汪子文 桂錚 盧學(xué)剛 郭棟 朱明 李臻 韓新巍 于耀宇

(1鄭州大學(xué)第一附屬醫(yī)院介入科，河南 鄭州 450001; 2西安交通大學(xué)理學(xué)院材料物理系，陜西 西安 710049)

·腦血管疾病診治研究·

磁導(dǎo)向納米磁性藥物載體對(duì)遲發(fā)型腦血管痙攣干預(yù)的實(shí)驗(yàn)研究

汪子文1桂錚1盧學(xué)剛2郭棟1朱明1李臻1韓新巍1于耀宇1*

(1鄭州大學(xué)第一附屬醫(yī)院介入科，河南 鄭州 450001;2西安交通大學(xué)理學(xué)院材料物理系，陜西 西安 710049)

目的探討在磁導(dǎo)向下納米磁性藥物載體(MNA-DPD)對(duì)遲發(fā)型腦血管痙攣(DCVS)干預(yù)的有效性。方法健康成年新西蘭白兔100只，隨機(jī)分為5組：正常組(n=20)，處理組(n=20)：溶劑對(duì)照組(DMSO)、納米磁性蛋白組(MNA)、2-2'聯(lián)吡啶組(DPD)和納米磁性藥物載體組(MNA-DPD)。處理組采用二次注血法制作DCVS模型，然后每天分別通過(guò)腰大池給予0.01% DMSO、MNA、DPD和MNA-DPD，劑量50 mg/kg。MNA組、MNA-DPD組給予外加磁場(chǎng)磁導(dǎo)向。在給藥7 d時(shí)處死動(dòng)物，取其基底動(dòng)脈行HE染色并測(cè)定管徑，同時(shí)行普魯士藍(lán)染色。Western Blot定量分析給藥后7 d動(dòng)物模型基底動(dòng)脈增殖細(xì)胞核抗原(PCNA)、血管內(nèi)皮生長(zhǎng)因子(VEGF)、c-Myc蛋白和p53蛋白表達(dá)水平。結(jié)果HE染色和普魯士藍(lán)染色顯示DMSO組、MNA組基底動(dòng)脈明顯痙攣，DPD組基底動(dòng)脈可見(jiàn)痙攣，MNA-DPD組基底動(dòng)脈基本正常的形態(tài)(Plt;0.05)。并可見(jiàn)蛛網(wǎng)膜下腔的納米磁性微粒被染成藍(lán)色。Western Blot定量分析PCNA、p53、c-Myc、VEGF蛋白在DMSO組、MNA組和DPD組呈現(xiàn)高表達(dá)，MNA-DPD組呈現(xiàn)低表達(dá)(Plt;0.05)。結(jié)論在磁導(dǎo)向下，腰大池給予納米磁性藥物載體，能夠有效地抑制DCVS。

磁導(dǎo)向納米磁性藥物載體; 遲發(fā)性腦血管痙攣; 蛛網(wǎng)膜下腔出血

自發(fā)性蛛網(wǎng)膜下腔出血(subarachnoid hemorrhage, SAH)85%以上是腦動(dòng)脈瘤破裂所致，遲發(fā)性腦血管痙攣(delayed cerebral vasospasm, DCVS)是SAH后病情加重，致殘、致死的主要原因。約70%以上SAH出現(xiàn)DCVS[1-2]。目前流行的DCVS治療是應(yīng)用Ca2+拮抗劑，療效不理想，至今仍存在爭(zhēng)議。本研究根據(jù)DCVS相對(duì)肯定的病理機(jī)制，經(jīng)過(guò)DCVS模型腰大池途徑，利用磁導(dǎo)向?qū)⒓{米磁性藥物載體靶向聚集于DCVS部位緩釋藥物，增加藥物的有效濃度。有效改善并阻止DCVS病理進(jìn)程。

