郭　琦，劉　燦，?！?，馬　騰，王　紅，宋純理，徐迎勝

(1.北京大學(xué)第三醫(yī)院神經(jīng)內(nèi)科, 北京　100191；2.北京大學(xué)第三醫(yī)院骨科, 北京　100191)

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殼聚糖導(dǎo)管復(fù)合辛伐他汀/泊洛沙姆407水凝膠修復(fù)大鼠外周神經(jīng)缺損的研究

郭琦1，劉燦2，海寶2，馬騰2，王紅2，宋純理2，徐迎勝1

(1.北京大學(xué)第三醫(yī)院神經(jīng)內(nèi)科, 北京100191；2.北京大學(xué)第三醫(yī)院骨科, 北京100191)

目的探討殼聚糖導(dǎo)管復(fù)合辛伐他汀/泊洛沙姆407水凝膠作為無細(xì)胞人工神經(jīng)支架修復(fù)坐骨神經(jīng)缺損的可行性。方法制備殼聚糖導(dǎo)管和辛伐他汀/泊洛沙姆407水凝膠, 通過體視顯微鏡、掃描電鏡、體外降解實驗和流變學(xué)檢測來觀察復(fù)合材料的性能。選取SPF級SD大鼠40只, 分為單純導(dǎo)管組、導(dǎo)管復(fù)合辛伐他汀0 mg組、導(dǎo)管復(fù)合辛伐他汀0.5 mg組, 和導(dǎo)管復(fù)合辛伐他汀1 mg組共4組其中前2組為對照組，后2組為辛伐他汀治療組, 每組10只, 造成左側(cè)坐骨神經(jīng)10 mm缺損模型, 用殼聚糖導(dǎo)管橋接缺損, 其內(nèi)填充不同濃度的辛伐他汀水凝膠。移植后10周, 取再生神經(jīng)的中間段進(jìn)行HE染色、透射電鏡觀察再生神經(jīng)的形態(tài)學(xué)改變, 并對再生神經(jīng)的軸突數(shù)量、髓鞘厚度、G-ratio等進(jìn)行統(tǒng)計學(xué)分析；免疫組化觀察再生神經(jīng)中NF200和S100蛋白的表達(dá)和神經(jīng)營養(yǎng)因子PTN、HGF、GDNF和VEGF的表達(dá)。結(jié)果殼聚糖導(dǎo)管和辛伐他汀/泊洛沙姆407水凝膠是適合神經(jīng)缺損的無細(xì)胞修復(fù)材料。植入10周后四組均可見再生神經(jīng), 但HE染色顯示辛伐他汀治療組的神經(jīng)干明顯較對照組粗；透射電鏡顯示辛伐他汀治療組的再生軸突數(shù)量顯著增多, 髓鞘顯著增厚, G-ratio顯示髓鞘化程度亦明顯好于對照組；免疫組化顯示辛伐他汀治療組再生神經(jīng)中標(biāo)記軸突的NF200和標(biāo)記雪旺細(xì)胞的S100的陽性表達(dá)明顯增強(qiáng), 且內(nèi)源性神經(jīng)營養(yǎng)因子PTN、HGF、VEGF和GDNF呈現(xiàn)高表達(dá)。結(jié)論殼聚糖導(dǎo)管復(fù)合辛伐他汀/泊洛沙姆407水凝膠明顯促進(jìn)神經(jīng)缺損組織學(xué)的重建, 可用于修復(fù)坐骨神經(jīng)缺損。

外周神經(jīng)損傷；辛伐他??；殼聚糖導(dǎo)管；泊洛沙姆407水凝膠；外周神經(jīng)再生

外周神經(jīng)缺損一直以來對于基礎(chǔ)和臨床學(xué)者來說都是嚴(yán)格的挑戰(zhàn)[1]。短距離缺損通過端端吻合即可達(dá)到治愈的目的[2], 而對于長距離神經(jīng)缺損, 自體神經(jīng)移植雖然被認(rèn)為是“金標(biāo)準(zhǔn)”, 卻也面臨著一系列的困擾[3]?，F(xiàn)今, 人工神經(jīng)導(dǎo)管復(fù)合神經(jīng)營養(yǎng)因子被認(rèn)為是自體神經(jīng)的理想的替代物[4-5]。然而, 單純應(yīng)用一種外源性神經(jīng)營養(yǎng)因子不足以支撐外周神經(jīng)再生的全過程；而且, 外源性神經(jīng)營養(yǎng)因子大部分都是蛋白質(zhì), 存在半衰期短和免疫排斥反應(yīng)[5]。因此, 利用某種可以誘導(dǎo)內(nèi)源性神經(jīng)營養(yǎng)因子分泌的小分子化合物能夠顯著提高促進(jìn)外周神經(jīng)再生的效果。

