神經(jīng)生物學(xué)相關(guān)論文摘要

1、神經(jīng)生物學(xué)相關(guān)論文摘要1. 在MN9D細(xì)胞-synuclein N、NAC、C端結(jié)構(gòu)域與線粒體關(guān)系的研究張韜 碩士2009帕金森?。≒D）是最常見(jiàn)的神經(jīng)退行性疾病之一，a-突觸核蛋白（a-synuclein）及線粒體與PD發(fā)病密切相關(guān)，但作用機(jī)制尚不明確。a-synuclein是PD病理特征Lewy體的主要成分, a-synuclein基因突變可導(dǎo)致家族性PD，大多數(shù)PD患者存在線粒體功能障礙。本課題前期工作證實(shí)a-synuclein可位于線粒體，其在大鼠體內(nèi)長(zhǎng)期過(guò)表達(dá)可引起神經(jīng)元線粒體結(jié)構(gòu)變化，導(dǎo)致線粒體退行性變。本實(shí)驗(yàn)的目的是研究a-synuclein各結(jié)構(gòu)域與線粒體結(jié)構(gòu)功能的關(guān)系。我們成

2、功構(gòu)建了a-synuclein 基因的各結(jié)構(gòu)域：pLNCX2/N，pLNCX2/NAC及pLNCX2/C的逆轉(zhuǎn)錄病毒的表達(dá)質(zhì)粒，通過(guò)病毒包裝獲得了可表達(dá)a-synuclein各結(jié)構(gòu)域的MN9D細(xì)胞。利用免疫細(xì)胞化學(xué)方法，通過(guò)激光掃描共焦顯微鏡發(fā)現(xiàn)a-synuclein/N端與線粒體存在共定位關(guān)系；a-synuclein/NAC主要在核內(nèi)聚集表達(dá)；a-synuclein/C端在胞漿和胞核內(nèi)均有表達(dá)。JC1染色流式細(xì)胞儀檢測(cè)顯示，過(guò)表達(dá)a-synuclein/N實(shí)驗(yàn)組細(xì)胞線粒體膜電位降低，同時(shí)CCK8檢測(cè)結(jié)果顯示該組細(xì)胞活力較正常組及其它處理組明顯下降。凋亡檢測(cè)顯示，過(guò)表達(dá)a-synuclein/

3、N可引起AIF總量增加及核轉(zhuǎn)位，刺激細(xì)胞色素C釋放，吖啶橙檢測(cè)發(fā)現(xiàn)引起核DNA斷裂以及線粒體形態(tài)發(fā)生變化。Annexin V/PI染色的直接證據(jù)表明a-synuclein/N參與凋亡并最終導(dǎo)致細(xì)胞死亡。電鏡形態(tài)學(xué)觀察發(fā)現(xiàn)a-synuclein/N可導(dǎo)致線粒體形態(tài)發(fā)生明顯改變。因此我們認(rèn)為a-synuclein的 N端可定位于線粒體，具有細(xì)胞毒性作用，并參與調(diào)節(jié)線粒體功能；a-synuclein/NAC雖具有疏水特性但卻能穿過(guò)核膜，聚集在核仁周?chē)?，發(fā)揮著核內(nèi)調(diào)控作用；a-synuclein/C端定位于胞漿和核內(nèi)可能參與多種細(xì)胞功能且不排除其參與細(xì)胞保護(hù)作用。Mitochondrial dysfu

4、nction is implicated in the pathophysiology of Parkinsons disease characterized by formation of intraneuronal inclusions, Lewy bodies and progressive degeneration of the nigrostriatal dopamine system. Recently, pathophysiological functions of a-synuclein, a major constituent of Lewy bodies, have bee

5、n shown in its interaction with mitochondria. Our present study is to identify which domain of a-synuclein is to be the key in affecting mitochondrial function, and how the function is to be impaired by alteration of a-synuclein. For this purpose, an N-terminal (amino acids 1-65), NAC (61-95), and C

