性激素对成年小鼠脑微管蛋白合成的影响

以＾３Ｈ－秋水仙碱为探针,测定小鼠脑微管蛋白含量,结果表明雌性激素具有显的促进成年离微管蛋白合成的作用,与雌性激素相比,雄性激素促进脑微管蛋白合成的作用较弱,特别值得指出的是雌性激素促进脑微管蛋白合成的作用发生在发育的临界期之外,而此时甲状腺激素早已丧失了促进脑微管收白合成的作用,因此雌性激素在维护成年脑结构和功能的完整完善方面起着重要作用,而且这种作用可能会获得新的应用。     

性类固醇激素对去势和未去势雄鼠脑微管蛋白合成的影响

雌性激素在某些方面改变着神经功能,在雌性激素敏感的神经元中,雌二醇调节各种PNA的表达,包括rRNA、神经多肽和神经传导物受体。这咎影响可能伴有在基因产物合成方面的相应改变或翻译后修饰。最近的研究已经揭示雌性激素能改变轴突生长以及突触器度。在我们以前的实验中注意到性别对脑微管蛋白合成的影响,也发现雌性激素可刺激雄鼠脑微管蛋白合成。雌性激素可能是成年动物神经突生长的一种调节物。这种假设已经得到深入研究和证实;发现性激素对大脑皮质、海马、中脑和间脑神经元的形态和数量有着重要的影响,并改变神经突生长和突触的连接。实验分为去势（GM）和未去势（IM）两组。第一组（GM）为1月龄鼠,去势后10天分为睾丸酮（GMT）、雌二醇（GME）、雌二醇加孕酮（GMEP）三个治疗实验组和一个对照组（GMC）。第二组（IM）为老年雄鼠,分为睾丸酮（IMT）、雌二醇（IME）、雌二醇加孕酮（IMEP）三个治疗组二组（IM）为老年雄鼠,分为睾丸酮（IMT）、雌二醇（IME）、雌二醇和孕酮（IMEP0三个治疗组和对照组（IMC）。对照组注射芝麻油。处理结束,迅速断头,制备脑匀浆液。取上清液进行^H秋水仙碱标记反应,液闪计数,离微管蛋白的生成量。实验结果如表一：微管蛋白每分钟的放射量在GM组中,GME比GMC增加37％－43％,GMT与GMC增加18％－23％;IM组中,IME比IMC增加34％,IMT比IMC增加13％－15％。睾丸酮和雌二醇均能刺激6月龄鼠脑微管蛋白的合成,但两者作用有明显差异,雌二醇作用强于睾丸酮。而且对雌雄鼠具有相同作用,对成年和老年鼠均能促进脑微管蛋白合成。微管蛋白是神经轴突和树突生长必需的成分。对于脑组织和脑功能的至关重要。雌性激素能促进雌雄鼠脑发育临界期以后脑微管蛋白的合成。因而,在临床上可以用于治疗脑发育缺隐和延缓衰老。甲状腺激素也具有促进微管蛋白合成的作用。甲状腺激素缺乏会引起神经细胞分化和神经突生长的停滞。但是,它的作用仅限于胎儿至新生儿早期。雌性激素可以于脑发育时限之外替代甲状腺。衰老首先开始于神经系统,然而波及其它器官或组织。脑衰老的原因是由于一些神经元死亡,突触未端减少,轴突、树突萎缩。外源雌性激素可以促进脑微管蛋白的合成,因而可以延缓衰老。对于微管蛋白的合成,与雌二醇相比,睾丸酮有较弱的促进作用。这被解释为：睾丸酮是通过它的芳香化作用转变成雌二醇而发挥作用的。因而,睾丸酮能否起作用槿起作用的大小,取决于其芳香化程度的大小。如果受到如雌二醇拮抗剂的抑制,睾丸酮的作用甚至会消失。     

