本文選題：多巴胺 + 樹突重塑�。� 參考：《南方醫(yī)科大學(xué)》2010年碩士論文

【摘要】： 吸毒是全世界廣泛關(guān)注的問題,毒品的泛濫直接危害人民的身心健康,并給經(jīng)濟(jì)發(fā)展和社會(huì)進(jìn)步帶來巨大威脅。吸毒最大的危害在于其成癮性。毒品成癮(drug addiction)指不擇手段、不計(jì)后果地強(qiáng)制性獲取、使用某種毒品。毒品成癮一旦形成,即可能成為一種終身性狀態(tài),成癮者不擇手段地獲取毒品,具有極高的復(fù)發(fā)性,甚至在戒斷多年后仍有可能復(fù)發(fā),造成很嚴(yán)重的社會(huì)問題。毒品成癮的形成是一個(gè)循序漸進(jìn)的過程,毒品成癮過程中,機(jī)體在分子、細(xì)胞水平發(fā)生代償性變化,這些包括基因表達(dá)和神經(jīng)元形態(tài)上的改變,會(huì)影響神經(jīng)元功能和神經(jīng)通路,進(jìn)而導(dǎo)致行為學(xué)上的異常。 可卡因成癮的過程中,大腦多個(gè)部位的結(jié)構(gòu)和功能發(fā)生改變,其中重要的是神經(jīng)元樹突發(fā)生了重塑(dendrite remodeling),這是可卡因(cocaine)成癮發(fā)生的結(jié)構(gòu)基礎(chǔ)。許多研究報(bào)道多巴胺信號(hào)通路在可卡因介導(dǎo)的神經(jīng)元樹突重塑中發(fā)揮重要的調(diào)節(jié)作用,并且有研究發(fā)現(xiàn)發(fā)現(xiàn)D1和D3多巴胺受體對(duì)下游的長(zhǎng)期誘導(dǎo)的靶基因Neogenin和SynaptotagminⅦ起到相反的調(diào)節(jié)作用；這些基因的長(zhǎng)期變化,參與神經(jīng)元可塑性的形成,并進(jìn)一步參與毒品給藥后的行為學(xué)變化。在典型的神經(jīng)元中,來自胞體的初級(jí)樹突重復(fù)分支建立起一個(gè)有特色的樹突樹(dendritic tree),樹突分支會(huì)生出微小的突起,稱之為樹突棘(spine),它是大多數(shù)興奮性突觸接受信息的位點(diǎn),而相應(yīng)的抑制性突觸主要分布在樹突樹的主干上,樹突結(jié)構(gòu)的改變會(huì)導(dǎo)致突觸信息的變化。Robinson等報(bào)道重復(fù)施用可卡因會(huì)造成紋狀體伏核區(qū)(nucleus accumbens, NAc)中度棘神經(jīng)元(medium spiny neuron,MSN)樹突增長(zhǎng)、分支增加和樹突棘的密度增加,而且這種形態(tài)變化會(huì)在停藥后至少維持一個(gè)月。結(jié)構(gòu)的改變和學(xué)習(xí)、長(zhǎng)時(shí)程增強(qiáng)均有密切聯(lián)系。結(jié)構(gòu)的改變會(huì)帶來神經(jīng)環(huán)路的改變、影響神經(jīng)遞質(zhì)的傳遞,更重要的是帶來突觸組織形式的改變。 多種信號(hào)分子參與了神經(jīng)元的可塑性變化,多巴胺受體(dopamine receptor)、NMDA (N-methyl-D-aspartate, NMDA)受體、ERK(extracell-ular signal-regulated kinase, ERK)以及Rho蛋白家族等等都可能在其中有重要的調(diào)節(jié)作用,但是它們?nèi)绾握{(diào)節(jié)可卡因介導(dǎo)的樹突重構(gòu)的分子機(jī)制尚存在很多值得我們研究的地方。我們的策略是通過特異性阻斷或者特異性激活上述信號(hào)通路,以探究該信號(hào)通路是否參與多巴胺誘導(dǎo)的神經(jīng)元樹突重構(gòu)以及其可能的下游信號(hào)分子,進(jìn)而揭示可卡因成癮過程中多巴胺信號(hào)通路誘導(dǎo)的神經(jīng)元樹突發(fā)生重塑可能的分子機(jī)制。 本課題首先構(gòu)建了Rho蛋白家族中兩個(gè)亞型Rac1和RhoA的顯性負(fù)效突變體(Rac1N17和RhoAN19)和組成型活性突變體(Rac1L61和RhoAL63)的慢病毒,通過感染原代培養(yǎng)的前皮質(zhì)神經(jīng)元細(xì)胞(Profrontal Cortex Neurons, PFC)驗(yàn)證Rac1和RhoA-GTPasess的生物學(xué)活性；進(jìn)而,在原代培養(yǎng)的前皮質(zhì)神經(jīng)元細(xì)胞中重復(fù)給與多巴胺,在最后一次刺激4d后,固定細(xì)胞做免疫熒光,結(jié)果顯示：多巴胺重復(fù)刺激下前皮質(zhì)區(qū)域椎體神經(jīng)元細(xì)胞樹突分支總數(shù)和樹突棘數(shù)目增多,神經(jīng)突觸密度增加。