趙鑫，王興文，寇江力，李忠浩，郭永強(qiáng)，伍亞民，張海鴻

·基礎(chǔ)研究·

沉默ski基因?qū)Υ笫笮切文z質(zhì)細(xì)胞遷移的影響

趙鑫1,2，王興文1,2，寇江力1,2，李忠浩1,2，郭永強(qiáng)1,2，伍亞民3，張海鴻1

目的研究ski基因在大鼠星形膠質(zhì)細(xì)胞遷移過程中的作用。方法分離大鼠腦皮質(zhì)星形膠質(zhì)細(xì)胞，體外培養(yǎng)。合成ski基因和陰性對(duì)照小干擾片段。設(shè)置ski-siRNA轉(zhuǎn)染組、陰性對(duì)照組和空白對(duì)照組。采用脂質(zhì)體法將特異性針對(duì)ski基因的siRNA和陰性對(duì)照siRNA轉(zhuǎn)入大鼠星形膠質(zhì)細(xì)胞。采用Western blotting檢測(cè)各組ski蛋白的表達(dá)水平；采用Transwell細(xì)胞遷移實(shí)驗(yàn)和細(xì)胞劃痕實(shí)驗(yàn)檢測(cè)ski-siRNA轉(zhuǎn)染后星形膠質(zhì)細(xì)胞遷移能力的變化。結(jié)果與空白對(duì)照組和陰性對(duì)照組比較，ski-siRNA轉(zhuǎn)染組細(xì)胞內(nèi)ski蛋白的相對(duì)表達(dá)水平顯著降低(F=132.957,P＜0.001)。Transwell細(xì)胞遷移實(shí)驗(yàn)發(fā)現(xiàn)，ski-siRNA轉(zhuǎn)染組每孔遷移細(xì)胞數(shù)少于空白對(duì)照組和陰性對(duì)照組(F＞47.197,P＜0.05)。細(xì)胞劃痕實(shí)驗(yàn)顯示，轉(zhuǎn)染組細(xì)胞1～5 d的劃痕愈合率均明顯低于對(duì)照組(F＞69.187,P＜0.01)。結(jié)論沉默ski基因能顯著抑制星形膠質(zhì)細(xì)胞的遷移能力，ski可能參與星形膠質(zhì)細(xì)胞的轉(zhuǎn)移過程，進(jìn)而調(diào)控膠質(zhì)瘢痕的形成。

ski；RNA干擾；星形膠質(zhì)細(xì)胞；Transwell細(xì)胞遷移實(shí)驗(yàn)；劃痕實(shí)驗(yàn)；大鼠

[本文著錄格式]趙鑫，王興文，寇江力，等.沉默ski基因?qū)Υ笫笮切文z質(zhì)細(xì)胞遷移的影響[J].中國康復(fù)理論與實(shí)踐,2017,23 (8):905-911.

CITED AS:Zhao X,Wang XW,Kou JL,et al.Effect of knocking down ski on migration of astrocytes in rats[J].Zhongguo Kangfu Li-lun Yu Shijian,2017,23(8):905-911.

星形膠質(zhì)細(xì)胞是中樞神經(jīng)系統(tǒng)(central nervous system,CNS)中數(shù)量最多、分布最廣的膠質(zhì)細(xì)胞，在維持CNS內(nèi)環(huán)境穩(wěn)定、營養(yǎng)支持神經(jīng)元、CNS創(chuàng)傷后的功能修復(fù)等方面具有重要調(diào)節(jié)作用[1-3]。反應(yīng)性星形膠質(zhì)細(xì)胞增生早期可分泌某些神經(jīng)元營養(yǎng)因子[4-5]，對(duì)維持和促進(jìn)神經(jīng)元的生長、發(fā)育、分化具有重要作用；但在晚期形成的膠質(zhì)瘢痕可作為物理屏障，妨礙軸突的再生、延長[6-7]。膠質(zhì)瘢痕的形成是影響CNS損傷功能恢復(fù)的重要因素，而膠質(zhì)瘢痕的形成與星形膠質(zhì)細(xì)胞遷移特性密不可分[8-9]，研究星形膠質(zhì)細(xì)胞的遷移功能對(duì)CNS損傷后的功能恢復(fù)具有重要意義。