性类固醇激素对去势和未去势雄鼠脑微管蛋白合成的影响

雌性激素在某些方面改变着神经功能。在雌性激素敏感的神经元中,雌二醇调节各种ＲＮＡ的表达,包括ｒＲＮＡ、神经多肽和神经传导物受体。这种影响可能伴有在基因产物合成方面的相应改变或翻译后修饰。最近的研究已经揭示雌性激素能改变轴突生长以及突触密度。在我们以前的实验中注意到性别对脑微管蛋白合成的影响,也发现雌性激素可刺激雄鼠脑微管蛋白合成。雌性激素可能是成年动物神经突生长的一种调节物。这种假设已经得到深入研究和证实;发现性激素对大脑皮质、海马、中脑和间脑神经元的形态和数量有着重要的影响,并改变神经突生长和突触的连接。实验分为去势（ＧＭ）和未去势（ＩＭ）两组。第一组（ＧＭ）为１月龄雄鼠,去势后１０天分为睾丸酮（ＧＭＴ）、雌二醇（ＧＭＥ）、雌二醇加孕酮（ＧＭＥＰ）三个治疗实验组和一个对照组（ＧＭＣ）。第二组（ＩＭ）为老年雄鼠,分为睾丸酮（ＩＭＴ）、雌二醇（ＩＭＥ）、雌二醇加孕酮（ＩＭＥＰ）三个治疗组和对照组（ＩＭＣ）。对照组注射芝麻油。处理结束,迅速断头,制备脑匀浆液。取上清液进行３Ｈ秋水仙碱标记反应,液闪计数,测定脑微管蛋白的生成量。实验结果如表一：微管蛋白每分钟的放射量在ＧＭ组中,ＧＭＥ比ＧＭＣ增加３７％～４３％,Ｇ     

发育中小鼠和鸡胚脑微管蛋白的合成与甲状腺功能间的关系

利用 ³H-秋水仙碱与微管蛋白间的特异结合及DEAE纤维素对微管蛋白的离子交换作用,连续测定小鼠、鸡胚脑发育过程中的脑微管蛋白的合成变化。结果表明脑微管蛋白的合成速度均在其脑发育的临界期时达到最高峰。此时恰是甲状腺功能逐渐完善的时期。当小鼠进入育龄期时,雌雄鼠脑微管蛋白含量差异显著。可能说明性激素对微管蛋白的合成有重要影响。     

调节因子IGFⅡ、TNFα、VRF促细胞增殖与β微管蛋白表达的相关性

细胞增殖必伴有染色体的一分为二及细胞质的增生,β微管蛋白则参与细胞的增殖过程．正性和负性调节因子对β微管蛋白的表达及细胞增殖间的相关性研究显示,不同生理剂量的正性调节因子ＩＧＦⅡ、Ｔ３／Ｔ４处理ＵＭＲ１０６细胞１２ｈ,Ｎｏｒｔｈｅｒｎｂｌｏｔ实验发现它们在促进细胞ＤＮＡ合成的同时,可使β微管蛋白ｍＲＮＡ表达增加,呈剂量依赖关系．而负性调节因子ＴＮＦα则相反地在抑制细胞ＤＮＡ合成的同时,使β微管蛋白ｍＲＮＡ表达降低,也呈剂量依赖关系．Ｗｅｓｔｅｒｎｂｌｏｔ实验进一步表明,ＩＧＦⅡ可使β微管蛋白表达增加,而ＴＮＦα使β微管蛋白表达降低．由此可见,β微管蛋白的合成与细胞增殖间存在着一定的相互联系．     

EGF及IGF－Ⅰ对UMR106细胞增殖及β微管蛋白表达的影响

应用３Ｈ－ＴｄＲ参入,流式细胞技术和Ｎｏｒｔｈｅｒｎｂｌｏｔ等方法,观察了ＥＧＦ和ＩＧＦ－Ⅰ对ＵＭＲ１０６细胞增殖及β微管蛋白表达的影响。结果显示,两种生长因子分别处理ＵＭＲ１０６细胞１２ｈ,在促进细胞ＤＮＡ合成的同时,β微管蛋白ｍＲＮＡ的表达量明显提高。运用间接免疫荧光技术及Ｗｅｓｔｅｒｎｂｌｏｔｔｉｎｇ方法,研究发现两种生长因子可使微管聚合及微管蛋白的表达有所增加。提示β微管蛋白的合成及聚合与细胞增殖间可能存在着一定的相互联系．     