進(jìn)一步,我們采用D1和D3受體的抑制劑與激動(dòng)劑、Rac1和RhoA的顯性負(fù)效突變體和組成型活性突變體的慢病毒及Rac1和RhoA的抑制劑,通過特異性的阻斷或者激活相應(yīng)的信號(hào)分子,以探究該信號(hào)分子是否參與多巴胺誘導(dǎo)的神經(jīng)元樹突重構(gòu)以及在樹突重構(gòu)中的作用。本研究主要結(jié)果如下： 1.構(gòu)建了Rho家族重組質(zhì)粒Plenti6/v5-Rac1N17, Plenti6/v5-Rac1L61, Plenti6/v5-RhoAL63, Plenti6/v5-RhoAN19,并且包裝制備了相應(yīng)的慢病毒,感染原代培養(yǎng)的前皮質(zhì)神經(jīng)元細(xì)胞后,采用G-LISATM系統(tǒng)對(duì)慢病毒Plenti6/v5-Rac1N17,Plenti6/v5-Rac1L61, Plenti6/v5-RhoAN19, Plenti6/v5-RhoA-L63進(jìn)行了功能活性鑒定,經(jīng)表皮生長(zhǎng)因子(Epidermal Growth Factor, EGF)(Rac1-GTPase測(cè)定)刺激或者溶血磷脂酸(Lysophosphatidic acid, LPS)(RhoA-GTPase測(cè)定)刺激2 min后收集細(xì)胞檢測(cè)相應(yīng)蛋白活性,結(jié)果顯示：與Plenti6/v5-EGFP組相比,Plenti6/v5-Rac1N17組Rac1活性降低了29.3%,而Plenti6/v5-Rac1L61組Racl活性則增高了2.3倍；與Plenti6/v5-EGFP組相比,Plenti6/v5-RhoA1N19組RhoA活性降低了42.8%,而Plenti6/v5-RhoA1L63組RhoA活性則增高了1.7倍；結(jié)果都具有顯著的統(tǒng)計(jì)學(xué)差異,提示構(gòu)建的慢病毒具備了特定的生物學(xué)活性。 2.建立了多巴胺重復(fù)刺激前皮質(zhì)神經(jīng)元細(xì)胞模型：在原代培養(yǎng)前皮質(zhì)神經(jīng)元細(xì)胞的第11d,13d,15d應(yīng)用1μmol/L多巴胺重復(fù)刺激。并應(yīng)用該模型探討了多巴胺重復(fù)刺激對(duì)于神經(jīng)元細(xì)胞的樹突分支數(shù)目、樹突棘數(shù)目和神經(jīng)突觸密度的影響。結(jié)果顯示：多巴胺重復(fù)刺激后神經(jīng)元細(xì)胞的樹突分支數(shù)目和樹突棘數(shù)目增多、神經(jīng)突觸密度增加,其結(jié)果與PBS對(duì)照組相比差異具有統(tǒng)計(jì)學(xué)意義。 3.應(yīng)用D1多巴胺受體抑制劑SCH23390(10μmol/L)、D1多巴胺受體激動(dòng)劑SKF81297(1μmol/L)和D3多巴胺受體抑制劑NGB2904(50μmol/L)以探究D1多巴胺受體和D3多巴胺受體在調(diào)節(jié)PFC細(xì)胞重塑中的作用。在每次加多巴胺刺激前5 min加SCH23390,10 min加NGB2904,制備多巴胺重復(fù)刺激模型；同時(shí)單獨(dú)應(yīng)用D1多巴胺受體激動(dòng)劑SKF81297刺激15 min,固定細(xì)胞做免疫熒光,結(jié)果顯示：在前皮質(zhì)區(qū)域先應(yīng)用D1多巴胺受體抑制劑SCH23390后,多巴胺重復(fù)刺激引起的神經(jīng)元形態(tài)結(jié)構(gòu)的改變會(huì)被顯著抑制,即：神經(jīng)元細(xì)胞樹突數(shù)目和樹突棘數(shù)目減少,神經(jīng)突觸密度降低；而SKF81297刺激后神經(jīng)元樹突數(shù)目、樹突棘數(shù)目、神經(jīng)突觸密度都顯著增加,與多巴胺重復(fù)刺激類似,提示D1多巴胺受體在多巴胺重復(fù)刺激誘導(dǎo)神經(jīng)元樹突重構(gòu)中發(fā)揮正性調(diào)控作用。而先應(yīng)用D3多巴胺受體抑制劑NGB2904后,神經(jīng)元細(xì)胞樹突數(shù)目和樹突棘數(shù)目相比單純的多巴胺組有所增高,差異具有統(tǒng)計(jì)學(xué)意義,提示D3多巴胺受體在神經(jīng)元樹突重塑中發(fā)揮負(fù)性的調(diào)控作用,即D1多巴胺受體和D3多巴胺受體在神經(jīng)元樹突重塑中發(fā)揮重要的調(diào)控作用,并且兩者的調(diào)控作用是相反的。 4.應(yīng)用Racl和RhoA的顯性負(fù)效突變體的慢病毒在前皮質(zhì)神經(jīng)元細(xì)胞培養(yǎng)的第7d感染細(xì)胞,次日換去全液,同樣給與多巴胺重復(fù)刺激,探討Rac1和RhoA蛋白是否參與了慢性多巴胺刺激誘導(dǎo)的神經(jīng)元樹突重塑以及其所發(fā)揮的作用。