ski作為進(jìn)化保守蛋白，在不同物種中廣泛參與并調(diào)節(jié)多種細(xì)胞的增殖及分化等過程[10-11]。本課題組已明確ski在正常脊髓組織中表達(dá)很低，而在脊髓損傷后表達(dá)逐漸增高，推測(cè)ski可能作為新型信號(hào)分子影響星形膠質(zhì)細(xì)胞的遷移，進(jìn)而調(diào)節(jié)膠質(zhì)瘢痕的形成[12-13]?；谶@一假設(shè)，本實(shí)驗(yàn)采用RNA干擾技術(shù)，轉(zhuǎn)染大鼠星形膠質(zhì)細(xì)胞，觀察ski對(duì)大鼠星形膠質(zhì)細(xì)胞遷移及其相關(guān)蛋白的影響。

1 材料與方法

1.1 實(shí)驗(yàn)動(dòng)物、主要試劑及來源

3日齡內(nèi)的Sprague-Dawley大鼠，購于甘肅省中醫(yī)藥大學(xué)動(dòng)物實(shí)驗(yàn)中心。

DMEM/F12培養(yǎng)基、胰蛋白酶：GIBCO。胎牛血清(fetal bovine serum,FBS)：PAN-Biotech GmbH。TritionX-100、SDS、小鼠抗大鼠及兔抗大鼠膠質(zhì)纖絲酸性蛋白(glial fibrillary acidic protein,GFAP)多克隆抗體(G3893)：SIGMA。RIPA裂解液和BCA蛋白定量檢測(cè)試劑盒：碧云天。兔抗大鼠ski多克隆抗體(H-329、sc-9140)：SANTA CRUZ。ski干擾RNA及陰性對(duì)照：THERMOFISHER公司合成。Lipofectamine?RNAiMAXReagent：THERMOFISHER。小鼠抗大鼠β-actin單克隆抗體：PROTEINTECH。山羊抗小鼠lgG、異硫氰酸熒光素(fluorescein isothiocyanate,FITC)標(biāo)記山羊抗兔lgG、山羊封閉血清、PVDF膜(0.45 μm)：SOLARBIO。辣根過氧化物酶標(biāo)記山羊抗兔或抗小鼠lgG：北京中杉金橋。ECL試劑盒：美國MILLOPORE。

本實(shí)驗(yàn)已經(jīng)本院倫理委員會(huì)審批通過。

1.2 方法

1.2.1 原代大鼠星形膠質(zhì)細(xì)胞的培養(yǎng)、純化和鑒定

大鼠在75%酒精中浸泡消毒，-20℃冰箱冷凍麻醉5 min。采用無菌方法快速取出雙側(cè)大腦皮質(zhì)，放入DMEM/F12液中，漂洗兩遍。用眼科尖鑷仔細(xì)剝離腦膜。把剝離好的腦皮質(zhì)置于盛有胰酶的皿器中，剪碎腦皮質(zhì)，用吸管輕柔吹打至組織塊消散，置入37℃溫箱輔助胰酶充分消化腦皮質(zhì)3 min。將消化好的腦皮質(zhì)移至15 ml離心管中，向離心管補(bǔ)加完全培養(yǎng)基3 ml，1500 r/min離心8min。棄上清液，再加入10%完全培養(yǎng)基(DMEM/FBS)4 ml，制成細(xì)胞懸液，將其移至培養(yǎng)瓶中。放入37℃、5%CO2培養(yǎng)箱中孵育。之后每隔3天換液，培養(yǎng)6～8 d，待細(xì)胞融合貼滿瓶壁，封口后放入37℃恒溫旋轉(zhuǎn)搖床190 r/min，除去懸浮細(xì)胞和貼壁不牢的細(xì)胞，6 h后取出，進(jìn)行傳代培養(yǎng)即可獲得純化的星形膠質(zhì)細(xì)胞。

