本文選題：β-淀粉肽(Aβ) + 神經(jīng)再生�。� 參考：《南京醫(yī)科大學(xué)》2012年博士論文

【摘要】：隨著人口老齡化的出現(xiàn),阿爾茨海默病(Alzheimer's disease, AD)的發(fā)病率呈明顯上升趨勢(shì),預(yù)測(cè)到2050年將超過9000萬。由于缺乏有效的預(yù)防、診斷和治療措施,AD已成為死亡病因的第四位。 AD是以進(jìn)行性認(rèn)知功能減退為關(guān)鍵臨床特征,以大量老年斑形成、神經(jīng)元纖維纏結(jié)和膽堿能神經(jīng)元缺失為病理學(xué)特征的神經(jīng)系統(tǒng)退行性疾病。β淀粉肽(β-amyloid peptide, Aβ)是老年斑的主要成分。Aβ?lián)p害膽堿能神經(jīng)系統(tǒng)被認(rèn)為是AD早期認(rèn)知障礙的病理機(jī)制之一。研究證明,α7煙堿型乙酰膽堿能受體(α7nicotinic acetylcholine receptor, α7nAChR)是Ap的靶蛋白。Ap與α7nAChR結(jié)合,不僅能損害α7nAChR的功能,同時(shí)也能增加Ap的積聚和沉積,增強(qiáng)Ap的神經(jīng)毒作用。本實(shí)驗(yàn)室的前期研究報(bào)道,Aβ25-35側(cè)腦室注射能損傷海馬的α7nAChR,導(dǎo)致認(rèn)知功能障礙。此外,Aβ還能通過破壞神經(jīng)細(xì)胞內(nèi)鈣穩(wěn)態(tài),產(chǎn)生大量自由基,激活細(xì)胞凋亡信號(hào)通路等引起神經(jīng)細(xì)胞的大量死亡 成年哺乳動(dòng)物的神經(jīng)干細(xì)胞可以分化為神經(jīng)細(xì)胞——成年神經(jīng)發(fā)生。海馬齒狀回(dentate gyrus, DG)的新生神經(jīng)細(xì)胞與成熟顆粒細(xì)胞具有相同的形態(tài)和功能特征,能與CA3區(qū)神經(jīng)元和內(nèi)嗅皮層的傳入纖維建立突觸聯(lián)系,產(chǎn)生突觸傳遞功能。海馬DG的神經(jīng)發(fā)生已被證實(shí)與空間認(rèn)知行為密切相關(guān),即增加新生神經(jīng)細(xì)胞數(shù)量能提高學(xué)習(xí)記憶功能,而阻礙成年神經(jīng)發(fā)生則造成記憶功能減退。我們的前期研究結(jié)果顯示,Aβ能損傷新生神經(jīng)細(xì)胞的突起生長(zhǎng),導(dǎo)致成熟的新生神經(jīng)細(xì)胞數(shù)量明顯減少。我們還報(bào)道了Aβ通過下調(diào)信號(hào)分子1mTOR(mammalian target of rapamycin),抑制新生神經(jīng)細(xì)胞的突起生長(zhǎng)。這些結(jié)果提示,Aβ?lián)p害神經(jīng)再生可能是AD患者認(rèn)知行為進(jìn)行性減退的重要病理機(jī)制之一。 神經(jīng)甾體激素硫化孕烯醇酮(pregnenolone sulfate, PREGS)的腦內(nèi)水平被認(rèn)為與AD的發(fā)病率、老年斑的形成呈負(fù)性相關(guān)。AD患者腦內(nèi)PREGS水平明顯低于同齡非認(rèn)知障礙人群。我們的前期研究結(jié)果發(fā)現(xiàn),PREGS體外給藥能改善Aβ大鼠的認(rèn)知功能,但是有關(guān)其機(jī)制尚不清楚。大量的研究報(bào)道,PREGS是N-甲基-D-天冬氨酸受體(N-methyl-D-aspartic acid receptor, NMDA-R)的正性調(diào)節(jié)劑,γ-氨基丁酸A型受體(y-aminobutyric acid type A receptor, GABAAR)的負(fù)性調(diào)節(jié)劑。PREGS能夠增強(qiáng)α7nAChR功能,促進(jìn)海馬神經(jīng)元的谷氨酸釋放。此外,PREGS還能激活sigma-1受體(sigma-1receptor, σ1R)。 σlR激動(dòng)劑PRE084通過上調(diào)P13K(phosphatidylinositol-3-kinase)—Akt信號(hào)通路,能阻止Aβ下調(diào)mTOR—p70S6k(70kDa ribosomal protein S6kinase)信號(hào)分子的磷酸化水平,保護(hù)新生神經(jīng)細(xì)胞的突起生長(zhǎng)。早期的研究已報(bào)道,PREGS通過抑制GABAAR活性,能促進(jìn)神經(jīng)干細(xì)胞的增殖。此外,NMDA-R的功能活性也已經(jīng)被證明與新生神經(jīng)元的存活、成熟和神經(jīng)回路的整合密切相關(guān)。