6、-terminal (91-140) fragments of a-synuclein were generated, and retroviral expression system was employed for transfection. Confocal microscopy, flow cytometry, Western blot analysis and electron microscope techniques were employed to evaluate co-localization of target protein and mitochondrion, mit

7、ochondrial membrane potential, mitochondrial modality, and direct interaction of interested proteins in MN9D cells. The results demonstrated that only the N-terminal fragment of a-synuclein was localized on mitochondria, causing the decline of mitochondrial membrane potential. Upon over-expressing o

8、f the N-terminus, cell viability was significantly decreased and the mitochondria were dramatically changed in shape. These results suggested that under altered conditions of a-synuclein expression, a-synuclein on mitochondria might produce enhanced toxicity leading to the eventual neuronal cell dea

9、th. Moreover, in order to examine apoptosis-related molecular events, apoptosis-inducing factor, cytochrome c release, together with Annexin V staining were employed to this cell model to analyze the cell death mechanism with alteration of a-synuclein, which revealed apoptotic events were apparently

10、 involved in the toxicity of N-terminal of a-synuclein2.  在多巴胺能神經(jīng)元磷酸化的 -SYN 參與保護(hù)作用及 TH 的調(diào)節(jié)機(jī)制劉琦 碩士2009帕金森?。≒arkinsons Disease, PD）是臨床上最常見(jiàn)的老年神經(jīng)退行性疾病之一。其典型的神經(jīng)病理學(xué)特征為中腦黑質(zhì)多巴胺 (dopamine, DA) 能神經(jīng)元選擇性進(jìn)行性缺失，同時(shí)殘存的神經(jīng)元胞漿和軸突內(nèi)出現(xiàn)路易氏體（Lewy body）和路易氏神經(jīng)索。-突觸核蛋白（-SYNuclein，-SYN）是一種與遺傳性和散發(fā)性PD均密切相關(guān)的磷蛋白，-SYN的異常表達(dá)和突變均

11、能導(dǎo)致PD的發(fā)生。已有研究發(fā)現(xiàn)，在路易氏體（Lewy body）中，-SYN磷酸化程度可達(dá)90%，遠(yuǎn)遠(yuǎn)超出正常生理情況下4%的磷酸化水平，且修飾位點(diǎn)僅為C端129位的絲氨酸。但到目前為止，磷酸化-SYN在PD發(fā)病過(guò)程中的作用仍不是很清楚，為了進(jìn)一步明確磷酸化-SYN對(duì)多巴胺能神經(jīng)元的影響及參與TH調(diào)控的機(jī)制，進(jìn)行了如下實(shí)驗(yàn)。 本 實(shí) 驗(yàn) 室 已 經(jīng) 成 功 構(gòu) 建 了 逆 轉(zhuǎn) 錄 病 毒 載 體 攜 帶 的 野 生 型 -SYN (WT/-SYN) ， 模 擬 磷 酸 化 -SYN （ S129D/-SYN ） 和 抑 制 磷 酸 化 -SYN（S129A/-SYN），通過(guò)病毒包裝和篩選，收集

12、病毒上清，感染 MN9D 細(xì)胞，通過(guò) real-time PCR 檢測(cè)細(xì)胞內(nèi)各基因的表達(dá)水平，結(jié)果顯示各組細(xì)胞內(nèi)，-SYN的表達(dá)較正常組明顯增加。感染細(xì)胞 24 和 48h 后，光鏡下觀察細(xì)胞形態(tài)，結(jié)果顯示過(guò)表達(dá) WT/-SYN 組細(xì)胞形態(tài)不規(guī)則，突起減少，胞體腫脹，且增殖減慢。Confocal 觀察感染 48h 后的細(xì)胞，發(fā)現(xiàn)過(guò)表達(dá) S129D/-SYN 組胞核及胞漿內(nèi)出現(xiàn)了較為明顯的 -SYN 聚集，過(guò)表達(dá) S129A/-SYN 組細(xì)胞內(nèi) -SYN 呈聚集趨勢(shì)，而過(guò)表達(dá) WT/-SYN 組細(xì)胞內(nèi)未見(jiàn) -SYN 聚集現(xiàn)象。CCK-8 檢測(cè)感染后 48h細(xì)胞活力，發(fā)現(xiàn)各組 MN9D 細(xì)胞活力較