采用GFAP多克隆抗體對(duì)純化后的星形膠質(zhì)細(xì)胞進(jìn)行免疫細(xì)胞化學(xué)染色。在熒光顯微鏡200倍視野下隨機(jī)選取6個(gè)互不重疊的視野進(jìn)行純度計(jì)數(shù)，計(jì)數(shù)GFAP陽性細(xì)胞數(shù)和總細(xì)胞數(shù)，陽性細(xì)胞數(shù)占總細(xì)胞數(shù)的百分?jǐn)?shù)即為星形膠質(zhì)細(xì)胞的純度。純度達(dá)到96%以上的星形膠質(zhì)細(xì)胞符合本實(shí)驗(yàn)要求。

1.2.2 免疫熒光染色

將原代細(xì)胞接種到蓋玻片上，貼壁后用PBS洗滌2遍，多聚甲醛固定30 min，PBS洗滌2遍，TritionX-100通透20 min，PBS洗滌2遍，滴加山羊封閉血清，37℃下封閉30 min，然后吸凈封閉液，勿洗，加入小鼠抗大鼠GFAP多克隆抗體(1∶300)，玻片置于濕盒中，于4℃冰箱中孵育過夜。PBS洗滌2遍，避光下加山羊抗小鼠lgG(1∶300)，37℃溫箱中避光孵育90 min，PBS洗滌2遍，避光下附加DAPI，室溫孵育20 min，PBS洗滌2遍，甘油封片。熒光顯微鏡下觀察。陰性對(duì)照用PBS代替一抗。

1.2.3 siRNA轉(zhuǎn)染

將純化的星形膠質(zhì)細(xì)胞平均分為ski-siRNA轉(zhuǎn)染組、陰性對(duì)照組和空白對(duì)照組。ski-siRNA轉(zhuǎn)染組采用siRNA Lipofectamine?RNAiMAX Reagent將ski-siRNA轉(zhuǎn)入星形膠質(zhì)細(xì)胞；陰性對(duì)照組采用siRNA Lipofectamine?RNAiMAX Reagent將陰性對(duì)照-siRNA(negative control-siRNA,NC-siRNA)轉(zhuǎn)入星形膠質(zhì)細(xì)胞；空白對(duì)照組僅加入siRNA Lipofectamine?RNAiMAXReagent。ski-siRNA正義鏈的序列為5'-GCC CUG AUU CGA GAC AGC UUC UAC U-3'，反義鏈的序列為5'-AGU AGA AGC UGU CUC GAA UCA GGG C-3'；NC-siRNA正義鏈的序列為5'-UUG UGG CCU GUU AGC UUC AGA GCG A-3'，反義鏈的序列為5'-UCG CUC UGAAGC UAACAG GCCACAA-3'。

取對(duì)數(shù)生長期的星形膠質(zhì)細(xì)胞接種于6孔板中，待細(xì)胞匯合度為80%時(shí)，進(jìn)行siRNA轉(zhuǎn)染，具體步驟依照siRNA轉(zhuǎn)染試劑盒說明書提供的方法進(jìn)行。待細(xì)胞轉(zhuǎn)染72 h后，于倒置光學(xué)顯微鏡下觀察細(xì)胞形態(tài)學(xué)的變化，隨后提取細(xì)胞總蛋白，采用Western blotting法檢測(cè)沉默效果。siRNA轉(zhuǎn)染效率的檢測(cè)：將FITC-control-siRNA(終濃度為100 nmol/L)6 μl與siRNA Lipofectamine?RNAiMAX Reagent 8 μl混勻后轉(zhuǎn)染星形膠質(zhì)細(xì)胞，12 h后在熒光顯微鏡下觀察并計(jì)數(shù)出現(xiàn)綠色熒光的細(xì)胞數(shù)。