银杏花粉微管蛋白的纯化及鉴定

采用丙酮粉抽提,DEAE-SephadexA-50、SephacrylS-300、MonoQ柱层析,从银杏（GinkgobilobaL．）花粉中分高纯化出微管蛋白（tubulin）,其两个亚基（α、β）的分子量分别为54kD和52kD．纯化的微管蛋白可与鸡脑微管蛋白抗体发生免疫交叉反应．     

靶向微管抗肿瘤药物研究进展

微管由微管蛋白组成,在细胞分裂、细胞内物质运输、信号传递、维持细胞形态等过程中起着重要作用.一些干扰微管功能的化合物可使细胞停滞在有丝分裂期而抑制细胞增殖.相对于正常细胞,肿瘤细胞有丝分裂异常频繁,以微管作为抗肿瘤的靶点已成为研究热点.作用于微管的微管蛋白抑制剂通过抑制微管蛋白的聚合促进微管解聚或者抑制微管解聚促进微管蛋白聚合来破坏微管动态平衡、干扰肿瘤细胞纺锤体形成、阻断细胞分裂、抑制肿瘤增殖,现就微管蛋白抑制剂的研究进展作一综述.     

综述:Tau蛋白过度磷酸化机制及其在阿尔茨海默病神经元变性中的作用

Tau蛋白是神经元中含量最高的微管相关蛋白,其经典生物学功能是促进微管组装和维持微管的稳定性．在阿尔茨海默病(Alzheimer's disease,AD)患者,异常过度磷酸化的Tau蛋白以配对螺旋丝结构形成神经原纤维缠结并在神经元内聚积．大量研究提示,Tau蛋白异常在AD患者神经变性和学习记忆障碍的发生发展中起重要作用．本课题组对Tau蛋白异常磷酸化的机制及其对细胞的影响进行了系列研究,发现Tau蛋白表达和磷酸化具有调节细胞生存命运的新功能,并由此对AD神经细胞变性的本质提出了新见解．本文主要综述作者实验室有关Tau蛋白的部分研究结果．     

鼠脑驱动蛋白ATP水解机制的晶体学分析

鼠脑驱动蛋白是一类利用ATP水解释放的能量在微管系统上高连续性运动的常规驱动蛋白。了解ATP水解的化学能如何转化为机械动能是驱动蛋白研究中的重大课题。为此,鼠脑驱动蛋白单体(rK354)的晶体通过浸泡的方式引入ATP的结构类似物AMPPNP。rK354-AMPPNP复合物和rK354-ADP复合物结构的比较,揭示了开关区域Ⅱ的Glu237起连接ATP的γ-磷酸和驱动蛋白微管结合区的枢纽作用。     

Effects of the Development of Thyroid and Sex Glands on the Synthesis of Brain Tubulin in Chick and Mouse Embryos

Using the specific affinity of tubulin for colchicine and the strong absorption of tubulin to DEAEion exchangers at neutral pH and moderate ionic strength,the amounts of tubulin in the brain fromboth mice and chicks during different developing stages were measured by ~3H-colchicine assay(expressed as colchicine binding activity).The results show that the rate oftubulin synthesis reacheda peak value during the critical period of brain development.This is exactly the period during whichthe organization and function of thyroid are being perfected.Besides,during breeding period,thedifference of tubulin contents between male and female is significant(P<0.001).The synthesis oftubulin is strictly sex dependent(this phenomenon appeared only during sex maturation stage).It issuggested that sexual hormones might exert their effect on tubulin synthesis.     