13、對(duì)照組相比均明顯下降（*p<0.05，*p<0.01），且過(guò)表達(dá) WT/-SYN 組細(xì)胞活力明顯低于過(guò)表達(dá) S129D/-SYN 組和S129A/-SYN 組（#p<0.01)，以上結(jié)果提示過(guò)表達(dá)野生型 -SYN 可以引起細(xì)胞內(nèi)寡聚體形式的 -SYN 增加，其毒性損傷多巴胺能神經(jīng)元，磷酸化 -SYN 可能通過(guò)形成不可溶的蛋白聚集體，使寡聚狀態(tài)的 -SYN 聚集，抑制對(duì)細(xì)胞的毒性損傷，從而保護(hù)細(xì)胞活力。阻抑磷酸化的 -SYN 亦可引起 -SYN 呈聚集狀態(tài)，可能由于聚集體形成較磷酸化晚，故保護(hù)作用不如磷酸化明顯。 酪氨酸羥化酶（TH）是多巴胺合成的限速酶，磷酸化 -突觸核蛋白是

14、否對(duì)TH 的表達(dá)和活性具有調(diào)節(jié)作用，可能的作用機(jī)制以及二者之間是否有直接或間接相互作用關(guān)系目前尚無(wú)明確報(bào)道。 本 實(shí) 驗(yàn) 將 構(gòu) 建 好 的 野 生 型 及 兩 種 突 變 的 myc-SYN 標(biāo) 簽 蛋 白 和pcDNA3.0-TH 共同轉(zhuǎn)染于 HEK293T 細(xì)胞內(nèi)，通過(guò)免疫共沉淀的方法，檢測(cè)各組-SYN 和 TH 之間是否存在相互作用關(guān)系，結(jié)果均發(fā)現(xiàn)陽(yáng)性條帶。將構(gòu)建好的野生型及兩種突變的熒光標(biāo)簽蛋白 HaloTag-SYN 和 pcDNA3.0-TH 共轉(zhuǎn)染于HEK293T 細(xì)胞，通過(guò)免疫細(xì)胞化學(xué)熒光方法，Confocal 檢測(cè)，可見(jiàn)各組 -SYN和 TH 之間存在共定位。Western

15、-blot 結(jié)果證實(shí)過(guò)表達(dá) S129D/-SYN 可以上調(diào)TH 的活性（*p<0.05），同時(shí)對(duì) TH 的表達(dá)水平無(wú)影響；過(guò)表達(dá) S129D/-SYN，PP2A 表達(dá)水平明顯下降（*p<0.01），磷酸化 ERK1/2 水平增強(qiáng)（*p<0.01）；而過(guò)表達(dá) WT/-SYN 結(jié)果正好相反，同時(shí)蛋白激酶 A 水平并無(wú)顯著性改變。以上試驗(yàn)結(jié)果提示，磷酸化 -SYN 可以上調(diào) TH 的活性。其調(diào)節(jié)機(jī)制可能不是通過(guò)與其相互作用，而是通過(guò)調(diào)節(jié) PP2A 和 ERK 通路實(shí)現(xiàn)的。 綜合以上實(shí)驗(yàn)結(jié)果，本實(shí)驗(yàn)室認(rèn)為，在 PD 的發(fā)展過(guò)程中，寡聚狀態(tài)可溶的-SYN 對(duì)多巴胺能神經(jīng)元具有毒性損傷作

16、用，磷酸化 -SYN 通過(guò)促進(jìn)形成不溶性的 -SYN 聚集體，局限了寡聚體 -SYN 引起的細(xì)胞毒性，并進(jìn)一步促進(jìn)蛋白酶體系統(tǒng)對(duì)其清除，從而在一定程度上抑制了野生型 -SYN 過(guò)表達(dá)引起的細(xì)胞毒性損傷。磷酸化 -SYN 水平增加，通過(guò)調(diào)節(jié) PP2A 和 ERK 的表達(dá)和活性，提高了 TH 磷酸化水平，增強(qiáng)了 TH 活性，促進(jìn)多巴胺的合成和分泌，彌補(bǔ)了野生 型 -SYN 過(guò)表達(dá)導(dǎo)致的 TH 活性抑制和多巴胺合成不足。Parkinsons disease is one of the most common progressive neurodegenerative disorders. The t