1.2.4 Western blotting

觀察上述各組細(xì)胞生長情況，收集轉(zhuǎn)染72 h后的各組細(xì)胞，用預(yù)冷的PBS清洗細(xì)胞2遍，用RIPA裂解液提取細(xì)胞總蛋白，BCA試劑盒測(cè)定總蛋白濃度。提取的蛋白質(zhì)行10%SDS-PAGE電泳(濃縮膠90 V、分離膠120 V)，將電泳分離好的蛋白電轉(zhuǎn)至甲醇浸透好的PVDF膜上，采用5%脫脂奶粉室溫封閉2 h，TBST(10 mmol/L Tris-HCl,pH7.5,150 mmol/L NaCl，0.5%Tween 20)洗滌10 min，共3次，加兔抗大鼠ski多克隆抗體(1∶100)、兔抗大鼠GFAP多克隆抗體(1∶2000)、小鼠抗大鼠β-actin單克隆抗體(1∶5000)，4℃冰箱中反應(yīng)過夜；TBST洗膜(方法同前)；加辣根過氧化物標(biāo)記的山羊抗兔或抗小鼠lgG(1∶5000)，室溫下反應(yīng)2 h；TBST洗膜3次(方法同前)；滴加ECL發(fā)光液，在GE凝膠成像系統(tǒng)中測(cè)量各目的條帶的灰度值，以目的蛋白的灰度值與內(nèi)參β-actin蛋白條帶的灰度值之比代表目的蛋白的表達(dá)水平。

1.2.5 Transwell細(xì)胞遷移實(shí)驗(yàn)

采用胰蛋白酶消化并分別收集轉(zhuǎn)染72 h后的ski-siRNA轉(zhuǎn)染組、陰性對(duì)照組及空白對(duì)照組的星形膠質(zhì)細(xì)胞，調(diào)整細(xì)胞密度為1×105/ml。上室中加入細(xì)胞懸液200 μl，下室加入含有10%胎牛血清的培養(yǎng)基500 μl。置于37℃、5%CO2的培養(yǎng)箱中培養(yǎng)24 h后取出小室，棉簽拭去上室膜上未穿過小室膜的細(xì)胞，PBS清洗5 min，共3次，4%多聚甲醛溶液固定細(xì)胞30 min，風(fēng)干后加入0.25%結(jié)晶紫染色10 min，PBS清洗3次，倒置光學(xué)顯微鏡下隨機(jī)選取6個(gè)視野(放大倍數(shù)為200)計(jì)數(shù)遷移到下室的細(xì)胞數(shù)。

1.2.6 細(xì)胞劃痕實(shí)驗(yàn)

實(shí)驗(yàn)操作前對(duì)所用物品進(jìn)行滅菌，直尺和記號(hào)筆等物品操作前應(yīng)在超凈臺(tái)內(nèi)紫外線照射30 min。常規(guī)消化呈指數(shù)生長的星形膠質(zhì)細(xì)胞，以5×105/孔接種至6孔板中，按前述轉(zhuǎn)染方法將細(xì)胞分為轉(zhuǎn)染組和對(duì)照組。待細(xì)胞貼壁密度達(dá)到80%時(shí)將6孔板放置在預(yù)先設(shè)計(jì)好的劃痕模版(模版上均勻劃6條橫線，線間距離1 cm)上，借助直尺，采用200 μl移液槍頭在板底依照模版在對(duì)應(yīng)孔中劃直線，用PBS漂洗2次以除去劃下的懸浮細(xì)胞，繼續(xù)培養(yǎng)相應(yīng)時(shí)間(1 d、2 d、3 d、4 d、5 d)后在倒置光學(xué)顯微鏡下觀察細(xì)胞遷移情況。用Image-Pro Plus 6.0軟件測(cè)各個(gè)時(shí)間點(diǎn)細(xì)胞未覆蓋的面積。