固定細(xì)胞做免疫熒光,結(jié)果顯示：在前皮質(zhì)區(qū)域,感染Rac1N17顯著抑制了多巴胺重復(fù)刺激引起的神經(jīng)元樹突分支數(shù)目和樹突棘數(shù)目的增多以及突觸密度的增加,差異具有統(tǒng)計(jì)學(xué)意義,這提示Rac1在多巴胺刺激誘導(dǎo)的神經(jīng)元樹突重構(gòu)過程中發(fā)揮重要的正性調(diào)控作用；相反感染RhoAN19慢病毒后增強(qiáng)了多巴胺刺激引起的神經(jīng)元樹突分支數(shù)目、樹突棘數(shù)目和突觸密度的增加,差異具有統(tǒng)計(jì)學(xué)意義,提示RhoA在多巴胺介導(dǎo)的神經(jīng)元樹突重塑中發(fā)揮負(fù)性調(diào)控作用。 5.同樣應(yīng)用Racl的抑制劑NSC23766以及RhoA的抑制劑Y27632以探究Racl和RhoA是否參與了多巴胺重復(fù)刺激誘導(dǎo)的神經(jīng)元樹突重塑。該組前皮質(zhì)細(xì)胞在每次加多巴胺刺激前3 min加NSC23766(100μmol/L)或者Y27632(100μmol/L)。細(xì)胞固定后進(jìn)行免疫熒光染色,結(jié)果顯示：在前皮質(zhì)區(qū)域,NSC23766顯著逆轉(zhuǎn)多巴胺重復(fù)刺激引起的樹突分支數(shù)目、樹突棘數(shù)目增多和神經(jīng)突觸密度增加,這與病毒感染的結(jié)果相同,差異具有統(tǒng)計(jì)學(xué)意義；而Y27632組與多巴胺組比較則尚無統(tǒng)計(jì)學(xué)差異。 通過上述研究,我們得出以下結(jié)論：一、成功構(gòu)建了Rac1和RhoA蛋白的顯性負(fù)效突變體和組成型活性突變體的慢病毒,并驗(yàn)證了相應(yīng)的生物學(xué)活性,為下一步研究奠定了基礎(chǔ)。二、發(fā)現(xiàn)多巴胺重復(fù)刺激介導(dǎo)神經(jīng)元細(xì)胞樹突分支數(shù)目、樹突棘數(shù)目增多和神經(jīng)突觸數(shù)目增加。三、多巴胺誘導(dǎo)的樹突重塑過程中,D1、D3多巴胺受體發(fā)揮了相反的調(diào)控作用,刺激多巴胺受體誘導(dǎo)的神經(jīng)元樹突增長(zhǎng)、分支增加和樹突棘密度增加主要是通過D1多巴胺受體,而D3多巴胺受體主要起抑制作用。四、多巴胺誘導(dǎo)的樹突重塑過程中,Rac1和RhoA蛋白參與介導(dǎo)神經(jīng)元樹突重塑,并且Rac1促進(jìn)樹突分支數(shù)目、樹突棘數(shù)目的增多以及神經(jīng)突觸密度的增加,而RhoA則發(fā)揮了相反的調(diào)節(jié)作用。 綜上所述,本研究成功構(gòu)建了Rho蛋白家族中Rac1和RhoA的顯性負(fù)效突變體和組成型活性突變體,制備了多巴胺重復(fù)刺激細(xì)胞模型,采用一系列特異性抑制劑、激動(dòng)劑及突變體,通過阻斷多巴胺受體通路、Rho家族信號(hào)通路,探究多巴胺誘導(dǎo)的神經(jīng)元樹突重構(gòu)可能的分子機(jī)制。結(jié)果表明,D1、D3多巴胺受體和Rac1、RhoA信號(hào)通路均參與了多巴胺誘導(dǎo)的神經(jīng)元樹突重塑,并且D1多巴胺受體和Rac1蛋白發(fā)揮正性調(diào)節(jié)作用,而D3多巴胺受體和RhoA蛋白則為負(fù)性調(diào)節(jié),這提示多巴胺誘導(dǎo)的神經(jīng)元樹突重構(gòu)是多種信號(hào)分子共同作用的結(jié)果。上述研究對(duì)于我們進(jìn)一步揭示多巴胺誘導(dǎo)的神經(jīng)元樹突重塑分子機(jī)制具有重要的意義,同時(shí)也為其臨床治療提供了諸多啟示。
[Abstract]:Drug abuse is a widespread concern in the world. The flood of drugs directly endangers the people's physical and mental health and poses a great threat to economic development and social progress. The greatest harm of drug addiction is its addiction. Drug addiction (drug addiction) refers to unscrupulous means, unreckless and strong acquisition, and the use of some kind of drug. Once the drug addiction is formed It can become a life-long state. The addicts obtain drugs by means of unscrupulous means of relapse. Even after many years of