17、ypical neuropathologic characters are the selectively progressive deletion of dopaminergic neurons and formation of Lewys bodys in lived neuro endochylema and axon. lpha-synuclein（-SYN） is a protein associated with familial and sporadic PD. The abnormal expression and mutation of -SYN can induce PD.

18、 Most researche has found that -SYN is hyperphosphorylated at 129 serine, approximately 90% in Lewy bodys, while remains only 4% in normal. But by now, it is still unclear that the mechenism of phosphorylated -SYN in PD. In order to indentify how phosphorylated -SYN involved in dopaminergic neuron a

19、nd regulates the expression and activity of TH, we design experiments as follow. WT/-SYN, S129D/-SYN and S129A/-SYN have constructed into retrovirus vector. Virus supernatant gathered and infected in mouse dopaminergic cells MN9D, then genes expression of transfected cells was detected by real-time

20、PCR and the results show that the genes level of -SYN in all groups are obviously higher than normal. Cell morphology was observed by microscope after infected 24h and 48h. The morphology of cells WT/-SYN over-expressed was irregular, the number of project from cell body was reduced, cell bodies wer

21、e swallow and proliferation turned slowly. By confocal, protein aggregation of insoluble -SYN was found in cells S129D/-SYN over expressed, in which both were in endochylema and nucleus and -SYN acted as prone to aggregate in over expression cells of S129A/-SYN, but -SYN uniformly distributed in cel

22、ls of WT/-SYN over expressed. The cell viability was obviously reduced in all groups than contral cells after 48h infection (*p<0.05，*p<0.01). And the viability of cells infected by WT/-SYN was much lower than the other two -SYN over-expressing groups (#p<0.01). These results apply that wil

23、d type -SYN over expression could up regulate the level of oligo -SYN, which is toxic to dopaminergic neuron, and -SYN phosphorylated at 129 Ser may protect the cells through formation of insoluble -SYN aggregates. Tyrosine hydroxylase (TH) is the rate-limiting enzyme of DA synthesis, so far, there

24、are no definitly reports about whether phosphorylated -SYN can regulate the expression and the activity of TH and the possibility regulation mechanism, it is still no clear that if any directe or indirecte interaction between them. Wild type and two other mutation -SYN fusion-expressing vectors were

25、 constructed, and co-transfected to human embryo kidney cells HEK293T with pcDNA3-TH. The relation between -SYN and TH was detected by immunocytochemistry, CO-IP and GST pull-down the co-localization detected and the positive belts were found in all groups. Anterior reseache in our lab already prove

26、d that phosphorylated -SYN up-regulated the activity of TH without any change of the expression levels (*p<0.05). By Western-blot, the results showed that the level of phosphorylated ERK1/2 increased and PP2A decreased (*p<0.05, *p<0.01), while without any change of PKA. Those results indic

27、ated that the activity of TH may be up-regulated by phophosphorylated -SYN not through the direct interaction but via cell signaling pathway of ERK1/2 and PP2A. Take these into consideration, in the developing of PD, oligo -SYN causes toxic function to dopaminergic neuron. Phosphorylated -SYN promot

28、es to form insoluble -SYN aggregation and constrains the cell toxicity of oligo -SYN, then facilitates the proteasome system to constrain the toxicity by wild type -SYN. Phosphorylated -SYN elevates the activity of TH through regulating cell signaling pathway of ERK1/2 and PP2A, could increase dopam