1.3 統(tǒng)計(jì)學(xué)分析

采用SPSS 21.0軟件進(jìn)行統(tǒng)計(jì)分析。每組數(shù)據(jù)來自3次獨(dú)立的實(shí)驗(yàn)，以(xˉ±s)表示，采用單因素方差分析(One-wayANOVA)。顯著性水平α=0.05。

2 結(jié)果

2.1 體外培養(yǎng)星形膠質(zhì)細(xì)胞形態(tài)及純度鑒定

星形膠質(zhì)細(xì)胞的胞核呈圓形或橢圓形。星形膠質(zhì)細(xì)胞純度為96%(n=5)。特異性標(biāo)記抗原GFAP為紅色熒光，DAPI標(biāo)記細(xì)胞核為藍(lán)色熒光。見圖1。

2.2 轉(zhuǎn)染后細(xì)胞形態(tài)觀察

未轉(zhuǎn)染的星形膠質(zhì)細(xì)胞均貼壁生長，呈鋪路石狀，可見細(xì)胞輪廓清晰，突起較多，且胞突之間有交叉連接，胞體多數(shù)呈星狀或不規(guī)則狀，細(xì)胞核清晰可見，呈圓形或橢圓形，核仁明顯可見，細(xì)胞胞膜完整(圖2a)；轉(zhuǎn)染ski-siRNA72 h后，可見星形膠質(zhì)細(xì)胞體積較轉(zhuǎn)染前有所增大，胞突增粗，但細(xì)胞集落減少，未貼壁細(xì)胞增多，細(xì)胞碎片增多(圖2b)。轉(zhuǎn)染FITC-control-siRNA后星形膠質(zhì)細(xì)胞中出現(xiàn)FITC綠色熒光，轉(zhuǎn)染效率為(82.78±3.78)%。見圖2c。

2.3 ski蛋白

Western blotting結(jié)果顯示，與空白對(duì)照組和陰性對(duì)照組比較，ski-siRNA轉(zhuǎn)染組細(xì)胞內(nèi)ski蛋白的相對(duì)表達(dá)水平顯著降低(P＜0.001)；而陰性對(duì)照組與空白對(duì)照組相比無顯著性差異(P＞0.05)。見表1、圖3。

2.4 GFAP

與空白對(duì)照組和陰性對(duì)照組相比，ski-siRNA轉(zhuǎn)染組細(xì)胞中GFAP的表達(dá)水平明顯下調(diào)(P＜0.01)。見表2、圖4。

2.5 星形膠質(zhì)細(xì)胞遷移能力

ski-siRNA轉(zhuǎn)染組每孔遷移細(xì)胞數(shù)均明顯少于空白對(duì)照組和陰性對(duì)照組(P＜0.01)，空白對(duì)照組與陰性對(duì)照組間無顯著性差異(P＞0.05)。見表3、圖5。

細(xì)胞培養(yǎng)1～5 d，轉(zhuǎn)染ski-siRNA組細(xì)胞劃痕愈合率均明顯小于對(duì)照組(P＜0.01)。見表4、圖6。

3 討論

ski基因是Stavnezer等[14]于1986年首次在感染病毒Bratislava77的雞胚細(xì)胞中發(fā)現(xiàn)的。研究表明ski與多種細(xì)胞的增殖、分化、轉(zhuǎn)化及腫瘤發(fā)生發(fā)展相關(guān)[15-17]。目前關(guān)于ski分子的調(diào)控研究主要集中在胚胎發(fā)育、組織創(chuàng)傷修復(fù)及對(duì)腫瘤細(xì)胞發(fā)生發(fā)展的調(diào)節(jié)等方面[16,18]。本研究組前期實(shí)驗(yàn)首次證實(shí)，ski在星形膠質(zhì)細(xì)胞生物學(xué)功能方面起到一定的作用[2]。