我們的前期研究已證明了a7nAChR激動(dòng)劑DMXB能劑量依賴地阻止Aβ?lián)p害a7nAChR功能,保護(hù)海馬的突觸傳遞功能和可塑性,改善Aβ癡呆小鼠的認(rèn)知功能。本課題將重點(diǎn)研究PREGS對(duì)Aβ?lián)p害神經(jīng)再生、Aβ誘導(dǎo)神經(jīng)細(xì)胞凋亡的影響及其分子機(jī)制,以系統(tǒng)地闡明PREGS改善Aβ癡呆小鼠認(rèn)知行為的機(jī)制,為PREGS能用于AD的預(yù)防和治療提供理論依據(jù)。 研究目的 1.明確PREGS對(duì)Aβ?lián)p害神經(jīng)再生的作用及其調(diào)控機(jī)制； 2.明確PREGS對(duì)Aβ誘導(dǎo)神經(jīng)細(xì)胞凋亡的作用及其分子機(jī)制。 第一部分PREGS對(duì)Aβ?lián)p害神經(jīng)再生的作用及其調(diào)控機(jī)制 材料與方法 1.動(dòng)物模型：8月齡雄性APPswe/PS1dE9轉(zhuǎn)基因(APP/PS1)小鼠的基因型鑒定。 部分機(jī)制研究采用雄性ICR小鼠(25-30g)。 2. PREGS處理-新生細(xì)胞標(biāo)記： (1) APP/PS1小鼠：用5-溴脫氧尿嘧啶核苷(bromodeoxyuridine, BrdU)連續(xù)12天腹腔注射標(biāo)記有絲分裂細(xì)胞。從BrdU注射前7天開始給予PREGS(20mg/kg/day)的皮下注射,連續(xù)34天。分別在BrdU末次注射后24小時(shí)(1天齡)或第28天(28天齡),進(jìn)行BrdU免疫組織化學(xué)染色。 (2)ICR小鼠：BrdU間隔6小時(shí)連續(xù)3次注射。BrdU給藥的當(dāng)天記為BrdU的第0天。在BrdU給藥后的第0-1天、5-6天、10-11天、15-16天或20-21天分別給予連續(xù)2天的PREGS(3nmol)側(cè)腦室注射。在BrdU給藥后的第22天(22天齡)進(jìn)行BrdU免疫組織化學(xué)染色。 3.增殖細(xì)胞核抗原(proliferating cell nuclear antigen, Ki67)免疫染色：檢測(cè)干細(xì)胞增殖。 4. DCX (Doublecortin)免疫染色：測(cè)量新生神經(jīng)細(xì)胞的突起長(zhǎng)度。 5. BrdU與神經(jīng)特異性核蛋白(neuron specific protein, NeuN)或膠質(zhì)纖維酸性蛋白(glial fibrillary acidic protein, GFAP)雙標(biāo)記免疫熒光染色：檢測(cè)神經(jīng)前體細(xì)胞的分化和新生神經(jīng)元的存活和成熟。 6.Aβ免疫染色：檢測(cè)老年斑。 7.酶聯(lián)免疫吸附實(shí)驗(yàn)(enzyme linked immunosorbent assay, ELISA):檢測(cè)海馬腦源性生長(zhǎng)因子(brain derived neurotrophic factor, BDNF)。 8.場(chǎng)電位記錄：ICR小鼠給予PREGS腹腔注射60min后取腦,制作海馬腦片,檢測(cè)海馬DG的興奮性突觸后電位(excitatory post-synaptic potentiation, EPSP)和雙脈沖易化(paired-pulse facilitation, PPF)。 9. Morris水迷宮：檢測(cè)空間認(rèn)知功能。 結(jié)果 1.與同窩或同周齡的野生型對(duì)照組小鼠相比,8月齡APP/PS1小鼠的海馬區(qū)域出現(xiàn)大量的老年斑,并表現(xiàn)水迷宮登臺(tái)潛伏期的明顯延長(zhǎng)。 2.與對(duì)照組小鼠相比,APP/PS1小鼠海馬DG的1天齡BrdU免疫陽性(BrdU+)細(xì)胞數(shù)量增加約30%,Ki67免疫陽性(Ki67+)細(xì)胞數(shù)量顯著增加,提示Aβ刺激干細(xì)胞的增殖。PREGS處理不影響APP/PS1小鼠的神經(jīng)干細(xì)胞過增殖。 3.與對(duì)照組小鼠相比,APP/PS1小鼠DCX免疫陽性(DCX+)細(xì)胞突起長(zhǎng)度顯著減小。PREGS處理能保護(hù)APP/PS1小鼠新生神經(jīng)元的突起生長(zhǎng)。 4.與對(duì)照組小鼠相比,APP/PS1小鼠28天齡BrdU+細(xì)胞數(shù)量減少約50%,BrdU和NeuN免疫雙陽性(BrdU+/NeuN+)細(xì)胞數(shù)量顯著減少,而BrdU和GFAP免疫雙陽性(BrdU+/GFAP+)細(xì)胞數(shù)量沒有改變,提示Aβ?lián)p害新生神經(jīng)元的存活和成熟。