PHBHHx膜表面親水改性及其與神經(jīng)干細(xì)胞生物相容性

呂海俠，楊志倩，盧曉云，李明川，焦倩，陳新林，王媛媛，張雅利

1 西安交通大學(xué)醫(yī)學(xué)院神經(jīng)生物學(xué)研究所，陜西 西安 710061

2 西安交通大學(xué)生命科學(xué)與技術(shù)學(xué)院 生物醫(yī)學(xué)信息工程教育部重點(diǎn)實(shí)驗(yàn)室，陜西 西安 710049

組織工程與細(xì)胞培養(yǎng)

PHBHHx膜表面親水改性及其與神經(jīng)干細(xì)胞生物相容性

呂海俠1，楊志倩2，盧曉云2，李明川2，焦倩1，陳新林1，王媛媛1，張雅利2

1 西安交通大學(xué)醫(yī)學(xué)院神經(jīng)生物學(xué)研究所，陜西 西安 710061

2 西安交通大學(xué)生命科學(xué)與技術(shù)學(xué)院 生物醫(yī)學(xué)信息工程教育部重點(diǎn)實(shí)驗(yàn)室，陜西 西安 710049

對(duì)羥基丁酸-羥基己酸共聚酯 (PHBHHx) 膜進(jìn)行表面改性，研究神經(jīng)干細(xì)胞 (NSCs) 在改性后的PHBHHx膜表面的貼附、增殖及分化情況，為開(kāi)發(fā)新型腦組織工程支架材料奠定基礎(chǔ)。采用溶劑揮發(fā)法制備PHBHHx膜，掃描電鏡觀察其表面性狀；分別通過(guò)脂肪酶處理，NaOH處理的方法對(duì)PHBHHx膜進(jìn)行表面改性，測(cè)量接觸角以檢測(cè)膜表面親水性。分離培養(yǎng)孕 14.5 d大鼠胚胎大腦皮質(zhì) NSCs，接種在表面改性后的PHBHHx膜表面進(jìn)行體外培養(yǎng)，掃描電鏡觀察膜表面細(xì)胞形態(tài)，MTT法檢測(cè)細(xì)胞活力，免疫細(xì)胞化學(xué)染色觀察NSCs存活和分化情況。結(jié)果顯示，與未處理的PHBHHx膜相比，脂肪酶、NaOH處理能夠顯著提高PHBHHx膜表面親水性，增加NSCs在PHBHHx膜表面貼附數(shù)量；NSCs在改性后的PHBHHx膜表面能夠良好地存活并分化為神經(jīng)元和膠質(zhì)細(xì)胞。結(jié)果提示PHBHHx膜表面堿處理通過(guò)提高材料表面親水性和粗糙程度，增加其與NSCs的生物相容性，改性后的PHBHHx材料是一種非常有潛力的新型腦組織工程支架材料，有望在NSCs移植修復(fù)腦損傷中發(fā)揮作用。

羥基丁酸-羥基己酸共聚酯，神經(jīng)干細(xì)胞，表面改性，生物相容性

Abstract:To study the attachment, proliferation and differenciation of neural stem cells (NSCs) on surface modified PHBHHx films and to establish the theory of PHBHHx application in NSCs-based brain tissue engineering. PHBHHx film was fabricated by a solution-casting method, and the morphology of the fi lm was observed under scanning electron microscopy(SEM). The films were treated by NaOH or lipase, then the surface hydrophilic property was characterized using water contact angle measurement. NSCs were isolated from the cerebral cortex of rat embryos on embryonic day 14.5, and cultured on surface treated PHBHHx films. The morphology of NSCs attached on the film was visualized under SEM, and the survival and differentiation of NSCs were observed through immunocytochemical staining. Compared with the untreated PHBHHx films, the water contact angle of NaOH or lipase treated PHBHHx films decreased dramatically, and the number of NSCs attached significantly increased. NSCs survived well on treated PHBHHx films and differentiated into neurons and glial cells. The amelioration of hydrophilic property of PHBHHx film improved its biocompatibility with NSCs. PHBHHx can serve as a novel CNS tissue engineering biomaterial applied for NSCs transplantation, brain repairing and regeneration.