他汀類藥物作為降脂藥, 由于它的抗炎、抗氧化、調(diào)節(jié)免疫和神經(jīng)保護(hù)作用而被應(yīng)用在神經(jīng)系統(tǒng)疾病[6-8]。我們先前的實驗結(jié)果表明辛伐他汀能夠顯著促進(jìn)脊髓損傷大鼠神經(jīng)功能的恢復(fù), 上調(diào)腦源性神經(jīng)營養(yǎng)因子(brain derived neurotrophic factor, BDNF)、神經(jīng)膠質(zhì)細(xì)胞源性的神經(jīng)營養(yǎng)因子(glial cell line-derived neurotrophic factor, GDNF)表達(dá)[9-10]。但辛伐他汀作為降脂藥, 其靶器官是肝臟, 口服后由于肝臟的首過效應(yīng), 口服他汀只有不足5%能夠進(jìn)入血液循環(huán)[11], 因此我們期待利用一種緩釋他汀, 提高其生物利用度的載體可以顯著提高受損神經(jīng)再生的效果。

目前, 關(guān)于局部應(yīng)用辛伐他汀對于外周神經(jīng)再生的效果仍無報道。我們研究的目的是探索一種辛伐他汀/泊洛沙姆407溫敏型水凝膠復(fù)合殼聚糖導(dǎo)管促進(jìn)坐骨神經(jīng)缺損10 mm大鼠的外周神經(jīng)再生的效果。從形態(tài)學(xué)角度觀察橋接后缺損神經(jīng)的再生修復(fù)情況, 為研究神經(jīng)缺損的橋接材料提供形態(tài)學(xué)基礎(chǔ)。

1　材料和方法

1.1材料

1.1.1實驗動物及飼養(yǎng)環(huán)境： SPF級SD大鼠, 40只, 雄性, 6～7周齡, 體重250～300 g。均購自于北京維通利華實驗動物技術(shù)有限公司【SCXK(京)2012-0001】。飼養(yǎng)于北京大學(xué)醫(yī)學(xué)部實驗動物科學(xué)部【SYXK(京)2011-0039】。按照實驗動物適用的“3R”原則給與人道關(guān)懷。實驗前適應(yīng)性飼養(yǎng)1周, 自由攝取食水。

1.1.2藥品與試劑：辛伐他汀原料藥(中國藥品生物制品檢定所)；殼聚糖(中國浙江金殼藥業(yè))；泊洛沙姆407(德國BASF公司)；一抗NF200和S100(英國Abcam公司)；一抗PTN、HGF、VEGF和GDNF(北京博奧森生物技術(shù)有限公司)；一步法聚合物檢測系統(tǒng)、DAB顯色試劑盒(北京中杉金橋生物技術(shù)有限公司)。

1.1.3主要儀器：體視顯微鏡(德國Leica公司)；光學(xué)顯微鏡(日本Nikon公司)；掃描電子顯微鏡(日本JEOL公司)；透射電子顯微鏡(日本JEOL公司)；流變儀(奧利地Anton Paar公司)。

1.2方法

1.2.1殼聚糖導(dǎo)管和辛伐他汀/泊洛沙姆407水凝膠的制備與特性檢測

殼聚糖導(dǎo)管的制備參考文獻(xiàn)[12], 略微改良。殼聚糖粉末溶解在冰醋酸中達(dá)到8%的濃度。外徑為1.6 mm的玻璃毛細(xì)管豎直插入殼聚糖溶液中, 豎直拔出, 再浸入5%的NaOH溶液中30 min, 雙蒸水反復(fù)沖洗直至pH值為中性。最后, 拔出玻璃毛細(xì)管留下殼聚糖導(dǎo)管, 切成14 mm長度的節(jié)段備用。

泊洛沙姆407 粉末溶解在雙蒸水中達(dá)到25%的濃度, 添加辛伐他汀至三種劑量, 分別為每20 μL混懸液中含有0 mg, 0.5 mg和1 mg辛伐他汀。水凝膠的流變學(xué)檢測利用流變儀, 設(shè)置參數(shù)為頻率為1 Hz, 幅度為0.1%，彈性模量(G′)和黏性模量(G″)的溫度范圍為4℃～45℃, 以0.5℃/min的速率加溫。