PREGS處理能增加APP/PS1小鼠成熟新生神經(jīng)元的數(shù)量。 5. PREGS能減少APP/PS1小鼠腦Ap的沉積。 6. PREGS能改善APP/PS1小鼠海馬BDNF水平的降低。 7.在ICR小鼠,BrdU注射后的第10-16天PREGS處理能引起22天齡BrdU+細(xì)胞數(shù)量增加。PREGS能引起EPSP斜率的持續(xù)性增加和雙脈沖比率(paired-pulse ratio, PPR)的降低,提示突觸前神經(jīng)遞質(zhì)的釋放增加。a7nAChR拮抗劑、σ1R拮抗劑或NMDA-R拮抗劑的前處理都能抑制PREGS促進(jìn)突觸前神經(jīng)遞質(zhì)釋放和促新生神經(jīng)元存活的作用,提示PREGS通過增強(qiáng)向新生神經(jīng)元的興奮性傳入,減少非活性化新生神經(jīng)元的死亡。 8. PREGS能改善APP/PS1小鼠的水迷宮登臺(tái)潛伏期延長(zhǎng)。 結(jié)論 1. PREGS通過減少Aβ沉積和提高BDNF水平,能阻止Aβ?lián)p害新生神經(jīng)元突起生長(zhǎng)和存活。 2. PREGS增加向新生神經(jīng)元的傳入神經(jīng)興奮,減少非活性新生神經(jīng)元的死亡,促進(jìn)新生神經(jīng)元的存活和成熟。 3. PREGS通過保護(hù)Aβ小鼠的神經(jīng)再生,可以改善Aβ癡呆小鼠的認(rèn)知行為。 第二部分PREGS對(duì)Aβ誘導(dǎo)神經(jīng)細(xì)胞凋亡的作用及其分子機(jī)制 材料與方法 1.動(dòng)物模型：Aβ25-35(9nmol)進(jìn)行側(cè)腦室注射制備Aβ癡呆小鼠模型。 2.藥物處理：從Aβ給藥后第二天開始連續(xù)7天腹腔注射PREGS(20mg/kg/day)。 3. Morris水迷宮：檢測(cè)空間記憶功能。 4.組織細(xì)胞檢查：海馬CA1區(qū)神經(jīng)細(xì)胞計(jì)數(shù)。 5. TUNEL染色：檢查細(xì)胞凋亡 6. Western blot:分析ERK1/2、Akt磷酸化水平和caspase-3。 結(jié)果 1.與對(duì)照組小鼠相比,Aβ25-35小鼠水迷宮登臺(tái)潛伏期明顯延長(zhǎng),海馬CA1區(qū)神經(jīng)細(xì)胞數(shù)量減少和TUNEL陽性細(xì)胞明顯增加。 2. PREGS處理能改善Aβ25-35小鼠的登臺(tái)潛伏期延長(zhǎng),減少海馬CA1區(qū)神經(jīng)細(xì)胞的凋亡 3.σ1R拮抗劑NE100、a7nAChR拮抗劑MLA能阻止PREGS的抗凋亡作用。 4.與對(duì)照組相比,Aβ25-35小鼠海馬的ERK2和Akt磷酸化水平減少,caspase-3增加。PREGS處理能增加Aβ25-35小鼠的ERK2和Akt磷酸化水平,降低caspase-3。 5.σ1R拮抗劑NE100能阻斷PREGS對(duì)ERK2、Akt和caspase-3的調(diào)節(jié),而a7nAChR拮抗劑MLA只能阻斷PREGS對(duì)Akt和caspase-3調(diào)節(jié)。 6.ERK抑制劑U0126.PI3K抑制劑LY294002能阻斷PREGS對(duì)Aβ25-35小鼠的抗凋亡作用。 7.P13K抑制劑LY294002能阻止PREGS改善Aβ25-35癡呆小鼠的認(rèn)知行為。 結(jié)論 1.Aβ抑制細(xì)胞保護(hù)因子ERK2和抗凋亡因子Akt的活性,激活caspase-3促進(jìn)海馬神經(jīng)元凋亡 2. PREGS通過激活σ1R介導(dǎo)的PI3k-Akt和ERK信號(hào)通路,同時(shí)啟動(dòng)a7nAChR介導(dǎo)PI3k-Akt的信號(hào)通路,阻止Ap的神經(jīng)毒性,以改善Aβ25-35小鼠的認(rèn)知行為。
[Abstract]:With the aging of the population, the incidence of Alzheimer's disease (AD) is on the rise, predicted by more than 90 million in 2050. Due to the lack of effective prevention, diagnosis and treatment, AD has become the fourth cause of death.