Triiodothyronine regulated tubulin biosynthesis in oligodendrocytes during myelinogenesis

l-Triiodothyronine (T₃) is shown to stimulate the biosynthesis of tubulin in oligodendrocytes isolated from rat brains at different stages of myelinogenesis. The hormone sensitivity appears around day 11, reaches a maximum at day 15 and disappears by day 25 after birth. Dose response studies show that the optimal stimulation of tubulin in the oligodendrocytes from 15 day rat brain occurs with 0.045 nM T₃ in contrast to 4.5 nM T₃ that was previously shown to promote a similar age-dependent induction of tubulin in the astroglial cells from neonatal rat brain. Exposure of the oligodendrocytes to optimal dose of T₃ (0.045 nM) for 2 h elicits a marked and almost selective increase in the level of tubulin. Higher concentrations of T₃ induce additional proteins resulting in a loss of this sensitivity. Studies on the synthesis and turnover of ³⁵S-labeled tubulin show that the stimulation of tubulin by T₃ is primarily due to enhanced synthesis of the protein. Pulse chase experiments reveal that the half life of tubulin is about 5.5 h and that it remains unaffected by T₃. The crucial role of thyroid hormones and the possible function of tubulin in the two most critical phases of brain maturation viz. synaptogenesis and myelinogenesis is discussed.     

Estrogen synthesis in the hippocampus

Estradiol plays essential roles in the modulation of synaptic plasticity and neuroprotection in males as well as in females, as has been shown particularly in the hippocampus. Although it has long been known that aromatase, the final enzyme in estrogen synthesis, is expressed in the hippocampus, a new paradigm emerged when it was shown that estradiol is actually synthesized de novo in this part of the brain. Increasing evidence indicates that hippocampus-derived estradiol plays a role in synaptic plasticity and neuroptrotection, rather than estradiol originating from the gonads. In recent years, a number of in vivo and in vitro studies have shown that hippocampus-derived estradiol substantially contributes to hippocampal function, in particular to structural synaptic plasticity.     

In vivo and in vitro regulation of Akt activation in human endometrial cells is estrogen dependent

Estrogen-bound estrogen receptors (ER) alpha and beta classically activate gene expression after binding to the estrogen response element in the promoter regions of target genes. Estrogen also has rapid, nongenomic effects. It activates several membranous or cytoplasmic kinase cascades, including the phosphatidylinositol 3-phosphate (PI3K/Akt) cascade, a signaling pathway that plays a key role in cell survival and apoptosis. Normal human endometrium is exposed to variable levels of steroid hormones throughout the menstrual cycle. We hypothesized that Akt phosphorylation in human endometrium may vary with the menstrual cycle and in early pregnancy and that fluctuations in estrogen level may play a role in Akt activation in endometrial cells. We analyzed Akt phosphorylation using in vivo and in vitro techniques, including Western blot, immunohistochemistry, and immunocytochemistry. Estradiol significantly increased Akt phosphorylation in endometrial cells. Rapid stimulation of Akt activation in cultured stromal cells was observed. Akt phosphorylation by estradiol was inhibited by the PI3K inhibitor, wortmannin, but not by the ER antagonist, ICI 182 780. The maximal effect on Akt activity was observed following 5-15 min of estradiol treatment. Our results suggest that estradiol may directly affect PI3K-related signaling pathway by increasing the phosphorylation of Akt in endometrial cells. Thus, estradiol may exert part of its proliferative and antiapoptotic effects by a nongenomic manner through the Akt signaling pathway.     

Regulation of tubulin by triiodothyronine in hypothyroid rat brain

The effect of T₃ (triiodothyronine) on the induction of tubulin in hypothyroid developing rat brain has been examined using organ cultures of brains from late fetal, neonatal and postnatalrats. The neonatal brain displayed maximum sensitivity to T₃. Hypothyroidism resulted in a 26% decline in the level of tubulin in the neonatal brain as opposed to a 5–15% decline in the fetal or postnatal brain. Exposure of the hypothyroi d neonatal brain to T₃ for 2 h in culture led to a 61% rise in the level of tubulin in contrast to a 41% increase seen in the case of normal brain. Total protein synthesis was not significantly affected . The preferential decline of tubulin in the neonatal hypothyroid brain, its enhanced sensitivity to T₃ compared to normal brain, and the coincidence of the period of sensitivity to that of brain maturation indicate that the regulation of the level of tubulin by T₃ in the developing brain is a natural ontogenic phenomenon.     