CNS正常結(jié)構(gòu)和功能的維持離不開膠質(zhì)細(xì)胞，而星形膠質(zhì)細(xì)胞作為CNS中數(shù)量最多、分布最廣的膠質(zhì)細(xì)胞[2]，其對(duì)神經(jīng)遞質(zhì)調(diào)節(jié)、內(nèi)環(huán)境穩(wěn)態(tài)、血腦屏障的維持起著重要的作用。CNS損傷后，星形膠質(zhì)細(xì)胞作為主要的膠質(zhì)細(xì)胞參與CNS損傷后的病理生理過程，其最具特征性的表現(xiàn)是反應(yīng)性星形膠質(zhì)細(xì)胞增生(reactive astrogliosis,RA)[19-20]，RA又表現(xiàn)為神經(jīng)GFAP表達(dá)上調(diào)[1,21]。而嚴(yán)重的膠質(zhì)細(xì)胞增生不僅出現(xiàn)GFAP上調(diào)及胞體肥大增生等表現(xiàn)，還可以見到致密膠質(zhì)瘢痕形成[19]。以往研究表明，膠質(zhì)瘢痕的形成會(huì)阻止軸突的再生，進(jìn)而妨礙CNS損傷后神經(jīng)功能恢復(fù)；但近幾年有研究表明[22-24]，膠質(zhì)瘢痕的形成不但可以加快損傷的神經(jīng)組織愈合，還可以作為一道物理屏障限制損傷局部炎癥的進(jìn)一步擴(kuò)散，避免損傷周圍組織的損傷。由此可見，RA對(duì)CNS損傷既是不利因素又有保護(hù)作用。因此，深入探討調(diào)控膠質(zhì)瘢痕形成的分子機(jī)制，以保存或放大RA的有利作用，同時(shí)減低其不利作用至關(guān)重要。

本實(shí)驗(yàn)利用脂質(zhì)體轉(zhuǎn)染法將ski-siRNA轉(zhuǎn)染至大鼠星形膠質(zhì)細(xì)胞中抑制ski的表達(dá)，初步探討沉默ski基因表達(dá)對(duì)星形膠質(zhì)細(xì)胞遷移特性的影響。本研究結(jié)果顯示，轉(zhuǎn)染ski-siRNA后成功抑制星形膠質(zhì)細(xì)胞中ski蛋白的表達(dá)以及細(xì)胞遷移能力，證明ski在促進(jìn)星形膠質(zhì)細(xì)胞遷移特性方面起到重要調(diào)控作用。

本研究還發(fā)現(xiàn)，ski-siRNA轉(zhuǎn)染組細(xì)胞中GFAP表達(dá)水平與ski的表達(dá)水平同時(shí)下調(diào)。GFAP作為星形膠質(zhì)細(xì)胞特有標(biāo)志蛋白[25]，參與星形膠質(zhì)細(xì)胞生物學(xué)活性的調(diào)節(jié)[21]。已有研究報(bào)道[26-27]，GFAP與星形膠質(zhì)細(xì)胞遷移能力呈正相關(guān)，而本研究發(fā)現(xiàn)在ski基因表達(dá)沉默后，GFAP表達(dá)量也相應(yīng)下降，因此細(xì)胞遷移能力下降可能與此有關(guān)。但抑制ski表達(dá)后是否還會(huì)抑制一些其他遷移相關(guān)蛋白的表達(dá)，如基質(zhì)金屬蛋白酶(matrix metalloproteinase,MMP2)[28]、整合素[29]等，進(jìn)而調(diào)節(jié)星形膠質(zhì)細(xì)胞的遷移及侵襲能力，還需進(jìn)一步研究。