材料與方法

一、實(shí)驗(yàn)動(dòng)物

健康成年新西蘭大白兔100只 (鄭州大學(xué)實(shí)驗(yàn)動(dòng)物中心提供), 雌雄不限，平均體重2.5 kg。

二、實(shí)驗(yàn)試劑和儀器

兔抗抑癌基因p53抗體、兔抗血管內(nèi)皮生長(zhǎng)因子(vascular endothelial growth factor, VEGF)抗體、小鼠抗增殖細(xì)胞核抗原(proliferating cell nuclear antigen, PCNA)抗體和兔抗原癌基c-myc抗體(Chemicon, Temecula, CA, USA)；蘇木素伊紅染色試劑盒(中國(guó)碧云天)；納米磁性藥物載體(MNA-DPD)自制。馬蹄形梯度磁場(chǎng)(0.1T)；恒冷箱切片機(jī)(Leica, CM-1800, USA)等。

三、研究方法

實(shí)驗(yàn)動(dòng)物隨機(jī)分為5組：正常組(n=20)，處理組(n=20)：溶劑對(duì)照組(dimethyl sulphoxide group, DMSO)、納米磁性蛋白組(magnetic nanoparticle-albumin, MNA)、2-2' 聯(lián)吡啶組(2, 2'-dipyridyl, DPD)和納米磁性藥物載體組(magnetic nanoparticle-albumin-2, 2'-dipyridyl, MNA-DPD)。四個(gè)處理組DCVS模型采用二次注血法制作[3]，造模后每天分別通過(guò)腰大池給予0.01% DMSO(溶劑)、MNA、DPD和MNA-DPD，劑量50 mg/kg。MNA-DPD和MNA組給予外加磁場(chǎng)磁導(dǎo)向(0.1T)。給藥第7天分批處死動(dòng)物，取基底動(dòng)脈行HE染色并測(cè)定管徑，同時(shí)行普魯士藍(lán)染色。Western Blot蛋白定量觀察第7天PCNA、VEGF、c-Myc和p53蛋白表達(dá)水平。

四、統(tǒng)計(jì)學(xué)處理

圖1 各組基底動(dòng)脈直徑量化和基底動(dòng)脈壁厚度量化變化 (HE, ×40)
Fig 1 Quantification of basilar artery diameter and basilar artery wall thickness were performed in each group (HE, ×40)
A: Normal basilar artery morphology in sham group; B: Basilar artery spasm in DMSO group; C: Basilar artery spasm in MNA group; D: Basilar artery spasm was slightly improved in DPD group; E: Basilar artery spasm was improved significantly in MNA-DPD group; F: Quantification histogram of basilar artery diameter in each group; G: Histogram of basilar artery wall thickness.

aPlt;0.05,vsMNA group;bPlt;0.05,vsDPD group.

Bar=100 μm.

結(jié) 果

一、HE染色

第7天時(shí)：對(duì)照組正常基底動(dòng)脈形態(tài)，DMSO組基底動(dòng)脈明顯痙攣，MNA組基底動(dòng)脈明顯痙攣，DPD組基底動(dòng)脈痙攣稍有改善，MNA-DPD組基底動(dòng)脈痙攣明顯改善，呈現(xiàn)差異顯著(Plt;0.05)。各組基底動(dòng)脈直徑量化和基底動(dòng)脈壁厚度量化變化見(jiàn)圖1(HE, ×40)。

二、普魯士藍(lán)染色

40倍鏡下DMSO組、MNA組基底動(dòng)脈明顯痙攣，MNA-DPD組基底動(dòng)脈基本正常的形態(tài)。并可見(jiàn)到達(dá)蛛網(wǎng)膜下腔的納米磁性微粒被染成藍(lán)色(鐵離子)(見(jiàn)圖2)。Western Blot第7天時(shí)模型基底動(dòng)脈PCNA、P53、c-Myc和VEGF蛋白在DMSO組、MNA組和DPD組呈現(xiàn)高表達(dá)，MNA-DPD組呈現(xiàn)低表達(dá)，MNA-DPD組與DPD組比較有顯著差異(Plt;0.05)，MNA-DPD與MNA組比較有顯著差異(Plt;0.05，圖3)。

圖2 各組血管普魯士藍(lán)染色結(jié)果(×40)
Fig 2 Prussian blue staining results for each group of blood vessels (×40)
A: The basilar artery in sham group showed the morphology; B: Basilar artery spasm in DMSO group; C: Basilar artery spasm in MNA group; D: Basilar artery spasm was slightly improved in DPD group; E: Basilar artery spasm was improved significantly in MNA-DPD group.

