本文選題：腦卒中 + 雌激素替代療法��； 參考：《第四軍醫(yī)大學(xué)》2017年博士論文

【摘要】：【背景】腦卒中已成為導(dǎo)致老年人死亡和殘疾的最主要原因。前期大量流行病學(xué)調(diào)查和基礎(chǔ)研究均證實(shí)雌激素具有顯著的腦卒中神經(jīng)保護(hù)作用。相比于同年齡段男性,絕經(jīng)后女性腦卒中發(fā)病率明顯增高且預(yù)后更差。但大規(guī)模臨床試驗(yàn)卻發(fā)現(xiàn),雌激素替代療法并未降低絕經(jīng)女性腦卒中的發(fā)生率,甚至加劇了神經(jīng)損傷,并增加了乳腺癌和子宮肌瘤的發(fā)病風(fēng)險(xiǎn)。因此,研究者回歸基礎(chǔ)實(shí)驗(yàn),深入探究如何才能有效發(fā)揮雌激素的腦卒中神經(jīng)保護(hù)作用及其機(jī)制,并提出了一系列假說,包括劑量依賴假說,關(guān)鍵期假說和受體選擇性假說。前期研究發(fā)現(xiàn),星形膠質(zhì)細(xì)胞大量表達(dá)雌激素受體,是介導(dǎo)雌激素神經(jīng)保護(hù)作用的重要靶點(diǎn)。ndrg2是我校生化教研室于2001年首先克隆的新基因,在腦內(nèi)ndrg2特異性表達(dá)于星形膠質(zhì)細(xì)胞,參與多種神經(jīng)退行性疾病的發(fā)生發(fā)展,但其在腦缺血損傷中的作用還知之甚少。前期研究發(fā)現(xiàn)ndrg2是雌激素及其受體β的靶基因。然而,ndrg2在雌激素替代療法防治腦卒中損傷中的作用還未見研究報(bào)道�！灸康摹�1.探究雌激素替代療法防治去卵巢動(dòng)物腦缺血損傷的劑量選擇效應(yīng)和機(jī)制;2.探究雌激素替代療法防治去卵巢動(dòng)物腦缺血損傷是否存在治療時(shí)間窗和機(jī)制;3.探究激活雌激素受體β(ERβ)能否減輕去卵巢動(dòng)物腦缺血損傷及分子機(jī)制;4.探究ndrg2在雌激素替代療法防治去卵巢動(dòng)物腦缺血損傷的作用�！痉椒ā�1.在體:給予SD雌性大鼠、C57雌性小鼠和ndrg2基因敲除雌性小鼠去卵巢手術(shù)(OVX)構(gòu)建雌性動(dòng)物絕經(jīng)模型,給予不同劑量的E2以及雌激素受體β激動(dòng)劑DPN替代治療,利用實(shí)時(shí)熒光定量PCR(Real Time PCR)檢測(cè)腦內(nèi)ndrg2 mRNA表達(dá)情況,利用免疫印跡方法(Western blot)檢測(cè)腦內(nèi)GFAP、NDRG2表達(dá)情況;給予ndrg2Loxp小鼠側(cè)腦室注射腺相關(guān)病毒,構(gòu)建選擇性下調(diào)成年雌鼠腦內(nèi)ndrg2表達(dá)的工具鼠;對(duì)正常動(dòng)物和接受激素替代治療的OVX雌性動(dòng)物構(gòu)建大腦中動(dòng)脈缺血(MCAO)和全腦缺血(GCI)模型,利用Garcia和Longa評(píng)分檢測(cè)神經(jīng)功能情況,利用RT-PCR檢測(cè)ndrg2 mRNA表達(dá)情況,利用Western blot檢測(cè)NDRG2、凋亡蛋白的表達(dá),利用TTC染色、TUNEL染色、尼氏染色和Neun染色檢測(cè)神經(jīng)損傷情況;2.離體:利用PC12細(xì)胞系,原代培養(yǎng)的星形膠質(zhì)細(xì)胞、神經(jīng)元和二者共培養(yǎng),給予不同劑量E2、ERα激動(dòng)劑PPT和ERβ激動(dòng)劑DPN處理,利用相差顯微鏡觀察細(xì)胞形態(tài)改變,利用MTT法檢測(cè)細(xì)胞活力,利用LDH測(cè)試檢測(cè)細(xì)胞損傷,利用流式細(xì)胞儀檢測(cè)細(xì)胞周期,利用細(xì)胞免疫熒光檢測(cè)細(xì)胞增殖,利用Real Time PCR檢測(cè)ndrg2 mRNA表達(dá)情況,利用Western blot檢測(cè)GFAP、NDRG2表達(dá)情況;構(gòu)建氧糖剝奪(OGD)模型,利用流式細(xì)胞儀檢測(cè)神經(jīng)細(xì)胞凋亡,利用RT-PCR檢測(cè)ndrg2m RNA表達(dá)情況,利用Western blot和細(xì)胞免疫熒光檢測(cè)NDRG2和凋亡蛋白表達(dá)情況�！窘Y(jié)果】1.(1)生理劑量(10 nM或20 nM)的E2(E2)促進(jìn)PC12細(xì)胞增殖,減輕OGD后PC12細(xì)胞的損傷,藥理劑量(10μM或20μM)的E2抑制PC12細(xì)胞增殖,加重OGD后PC12細(xì)胞的損傷;(2)OVX顯著增加了雌性大鼠腦缺血損傷,生理劑量(6μg/kg和20μg/kg)的E2替代治療顯著減輕OVX雌性大鼠腦缺血神經(jīng)損傷,超生理劑量(50μg/kg)的E2替代治療并沒有減輕OVX雌性大鼠腦缺血神經(jīng)損傷。2.