Keywords:PHBHHx, neural stem cells, surface treatment, biocompatibility

神經(jīng)干細(xì)胞 (NSCs) 是具有自我更新和多向分化潛能的細(xì)胞，它能不斷增殖，并分化為神經(jīng)系統(tǒng)主要的細(xì)胞類(lèi)型 (神經(jīng)元、星形膠質(zhì)細(xì)胞和少突膠質(zhì)細(xì)胞)[1]。NSCs移植已成為目前促進(jìn)神經(jīng)損傷修復(fù)最有潛力的手段[2-4]。然而，單純的NSCs移植效果并不理想[5-7]。因此，有需要、有必要在深入了解NSCs特性以及對(duì)于腦組織的修復(fù)作用的同時(shí)，積極尋求能夠促進(jìn)NSCs在移植部位更好地存活并向損傷修復(fù)所需的細(xì)胞分化的策略。目前基于NSCs修飾[8-10]、NSCs體外定向誘導(dǎo)分化后移植[11]、混合細(xì)胞共移植[12]以及干細(xì)胞-生物材料共移植[13-14]等的一些嘗試取得了比較明顯的效果，在一定程度上提高了移植細(xì)胞在移植部位存活及分化能力。特別是隨著組織工程技術(shù)的快速發(fā)展，以生物可降解材料為支架進(jìn)行神經(jīng)細(xì)胞培養(yǎng)的腦組織工程及其在腦組織損傷修復(fù)和神經(jīng)系統(tǒng)重建中的應(yīng)用成為一新興的前沿領(lǐng)域[15-17]。新型腦組織工程支架材料的開(kāi)發(fā)及生物相容性研究成為這一領(lǐng)域的研究熱點(diǎn)之一。

聚羥基脂肪酸酯 (PHA) 是一類(lèi)可由微生物合成的具有良好生物相容性的天然生物可降解高分子材料[18-19]，羥基丁酸-羥基己酸共聚酯(poly (3-hydroxybutyrate-co-3-hydroxhexanoate)，PHBHHx) 是PHA家族中的一員[20-21]，在生物相容性、生物活性及機(jī)械性能方面的性能優(yōu)良，與多種細(xì)胞都具有良好的生物相容性[22-25]，是近年來(lái)在組織工程中被廣泛關(guān)注的新型生物材料[19,26]。PHBHHx作為外周神經(jīng)修復(fù)支架材料和NSCs體外培養(yǎng)支架材料的研究已有報(bào)道[27-28]。但由于PHBHHx的聚酯性質(zhì)，其表面疏水性強(qiáng)，阻礙了細(xì)胞在其表面的貼附生長(zhǎng)，因此也局限了其應(yīng)用范圍[29]。有學(xué)者通過(guò)采用紫外輻照、絲素蛋白修飾等方法對(duì) PHBHHx材料進(jìn)行改性處理[30-32]，提高了 PHBHHx材料與成纖維細(xì)胞、血管內(nèi)皮細(xì)胞等的生物相容性。本研究進(jìn)行了 PHBHHx膜材料表面改性條件的摸索，并研究了改性后PHBHHx膜與NSCs的生物相容性，為優(yōu)化基于 PHA材料的新型腦組織工程支架奠定實(shí)驗(yàn)基礎(chǔ)。

1 材料與方法

1.1 PHBHHx膜的制備及表面改性

1.1.1 PHBHHx膜的制備

溶劑揮發(fā)法制備PHBHHx膜[33]。15 mL氯仿溶解0.5 g PHBHHx，60 ℃水浴助溶1 h。通風(fēng)櫥中，將液體傾入9 cm玻璃培養(yǎng)皿中，室溫下使氯仿?lián)]發(fā)1～2 d，待膜形成后置于4 ℃老化2周。