以上實(shí)驗(yàn)結(jié)果證明，采用ski-siRNA可以成功沉默ski基因表達(dá)，沉默ski基因后的星形膠質(zhì)細(xì)胞遷移能力明顯受抑制。由此，我們推測(cè)ski可能調(diào)控膠質(zhì)瘢痕的生成過程，但其具體分子機(jī)制有待于進(jìn)一步研究證實(shí)。深入研究ski對(duì)星形膠質(zhì)細(xì)胞生物學(xué)功能的作用，可為CNS損傷后神經(jīng)功能的康復(fù)提出一條新的途徑。

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Effect of Knocking Down ski on Migration ofAstrocytes in Rats

ZHAO Xin1,2,WANG Xing-wen1,2,KOU Jiang-li1,2,LI Zhong-hao1,2,GUO Yong-qiang1,2,WU Ya-min3,ZHANG Hai-hong1
1.Department of Orthopedics,Second Clinical Medical College of Lanzhou University,Lanzhou,Gansu 730030, China;2.Key Laboratory of Orthopedics of Gansu Province,Lanzhou,Gansu 730030,China;3.State Key Laboratory of Trauma,Burns and Combined Injury,the Third Department of Research Institute of Surgery,Daping Hospital,Third Military University,Chongqing 400042,China

ZHANG Hai-hong.E-mail:zhanghaihong1968@sina.com

ObjectiveTo investigate the effect of ski gene in migration process of astrocytes in rats.Methods Astrocytes were obtained from rats'cerebral cortex and cultured in vitro.siRNA targeting ski gene and negative control sequences were prepared.The ski-siRNA group,siRNA negative control group and untreated group were set in this experiment.The specific siRNA targeting ski gene was transfected into astrocytes with Lipofectamine?RNAiMAX Reagent.Then the ski protein levels were determined with Western blotting.After transfection,the changes in migration of astrocytes were measured with wound scratch assay and Transwell migration assay.Results Western blotting showed that the expression of ski protein was significantly lower in the ski-siRNA group than in the siRNA negative control group and untreated group(F=132.957,P＜0.001).Transwell migration assay showed that the number of astrocytes crossing through chambers was less in the ski-siRNA group than in the siRNA negative control group and untreated group(F＞47.197,P＜0.05).Wound scratch assay showed that the wound healing rate was lower in the ski-siRNA group than in the control group one,two,three,four and five days after transfection (F＞69.187,P＜0.001).Conclusion Ski knocked down by siRNA could inhibit the migration ability of astrocytes.It is a reminding that ski may take part in the migration process of astrocytes,and moreover,ski may play an important role in the formation of glial scar.

ski;siRNA;astrocyte;Transwell migration assay;wound scratch assay;rats

R364.5

A

1006-9771(2017)08-0905-07

2017-04-10

2017-05-05)

10.3969/j.issn.1006-9771.2017.08.008

1.國家自然科學(xué)基金項(xiàng)目(No.30772299)；2.蘭州大學(xué)第二醫(yī)院科研基金項(xiàng)目(No.sdkyjj-04)。

1.蘭州大學(xué)第二醫(yī)院骨科，甘肅蘭州市730030；2.甘肅省骨關(guān)節(jié)疾病研究重點(diǎn)實(shí)驗(yàn)室，甘肅蘭州市730030；3.第三軍醫(yī)大學(xué)大坪醫(yī)院野戰(zhàn)外科研究所三室，創(chuàng)傷、燒傷與復(fù)合傷國家重點(diǎn)實(shí)驗(yàn)室，重慶市400042。作者簡介：趙鑫(1991-)，男，漢族，山東濰坊市人，碩士研究生，主要研究方向：脊柱脊髓損傷。通訊作者：張海鴻，主任醫(yī)師，副教授，碩士研究生導(dǎo)師，主要研究方向：脊柱外科。E-mail:zhanghaihong1968@sina.com。