29、ine synthesis and secretion, thus make up dopaminal quantity which has been downregulated by inhibition of TH activity through oligo -SYN formation.帕金森病相關(guān)基因PINK1和-突觸核蛋白的相互作用范春香 碩士 2014帕金森病（Parkinsons Disease, PD）是臨床上最常見(jiàn)的神經(jīng)退行性疾病之一。它的主要特點(diǎn)是中腦黑質(zhì)多巴胺能神經(jīng)元的進(jìn)行性缺失和胞質(zhì)內(nèi)路易體的形成，其中的主要成分為-synuclein，近期報(bào)導(dǎo) PINK1等蛋白也在其

30、中。PD的發(fā)病機(jī)制涉及到多方面，包括：蛋白降解，氧化應(yīng)激，蛋白磷酸化以及線粒體功能障礙等，具體機(jī)制仍不清楚。-synuclein和PINK1均與家族性PD有關(guān)。兩者均能定位于線粒體，兩者的突變體都能影響線粒體的活力。但兩者之間的關(guān)系至今仍不清楚。為了研究-synuclein和PINK1之間的關(guān)系，我們首先利用多巴胺能神經(jīng)元MN9D細(xì)胞，進(jìn)行免疫細(xì)胞化學(xué)染色，通過(guò)共聚焦顯微鏡觀察-synuclein和PINK1兩種蛋白在細(xì)胞內(nèi)的分布及共定位狀態(tài)。結(jié)果顯示兩者主要分布于細(xì)胞漿中并且能夠部分共定位，這在空間上提供了兩者能夠相互作用的可能性。我們進(jìn)一步克隆了野生型PINK1及其突變體G309D，C14

31、5 及N35，并成功構(gòu)建了含有Flag-tag的蛋白質(zhì)表達(dá)載體。將本室構(gòu)建的GST-synuclein經(jīng)大腸桿菌BL-2l表達(dá)純化后，與MN9D細(xì)胞裂解液共孵育，通過(guò)GST-Pull down 技術(shù)，檢測(cè)到PINK1與-synuclein蛋白間的相互作用。并應(yīng)用MN9D細(xì)胞的內(nèi)源性PINK1與-synuclein進(jìn)行免疫沉淀實(shí)驗(yàn)，來(lái)進(jìn)一步驗(yàn)證兩蛋白之間的相互作用。結(jié)果發(fā)現(xiàn)利用GST-synuclein融合蛋白捕獲及免疫沉淀技術(shù)，均檢測(cè)到特異的PINK1條帶。采用脂質(zhì)體轉(zhuǎn)染的方法將本室構(gòu)建的myc-synuclein及pCMV-myc，pcDNA3-Flag-PINK1及pcDNA3-Flag分

32、別單獨(dú)瞬時(shí)轉(zhuǎn)染293T細(xì)胞，利用myc抗體及anti-Flag M2 affinity gel進(jìn)行免疫共沉淀，進(jìn)一步確定了兩者之間的相互作用。有研究表明PINK1突變可導(dǎo)致-synuclein表達(dá)量的上調(diào)和在細(xì)胞內(nèi)的聚集。在本室成功干擾掉PINK1的MN9D細(xì)胞株中，我們檢測(cè)到-synuclein的蛋白水平是明顯增加的。此外，利用本室收集的表達(dá)-synuclein的逆轉(zhuǎn)錄病毒上清感染MN9D細(xì)胞，實(shí)時(shí)定量檢測(cè)證明-synuclein的基因表達(dá)量顯著上調(diào)，我們發(fā)現(xiàn)該種細(xì)胞中PINK1的蛋白水平是明顯增加的。這些結(jié)果證明PINK1與-synuclein在細(xì)胞中能夠共定位并相互作用，并能相互影響細(xì)胞

33、中的蛋白水平。這可能為PD相關(guān)基因的關(guān)系及機(jī)制研究提供一定的依據(jù)。Parkinsons Disease (PD) is a neurodegenerative disorder with pathological hallmarks of dopaminergic neuron degeneration in the substantia nigra pars compacta and cytoplasmic inclusion Lewy bodies that contain -synuclein, PINK1. The PD pathogenic pathways involve defe