AD is a neurodegenerative disease which is characterized by progressive cognitive impairment, with a large number of senile plaques, neurofibrillary tangles and cholinergic neurons missing. Beta amyloid (beta -amyloid peptide, A beta) is the main component of the senile plaques, the.A beta damage to the cholinergic nervous system is considered to be early AD cognition. One of the pathological mechanisms of the obstacle has proved that alpha 7 nicotinic acetylcholinergic receptor (alpha 7nicotinic acetylcholine receptor, alpha 7nAChR) is the binding of Ap target protein.Ap to alpha 7nAChR, not only to damage the function of alpha 7nAChR, but also to increase the accumulation and deposition of Ap, and to enhance the neurotoxicity of Ap. The previous study in this laboratory, A beta 25-3 5 lateral ventricle injections can damage the alpha 7nAChR of the hippocampus, which leads to cognitive dysfunction. In addition, A beta can also cause a large number of free radicals by destroying the calcium homeostasis in the nerve cells, activating the apoptotic signaling pathway and so on, causing a large number of death of the nerve cells.
The neural stem cells of adult mammals can differentiate into neural cells - adult neurogenesis. The newborn neural cells of the dentate gyrus (DG) have the same morphological and functional characteristics as the mature granular cells, and can establish synaptic connections with the afferent fibers of the CA3 and the olfactory cortex and produce synaptic transmission. The neurogenesis of horse DG has been proved to be closely related to spatial cognitive behavior, that is, increasing the number of new nerve cells can improve the learning and memory function, while hindering the adult neurogenesis can cause memory impairment. Our previous study showed that A beta could damage the growth of the new neural cells and lead to the number of mature new nerve cells. We also reported that A beta, through the down regulation of the signal molecule 1mTOR (mammalian target of rapamycin), inhibits the protuberance growth of newborn nerve cells. These results suggest that A beta damage to the nerve may be one of the important pathological mechanisms of progressive degeneration of cognitive behavior in AD patients.