Bar=100 μm.

圖3 基底動(dòng)脈中PCNA、P53、c-Myc和VEGF的Western Blot定量分析
Fig 3 Western Blot analysis of PCNA, P53, c-Myc and VEGF in basilar artery
A: Western Blot analysis of PCNA in basilar artery; B: Western Blot analysis of P53 in basilar artery; C: Gray scale ratio of PCNA compared with actin control; D: Gray scale ratio of P53 compared with actin control; E: Western Blot analysis of c-Myc in basilar artery; F: Western Blot analysis of VEGF in basilar artery; G: Gray scale ratio of c-Myc compared with actin control; H: Gray scale ratio of VEGF compared with actin control.

aPlt;0.05,vsMNA group;bPlt;0.05,vsDPD group.

討 論

遲發(fā)性腦血管痙攣(DCVS)機(jī)制錯(cuò)綜復(fù)雜，治療方法眾多，但療效甚微[4-6]。目前流行的DCVS治療是應(yīng)用Ca2+拮抗劑(如尼莫通等)，價(jià)格昂貴且臨床療效不理想，因此DCVS治療的途徑或體系值得分析考慮。本研究根據(jù)DCVS相對(duì)肯定的病理機(jī)制，經(jīng)過(guò)DCVS模型腰大池途徑，利用磁導(dǎo)向?qū)⒓{米磁性藥物載體(MNA-DPD)靶向聚集于DCVS部位，并緩釋藥物，大大增加藥物的有效濃度。同時(shí)還具有抗氧化、抗自由基(阻止Fe2+參與自由基的形成)、抗免疫、抗增殖和抗細(xì)胞水腫的作用。抑制內(nèi)皮細(xì)胞和平滑肌細(xì)胞的異常增殖和/或遷移，防止腦血管的痙攣后期出現(xiàn)的器質(zhì)性痙攣[7-9]。

實(shí)驗(yàn)證明MNA-DPD中，MNA具有良好的生物相溶性、水溶性、磁響應(yīng)性、穩(wěn)定性和靶向性[10-11]。DPD對(duì)Fe2+有高度親和力，可以螯合Fe2+。MNA-DPD可以在細(xì)胞內(nèi)外通過(guò)藥物濃度梯度或者在外在磁場(chǎng)作用下緩慢釋放DPD。通過(guò)腰大池給藥給予MNA-DPD，在外在聚焦梯度磁場(chǎng)作用下，由于納米磁性藥物載體具有磁響應(yīng)性和靶向性，可以通過(guò)腦脊液循環(huán)靶向聚集于位局部出血的蛛網(wǎng)膜下腔，緩慢釋放DPD，DPD 通過(guò)其螯合環(huán)結(jié)合OxyHb降解的 Fe2+，阻斷Fe2+導(dǎo)致DCVS的一系列病理過(guò)程，有效阻止腦血管痙攣的發(fā)生；鐵離子螯合劑還可以減輕腦水腫，減輕OxyHb對(duì)平滑肌的副作用[12]。

c-Myc和P53基因表達(dá)與PCNA有明顯正相關(guān)性，是促進(jìn)血管平滑肌細(xì)胞表型轉(zhuǎn)換及增殖，加速新生內(nèi)膜形成的重要原因。VEGF在新生內(nèi)膜形成中所起的作用最為重要，VEGF還是血管平滑肌細(xì)胞重要的化學(xué)趨化因子，促進(jìn)血管平滑肌細(xì)胞增殖。P53還可以促進(jìn)細(xì)胞的凋亡，導(dǎo)致或加重血管痙攣[13]。因此上述研究基本證實(shí)了MNA-DPD定向干預(yù)DCVS治療的有效性。