34、cts in many cellular processes such as protein degradation, oxidative stress, phosphorylation signaling, and mitochondrial function. -synuclein and PINK1 are both implicated in genetic forms of familial PD. They both can localize to mitochondria, and their mutations have been shown to disrupt mitoch

35、ondrial fuction. Despite this wealth of evidence, its still unclear whether -synuclein and PINK1 have relationship between each other.To answer this question we first detected the location of -synuclein and PINK1 in MN9D cells by immunohistochemical staining. We found that they could be partly co-lo

36、calized in cytoplasm. This provided the possibility that they can interact with each other. We constructed the pcDNA3-Flag-PINK1 (G309D, C145, N35) eukaryotic expression vectors encoding wild-type and its mutants for further research. The GST-synuclein fusion protein was expressed in Escherichia col

37、i and purified on Glutathione-Sepharose 4B by standard methods, and incubated with MN9D cells lysate to detect the interaction of PINK1 with -synuclein. In addition, the full length of PINK1 robustly associated with -synuclein was determined by immunoprecipitation experiment in MN9D cells. We detect

38、ed that GST-synuclein interacted specifically with PINK1, but not GST. Immunoprecipitation with an anti-synuclein antibody and subsequent Western Blotting with an anti-PINK1 antibody confirmed the specific interaction of -synuclein with PINK1. We transiently transfected myc-synuclein/pCMV-myc or pcD

39、NA3-Flag-PINK1/pcDNA3-Flag into 293T cells respectively, and then repeated the immunoprecipitation with anti-myc or anti-Flag M2 affinity gel. The results confirmed the association of PINK1 and -synuclein.It has been proved that PINK1 mutations can upregulate the expression of -synuclein and induce

40、its aggregation. We found that MN9D cells knock-down PINK1 increased -synuclein protein level while overexpression of -synuclein evaluated the protein level of PINK1.These finds indicate that PINK1 and -synuclein can co-localize in cytoplasm and interact with each other, and they also can affect eac

41、h other at protein level.聚酰胺-胺樹(shù)枝狀分子修飾的硅殼熒光磁性納米粒趙煥瑛 碩士 2009 人類(lèi)腦部疾患的發(fā)病率正呈逐年上升的趨勢(shì)，對(duì)人類(lèi)生命和健康造成嚴(yán)重的危害。由于血腦屏障( blood brain barrier, BBB) 的存在，使腦部疾病無(wú)論采取基因治療還是藥物治療都很難使有效成分導(dǎo)入中樞神經(jīng)系統(tǒng)發(fā)揮作用。目前,探索利用載體經(jīng)血管途徑透過(guò)BBB治療腦部疾病成為研究的熱點(diǎn)。在協(xié)助藥物或基因入腦的方法中，磁性納米載體(又稱(chēng)磁納米粒，magnetic nanoparticles，MNP)因其既具有納米材料的特性，又具有磁響應(yīng)性及超順磁性，而且，這類(lèi)藥物載體還能延

42、緩藥物在腦內(nèi)的釋放，降低外周毒性。因此，成為最有前景的靶向性藥物或基因載體。本課題設(shè)計(jì)了聚酰胺-胺(PAMAM)樹(shù)狀分子修飾的可熒光示蹤的硅殼磁性納米載體作為治療中樞神經(jīng)系統(tǒng)疾病的藥物或基因載體。該載體不僅具有熒光性、超順磁性等特點(diǎn)，其外表面由于有PAMAM樹(shù)狀分子覆蓋，具有大量官能團(tuán)可再進(jìn)一步修飾(如連接跨膜肽TAT、抗體、受體等)，而且能阻止包埋的熒光分子泄露所帶來(lái)的毒副作用。本論文對(duì)所制備的系列載體進(jìn)行了體內(nèi)外的分布、毒性及穿越BBB能力評(píng)估，同時(shí)篩選出可攜帶基因的納米載體。主要研究?jī)?nèi)容如下： (1) A549 和9L細(xì)胞吞噬納米粒的體外實(shí)驗(yàn)：首次對(duì)納米粒進(jìn)入膠質(zhì)瘤9L細(xì)胞、人肺腺癌A5

