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        Fasudil聯(lián)合骨髓源神經(jīng)干細(xì)胞對(duì)EAE小鼠的神經(jīng)保護(hù)作用*

        2017-12-22 09:09:12宋國(guó)斌席國(guó)萍李艷花李加善劉建春肖保國(guó)張光先馬存根
        中國(guó)病理生理雜志 2017年12期
        關(guān)鍵詞:膠質(zhì)干細(xì)胞分化

        宋國(guó)斌, 席國(guó)萍, 李艷花, 李加善, 劉建春, 柴 智, 肖保國(guó), 張光先, 4△, 馬存根, △

        (1山西大同大學(xué)腦科學(xué)研究所, 山西 大同 037009; 2山西中醫(yī)學(xué)院神經(jīng)生物學(xué)研究中心, 山西 太原 030024;3復(fù)旦大學(xué)華山醫(yī)院神經(jīng)病學(xué)研究所, 上海 200025; 4托馬斯·杰弗遜大學(xué)神經(jīng)學(xué)系, 美國(guó) 賓夕法尼亞州 費(fèi)城 19107)

        ·論著·

        Fasudil聯(lián)合骨髓源神經(jīng)干細(xì)胞對(duì)EAE小鼠的神經(jīng)保護(hù)作用*

        宋國(guó)斌1, 席國(guó)萍1, 李艷花1, 李加善1, 劉建春2, 柴 智2, 肖保國(guó)3, 張光先1, 4△, 馬存根1, 2△

        (1山西大同大學(xué)腦科學(xué)研究所, 山西 大同 037009;2山西中醫(yī)學(xué)院神經(jīng)生物學(xué)研究中心, 山西 太原 030024;3復(fù)旦大學(xué)華山醫(yī)院神經(jīng)病學(xué)研究所, 上海 200025;4托馬斯·杰弗遜大學(xué)神經(jīng)學(xué)系, 美國(guó) 賓夕法尼亞州 費(fèi)城 19107)

        目的探討Rho激酶抑制劑法舒地爾(fasudil)聯(lián)合骨髓源神經(jīng)干細(xì)胞(bone marrow-derived neural stem cells,BM-NSCs)對(duì)實(shí)驗(yàn)性自身免疫性腦脊髓炎(experimental autoimmune encephalomyelitis,EAE)小鼠的神經(jīng)保護(hù)作用。方法32只雌性C57BL/6小鼠(8~10周齡),用髓鞘少突膠質(zhì)細(xì)胞糖蛋白35-55(MOG35-55)免疫,制備EAE,隨機(jī)分為對(duì)照(ddH2O)組、fasudil組、BM-NSCs組和fasudil+BM-NSCs組,并分別給予相應(yīng)處理。免疫后檢測(cè)小鼠臨床癥狀和相關(guān)神經(jīng)營(yíng)養(yǎng)因子的表達(dá),Image-Pro Plus 6.0軟件進(jìn)行陽(yáng)性細(xì)胞計(jì)數(shù),GraphPad Prism 5軟件統(tǒng)計(jì)分析。結(jié)果與ddH2O組比較,fasudil+BM-NSCs處理組明顯延遲小鼠的平均起病時(shí)間,降低最高臨床評(píng)分,并緩解EAE小鼠的臨床癥狀;fasudil組、BM-NSCs組和fasudil+BM-NSCs組中腦源性神經(jīng)營(yíng)養(yǎng)因子、膠質(zhì)細(xì)胞源性神經(jīng)營(yíng)養(yǎng)因子、神經(jīng)生長(zhǎng)因子、神經(jīng)營(yíng)養(yǎng)因子3和睫狀神經(jīng)營(yíng)養(yǎng)因子陽(yáng)性細(xì)胞數(shù)均有不同程度的增加,其中,fasudil+BM-NSCs組上述神經(jīng)營(yíng)養(yǎng)因子的表達(dá)明顯多于ddH2O組、fasudil組和BM-NSCs組(P<0.01)。結(jié)論Fasudil聯(lián)合BM-NSCs通過(guò)協(xié)同和疊加效應(yīng)促進(jìn)神經(jīng)營(yíng)養(yǎng)因子表達(dá),改善中樞神經(jīng)系統(tǒng)微環(huán)境,發(fā)揮神經(jīng)保護(hù)作用,從而緩解EAE的臨床癥狀。