1.1.2 PHBHHx膜的表面改性及處理

經(jīng)老化后的PHBHHx膜用于表面改性處理，各組的處理方式分別為：對(duì)照組 (Control) 不做任何處理；堿處理組 (A)，用2 mol/L NaOH室溫處理1 h[29]；酶處理組 (L)，脂肪酶 (L3126，sigma) 37 ℃處理 24 h[34]；酶+堿處理組 (A+L)，脂肪酶37 ℃處理24 h后2 mol/L NaOH室溫處理1 h；堿+酶處理組 (L+A)，2 mol/L NaOH室溫1 h后脂肪酶37 ℃處理24 h。

細(xì)胞接種之前，PHBHHx膜經(jīng) 75%乙醇浸泡2 h，紫外線照射正、反兩面各1 h進(jìn)行消毒。PBS浸泡過(guò)夜，將膜裁剪成合適大小，放入 24孔板內(nèi)備用。

1.2 NSCs的分離和培養(yǎng)

采用本實(shí)驗(yàn)室常規(guī)方法[35-36]分離孕14.5 d大鼠 (購(gòu)自西安交通大學(xué)實(shí)驗(yàn)動(dòng)物中心) 胚胎大腦皮質(zhì)，細(xì)胞接種密度為1×105～2×105個(gè)細(xì)胞/mL，以完全培養(yǎng)基進(jìn)行常規(guī)培養(yǎng)，每5 d傳代1次，第3～8代的NSCs用于實(shí)驗(yàn)。完全培養(yǎng)基成分：無(wú)血清的 DMDEM/F12 (1:1) 培養(yǎng)基添加N2 (100×，GIBCO，USA)、B27 (50×，GIBCO，USA)、20 μg/L hEGF (GIBCO，USA) 和 10 μg/L bFGF (Sigma，USA)。NSCs分化培養(yǎng)基成分：去除完全培養(yǎng)基中hEGF和bFGF，并添加1%的胎牛血清 (Fetal bovine serum，F(xiàn)BS)。

1.3 在PHBHHx膜培養(yǎng)NSCs及細(xì)胞活力測(cè)定

傳代時(shí)，將細(xì)胞重懸于分化培養(yǎng)基中，并以4×104個(gè)細(xì)胞/mL的密度接種于預(yù)先放入培養(yǎng)板中的PHBHHx膜，每孔加培養(yǎng)基500 μL，細(xì)胞培養(yǎng)箱內(nèi)常規(guī)培養(yǎng)7 d，每隔3 d半量換液1次。MTT法測(cè)定NSCs細(xì)胞活力。

1.4 免疫細(xì)胞化學(xué)染色

細(xì)胞在膜表面貼附生長(zhǎng)7 d以后，經(jīng)PBS洗滌3次，0.3% Triton室溫處理30 min，再用PBS洗滌3次后以5%的正常山羊血清室溫封閉1 h，與一定比例稀釋的一抗 (Nestin 1:200，β tubulinⅢ 1:200，GFAP 1:500) 室溫孵育2 h后4 ℃過(guò)夜，次日以PBS洗滌3次后與FITC標(biāo)記二抗于暗環(huán)境室溫孵育2 h，以DAPI 標(biāo)記細(xì)胞核 (室溫，5 min)，以50%的甘油緩沖液封片，熒光顯微鏡下觀察。

1.5 掃描電鏡樣品制備

細(xì)胞在膜表面貼附生長(zhǎng)7 d，經(jīng)PBS洗滌2次后用2.5%的戊二醛4 ℃固定；再經(jīng)0.1 mol/L磷酸緩沖液浸洗10 min后用1%四氧化鋨固定液4 ℃固定 2 h；再用 0.1 mol/L磷酸緩沖液浸洗10 min后用乙醇梯度脫水并經(jīng)乙酸異戊酯浸泡10 min；臨界點(diǎn)干燥；噴金后掃描電鏡 (TM-1000)下觀察拍照。