43、49細(xì)胞的能力、機(jī)制及細(xì)胞內(nèi)定位進(jìn)行了研究。由流式細(xì)胞計(jì)數(shù)結(jié)果可知，納米粒進(jìn)入細(xì)胞的能力呈時(shí)間和濃度依賴(lài)性，且受表面電荷影響。細(xì)胞經(jīng)DAPI、Lysotracker blue染色結(jié)果表明納米粒主要分布于胞漿中，且能被溶酶體俘獲。細(xì)胞加入450mM蔗糖和0.2mg/mL 木黃酮兩種抑制劑來(lái)觀察細(xì)胞攝取納米粒的機(jī)制，結(jié)果證明A549細(xì)胞吞噬納米粒可通過(guò)網(wǎng)格蛋白有被小窩介導(dǎo)的內(nèi)吞及細(xì)胞膜穴樣內(nèi)陷介導(dǎo)的內(nèi)吞兩種途徑。9L細(xì)胞主要通過(guò)細(xì)胞膜穴樣內(nèi)陷介導(dǎo)的內(nèi)吞途徑。透射電鏡結(jié)果驗(yàn)證了納米粒進(jìn)入細(xì)胞是通過(guò)胞吞作用完成的，且主要分布在細(xì)胞漿，而帶有PEG作連接臂的，及整代PAMAM分子修飾的，納米粒更易進(jìn)入細(xì)

44、胞核。CCK-8毒性實(shí)驗(yàn)證實(shí)四種納米粒毒性都很小，但PAMAM修飾后毒性更小，尤其PAMAM(G2.5)修飾的對(duì)細(xì)胞活力的影響最小。(2) 體內(nèi)分布、毒性及跨血腦屏障能力評(píng)估實(shí)驗(yàn)：通過(guò)頸總動(dòng)脈將四種納米粒分別注射入大鼠體內(nèi)，在激光共聚焦顯微鏡下觀察各器官中納米粒分布情況，并進(jìn)行組織病理學(xué)HE染色分析。結(jié)果證明四種納米粒都在腦組織分布，但只有PEG-PAMAM(G2)修飾的納米粒分布的最多且聚集最少，其次為ICP-PAMAM(G2)修飾的納米粒，且四種納米粒都集中分布在網(wǎng)狀內(nèi)皮系統(tǒng)(RES)豐富的器官：肝臟、脾臟及肺臟。接著我們又將PEG-PAMAM(G2)修飾的納米粒從ICR小鼠尾靜脈進(jìn)行注射

45、研究，通過(guò)行為學(xué)、血清生化分析、血液學(xué)分析及組織病理學(xué)分析表明：納米?？煞植荚谌砀髌鞴伲夷艽┰窖X屏障和血睪屏障，可通過(guò)尿液代謝排除，同時(shí)表明納米粒對(duì)小鼠沒(méi)有急性毒性作用。(3) 硅殼熒光納米粒攜基因能力及轉(zhuǎn)染功能的評(píng)價(jià)：體外進(jìn)行了納米粒與DNA結(jié)合實(shí)驗(yàn)、納米粒/DNA復(fù)合物抵抗DNase和血清消化實(shí)驗(yàn)及在Cos7細(xì)胞中基因轉(zhuǎn)染等實(shí)驗(yàn)，結(jié)果表明只有PAMAM(G2)修飾的納米粒才具有攜帶DNA能力并能抵御核酸酶和血清的消化，PAMAM(G2)修飾的硅殼熒光納米粒攜帶的pDRsed monomer 基因在Cos7細(xì)胞中都表達(dá)了紅色熒光蛋白。本課題設(shè)計(jì)合成的目標(biāo)載體及其進(jìn)入血腦屏障的研究未見(jiàn)文

46、獻(xiàn)報(bào)道，結(jié)果表明PAMAM(G2)修飾納米粒不但可以穿越血腦屏障,而且容易被神經(jīng)元和膠質(zhì)細(xì)胞攝取。本研究結(jié)果為PAMAM修飾的硅殼熒光納米粒作為藥物/基因載體，用于腦部疾病的治療提供了基礎(chǔ)研究數(shù)據(jù)，因而具有理論研究?jī)r(jià)值和潛在的應(yīng)用價(jià)值。The diseases in human brain are rapidly increasing and threatening human health. The existence of BBB blocks most drugs or gene from entering the brain via blood system in the treatm

