雌激素替代防治去卵巢动物脑缺血损伤的作用及机制研究

发布时间：2018-05-13 12:48

  本文选题：脑卒中 + 雌激素替代疗法　； 参考：《第四军医大学》2017年博士论文

【摘要】：【背景】脑卒中已成为导致老年人死亡和残疾的最主要原因。前期大量流行病学调查和基础研究均证实雌激素具有显著的脑卒中神经保护作用。相比于同年龄段男性,绝经后女性脑卒中发病率明显增高且预后更差。但大规模临床试验却发现,雌激素替代疗法并未降低绝经女性脑卒中的发生率,甚至加剧了神经损伤,并增加了乳腺癌和子宫肌瘤的发病风险。因此,研究者回归基础实验,深入探究如何才能有效发挥雌激素的脑卒中神经保护作用及其机制,并提出了一系列假说,包括剂量依赖假说,关键期假说和受体选择性假说。前期研究发现,星形胶质细胞大量表达雌激素受体,是介导雌激素神经保护作用的重要靶点。ndrg2是我校生化教研室于2001年首先克隆的新基因,在脑内ndrg2特异性表达于星形胶质细胞,参与多种神经退行性疾病的发生发展,但其在脑缺血损伤中的作用还知之甚少。前期研究发现ndrg2是雌激素及其受体β的靶基因。然而,ndrg2在雌激素替代疗法防治脑卒中损伤中的作用还未见研究报道。【目的】1.探究雌激素替代疗法防治去卵巢动物脑缺血损伤的剂量选择效应和机制;2.探究雌激素替代疗法防治去卵巢动物脑缺血损伤是否存在治疗时间窗和机制;3.探究激活雌激素受体β(ERβ)能否减轻去卵巢动物脑缺血损伤及分子机制;4.探究ndrg2在雌激素替代疗法防治去卵巢动物脑缺血损伤的作用。【方法】1.在体:给予SD雌性大鼠、C57雌性小鼠和ndrg2基因敲除雌性小鼠去卵巢手术(OVX)构建雌性动物绝经模型,给予不同剂量的E2以及雌激素受体β激动剂DPN替代治疗,利用实时荧光定量PCR(Real Time PCR)检测脑内ndrg2 mRNA表达情况,利用免疫印迹方法(Western blot)检测脑内GFAP、NDRG2表达情况;给予ndrg2Loxp小鼠侧脑室注射腺相关病毒,构建选择性下调成年雌鼠脑内ndrg2表达的工具鼠;对正常动物和接受激素替代治疗的OVX雌性动物构建大脑中动脉缺血(MCAO)和全脑缺血(GCI)模型,利用Garcia和Longa评分检测神经功能情况,利用RT-PCR检测ndrg2 mRNA表达情况,利用Western blot检测NDRG2、凋亡蛋白的表达,利用TTC染色、TUNEL染色、尼氏染色和Neun染色检测神经损伤情况;2.离体:利用PC12细胞系,原代培养的星形胶质细胞、神经元和二者共培养,给予不同剂量E2、ERα激动剂PPT和ERβ激动剂DPN处理,利用相差显微镜观察细胞形态改变,利用MTT法检测细胞活力,利用LDH测试检测细胞损伤,利用流式细胞仪检测细胞周期,利用细胞免疫荧光检测细胞增殖,利用Real Time PCR检测ndrg2 mRNA表达情况,利用Western blot检测GFAP、NDRG2表达情况;构建氧糖剥夺(OGD)模型,利用流式细胞仪检测神经细胞凋亡,利用RT-PCR检测ndrg2m RNA表达情况,利用Western blot和细胞免疫荧光检测NDRG2和凋亡蛋白表达情况。【结果】1.(1)生理剂量(10 nM或20 nM)的E2(E2)促进PC12细胞增殖,减轻OGD后PC12细胞的损伤,药理剂量(10μM或20μM)的E2抑制PC12细胞增殖,加重OGD后PC12细胞的损伤;(2)OVX显著增加了雌性大鼠脑缺血损伤,生理剂量(6μg/kg和20μg/kg)的E2替代治疗显著减轻OVX雌性大鼠脑缺血神经损伤,超生理剂量(50μg/kg)的E2替代治疗并没有减轻OVX雌性大鼠脑缺血神经损伤。2.(1)短期去卵巢(OVX 1w)和长期去卵巢(OVX 10w)并没有改变雌性小鼠脑内ERα的表达;(2)OVX 10w雌性小鼠脑内ERβ表达显著减少,ERα-Ser118位点磷酸化水平显著降低,而OVX 1 w雌性小鼠脑内ERβ表达无明显改变,ERα-Ser118位点磷酸化水平无明显改变;(3)生理低剂量(50μg/kg)或高剂量(100μg/kg)E2替代治疗能够显著减轻OVX 1w雌性小鼠脑缺血神经损伤,但不能减轻OVX 10w雌性小鼠脑缺血神经损伤。3.(1)在雌性小鼠脑内,ERβ主要与星形胶质细胞标志物GFAP存在共定位;在原代培养的星形胶质细胞,ERβ的表达明显多于ERα;(2)OVX雌性小鼠脑内GFAP表达显著下降,50μg/kg E2和8 mg/kg DPN替代治疗能够显著增加OVX小鼠脑内星形胶质细胞GFAP的表达;(3)(2.5 nM、5 nM、10 nM、20 nM)E2剂量依赖性上调原代星形胶质细胞GFAP的表达,并于5 nM和10 nM时达到峰值;10 nM DPN显著上调原代星形胶质细胞GFAP的表达,而10 nM PPT不能上调原代星形胶质细胞GFAP的表达;(4)50μg/kg E2和8 mg/kg DPN替代治疗能够显著减轻短期OVX雌性小鼠脑缺血神经损伤;(5)10 nM E2和10 nM DPN预处理星形胶质细胞能够减轻OGD后神经元凋亡;4.(1)E2能够上调原代星形胶质细胞ndrg2 mRNA和蛋白的表达,并存在时间和剂量依赖效应;(2)10 nM DPN上调原代星形胶质细胞ndrg2 mRNA和蛋白表达,10nM PPT不能上调原代星形胶质细胞ndrg2 mRNA和蛋白表达;(3)OVX雌性小鼠脑内ndrg2表达显著下降,50μg/kg E2、100μg/kg E2及8 mg/kg DPN替代治疗显著增加OVX雌性小鼠脑内ndrg2 mRNA和蛋白的表达,而2 mg/kg PPT替代治疗并不能显著增加OVX雌性小鼠脑内ndrg2 mRNA和蛋白的表达;(4)脑缺血损伤后,缺血半暗带区域ndrg2 mRNA和蛋白的表达于再灌注6 h开始增高,到24 h达到峰值,并发生核转位现象;(5)原代星形胶质细胞OGD损伤后ndrg2 mRNA和蛋白的表达于复氧复糖2 h显著增加,于6 h即达到峰值,并发生核转位现象;(6)ndrg2基因敲除(ndrg2-/-)雌鼠脑缺血神经损伤明显重于野生型(ndrg2+/+)雌鼠;利用腺相关病毒和ndrg2 Loxp雌性小鼠成功地选择性下调了成年雌性小鼠脑内NDRG2的表达,并加重了脑缺血神经损伤;(7)50μg/kg E2或8 mg/kg DPN替代治疗能够显著减轻ndrg2+/+-OVX雌鼠脑缺血神经损伤,而50μg/kg E2或8 mg/kg DPN替代治疗不能够减轻ndrg2-/--OVX雌鼠脑缺血神经损伤。【结论】1.应选择生理剂量的雌激素替代防治去卵巢动物的脑缺血损伤;2.雌激素替代疗法防治去卵巢雌性动物脑缺血损伤存在“最佳治疗时间窗”,与长期去卵巢动物脑内ERβ表达显著降低和ERα-Ser118位点磷酸化水平显著降低有关;3.选择性激活ERβ的DPN替代治疗(DRT)通过恢复去卵巢雌性动物脑内星形胶质细胞的活性减轻脑缺血神经损伤,加之其无致癌副作用,因此,选择性激活ERβ有望成为防治绝经女性脑卒中的新策略;4.星形胶质细胞ndrg2是雌激素及ERβ的靶基因;5.星形胶质细胞ndrg2是介导雌激素及ERβ脑缺血神经保护作用的关键分子。