abstinence, it is possible to relapse and cause serious social problems. The formation of drug addiction is a gradual process. In the course of drug addiction, the body has compensatory changes at the molecular and cell levels in the drug addiction process. These include gene expression and changes in neuronal morphology, which can affect neuronal function and neural pathways, leading to behavioral abnormalities.
In the process of cocaine addiction, the structure and function of multiple parts of the brain are altered, and it is important that the neuron dendrites reshape (dendrite remodeling), which is the structural basis for the occurrence of cocaine (cocaine) addiction. Many studies have reported that dopamine signaling plays an important role in cocaine mediated remolding of neurons. We have found that the D1 and D3 dopamine receptors play the opposite role in the long-term induced target gene Neogenin and Synaptotagmin VII; the long-term changes in these genes participate in the formation of neuronal plasticity and further participate in the behavioral changes after drug delivery. In typical neurons, The primary dendrite repeating branch of the cell body builds a distinctive dendritic tree (dendritic tree). The branch of the dendrite produces a tiny protuberance called the dendritic spines (spine), which is the site for most excitatory synapses to receive information, and the corresponding inhibitory synapses are mainly distributed on the trunk of the tree, and the changes in the dendrite structure will lead to a change in the dendrite structure. Changes in synaptic information.Robinson and other reports that repeated use of cocaine can cause the growth of the dendritic neurons (medium spiny neuron, MSN) in the nucleus accumbens (nucleus accumbens, NAc), the increase of the branch and the density of the dendritic spines, and this morphological change will last for at least one month after the withdrawal of the drug. Structural changes and learning, long time The changes in the structure will bring about the changes in the neural circuits, affect the transmission of neurotransmitters, and more importantly, bring about changes in the form of synapses.