殼聚糖導(dǎo)管填充辛伐他汀/泊洛沙姆407溫敏型水凝膠使用體視顯微鏡觀察宏觀形態(tài)。另外, 取一片殼聚糖膜和水凝膠凍干、噴金、掃描電鏡觀察微觀結(jié)構(gòu)。

1.2.2辛伐他汀/泊洛沙姆407水凝膠的體外降解實驗

取2 mL辛伐他汀/泊洛沙姆407溫敏型水凝膠置于4.0 mL離心管內(nèi)并在37℃恒溫箱中孵育呈凝膠狀。再向離心管內(nèi)添加2 mL PBS。每24 h換一次緩沖液。在第0, 3, 5, 7, 10和14天拍圖觀察水凝膠的降解情況。

1.2.3外周神經(jīng)損傷模型制作

大鼠麻醉后, 消毒備皮, 于左側(cè)大腿外側(cè)切開皮膚, 皮下, 沿肌間隙分離暴露坐骨神經(jīng), 于梨狀肌下孔5 mm處完全橫斷坐骨神經(jīng), 造成10 mm缺口, 兩端用9-0縫線縫合殼聚糖導(dǎo)管, 根據(jù)實驗分組填充不同劑量的辛伐他汀水凝膠。傷口處灑青霉素粉末以防感染, 分層縫合肌肉與皮膚, 術(shù)后分籠飼養(yǎng), 每籠3只, 術(shù)后觀察大鼠精神狀態(tài)、術(shù)側(cè)肢體活動、飲食、傷口愈合情況等。手術(shù)操作過程見圖1。

1.2.4動物分組及給藥

標(biāo)準(zhǔn)分籠飼養(yǎng)的SD大鼠40只, SPF級, 雄性, 6～7周齡, 體重250～300 g, 由北京大學(xué)醫(yī)學(xué)部實驗動物科學(xué)部提供, 采用隨機(jī)數(shù)字表法將40只SD大鼠隨機(jī)分為A、B、C、D共 4組, 每組10只：A組：殼聚糖導(dǎo)管組(conduit)；B組：殼聚糖導(dǎo)管復(fù)合辛伐他汀水凝膠0 mg(c+sim 0 mg)；C組：殼聚糖導(dǎo)管復(fù)合辛伐他汀水凝膠0.5 mg(c+sim 0.5 mg)；D組：殼聚糖導(dǎo)管復(fù)合辛伐他汀水凝膠1 mg(c+sim 1 mg)。

1.2.5組織學(xué)觀察

術(shù)后10周進(jìn)行組織學(xué)觀察。取再生坐骨神經(jīng)置于10%福爾馬林固定, 脫水, 石蠟包埋。取再生神經(jīng)最中間段切片(5 μm)行HE染色, 光鏡下觀察。

1.2.6透射電鏡觀察

術(shù)后10周進(jìn)行透射電鏡觀察。取再生坐骨神經(jīng)置于2.5%戊二醛中固定, PBS沖洗, 鋨酸固定, 脫水, 樹脂包埋。半薄切片(100 nm)行甲苯胺藍(lán)染色, 超薄切片(50 nm)行鈾酸-檸檬酸鉛染色。透射電鏡下觀察, 計量髓鞘化軸突的直徑、髓鞘的厚度和G-ratio(軸突直徑與神經(jīng)纖維總直徑的比值)，并進(jìn)行統(tǒng)計分析。

1.2.7免疫組織化學(xué)觀察

術(shù)后10周進(jìn)行免疫組織化學(xué)觀察。取再生坐骨神經(jīng)置于10%福爾馬林固定, 脫水, 石蠟包埋, 切片(5 μm)孵育一抗NF200和S100, 分別標(biāo)記軸突和雪旺細(xì)胞, 4℃過夜, PBS水洗, 滴加山羊抗IgG/HRP聚合物, 室溫30 min, DAB顯色, 光鏡下觀察。以上一抗?jié)舛染鶠?∶200。