[Abstract]:[background] stroke has become the leading cause of death and disability in the elderly. A large number of early epidemiological investigations and basic studies have confirmed that estrogen has a significant neuroprotective effect on stroke. Compared to men of the same age, the incidence of stroke in postmenopausal women is significantly higher and the prognosis is worse. It was found that estrogen replacement therapy did not reduce the incidence of stroke in menopause women, even aggravated nerve damage, and increased the risk of breast cancer and uterine myoma. Therefore, the researchers returned to the basic experiment to explore how to effectively exert the protective effect and mechanism of estrogen in stroke and put forward a system. The column hypothesis, including the dose dependence hypothesis, the critical period hypothesis and the receptor selectivity hypothesis, has been found that astrocytes express estrogen receptors in large quantities and are an important target for the protection of estrogen neuroprotection,.Ndrg2, a new gene cloned first in the biochemistry teaching and Research Department of our school in 2001, and the specific expression of NDRG2 in the brain in astrocytes in the brain. Stromal cells are involved in the development of a variety of neurodegenerative diseases, but their role in cerebral ischemia is poorly understood. Earlier studies have found that NDRG2 is the target gene for estrogen and its receptor beta. However, the role of NDRG2 in the prevention and treatment of cerebral apoplexy by estrogen replacement therapy has not been reported. [Objective] 1. explore estrogen replacement. The therapeutic effect and mechanism of the treatment to prevent the cerebral ischemia injury of ovariectomized animals; 2. whether there is a time window and mechanism for the treatment of cerebral ischemia injury in ovariectomized animals by estrogen replacement therapy; 3. to explore whether the activation of estrogen receptor beta (ER beta) can reduce the damage of cerebral hemorrhage in the ovariectomized animals and the molecular mechanism; 4. explore NDRG2 in estrogen replacement. [Methods] 1. in vivo: 1. female rats, C57 female mice and NDRG2 gene knockout female mice were given to ovariectomy (OVX) to construct female menopause model of female animals, and different doses of E2 and estrogen receptor beta agonist DPN replacement therapy were given, and real-time fluorescent quantitative PCR (Real) was used. The expression of NDRG2 mRNA in the brain was detected by Time PCR, and the expression of GFAP and NDRG2 in the brain was detected by immunoblotting (Western blot), and the mice were injected with adeno-related virus in the lateral ventricle of the mice to construct a tool for selectively down-regulation of NDRG2 expression in the brain of adult female rats; the construction of OVX females and the OVX female animals receiving hormone replacement therapy were constructed. The model of middle cerebral artery ischemia (MCAO) and whole brain ischemia (GCI) was used to detect neural function by Garcia and Longa scores. RT-PCR was used to detect the expression of NDRG2 mRNA. Western blot