Many signal molecules are involved in the plasticity of neurons. The dopamine receptor (dopamine receptor), the NMDA (N-methyl-D-aspartate, NMDA) receptor, ERK (extracell-ular signal-regulated kinase, ERK), and the Rho protein family all have important regulatory effects, but how they regulate the weight of cocaine mediated dendrites Our strategy is to explore whether the signaling pathway participates in the dopamine induced neuronal dendrite remodeling and its possible downstream signal molecules by specific blocking or specific activation of the signal pathway, thus revealing the dopamine in the process of cocaine addiction. Signaling pathway induces neuronal remodeling in dendritic cells.
We first constructed the lentivirus of the dominant negative effects mutants (Rac1N17 and RhoAN19) and the constituent active mutants (Rac1L61 and RhoAL63) of two subtypes of the Rho protein family (Rac1N17 and RhoAN19) and the component type active mutants (Rac1L61 and RhoAL63), and verified the biological activity of the Rac1 and it through the primary cultured precultured cortical neurons (Profrontal Cortex Neurons, PFC). Furthermore, dopamine was repeated in the primary cultured cortical neuron cells. After the last stimulation of 4D, the fixed cells were immunofluorescent. The results showed that the number of dendritic branches and the number of dendritic spines in the vertebral neuron cells increased and the density of synapses increased in the anterior cortex of the anterior cortex. Further, we used D1. D3 receptor inhibitors and agonists, Rac1 and RhoA dominant negative effects mutants and constituent active mutants of lentivirus and Rac1 and RhoA inhibitors, through specific blocking or activation of the corresponding signal molecules to explore whether the signal molecules are involved in dopamine induced deity dendrites reconstruction and the reconfiguration of dendrites The main results of this study are as follows:
1. Rho family recombinant plasmids Plenti6/v5-Rac1N17, Plenti6/v5-Rac1L61, Plenti6/v5-RhoAL63, Plenti6/v5-RhoAN19 were constructed, and the corresponding lentivirus was packaged and infected with the primary cultured cortical neurons, and G-LISATM system was used for the lentivirus Plenti6/ v5-Rac1N17, Plenti6/v5-Rac1L61, Plenti6/v5-RhoAN19, Plenti6/. V5-RhoA-L63 performed functional activity identification, stimulated by epidermal growth factor (Epidermal Growth Factor, EGF) (Rac1-GTPase) or lysophosphatidic acid (Lysophosphatidic acid, LPS) (Lysophosphatidic acid, LPS) (RhoA-GTPase assay) stimulated 2 min cells to collect cells to detect the corresponding protein activity. The activity of AC1 was reduced by 29.3% and the activity of Racl in group Plenti6/v5-Rac1L61 increased by 2.3 times. Compared with the Plenti6/v5-EGFP group, the RhoA activity of Plenti6/v5-RhoA1N19 group decreased by 42.8% and the RhoA activity of the Plenti6/v5-RhoA1L63 group increased by 1.7 times; the results all had significant statistical differences, suggesting that the constructed lentivirus had a specific organism. Learning activity.
2. the model of cortical neurons before dopamine repeated stimulation was established: 11d, 13D, and 15d of cortical neurons before primary culture were repeated with 1 micron mol/L dopamine, and the effects of dopamine repeated stimulation on the number of dendritic branches, the number of dendritic spines and the synaptic density were investigated. The results showed that the number of dendritic branches and the number of dendritic spines increased and the synapse density increased after dopamine repeated stimulation, and the difference was statistically significant compared with the PBS control group.