1.2.8神經(jīng)營養(yǎng)因子的免疫組織化學(xué)觀察

術(shù)后10周進(jìn)行神經(jīng)營養(yǎng)因子免疫組織化學(xué)觀察。取再生坐骨神經(jīng)置于10%福爾馬林固定, 脫水, 石蠟包埋, 切片(5 μm),孵育一抗多效生長因子(pleiotrophin, PTN)、肝細(xì)胞生長因子(hepatocyte growth factor, HGF)、血管內(nèi)皮生長因子(vascular endothelial growth factor, VEGF)和膠質(zhì)細(xì)胞源性神經(jīng)營養(yǎng)因子(glial cell line derived neurotrophic factor, GDNF), 4℃過夜, PBS水洗, 滴加山羊抗IgG/HRP聚合物, 室溫30 min, DAB顯色, 光鏡下觀察。以上一抗?jié)舛染鶠?∶200。

1.3統(tǒng)計學(xué)分析

2　結(jié)果

2.1實驗動物數(shù)量分析

參加實驗40只SD大鼠均進(jìn)入結(jié)果分析。術(shù)后各組大鼠精神良好, 均成活, 進(jìn)食及飲水正常, 營養(yǎng)狀況良好, 切口愈合良好、無感染。

2.2殼聚糖導(dǎo)管的特性觀察

如圖2所示, 殼聚糖導(dǎo)管呈現(xiàn)為半透明狀, 直徑為1.6 mm, 長度為14 mm。通過掃描電子顯微鏡可以觀察到良好的微觀結(jié)構(gòu)。

2.3辛伐他汀/泊洛沙姆407水凝膠的特性觀察

如圖3，體外降解實驗顯示水凝膠在14d內(nèi)幾乎降解完全。且通過掃描電子顯微鏡可觀察到水凝膠的多孔狀微觀結(jié)構(gòu)。圖4可見辛伐他汀/泊洛沙姆407水凝膠呈現(xiàn)出溫敏性, 即在4℃呈現(xiàn)為液相, 37℃轉(zhuǎn)為固相。

注：A：暴露手術(shù)野；B：橫斷坐骨神經(jīng)；C：橋接殼聚糖導(dǎo)管；D：注射水凝膠。圖1　手術(shù)操作圖Note. A: Exposure of surgical site; B: Transection of sciatic nerve; C: Grafting of chitosan conduit; D: Injection of hydrogel.Fig.1　Pictures of surgical procedure

注：A：殼聚糖導(dǎo)管的大體結(jié)構(gòu)觀察；B：殼聚糖導(dǎo)管的掃描電鏡觀察。圖2　殼聚糖導(dǎo)管特性Note：A: The gross structure of the chitosan conduits; B: The SEM of the chitosan conduits.Fig.2　Characteristics of the chitosan conduits

注：A：水凝膠在體外不同時間的降解情況； B：水凝膠在掃描電鏡下觀察到的多孔狀結(jié)構(gòu)。圖3　辛伐他汀/泊洛沙姆水凝膠特性的觀察Note：A: Degradation of the simvastatin/pluronic F-127 hydrogel at 37℃ on days 0, 3, 5, 7, 10, and 14 in vitro; B: SEM of the simvastatin/pluronic F-127 hydrogel.Fig.3　Characteristics of the simvastatin/pluronic F-127 hydrogel

2.4再生神經(jīng)在體觀察

如圖5所示, 再生神經(jīng)在體觀察可發(fā)現(xiàn)填充辛伐他汀/泊洛沙姆407水凝膠組的再生坐骨神經(jīng)顯著粗于對照組, 表明其再生情況明顯優(yōu)于對照組。

注：在彈性模量(G′)和黏性模量(G″)相交接的溫度下, 實現(xiàn)液相-固相的相轉(zhuǎn)變。圖4　辛伐他汀/泊洛沙姆水凝膠流變學(xué)檢測結(jié)果Note：It underwent a phase transition from solution to gel at the temperature where the storage modulus (G′) and loss modulus (G″) cross.Fig.4　The rheological properties of the simvastatin/pluronic F-127 hydrogel

2.5再生神經(jīng)的組織學(xué)和免疫組化

如圖6所示, HE染色表明辛伐他汀治療組的再生神經(jīng)明顯粗于對照組, 且鏡下微觀結(jié)構(gòu)顯示再生神經(jīng)纖維多且直徑較粗大, 排列更為規(guī)則, 較接近正常坐骨神經(jīng)。

2.6再生神經(jīng)的組織學(xué)和免疫組化

如圖7所示, 辛伐他汀治療組中標(biāo)記再生軸突的NF(神經(jīng)絲)陽性細(xì)胞數(shù)和標(biāo)記雪旺細(xì)胞的S100陽性細(xì)胞數(shù)顯著多于對照組。