was used to detect NDRG2, the expression of apoptotic protein, TTC staining, TUNEL staining, Nissl staining and staining were used to detect nerve damage; 2. in vitro: use of fine fine. Cell lines, primary cultured astrocytes, neurons and two groups were co cultured and treated with different doses of E2, ER alpha agonist PPT and ER beta agonist DPN. Cell morphology changes were observed by phase contrast microscope, cell viability was detected by MTT method, cell injury was detected by LDH test, cell cycle was detected by flow cytometry, cell cycle was detected by flow cytometry, and cell exempts were used. The cell proliferation was detected by the immunofluorescence, the expression of NDRG2 mRNA was detected by Real Time PCR, the expression of GFAP and NDRG2 were detected by Western blot, the oxygen glucose deprivation (OGD) model was constructed, the apoptosis of the nerve cells was detected by flow cytometry, and the expression of ndrg2m was detected by RT-PCR, and apoptosis was detected and apoptosis was detected by cell immunofluorescence and apoptosis. [results] 1. (1) the physiological dose (10 nM or 20 nM) of E2 (E2) promoted the proliferation of PC12 cells, reduced the damage of PC12 cells after OGD, and the pharmacological dose (10 mu or 20 u M) E2 inhibited the proliferation of PC12 cells and aggravated the injury of the cells after OGD, and (2) it significantly increased the cerebral ischemia injury in the female rats, and the physiological dose (6 Mu and 20 mu) 2 replacement therapy significantly alleviated the cerebral ischemic nerve injury in OVX female rats. The super physiological dose (50 g/kg) E2 replacement therapy did not reduce the.2. (1) short-term ovariectomized (OVX 1W) and long-term ovariectomized (OVX 10W) of OVX female rats, and did not change the expression of ER alpha in the brain of female rats; (2) the expression of beta expression in the brain of OVX 10W female mice was significant. The level of phosphorylation of ER alpha -Ser118 site decreased significantly, while the expression of ER beta in the brain of OVX 1 W female mice was not significantly changed, and there was no significant change in the phosphorylation level of ER alpha -Ser118 loci. (3) the replacement therapy of low dose (50 u g/kg) or high dose (100 micron) E2 could significantly reduce the brain ischemic nerve injury in OVX 1W female mice, but it could not reduce it. 10W female mice brain ischemic nerve injury.3. (1) in the female mouse brain, ER beta is mainly located in the astrocyte marker GFAP. In the primary cultured astrocytes, the expression of ER beta is more than ER alpha; (2) the expression of GFAP in the brain of OVX female mice decreased significantly, and the replacement therapy of 50 mu g/kg E2 and 8 mg/kg DPN could significantly increase the smaller size. The expression of astrocyte GFAP in rat brain; (3) (2.5 nM, 5 nM, 10 nM, 20 nM) E2 dose dependent up regulation of the expression of GFAP in primary astrocytes, and peak value at 5 nM and 10 nM; 10 nM DPN significantly up-regulated the expression of primary astrocyte GFAP, and 10 failed