2 結(jié)果

2.1 表面改性處理增加PHBHHx膜親水性

分別對(duì)NaOH處理，脂肪酶處理以及NaOH聯(lián)合脂肪酶處理后 PHBHHx膜表面接觸角進(jìn)行測(cè)量，結(jié)果提示：各種處理方式處理后膜表面接觸角與未處理的對(duì)照組相比都有明顯降低(圖1，P＜0.01)，其中單純NaOH處理PHBHHx膜表面接觸角降低程度較單純脂肪酶處理組更為明顯 (P＜0.05)，與NaOH與脂肪酶聯(lián)合處理組間沒(méi)有顯著性差異 (P＞0.05)。

2.2 NSCs的培養(yǎng)與鑒定

由胚胎大鼠大腦皮質(zhì)分離的原代細(xì)胞在完全培養(yǎng)基中呈懸浮狀態(tài)生長(zhǎng)，培養(yǎng)3～5 d后可形成大小不等的神經(jīng)球 (圖 2A)，并隨培養(yǎng)時(shí)間延長(zhǎng)，神經(jīng)球直徑不斷增大；在含1% FBS的分化培養(yǎng)基連續(xù)培養(yǎng)7 d后，部分細(xì)胞仍然保持nestin陽(yáng)性的未分化NSCs，另有部分細(xì)胞分化為GFAP陽(yáng)性星形膠質(zhì)細(xì)胞和 β-tubulin Ⅲ陽(yáng)性的神經(jīng)元(圖 2B，C，D)。

2.3 PHBHHx膜表面改性對(duì) NSCs貼附的影響

24孔培養(yǎng)板中，將 NSCs接種在堿處理的PHBHHx膜表面，以分化培養(yǎng)基培養(yǎng)7 d，DAPI染色后進(jìn)行熒光顯微鏡觀察 (圖3)，結(jié)果顯示，與未處理組 (3A) 相比，堿處理顯著增加了NSCs在 PHBHHx膜表面的貼附生長(zhǎng) (3B)，掃描電鏡觀察膜表面細(xì)胞保持原有的神經(jīng)細(xì)胞形態(tài)特點(diǎn) (3C，D)。

圖1 表面改性對(duì)PHBHHx膜表面接觸角的影響Fig.1 Water contact angle after surface treatment. A: NaOH treatment; L: lipase treatment; A+L: NaOH treatment followed by lipase treatment; L+A: lipase treatmemt followed by NaOH treatment. *P<0.05; **P<0.01.

圖2 NSCs體外培養(yǎng)及鑒定Fig.2 Cultivation and identification of NSCsin vitro. (A) Neurosphers. (B) Nestin positive NSCs. (C) GFAP positive astrocytes. (D) β-tubulin Ⅲ positive neurons. Cell nucli stained with DAPI (blue). Nestin, GFAP and β-tubulin Ⅲwere in green. Scale bar=50 μm.

2.4 NSCs在改性 PHBHHx膜表面的增殖及分化情況

將單個(gè)NSCs接種于PHBHHx膜，并以經(jīng)多聚賴(lài)氨酸包被的蓋玻片為對(duì)照，分化培養(yǎng)基常規(guī)培養(yǎng)3 d后，MTT檢測(cè)PHBHHx膜表面貼附生長(zhǎng)的 NSCs活力，結(jié)果提示：在改性PHBHHx膜上生長(zhǎng)的 NSCs細(xì)胞活力明顯增強(qiáng)(圖 4)。

分化培養(yǎng)基中培養(yǎng)7 d，以免疫細(xì)胞化學(xué)染色法觀察 NSCs分化情況，結(jié)果顯示：細(xì)胞在PHBHHx膜表面發(fā)生多向分化 (圖5)，分化為星形膠質(zhì)細(xì)胞 (GFAP陽(yáng)性，A、B、C) 和神經(jīng)元 (btubulin Ⅲ陽(yáng)性細(xì)胞，D、E、F)。隨機(jī)選擇的視野中，顯微鏡下計(jì)數(shù)全部細(xì)胞、GFAP陽(yáng)性細(xì)胞、b tubulin Ⅲ陽(yáng)性和nestin陽(yáng)性細(xì)胞，計(jì)算分化細(xì)胞比例，結(jié)果顯示在改性的 PHBHHx膜表面生長(zhǎng)細(xì)胞約有30%發(fā)生分化，其中多數(shù)為b tubulinⅢ陽(yáng)性神經(jīng)元，而其余的 70%仍維持在未分化狀態(tài)；而在蓋玻片上培養(yǎng)的細(xì)胞則有約90%的細(xì)胞發(fā)生不同方向的分化 (圖6)，相對(duì)來(lái)講，分化為b tubulin Ⅲ陽(yáng)性神經(jīng)元和GFAP陽(yáng)性細(xì)胞膠質(zhì)細(xì)胞的比例 (1.37) 明顯低于其在PHBHHx膜上的分化 (2.14)。