(1)短期去卵巢(OVX 1w)和長(zhǎng)期去卵巢(OVX 10w)并沒有改變雌性小鼠腦內(nèi)ERα的表達(dá);(2)OVX 10w雌性小鼠腦內(nèi)ERβ表達(dá)顯著減少,ERα-Ser118位點(diǎn)磷酸化水平顯著降低,而OVX 1 w雌性小鼠腦內(nèi)ERβ表達(dá)無明顯改變,ERα-Ser118位點(diǎn)磷酸化水平無明顯改變;(3)生理低劑量(50μg/kg)或高劑量(100μg/kg)E2替代治療能夠顯著減輕OVX 1w雌性小鼠腦缺血神經(jīng)損傷,但不能減輕OVX 10w雌性小鼠腦缺血神經(jīng)損傷。3.(1)在雌性小鼠腦內(nèi),ERβ主要與星形膠質(zhì)細(xì)胞標(biāo)志物GFAP存在共定位;在原代培養(yǎng)的星形膠質(zhì)細(xì)胞,ERβ的表達(dá)明顯多于ERα;(2)OVX雌性小鼠腦內(nèi)GFAP表達(dá)顯著下降,50μg/kg E2和8 mg/kg DPN替代治療能夠顯著增加OVX小鼠腦內(nèi)星形膠質(zhì)細(xì)胞GFAP的表達(dá);(3)(2.5 nM、5 nM、10 nM、20 nM)E2劑量依賴性上調(diào)原代星形膠質(zhì)細(xì)胞GFAP的表達(dá),并于5 nM和10 nM時(shí)達(dá)到峰值;10 nM DPN顯著上調(diào)原代星形膠質(zhì)細(xì)胞GFAP的表達(dá),而10 nM PPT不能上調(diào)原代星形膠質(zhì)細(xì)胞GFAP的表達(dá);(4)50μg/kg E2和8 mg/kg DPN替代治療能夠顯著減輕短期OVX雌性小鼠腦缺血神經(jīng)損傷;(5)10 nM E2和10 nM DPN預(yù)處理星形膠質(zhì)細(xì)胞能夠減輕OGD后神經(jīng)元凋亡;4.(1)E2能夠上調(diào)原代星形膠質(zhì)細(xì)胞ndrg2 mRNA和蛋白的表達(dá),并存在時(shí)間和劑量依賴效應(yīng);(2)10 nM DPN上調(diào)原代星形膠質(zhì)細(xì)胞ndrg2 mRNA和蛋白表達(dá),10nM PPT不能上調(diào)原代星形膠質(zhì)細(xì)胞ndrg2 mRNA和蛋白表達(dá);(3)OVX雌性小鼠腦內(nèi)ndrg2表達(dá)顯著下降,50μg/kg E2、100μg/kg E2及8 mg/kg DPN替代治療顯著增加OVX雌性小鼠腦內(nèi)ndrg2 mRNA和蛋白的表達(dá),而2 mg/kg PPT替代治療并不能顯著增加OVX雌性小鼠腦內(nèi)ndrg2 mRNA和蛋白的表達(dá);(4)腦缺血損傷后,缺血半暗帶區(qū)域ndrg2 mRNA和蛋白的表達(dá)于再灌注6 h開始增高,到24 h達(dá)到峰值,并發(fā)生核轉(zhuǎn)位現(xiàn)象;(5)原代星形膠質(zhì)細(xì)胞OGD損傷后ndrg2 mRNA和蛋白的表達(dá)于復(fù)氧復(fù)糖2 h顯著增加,于6 h即達(dá)到峰值,并發(fā)生核轉(zhuǎn)位現(xiàn)象;(6)ndrg2基因敲除(ndrg2-/-)雌鼠腦缺血神經(jīng)損傷明顯重于野生型(ndrg2+/+)雌鼠;利用腺相關(guān)病毒和ndrg2 Loxp雌性小鼠成功地選擇性下調(diào)了成年雌性小鼠腦內(nèi)NDRG2的表達(dá),并加重了腦缺血神經(jīng)損傷;(7)50μg/kg E2或8 mg/kg DPN替代治療能夠顯著減輕ndrg2+/+-OVX雌鼠腦缺血神經(jīng)損傷,而50μg/kg E2或8 mg/kg DPN替代治療不能夠減輕ndrg2-/--OVX雌鼠腦缺血神經(jīng)損傷。【結(jié)論】1.應(yīng)選擇生理劑量的雌激素替代防治去卵巢動(dòng)物的腦缺血損傷;2.雌激素替代療法防治去卵巢雌性動(dòng)物腦缺血損傷存在“最佳治療時(shí)間窗”,與長(zhǎng)期去卵巢動(dòng)物腦內(nèi)ERβ表達(dá)顯著降低和ERα-Ser118位點(diǎn)磷酸化水平顯著降低有關(guān);3.選擇性激活ERβ的DPN替代治療(DRT)通過恢復(fù)去卵巢雌性動(dòng)物腦內(nèi)星形膠質(zhì)細(xì)胞的活性減輕腦缺血神經(jīng)損傷,加之其無致癌副作用,因此,選擇性激活ERβ有望成為防治絕經(jīng)女性腦卒中的新策略;4.星形膠質(zhì)細(xì)胞ndrg2是雌激素及ERβ的靶基因;5.星形膠質(zhì)細(xì)胞ndrg2是介導(dǎo)雌激素及ERβ腦缺血神經(jīng)保護(hù)作用的關(guān)鍵分子。
[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.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R743.3
，

本文編號(hào)：1883210