3. the use of D1 dopamine receptor inhibitor SCH23390 (10 micron mol/L), D1 dopamine receptor agonist SKF81297 (1 micron mol/L) and D3 dopamine receptor inhibitor NGB2904 (50 micron) to explore the role of D1 dopamine receptor and D3 dopamine receptor in regulating PFC cell remodeling. 5 doses of dopamine were added before each dopamine stimulation. The D1 dopamine receptor agonist SKF81297 stimulated 15 min and the immobilized cells were immunofluorescent. The results showed that the changes in the morphological structure of neurons caused by dopamine repeated stimulation were significantly inhibited in the precortical region by using the D1 dopamine receptor inhibitor SCH233 90, that is, neurons. The number of dendritic spines and the number of dendritic spines decreased, and the density of synapses decreased, while the number of dendrites, the number of dendritic spines and the synapse density increased significantly after SKF81297 stimulation, similar to the repetitive dopamine stimulation, suggesting that D1 dopamine receptors play a positive role in the reconstruction of neuron dendrites induced by repetitive dopamine stimulation. After using the D3 dopamine receptor inhibitor NGB2904, the number of dendritic cells and the number of dendritic spines increased in comparison with the number of dendritic spines. The difference was statistically significant, suggesting that D3 dopamine receptors play a negative role in the remolding of neuron dendrites, namely, D1 dopamine receptor and D3 dopamine receptor in neuron dendrite weight. Plastic plays an important regulatory role, and the regulation function of the two is the opposite.
4. using the dominant negative effect mutant of Racl and RhoA in the 7d infected cells cultured in the precortical neuron cells, the following day was changed to the full liquid, and the same was given to the dopamine repeated stimulation. It was discussed whether the Rac1 and RhoA proteins were involved in the dendrite remolding induced by chronic dopamine stimulation and the role it played. The results showed that in the anterior cortex, infection of Rac1N17 significantly inhibited the number of dendritic branches and the number of dendritic spines caused by dopamine repeated stimulation and the increase of the density of synapses. The difference was statistically significant, suggesting that Rac1 plays an important role in the process of dendritic reconstruction induced by dopamine. Positive regulation, and the number of dendritic branches caused by dopamine stimulation, the number of dendritic spines and the increase of synapse density, and the difference between the number of dendritic spines and the density of synapse were increased after RhoAN19 infection, suggesting that RhoA plays a negative role in the remolding of dopamine mediated dendrites.
5. the same Racl inhibitor NSC23766 and the inhibitor Y27632 of RhoA were used to explore whether Racl and RhoA were involved in the remolding of neuron dendrites induced by dopamine repetitive stimulation. In this group, the anterior cortical cells were 3 min plus NSC23766 (100 u mol/L) or Y27632 (100 mu mol/L) before each dopamine stimulation. Immunofluorescence staining was performed after the cells were fixed. The results showed that in the anterior cortex, NSC23766 significantly reversed the number of dendritic branches caused by dopamine repetitive stimulation, the increase of dendritic spines and the increase of synapse density, which was the same as the results of virus infection, and the difference was statistically significant, but there was no statistical difference between the Y27632 group and the dopamine group.
Through the above studies, we draw the following conclusions: first, the dominant negative and constituent active mutants of Rac1 and RhoA proteins were successfully constructed, and the corresponding biological activity was verified. Two, the number of dendritic branches, dendritic spines, and the number of dendritic spines were found. Increase in number and number of synapses increased. Three, during the remolding process of dopamine induced dendrites, D1, D3 dopamine receptors play an opposite role in stimulating the growth of neuron dendrites induced by dopamine receptors, and the increase in the density of branches and dendrites is mainly through the D1 dopamine receptor, while D3 dopamine receptors mainly inhibit the dendrites. Four, during the remolding process of dopamine induced dendrites, Rac1 and RhoA proteins are involved in mediating the remolding of neuron dendrites, and Rac1 promotes the number of dendritic branches, the number of dendritic spines and the increase of neural synapse density, while RhoA plays the opposite role in regulating the dendrite.
To sum up, this study successfully constructed the dominant negative and active mutants of Rac1 and RhoA in the Rho protein family, prepared a dopamine repetitive stimulation cell model, and used a series of specific inhibitors, agonists and mutants to explore dopamine induced by blocking the dopamine receptor pathway, the Rho family signal pathway. The possible molecular mechanism of neuron dendrite reconstruction shows that D1, D3 dopamine receptors and Rac1, RhoA signaling pathways are involved in dopamine induced dendritic remodeling, and D1 dopamine receptors and Rac1 proteins play a positive regulatory role, while D3 dopamine receptors and RhoA egg white are negatively regulated, which suggests dopamine induced nerves. The reconfiguration of the dendrites is the result of the interaction of various signal molecules. The above study is of great significance for us to further reveal the molecular mechanism of dopamine induced remolding of neuron dendrites, and also provides a great deal of inspiration for its clinical treatment.

【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R346

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本文編號(hào)：1773646