The brain levels of pregnenolone sulfate (PREGS) are considered to be associated with the incidence of AD, and the formation of the senile plaque is negatively correlated with the PREGS level in the brain of the.AD patients. Our previous study found that PREGS in vitro administration could improve the cognitive function of A beta rats. It is not clear about its mechanism. A large number of studies have reported that PREGS is a positive regulator of the N- methyl -D- aspartic acid receptor (N-methyl-D-aspartic acid receptor, NMDA-R), and the negative regulator of the A type receptor of gamma aminobutyric acid (Y-aminobutyric acid type A) can enhance the alpha function and promote the Valley of hippocampal neurons. In addition, PREGS also activates the sigma-1 receptor (sigma-1receptor, sigma 1R). The sigma lR agonist PRE084 can prevent the phosphorylation level of A beta by the P13K (phosphatidylinositol-3-kinase) - Akt signaling pathway, and protects the protuberance growth of newborn nerve cells. The study has reported that PREGS can promote the proliferation of neural stem cells by inhibiting GABAAR activity. In addition, the functional activity of NMDA-R has also been proved to be closely related to the survival of new neurons and the integration of maturation and neural circuits. Our previous studies have shown that the a7nAChR irritable agent DMXB can prevent A beta damage to a7nAChR dose in a dose dependent manner. To protect the synaptic transmission function and plasticity of the hippocampus and improve the cognitive function of A beta dementia mice. This topic will focus on the effect of PREGS on the regeneration of A beta injury and the effect of A beta induced neuronal apoptosis and its molecular mechanism, so as to systematically elucidate the mechanism of PREGS to improve the cognitive behavior of A beta dementia mice, and to provide PREGS for the prevention and treatment of AD. Provide a theoretical basis.
research objective
1. to clarify the role and regulation mechanism of PREGS on A beta damaging nerve regeneration.
2. to clarify the effect of PREGS on A beta induced neuronal apoptosis and its molecular mechanism.
Part one is the effect of PREGS on A beta damaging nerve regeneration and its regulatory mechanism.
Materials and methods
1. animal model: genotype identification of 8 month old male APPswe/PS1dE9 transgenic (APP/PS1) mice.
Male ICR mice (25-30g) were used in some mechanism studies.
2. PREGS treatment - new cell markers:
(1) APP/PS1 mice: 5- bromodeoxyuridine (bromodeoxyuridine, BrdU) was injected into the mitotic cells by intraperitoneal injection for 12 days. PREGS (20mg/kg/day) was injected subcutaneously from 7 days before BrdU injection, for 34 days for consecutive 24 hours (1 days of age) or twenty-eighth days (28 days of age) after the final injection of BrdU, respectively. Color.
(2) ICR mice: the day of 6 hours of BrdU interval of 3 consecutive injections of.BrdU was recorded as zeroth days of BrdU. The 0-1 day after BrdU, 5-6 days, 10-11 days, 15-16 days, and 20-21 days were given for 2 days of PREGS (3nmol) intraventricular injection respectively. BrdU immunohistochemical staining was performed on the day after BrdU Administration (22 days of age).
3. proliferating cell nuclear antigen (Ki67) immunostaining: detection of stem cell proliferation.
4. DCX (Doublecortin) immunostaining: the length of neurite protrusion was measured.
5. BrdU and neural specific nucleoprotein (neuron specific protein, NeuN) or glial fibrillary acidic protein (glial fibrillary acidic protein, GFAP) double labeled immunofluorescence staining: to detect the differentiation of neural precursor cells and the survival and maturation of newborn neurons.
6.A beta immunostaining: detection of senile plaques.
7. enzyme linked immunosorbent assay (ELISA): detection of hippocampus brain derived growth factor (brain derived neurotrophic factor, BDNF).
8. field potentials recording: ICR mice were given PREGS after 60min intraperitoneal injection to take the brain, make hippocampus brain slices, detect the excitatory postsynaptic potential of the hippocampus DG (excitatory post-synaptic potentiation, EPSP) and double pulse susceptibility (paired-pulse facilitation, PPF).
9. Morris water maze: detection of spatial cognitive function.
Result
1. a large number of senile plaques appeared in the hippocampus of 8 month old APP/PS1 mice compared with the wild type control group with the same or the same age, and the latency of the water maze was obviously prolonged.
2. compared with the control group, the number of BrdU immunoreactive (BrdU+) cells in the hippocampal DG of APP/PS1 mice increased by about 30%, and the number of Ki67 immunoreactive (Ki67+) cells increased significantly, suggesting that the proliferation of A beta stimulated stem cells did not affect the proliferation of neural stem cells in APP/PS1 mice.