Lever pressing for pups: Evidence for hormonal influence upon maternal behavior of mice

Five experiments were performed to determine the extent to which hormones influence maternal behavior of the female mouse. The number of pup-reinforced lever presses served as the measure of maternal behavior. In Experiment 1 it was found that postpartum mice performed significantly more pup-reinforced lever presses than intact or ovariectomized virgins. The second experiment showed that the augmentation of responding was unique to the postpartum period since responding was not facilitated during the last three days of pregnancy. Experiment 3 established that the postpartum augmentation of responding was mediated by the termination of pregnancy and not by parturition per se. It also was observed that the earlier during gestation pregnancy was terminated the greater the decline in response rate. Experiment 4 proved that ovarian secretions play a critical role in the pregnancy-terminated facilitation of pup-reinforced lever pressing. The final experiment demonstrated that the termination-of-pregnancy effect on responding in all likelihood is mediated by the synergistic actions of estrogen stimulation and progesterone withdrawal. The results show that the maternal behavior of the mouse, like that of the rat, is responsive to hormonal stimulation.     

Evidence for a direct effect of thyroid hormones on the hepatic synthesis of estrogen receptors in the rat

The role of thyroid hormones in the regulation of estrogen receptor turnover in the rat liver was studied. Animals subjected to thyroidectomy or hypophysectomy in combination with different hormone substitutions, were used. The receptor level in control animals was 53 fmol/mg cytosol protein. Thyroidectomy for 28 days caused a dramatic reduction to 20 fmol/mg, whereas hypophysectomy for 9 days resulted in an even more substantial reduction to 11 fmol/mg protein. If animals, hypophysectomized for 9 days, were given triiodothyronine (T3) for 9 days the hepatic estrogen receptor concentration was elevated to 22.5 fmol/mg protein. Estradiol given together with T3 did not cause any further increase in the receptor level. We conclude that thyroid hormones affect the hepatic synthesis of estrogen receptors on two levels, via a direct action on the liver and via an indirect modulation of the pituitary hormone synthesis/release.     

Prolactin and growth hormone synthesis: effects of perphenazine, alpha-methyltyrosine and estrogen in different thyroid states.

The effects of thyroid hormones on prolactin (PRL) and growth hormone (GH) synthesis by the rat anterior pituitary gland were assessed in vitro. A marked reduction (84-87%) in the rate of H3-leucine incorporation into GH was evident 2-4 weeks after thyroidectomy, while incorporation into PRL was 52-71% less than that measured in glands from intact rats. A single injection of T4 (200 mug/kg) administered to thyroidectomized (THX) rats 48 hr before sacrifice significantly increased incorporation into both pituitary hormones, although the stimulation of GH synthesis was much more dramatic. Perphenazine, alpha-methyltyrosine and estrogen enhanced the rate of PRL synthesis in intact rats. Thyroid ablation did not affect the response to perphenazine, but significantly increased the response to alpha-methyltyrosine and estrogen. On the other hand, administration of T4 to THX rats receiving perphenazine, alpha-methyltyrosine or estrogen diminished the stimulatory influence of these treatments on PRL synthesis. Perphenazine, alpha-methyltyrosine and estrogen had no effect on the rate of GH synthesis in THX rats, nor did they alter the ability of T4 to restore GH synthesis in these animals. These results indicate that GH synthesis in the rat is dependent upon thyroid hormones and support the concept that these hormones exert their stimulatory effect directly on pituitary somatotrophs. Pituitary lactotrophs, however, appear to retain much of their capacity to synthesize PRL under conditions of thyroid deficiency. The changes in pituitary PRL levels and synthesis rate induced by thyroid ablation might reflect differences in the number rather than the activity of these cells.