圖3 NSCs在改性處理后的PHBHHx膜表面的貼附生長(zhǎng)情況Fig.3 Attachment and survival of NSCs on alkali-treated PHBHHx film. Scale bar in A and B =100 μm.

圖4 在改性的PHBHHx膜表面貼附生長(zhǎng)的NSCs活力Fig.4 NSCs viability on the surface of modified PHBHHx films.

圖5 NSCs在改性PHBHHx膜表面的分化情況Fig.5 Differentiation of NSCs on the surface of PHBHHx films. DAPI was in blue, nestin was in green, GFAP and β-tubulin Ⅲ were in red. Scale bar=50 μm.

3 討論

圖6 NSCs在改性后PHBHHx表面分化比例Fig.6 Differentiation of NSCs on PHBHHx films.

生物可降解聚合物材料是組織工程學(xué)材料研究中最活躍的領(lǐng)域[37]。PHA家族是近年來(lái)在組織工程中被廣泛關(guān)注的新型生物材料[19,26]，由于各種 PHA材料單體組成的不同，其機(jī)械性能和生物相容性也有很大差異。PHBHHx是 PHA家族中具有很好生物相容性的一員，由于其主鏈含有短鏈的 3-羥基丁酸單體和中長(zhǎng)鏈的 3-羥基己酸單體，其機(jī)械性能和可加工性能均優(yōu)于只含有短鏈單體的 PHA材料，如聚羥基丁酸酯(Polyhydroxybutyrate，PHB) 和羥基丁酸-羥基戊酸共聚酯 (poly(3-hydroxybutyrate-co-3-hydroxvalerate)，PHBV)。但由于PHBHHx的聚酯性質(zhì)，其表面疏水性仍比較強(qiáng)，不利于細(xì)胞的貼附[29]。Zhao等采用NaOH和脂肪酶分別處理PHB膜，結(jié)果顯示脂肪酶處理能夠顯著改善 PHB膜表面與成纖維細(xì)胞的生物相容性，NaOH處理對(duì)PHB生物相容性的改善不明顯[38-39]。本實(shí)驗(yàn)中以堿(NaOH) 和脂肪酶對(duì)PHBHHx膜表面進(jìn)行處理，結(jié)果顯示兩種處理方式都可以通過(guò)打斷 PHA材料主鏈中的酯鍵，增加被處理表面的羥基和羧基含量，從而提高 PHBHHx膜表面的親水性。掃描電鏡的結(jié)果提示NaOH處理后PHBHHx膜表面出現(xiàn)很多細(xì)小的微孔，膜表面粗糙程度增加，最終促進(jìn)了細(xì)胞在膜表面的貼附和生長(zhǎng)。其中單純的NaOH處理效果更為顯著，而且處理方法簡(jiǎn)單，成本低廉，耗時(shí)較短，因此不失為一種簡(jiǎn)便而有效的PHBHHx膜表面改性手段。