47、ent of neurological or psychiatric disorders of central nervous system (CNS). Now more interests in drug carriers have been focused on treating CNS disease through intravascular ways. Among various strategies aimed to enhance the penetration of drugs or gene carrier into the brain tissue, magnetic n

48、anoparticles (MNPs) have become a promising targeting carrier for drug or gene delivery through BBB. MNPs which have size in the scale of nanometer and possess unique superparamagnetism, have been shown to release drugs in a controlled manner and possess reduced peripheral toxicity. In this study, p

49、olyamidoamine (PAMAM) dendrimer conjugated fluorescein isothiocyanate (FITC)-doped silica-Fe3O4 core-shell magnetic nanoparticle has been developed as carrier for drug or gene delivery across the BBB. This carrier exhibits a unique superparamagnetism and can be traced by fluoresce as well. The conju

50、gation of PAMAM provides to the carrier a number of primary amino surface functional groups which allow it to conjugate additional ligands, such as transmembrane peptide TAT, appropriate receptors and antibody, to enhance the delivery of drug/gene to the targeting region. Furthermore, the capped PAM

51、AM might reduce the toxicity caused by the leakage of FITC. In this thesis, the in vivo and ex vivo pattern of distribution and toxicity of four kinds of nanoparticles and its ability to penetrate the BBB were investigated.Among them, nanoparticles which can carrier gene were choosen. These results

52、are shown as follows: (1) Examination of the cellular uptake of nanoparticles by 9L cells and A549 cells: The ability, mechanism and intracellular localization of the nanoparticles for 9L and A549 cells were evaluated. Flow cytometry analysis suggested the uptake process of the carrier by cells was

53、a time- and dose-dependent. The result also showed that the efficiency of intracellular uptake was affected by the surface charge of the particales, with the positive charge enhancing the uptake of cells. TEM studies have indicated that the nanoparticles were uptake by the cells through endocytosis.

54、Combined with the DAPI and lysotracker blue costaining result, nanoparticles were mostly internalized into cytoplasm with subsequent intracellular localization in late endosomes/lysosomes. Additional, Only PEG-PAMAM (G2) modified nanoparticles can be detected occassionaly in nucleus. To investigate

55、the possible mechanism of endocytosis of four kinds of nanoparticles, we tested the cellular uptake of nanoparticles in presence of specific inhibitors of different endocytotic pathways. Our results indicated that nanoparticles were endocytosed by A549 cell via a clathrin-pitted mechanism and a cavo

56、lar-mediated mechanism, as the uptakes were inhibited by 450mM sucrose and 0.2mg/ml genistein. However, the uptake efficiency of 9L cells was only affected by a caveolar inhibitor, genistein, suggesting that these nanoparticles are endocytosed via a caveolae-mediated mechanism.(2) Animal stuides of

57、the biodistribution, biocompatibility of nanoparticles and the ability of crossing the BBB: Four kinds of nanoparticles were infused in rats through carotid artery. Subsequently, the biodistribution of nanoparticles in tissue slices stained with DAPI were investigated by confocal laser scanning micr

58、oscope (CLSM). Histopathological analysis in various organs was observed in tissue slices stained with hematoxilin and eosin (HE). HE result showed that no abnomal histopathological changes were observed. CLSM result showed that four type of nanoparticles all were found in the brain, but PEG-PAMAM(G

59、2) modified nanoparticles were distributed more widespread and lesser accumulative than other nanoparticles. Besides, nanoparticles were also found in the organs with plenty of reticulo endothelial system (RES), such as liver, spleen and lung. At the same time FMNPs-PEG-PAMAM (G2) were injected intravenously via the tail vein of mice at a dose of 10 mg /kg. The in vivo toxicity was evaluated. The results of serum biochemical analysis, hema