2.7透射電鏡觀察

圖8所示為再生神經(jīng)中段的甲苯胺藍(lán)和醋酸鈾-檸檬酸鉛雙染色結(jié)果。如圖9所示, 經(jīng)統(tǒng)計學(xué)分析，辛伐他汀1 mg組的髓鞘化軸突數(shù)目最多, 而導(dǎo)管組數(shù)目最少。相同的趨勢可在髓鞘化軸突的直徑和髓鞘厚度中可見。并且, 辛伐他汀治療組中再生神經(jīng)的G-ratio值(軸突直徑與纖維總直徑比值)顯著低于對照組，表明其髓鞘化程度更好。

2.8內(nèi)源性營養(yǎng)因子的表達(dá)情況

如圖10所示,免疫組化表明辛伐他汀誘導(dǎo)內(nèi)源性神經(jīng)營養(yǎng)因子包括PTN、HGF、VEGF和GDNF的高表達(dá)。

注：辛伐他汀組再生的坐骨神經(jīng)較對照組明顯增粗。圖5　每組于術(shù)后10周再生神經(jīng)的大體情況Note：The regenerated sciatic nerves in the rats whose defects were bridged by chitosan conduits filled with simvastatin/pluronic F-127 hydrogel were much thicker than that without simvastatin.Fig.5　Gross views of the regenerated sciatic nerves 10 weeks after surgery

圖6　再生坐骨神經(jīng)中段HE染色觀察Fig.6　The HE staining of the regenerated sciatic nerve

注：A：NF標(biāo)記再生軸突；B：S100標(biāo)記雪旺細(xì)胞。圖7　免疫組化染色Note： A: NF to lable the regenerated axons; B: S-100 to lable the Schwann cells.Fig.7　The immunohistochemical staining of the regenerated nerves

圖8　再生神經(jīng)軸突和髓鞘的透射電鏡觀察Fig.8　Observation of the regenerated axons and myelin sheaths by TEM

注：A：再生軸突直徑的統(tǒng)計學(xué)分析；B：髓鞘厚度的統(tǒng)計學(xué)分析；C：髓鞘化程度(G-ratio)的統(tǒng)計學(xué)分析。圖9　再生坐骨神經(jīng)中段的透射電鏡觀察Note：A: Quantifications of axonal diameter; B: Quantifications of the thickness of the myelin sheaths; C: Quantifications of the G-ratios of the myelinated nerve fibers. Fig.9　Observation of the regenerated nerves by TEM

組別Group軸突直徑(μm)Axonaldiameter髓鞘厚度(μm)Thicknessofthemyelinsheaths髓鞘化程度G-ratio正常組normal3.72±1.181.59±0.680.55±0.10殼聚糖導(dǎo)管組conduit1.67±0.500.39±0.160.68±0.10辛伐他汀0mg組c+sim0mg1.88±0.660.48±0.300.67±0.11辛伐他汀0.5mg組c+sim0.5mg2.17±0.68**0.64±0.29**0.63±0.11*/**辛伐他汀1mg組c+sim1mg2.70±0.89**0.75±0.25**0.64±0.08**

注：對于軸突直徑, 髓鞘厚度：與導(dǎo)管組和辛伐他汀0mg組比較,*P<0.05,**P<0.01；對于G-ratio：與導(dǎo)管組比較,*P<0.05,**P<0.01。

Note: For the axonal diameter and thickness of the myelin sheaths : Compared with the groups of conduit and c+sim 0mg,*P<0.05,**P<0.01; For G-ratio: Compared with the groups of conduit,*P<0.05,**P<0.01.

注：辛伐他汀治療組的局部神經(jīng)營養(yǎng)因子表達(dá)明顯增高。圖10　內(nèi)源性營養(yǎng)因子PTN、HGF、VEGF和GDNF的免疫組化結(jié)果Note：Simvastatin dramatically induces the secretion of PTN, HGF, VEGF and GDNF.Fig.10　The immunohistochemical staining for PTN, HGF, VEGF and GDNF