NSCs聯(lián)合生物材料移植治療神經(jīng)損傷已成為神經(jīng)損傷修復(fù)以及功能重建領(lǐng)域新的熱點(diǎn)。作為可用于促進(jìn)神經(jīng)損傷修復(fù)的生物材料，必須與其負(fù)載細(xì)胞NSCs有良好的相容性，既不產(chǎn)生細(xì)胞毒性，也不影響細(xì)胞功能。PHA家族中多個(gè)成員，包括PHB，PHBHHx和PHBVHHx均被嘗試用于中樞及外周神經(jīng)的支架材料[27-28,40-42]，其中 PHBHHx材料顯示出與神經(jīng)干細(xì)胞、神經(jīng)膠質(zhì)細(xì)胞的良好生物相容性[27]。最近，Xu等[27-28]的研究表明PHBHHx作為NSCs體外培養(yǎng)支架材料具有低細(xì)胞毒性、與NSCs有很好的相容性，并能夠誘導(dǎo)NSCs向神經(jīng)元方向分化。本研究結(jié)果提示，對(duì) PHBHHx膜表面的改性處理可以增加NSCs在其表面的貼附生長(zhǎng)，而且NSCs活力明顯高于玻璃片表面，細(xì)胞保持其原有的多向分化能力；在發(fā)生分化的細(xì)胞中以b tubulin Ⅲ陽(yáng)性的神經(jīng)元為主，這與Xu等報(bào)道的研究結(jié)果一致。除此之外，本研究還通過(guò)降低培養(yǎng)體系中的血清濃度，(由Xu等采用的 10%FBS降至1%)，能夠更好地保留 NSCs原有的增殖能力，使大約70%的細(xì)胞仍處于nestin陽(yáng)性的未分化狀態(tài)，這部分細(xì)胞能夠在體內(nèi)外進(jìn)一步增殖，一方面為獲得足量的移植細(xì)胞提供了保證，同時(shí)也為其在體內(nèi)分化成“神經(jīng)功能重建所需的特定細(xì)胞類(lèi)型”奠定了基礎(chǔ)。

本研究通過(guò)采用NaOH對(duì)PHBHHx膜進(jìn)行表面處理，增加了 PHBHHx膜表面親水性和與神經(jīng)干細(xì)胞的生物相容性，促進(jìn)NSCs的貼附生長(zhǎng)；在合適的培養(yǎng)條件下，NSCs在PHBHHx表面不僅可以很好的存活，而且能夠保持持久的增殖能力和多向分化能力。以上結(jié)果進(jìn)一步提示PHBHHx是一種非常有潛力的中樞神經(jīng)系統(tǒng)組織工程支架材料和神經(jīng)干細(xì)胞移植的細(xì)胞載體材料，在神經(jīng)損傷修復(fù)和功能重建方面有非常廣泛的應(yīng)用前景。

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Biocompatibility of surface modified PHBHHx with rat embryonic neural stem cells

Haixia Lü1, Zhiqian Yang2, Xiaoyun Lu2, Mingchuan Li2, Qian Jiao1, Xinlin Chen1,Yuanyuan Wang1, and Yali Zhang2

1Institute of Neurobiology,Xi’an Jiaotong University College of Medicine,Xi’an710061,Shaanxi,China
2Department of Biological Science and Bioengineering,Key Laboratory of Biomedical Information Engineering of Ministry of Education,School of Life Science and Technology,Xi’an Jiaotong University,Xi’an710049,Shaanxi,China

呂海俠, 楊志倩, 盧曉云, 等. PHBHHx膜表面親水改性及其與神經(jīng)干細(xì)胞生物相容性. 生物工程學(xué)報(bào), 2012, 28(10):1216?1226.

Lü HX, Yang ZQ, Lu XY, et al. Biocompatibility of surface modified PHBHHx with rat embryonic neural stem cells. Chin J Biotech, 2012, 28(10): 1216?1226.

Received:April 17, 2012;Accepted:June 6, 2012

Supported by:National Natural Science Foundation of China (Nos. 81070998, 31070943, 31101266), Fundamental Research Funds for the Central Universities.

Corresponding author:Xiaoyun Lu. Tel/Fax: +86-29-82668463-423; E-mail: luxy05@mail.xjtu.edu.cn

國(guó)家自然科學(xué)基金 (Nos. 81070998，31070943，31101266)，西安交通大學(xué)中央高?；究蒲袠I(yè)務(wù)費(fèi)專(zhuān)項(xiàng)資金資助。