonadal axis directly or indirectly, still needs more studies to clarity. Therefore, this experiment study on leptin’s probable function on the hypothalamus-pituitary axis, and expect to tentatively probe into leptin’s mechanism of regulating reproduction, offer certain experimental data for clinical practice, via observing leptin’s influence on the expression of POMC (Proopiomelanocortin) ih hypothalamus and the regulating function on the hypothalamus-pituitary axis hormone. Research contents and results are as follows:1 Regulation of leptin on POMC gene and its protein Objective: To observe the influence after injection of leptin into the cerebral ventricle on the female OEP(ovariectomized, estrogen primed) rat’s POMC mRNA and its protein.Methods: Select adult female Wistar rat to make OEP model , from the 8th day after the operation, hypodermic injection of Estradiol Benzoate about 25μg everyday for each rat, after 5 days injection, located the cerebral ventricle according to“The Rat Brain in stereotaxic coordinates”written by George Paxinos and Charles Watson. 5μl leptin was injected into the cerebral ventricle in the experimental group, the control group was injected with saline 5μl. After injection, the brain tissue was cut out between optic chiasma and mamillary body rapidly at different time then weigh and grind under the low temperature, centrifuged for 15 min at 4℃3000rpm, the supernatants were collected and stored at -80℃. According to the Trizol interpretation, hypothalamus tissues were stored at liquid nitrogen and RNA was extracted strictly. After detection and quantitation, expression of POMC mRNA after microinjection was measured by RT-PCR. According to the experimental step of Western blot, observing the change of POMC protein. All the results were analyzed by statistical analysis.Results: 1 After microinjection 1.0h later POMC mRNA expression in rats’hypothalamus increased slightly, but there was no significant difference in leptin-injected group when compared with control group(P>0.05). POMC mRNA expression in hypothalamus increased in leptin-injected rats both 2.0h later and 4.0h later, and there was significant difference between control group and leptin-injected group(P<0.05). And 6.0h later, there was no significant difference between control group and leptin-injected group(P>0.05). 2 Effects on POMC expression by lateral cerebral ventricle injection of leptin: 1.0h later after injection, POMC expression in rats’hypothalamus no significantly increased compared with control group, there was no significant difference in leptin-injected group(P > 0.05). Both 2.0h later and 4.0h later after microinjection, POMC expression in hypothalamus begun to increase and then reach its highest level, there was significant difference between leptin-injected group and control group(P<0.05). 6.0h later, expression of POMC in hypothalamus decreased in leptin-injected group, but there was also significant difference between the groups(P<0.05).Conclusions: 1 After the lateral cerebral ventricle injection of leptin, the expression of POMC mRNA in hypothalamus increased, leptin injection show a positive regulation to POMC mRNA. 2 After injection of leptin, POMC protein expression in hypothalamus also increased, leptin injection show a positive regulation to POMC protein expression. 3 Leptin could play a regulating role to energy balance and nutrition state through POMC neuron.2 Effects on the hormone secretion of hypothalamus-pituitary axis by lateral cerebral ventricle injection of leptin andα-MSHObjectives: To observe the influence on rats’plasma GnRH, LH, FSH by the lateral cerebral ventricle microinjection of leptin andα-MSH. Explore the effects of POMC neuron on hypothalamus-pituitary- reproduction axis.Methods: Adult female Wistar rats were divided into three groups randomly: control group, Leptin group andα-MSH group. All the rats were ovariectomized in sterile condition. Since the 8th day after operation, rats were injected s.c. with 25μg of estradiol benzoate everyday and were used for 5 days. Then according to the《The Rat Brain in Stereotaxic Coordinates》written by George Paxinos and Charles Watson, lateral cerebral ventricle was located: posterior to bregma point 1.0mm, lateral 1.5mm, deep 4.0mm. Injecting respectively leptin 5μl,α-MSH 5μl, saline 5μl into lateral cerebral ventricle according to the groups. 1h, 2h, 4h and 6h later after microinjection, open the thorax of the rat under anesthesia, blood samples were collected from the right atrium and stored at -20℃after centrifugation until measurements of GnRH、LH and FSH by ELISA kits.Results: 1 Effects on rats’serum GnRH by lateral ventricle microinjection of leptin: the concentration of GnRH increased significantly. after microinjection 1.0h later, After microinjection 2.0h later, the concentration of GnRH reached its highest level. Microinjection 4.0h later, the concentration of GnRH decreased significantly. Concentration of GnRH after microinjection 1.0h、2.0h、4.0h later, there was significant difference compared with control group(P<0.05). After microinjection 6.0h later, there was no significant difference between treated group and control group (P>0.05). 2 Results of rats serum LH level by lateral ventricle injection of leptin: measurements of ELISA were as follows: after microinjection 1.0h later, serum LH level increased significantly, and there were significant changes between treated group and control group(P<0.05). 2.0h later, compared with saline group, serum LH level increased continuously in leptin-injected group. After microinjection 4.0h later, serum LH level decreased significantly, and there was also significant difference between the two groups(P<0.05). However 6.0h later, rats serum LH level decreased to normal level in leptin-injected group, and there was no significant difference when compared with control group(P>0.05). 3 Effects on rats serum FSH by lateral ventricle injection of leptin: after microinjection 1.0h later, there was no significant changes on serum FSH level in treated group and control group(P>0.05). But 2.0h later, compared with control group, serum FSH level increased quickly in leptin-injected animals, and there was significant difference between the two groups(P<0.05). FSH level decreased significantly after microinjection 4.0h later, but there was still significant difference between control group and leptin-injected group(P<0.05). There was no significant difference between treated group and control group 6.0h later(P>0.05). 