to increase the expression of primary astrocytes; (4) 50 micron The replacement therapy of 8 mg/kg DPN can significantly reduce the cerebral ischemic nerve injury in short term OVX female mice; (5) 10 nM E2 and 10 nM DPN pretreated astrocytes can reduce neuronal apoptosis after OGD; 4. (1) E2 can up regulate the expression of NDRG2 mRNA and protein in primary astrocytes, and there is a time and dose dependence effect; (2) 10 nM up up. The expression of NDRG2 mRNA and protein in primary astrocytes, 10nM PPT could not increase the expression of NDRG2 mRNA and protein in the primary astrocytes. (3) the expression of NDRG2 in the brain of OVX female mice decreased significantly. The expression of 50 micron E2100 in g/kg E2 and 8 of the protein expression in the brain of female mice was significantly increased. 2 Treatment did not significantly increase the expression of NDRG2 mRNA and protein in the brain of OVX female mice; (4) after cerebral ischemia, the expression of NDRG2 mRNA and protein in the ischemic penumbra region began to increase, to the peak of 24 h, and the phenomenon of nuclear transposition, and (5) the expression of NDRG2 mRNA and protein in the original astrocytes after OGD injury. The 2 h was significantly increased and the peak was reached at 6 h, and the nuclear transposition occurred. (6) the cerebral ischemic nerve injury in NDRG2 gene knockout (ndrg2-/-) female rats was significantly heavier than that of the wild type (ndrg2+/+) female mice. The expression of NDRG2 in the brain of adult female mice was successfully selected by adeno-related virus and NDRG2 Loxp female mice, and the brain deficiency was aggravated. Blood nerve injury; (7) 50 g/kg E2 or 8 mg/kg DPN replacement therapy can significantly reduce the cerebral ischemic nerve injury in ndrg2+/+-OVX female rats, and 50 micron E2 or 8 mg/kg DPN replacement therapy can not reduce the cerebral ischemic nerve injury in ndrg2-/--OVX female rats. [Conclusion] 1. the physiological dose of estrogen replacement should be chosen to prevent the cerebral ischemia from the ovariectomized animals. 2. estrogen replacement therapy has a "best time window" for the prevention of cerebral ischemia in female ovariectomized animals, which is associated with a significant decrease in ER beta expression in the brain of long-term ovariectomized animals and a significant decrease in the level of phosphorylation of ER alpha -Ser118 loci; 3. DPN alternative therapy (DRT) selectively activates ER beta in the brain of female animals of ovariectomized animals. The activity of glial cells reduces the brain ischemic nerve damage and has no carcinogenic side effects. Therefore, the selective activation of ER beta is expected to be a new strategy for the prevention and treatment of menopause female stroke. 4. astrocyte NDRG2 is the target gene of estrogen and ER beta, and 5. astrocyte NDRG2 is the key to mediate the protective effect of estrogen and ER beta cerebral ischemia Molecules.

【学位授予单位】：第四军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R743.3
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