3　討論

尋找誘導(dǎo)性促進(jìn)神經(jīng)再生生物材料一直是神經(jīng)修復(fù)領(lǐng)域研究的熱點。有研究發(fā)現(xiàn), 殼聚糖降解產(chǎn)物可以促進(jìn)雪旺細(xì)胞增殖和神經(jīng)再生[13], 水凝膠作為可生物降解、良好生物相容性的高分子材料可作為緩釋載體[14-16], 并可填充殼聚糖空導(dǎo)管, 形成軸突再生的有效支架。

本研究中, 我們觀察到了殼聚糖導(dǎo)管中填充辛伐他汀/泊洛沙姆407水凝膠可以顯著促進(jìn)大鼠缺損10 mm的坐骨神經(jīng)再生, 在組織學(xué)上能夠明顯促進(jìn)神經(jīng)再生的結(jié)構(gòu)的修復(fù)。并且, 在辛伐他汀治療組中與神經(jīng)再生相關(guān)的內(nèi)源性神經(jīng)營養(yǎng)因子的分泌包括 PTN、HGF、VEGF 和GDNF也顯著增加。

有研究發(fā)現(xiàn), 短期口服大劑量阿托伐他汀可以預(yù)防坐骨神經(jīng)損傷, 促進(jìn)睫狀神經(jīng)營養(yǎng)因子(ciliary neurotrophic factor, CNTF)高表達(dá)[6]；腹腔注射給予辛伐他汀(20 mg/kg/d)能夠促進(jìn)大鼠坐骨神經(jīng)挫傷的恢復(fù)[17]。相似的, 也有報道稱連續(xù)14 d腹腔注射阿托伐他汀(5 mg/kg/d)有助于大鼠坐骨神經(jīng)完全橫斷后的神經(jīng)-肌肉再支配的重建[18]。與此相對, 另有報道稱口服給予低劑量辛伐他汀(2 mg/kg/d)連續(xù)14 d會延緩神經(jīng)纖維的再生, 但對電生理檢測并無影響[19]。這相反的結(jié)果在某種程度上與他汀在肝臟的首過效應(yīng)有關(guān), 口服給與的他汀只有少于5%能夠到達(dá)系統(tǒng)循環(huán)中[11], 而高劑量的辛伐他汀又會引起一系列的副作用[20]。因此, 局部應(yīng)用辛伐他汀既可以避免副作用, 又可以提高局部辛伐他汀的生物利用度從而有效地促進(jìn)神經(jīng)再生。本實驗的數(shù)據(jù)表明辛伐他汀對于神經(jīng)再生是很有前景的生物小分子化合物。

大量的神經(jīng)營養(yǎng)因子在中樞或外周神經(jīng)損傷中均有促進(jìn)神經(jīng)再生的作用[4-5]。人工神經(jīng)通過緩釋神經(jīng)營養(yǎng)因子作為神經(jīng)修復(fù)材料取得一定的效果, 但外源性神經(jīng)營養(yǎng)因子由于其價格昂貴、半衰期短、免疫源性、制備緩釋載體困難及儲備、存儲不便等因素限制了臨床的應(yīng)用；并且單一的神經(jīng)營養(yǎng)因子難以滿足神經(jīng)修復(fù)再生的全過程。國內(nèi)羅卓荊課題組[21]通過c-Jun基因修飾雪旺細(xì)胞, 可以促進(jìn)多種神經(jīng)營養(yǎng)因子如GDNF, BDNF, LIF等分泌。 同基因修飾等遺傳學(xué)方法及通過種子細(xì)胞和外源性神經(jīng)營養(yǎng)因子等組織工程學(xué)方法相比, 采用辛伐他汀這一小分子化合物的局部緩釋促進(jìn)內(nèi)源性神經(jīng)營養(yǎng)因子的表達(dá), 促進(jìn)周圍神經(jīng)再生, 無疑具有重要的研究價值。

本研究中, 采用的辛伐他汀劑量為0.5、1 mg, 均顯示可顯著促進(jìn)大鼠周圍神經(jīng)損傷修復(fù), 但兩者(0.5 mg vs. 1 mg)之間沒有顯著性差異, 結(jié)合泊洛沙姆水凝膠的體外降解實驗結(jié)果, 2周內(nèi)即基本完全降解, 提示更小劑量的辛伐他汀即能促進(jìn)神經(jīng)再生；同時延長緩釋時間或?qū)⒏玫匕l(fā)揮促進(jìn)神經(jīng)再生的作用。