3. compared with the control group, the DCX immunoreactive (DCX+) cell protuberance length of the APP/PS1 mice was significantly reduced by the.PREGS treatment to protect the growth of the new neurons in the APP/PS1 mice.
4. compared with the control group, the number of BrdU+ cells in APP/PS1 mice at 28 days of age decreased by about 50%, the number of BrdU and NeuN double positive (BrdU+/NeuN+) cells decreased significantly, while the number of BrdU and GFAP immunologic double positive (BrdU+/GFAP+) cells did not change, suggesting that A beta damage to the survival of newborn neurons and mature.PREGS treatment could increase the maturity of APP/PS1 mice. The number of newborn neurons.
5. PREGS can reduce the deposition of Ap in the brain of APP/PS1 mice.
6. PREGS could improve the level of BDNF in hippocampus of APP/PS1 mice.
7. in ICR mice, PREGS treatment on the 10-16 day after injection can cause an increase in the number of BrdU+ cells at 22 days of age to cause a continuous increase in the EPSP slope and the decrease of the double pulse ratio (paired-pulse ratio, PPR). It suggests that the release of the transmitter of the synapse increases the.A7nAChR antagonist, and the pretreatment of the sigma 1R antagonist or the NMDA-R antagonist can be used. Inhibition of PREGS promotes the release of neurotransmitters and the survival of newborn neurons, suggesting that PREGS reduces the death of inactive newborn neurons by enhancing the excitatory afferent to newborn neurons.
8. PREGS could improve the latency of water maze in APP/PS1 mice.
conclusion
1. PREGS, by reducing A beta deposition and raising BDNF level, can prevent A beta from damaging the growth and survival of new neurons.
2. PREGS increased the afferent nerve stimulation to neonatal neurons, reduced the death of inactive neonatal neurons, and promoted the survival and maturation of neonatal neurons.
3. PREGS can improve the cognitive behavior of A beta dementia mice by protecting nerve regeneration from A beta mice.
The second part is the effect of PREGS on A beta induced neuronal apoptosis and its molecular mechanism.
Materials and methods
1. animal model: A beta 25-35 (9nmol) was used to prepare A beta dementia mouse model by lateral ventricle injection.
2. drug treatment: PREGS (20mg/kg/day) was administered intraperitoneally for 7 consecutive days on the second day after administration of A beta.
3. Morris water maze: testing space memory function.
4. histiocytic examination: nerve cell count in hippocampal CA1 area.
5. TUNEL staining: examination of cell apoptosis
6. Western blot: analysis of ERK1/2, Akt phosphorylation levels and caspase-3.
Result
1. compared with the control group, the incubation period of the A maze 25-35 mice was significantly prolonged, and the number of nerve cells in the CA1 area of hippocampus decreased and the TUNEL positive cells increased significantly.
2. PREGS treatment could prolong the incubation period of A beta 25-35 mice and reduce the apoptosis of CA1 cells in hippocampus.
The 3. Sigma 1R antagonist NE100 and a7nAChR antagonist MLA can prevent the anti apoptotic effect of PREGS.
4. compared with the control group, the phosphorylation level of ERK2 and Akt in the hippocampus of A beta 25-35 mice decreased, and the caspase-3 increase.PREGS treatment could increase the level of phosphorylation of ERK2 and Akt in A beta 25-35 mice and reduce caspase-3..
The 5. Sigma 1R antagonist NE100 can block the regulation of PREGS on ERK2, Akt and Caspase-3, while a7nAChR antagonist MLA can only block the regulation of PREGS on Akt and caspase-3.
6.ERK inhibitor U0126.PI3K inhibitor LY294002 can block the anti apoptotic effect of PREGS on A beta 25-35 mice.
7.P13K inhibitor LY294002 can prevent PREGS from improving the cognitive behavior of A beta 25-35 dementia mice.
conclusion
1.A beta inhibits the activity of cytoprotective factor ERK2 and anti apoptotic factor Akt, and activates caspase-3 to promote apoptosis of hippocampal neurons.
2. PREGS activates the PI3k-Akt and ERK signaling pathway mediated by sigma 1R, and activates PI3k-Akt signaling pathway mediated by a7nAChR to prevent the neurotoxicity of Ap in order to improve the cognitive behavior of A beta 25-35 mice.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2012
【分類號(hào)】：R749.16

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