4 Results after lateral ventricle injection ofα-MSH were as follows: 1.0h later, rats’plasma GnRH concentration was decreased, compared with the control group, and there was significant difference between the two groups(P<0.05). After injection 2.0h, the plasma GnRH concentration continues reducing, compared with the control group, and there was also significant difference between the two groups(P<0.05). 4.0h and 6.0h later, GnRH concentration compared with the control group, there is no significant difference(P>0.05). 5 After the lateral ventricle injection ofα-MSH for 1.0h later, LH plasma concentration reduced obviously. 2.0h later, LH concentration decreased continuously and 4.0h later, the concentration of LH increased significantly. 1.0h、2.0h and 4.0h later, LH concentration compared with control group, there was significant difference. LH concentration returned to normal at the 6h, and there was no significant difference betweenα-MSH group and control group. 6 The resultes of FSH concentration after microinjection ofα-MSH were as follows: after microinjection ofα-MSH 1.0h later, the rats’plasma FSH concentration was slightly decreased, compared with the control group, but there was no significant difference between the tow groups(P>0.05). FSH concentration remarkably decreased after microinjection ofα-MSH 2.0h later. FSH concentration increased after 4.0h later, but also lower than the normal. FSH concentration returned to normal at 6.0h after microinjection ofα-MSH. FSH concentration after injection ofα-MSH 2.0h and 4.0h later, there was significant difference between experiment groups and control groups(P<0.05). 6.0h later, FSH concentration there was no significant difference compared with the control group.Conclusions: 1 There was a positive regulation effect on OEP rats’plasma GnRH、LH and FSH concentration after microinjection of leptin. 2 There was a negative regulation effect on OEP rats’plasma GnRH、LH and FSH concentration after microinjection ofα-MSH.3 Effect of leptin andα-MSH on the secretion of LH、FSH from cultured pituitary cells in vitroObjectives: To study the effect of leptin andα-MSH on reproductive at pituitary level through researching the influence of leptin andα-MSH on the secretion of LH、FSH from cultured anterior pituitary cells in vitro.Methods: Adult female Wistar rats, abdominal cavity anesthesia, skin was sterilized by alcohol, cut the skin to expose the skull, strip the skull and expose the brain, then took out the pituitary quickly. Put it into the balanced salt solution of D-Hanks. Separating posterior pituitary and anterior pituitary. Cut the anterior pituitary into small pieces( <1mm3), through digesting, centrifugalization, filtering, living cell counting, and observe cells growing. On the 4th day of cells cultured, added leptin andα-MSH of different concentration separately into culture medium. After continuing cells culture for 24h, collecting the culture liquid of every group, stored at -20℃, measuring LH、FSH concentration in the culture liquid with ELISA kits.Results: 1)The shape of cells was round at the beginning of culture and stick to the wall of the glass dish in 6h, some cells began to grow and protrude in 24h, after 48h cell stick to the wall well, then the cells began to get together. The shape of anterior pituitary cells appeared to be oval, triangle or the polygon, like epithelioid cell. In 7d, the shape of gland cell was not changed obviously except growing. From 5th day on, another cell began to grow day by day, and the quantity increased gradually, then developing into the fibroblast. From 7th day on, the fibroblast grew rapidly, and the gland cell began to lose the original shape and characteristic, from 10th day on, the gland cell was almost replaced by the fibroblast completely. 2 The releasing amount of LH increased with the increasing of leptin concentration. When leptin concentration was 10-9 mol/L, LH releasing amount reached its highest level, then the releasing amount of LH decreased with increasing of leptin concentration. Except that when leptin concentration was 10-12 mol/L, the releasing amount of LH was significantly difference compared with the control group(P<0.05). Between different groups of leptin, the releasing amount of LH was also significantly difference(P<0.05). 3 Under the stimulation of leptin, the releasing amount of FSH also increased with the increasing concentration of leptin. When leptin concentration was 10-9 mol/L, FSH releasing amount reached its highest level, then the releasing amount of FSH decreased with increasing concentration of leptin. Except that when leptin concentration were 10-12 mol/L and 10-6 mol/L, the releasing amount of FSH was significantly difference compared with the control group(P<0.05). Between different groups of leptin, except the two groups of 10-12 mol/L and 10-6 mol/L, the releasing amount of FSH was also significantly difference(P<0.05). 4 Under the stimulation ofα-MSH, the releasing amount of LH, FSH there was no significant difference compared between experiment groups and control groups. the releasing amount of LH, FSH there was also no significant difference compared between different experiment groups. Conclusions: 1 The cultured anterior pituitary cells in vitro stick to the wall of the glass dish in 6h, some cells began to grow and protrude in 24h, after 48h cell stick to the wall well. From 5th day on, another group cells began to grow and developing into the fibroblast. In 7d, the shape of gland cell was not changed obviously except growing, then the fibroblast grew rapidly, and the gland cell began to lose the original shape and characteristic. The shape of anterior pituitary cells appeared to be oval, triangle or the polygon. From 10th day on, the gland cell was almost replaced by the fibroblast completely. 2 There was obviously influence on the secretion of LH、FSH from cultured pituitary cells in vitro by leptin stimulation. When leptin concentration was 10-9 mol/L, LH、FSH releasing amount reached its highest level. The effect would be depressed when the concentration of leptin was higher or lower. 3α-MSH did not influence the secretion of LH、FSH from cultured pituitary cells in vitro.更多还原