本研究中, 我們首次報道辛伐他汀顯著增加大鼠外周神經(jīng)中PTN的分泌、PTN一直以來被認(rèn)為在體內(nèi)和體外實驗中具有促進(jìn)軸突再生的作用[22]。HGF被報道在大鼠坐骨神經(jīng)損傷[4]、面神經(jīng)損傷[23]和視神經(jīng)損傷[24]后均有促進(jìn)神經(jīng)再生和功能恢復(fù)的效果。最近, 更多的研究表明VEGF也有神經(jīng)營養(yǎng)和神經(jīng)保護(hù)的作用[25], 加速神經(jīng)再生和促進(jìn)神經(jīng)功能的恢復(fù)[26-27]。以上這些發(fā)現(xiàn)均表明局部應(yīng)用辛伐他汀通過促進(jìn)內(nèi)源性神經(jīng)營養(yǎng)因子的分泌來促進(jìn)缺損坐骨神經(jīng)的再生。

綜上, 我們的結(jié)果表明局部應(yīng)用辛伐他汀通過增加內(nèi)源性PTN、HGF、VEGF和GDNF的分泌, 從而促進(jìn)坐骨神經(jīng)缺損大鼠模型的神經(jīng)再生。

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Bridging sciatic nerve defect with simvastatin delivered by injectable thermosensitive pluronic F-127 hydrogel repairs peripheral nerve defect in rats

GUO Qi1, LIU Can2, HAI Bao2, MA Teng2, WANG Hong2, SONG Chun-li2, XU Ying-sheng1

(1. Department of Neurology, Peking University Third Hospital, Beijing 100191, China；2. Department of Orthopedics, Peking University Third Hospital, Beijing 100191, China)

ObjectiveTo explore the feasibility of combining chitosan conduit filled with simvastatin/ pluronic F-127 thermosensitive hydrogel to repair peripheral nerve defects in rats. Methods The chitosan conduits and simvastatin/ pluronic F-127 hydrogel were prepared and tested by stereomicroscope, SEM, vitro degradation testing and rheometer property. A total of 40 Sprague-Dawley rats were randomly divided into 4 groups (n=10 per group): the chitosan conduit alone (conduit group) or the conduit filled with pluronic F-127 hydrogel containing 0 (c+sim 0mg), 0.5 (c+sim 0.5mg), and 1 mg (c+sim 1mg) simvastatin. A 10mm nerve defect was created in the rat left sciatic nerve and bridged with the chitosan conduit filled with pluronic F-127 hydrogel containing different doses of simvastatin. At 10 weeks after surgery, the regenerated nerves were performed the H&E and TEM to observe the morphological change. The diameters of the myelinated axons, the thickness of the myelin sheath, and the G-ratios (the ratio of the axon diameter to the total fiber diameter) were calculated to make statistical analyses. And the immunohistochemical method was used to measure the expression of NF200 and S100 protein in the regenerated nerves and to detect the expression of neurotrophins including pleiotrophin (PTN), hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF) and glial cell line-derived neurotrophic factor (GDNF). Results The chitosan conduits filled with simvastatin/ pluronic F-127 promote nerve regeneration in rats. All groups showed the regenerated nerves, but H&E staining showed that the regenerated nerves in the simvastatin treated groups were much thicker than those without simvastatin; And the same trend was also found for the diameter of the myelinated axons, the thickness of the myelin sheath and G-ratio; In addition, the numbers of NF-positive cells indicating regenerated axons and S100-positive cells indicating Schwann cells were significantly larger in simvastatin treated groups than those without simvastatin. And immunohistochemical staining indicated that simvastatin induced an increased expression of PTN, HGF, VEGF and GDNF compared to those found in the control group. Conclusions Taken together, the simvastatin/ pluronic F-127 hydrogel filling in the chitosan conduits promoted the reconstruction of histological in peripheral nerve defects, which can be used for peripheral nerve regeneration.

Peripheral nerve injury; Simvastatin; Chitosan conduit; Pluronic F-127 hydrogel; Peripheral nerve regeneration

國家自然科學(xué)基金(81171693；81100895)；國家高技術(shù)研究發(fā)展計劃“863計劃”(SS2015AA020304)。

郭琦(1989-)，女，碩士生，研究方向：神經(jīng)損傷、修復(fù)與重建。

徐迎勝(1971-)女，主任醫(yī)師，副教授。 E-mail：xys@bjmu.edu.cn。

研究報告

R-332

A

1671-7856(2016) 05-0001-09

10.3969.j.issn.1671-7856. 2016.005.001

2016-03-28