本研究根據(jù)DCVS相對(duì)肯定的病理機(jī)制，經(jīng)過(guò)DCVS模型腰大池途徑，利用磁導(dǎo)向?qū)⒓{米磁性藥物載體靶向聚集于DCVS部位，并緩釋藥物，大大增加藥物的有效濃度。鐵螯合劑DPD對(duì)Fe2+具有的高度和親和力，能夠有效地螯合并清除蛛網(wǎng)膜下腔里OxyHb解離出的Fe2+，阻斷DCVS的病理過(guò)程，同時(shí)還具有抗自由基(Fe2+參與自由基的形成)的作用，最終達(dá)到有效地?cái)U(kuò)張痙攣的腦血管。

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Theinterventionaleffectofmagneticnanoparticle-albumin-2, 2'-dipyridyl(MNA-DPD)ondelayedcerebralvasospasm

WANGZiwen1,GUIZheng1,LUXuegang2,GUODong1,ZHUMing1,LIZhen1,HANXinwei1,YUYaoyu1

1DepartmentofInterventionalRadiology,FirstAffiliatedHospitalofZhengzhouUniversity,Zhengzhou450001;2DepartmentofMaterialPhysics,SchoolofScience,Xi'anJiaotongUniversity,Xi'an710049, China

ObjectiveThe interventional effect of magnetic nanoparticle-albumin-2, 2'-dipyridyl (MNA-DPD) on delayed cerebral vasospasm (DCVS) with magnetic field targeting was explored.MethodsThe experimental animals, 100 healthy adult New Zealand rabbits, were randomly divided into 5 groups: dimethyl sulphoxide group (DMSO), magnetic nanoparticle- albumin group (MNA), 2, 2'-dipyridyl group (DPD), magnetic nanoparticle-albumin-2, 2'-dipyridyl group (MNA-DPD), and control group. DCVS models were made by the secondary injection of blood in DMSO group, MNA group, DPD group and MNA-DPD group. 0.01% DMSO, MNA, DPD and MNA-DPD were administered respectively in each experimental group once every day (50 mg/kg). Focusing magnetic field was guided in MNA group and MNA-DPD group. In 7 d after injection, animals were killed and given perfusion fixation. Basilar arteries were extracted for HE staining, Prussian blue staining and Western Blot test. The expression of proliferating cell nuclear antigen (PCNA), vascular endothelial growth factor (VEGF), c-Myc protein and Tumor protein p53 in the seventh day were measured with Western Blot test.ResultsAt 7 d after injection, HE staining and Prussian blue staining showed obvious vasospasm in DMSO group and MNA group, moderate vasospasm in DPD group and nearly no vasospasm in MNA-DPD group. It was significantly different in MNA-DPD group compared with MNA group, DPD group and DMSO group (Plt;0.05). Prussian blue staining showed magnetic nanoparticles were dyed blue in the subarachnoid space of MNA group and MNA-DPD group. Western Blot test indicated high expression of PCNA, P53, c-Myc and VEGF in DMSO group, MNA group and DPD group, while low expression in MNA-DPD group (Plt;0.05).ConclusionMNA-DPD injection via lumbar cistern can effectively relieve DCVS in rabbits with magnetic field targeting.

MNA-DPD; Delayed cerebral vasospasm; Subarachnoid hemorrhage

1671-2897(2017)16-293-04

R 743.35

A

國(guó)家自然科學(xué)基金資助項(xiàng)目(81171116)

汪子文，碩士，住院醫(yī)師，E-mail: 492047008@qq.com

*通訊作者：于耀宇，副教授、主任醫(yī)師，E-mail: yuyaoyu666@aliyun.com

2016-08-30；

2016-12-25)