        法舒地爾; 骨髓源神經(jīng)干細(xì)胞; 實(shí)驗(yàn)性自身免疫性腦脊髓炎; 神經(jīng)營(yíng)養(yǎng)因子

        多發(fā)性硬化(multiple sclerosis,MS)是一種由T細(xì)胞介導(dǎo)的中樞神經(jīng)系統(tǒng)(central nervous system,CNS)自身免疫性疾病[1]。MS典型的病理特點(diǎn)包括慢性炎癥、髓鞘脫失、軸突損傷和神經(jīng)變性等[2]。實(shí)驗(yàn)性自身免疫性腦脊髓炎(experimental autoimmune encephalomyelitis,EAE)和MS的許多臨床癥狀及病理表現(xiàn)相似,被認(rèn)為是研究MS發(fā)病機(jī)制和治療方法的理想動(dòng)物模型[3]。

        近年來(lái),國(guó)內(nèi)外研究者發(fā)現(xiàn)骨髓來(lái)源的間充質(zhì)細(xì)胞在體外培養(yǎng)可分化為神經(jīng)干細(xì)胞(neural stem cells,NSCs),稱為骨髓源NSCs(bone marrow-derived NSCs,BM-NSCs),其具有和來(lái)源于腦組織的NSCs相同的特性,如培養(yǎng)增殖速度快,有分化為神經(jīng)元、少突膠質(zhì)細(xì)胞和星形膠質(zhì)細(xì)胞的能力,可通過(guò)血腦屏障,動(dòng)員自身內(nèi)源性神經(jīng)干細(xì)胞增殖[4]。目前已有動(dòng)物實(shí)驗(yàn)證明,靜脈注射BM-NSCs具有改善神經(jīng)功能的作用,但是細(xì)胞需要承受“首過(guò)效應(yīng)”,大量的細(xì)胞被肝臟、肺和脾臟過(guò)濾,影響了BM-NSCs進(jìn)入CNS的數(shù)量和效率[5]。“鼻腔給藥”途徑在過(guò)去幾年里已經(jīng)引起廣泛關(guān)注。研究表明,藥物可通過(guò)嗅黏膜上皮通路,繞過(guò)血腦屏障,進(jìn)入腦脊液,直接擴(kuò)散于CNS,發(fā)揮有效的治療作用;另外,也可通過(guò)血液循環(huán)或淋巴系統(tǒng)、三叉神經(jīng)和視神經(jīng)等間接通路進(jìn)入腦內(nèi)[6-8]。

        Rho激酶(Rho-associated coiled-coil containing kinases,ROCK)抑制劑法舒地爾(fasudil)具有外周和中樞免疫調(diào)節(jié)作用,可促進(jìn)中樞神經(jīng)營(yíng)養(yǎng)因子產(chǎn)生,改善CNS微環(huán)境并促進(jìn)干細(xì)胞增殖、存活和維持干細(xì)胞的低分化[9-11]。

        鑒于以上實(shí)驗(yàn)和理論基礎(chǔ),我們?cè)O(shè)想聯(lián)合BM-NSCs的神經(jīng)再生功能和fasudil的免疫調(diào)節(jié)作用。該法有望成為一種治療EAE的有效的“雞尾酒”療法。

        材 料 和 方 法

        1 實(shí)驗(yàn)動(dòng)物

        清潔級(jí)雌性C57BL/6小鼠32只(8~10周齡,體重18~20 g)及清潔級(jí)雌性3~4周齡C57BL/6小鼠10只(12~14 g),購(gòu)于北京維通利華實(shí)驗(yàn)動(dòng)物技術(shù)有限公司,動(dòng)物合格證編號(hào)為11400700146392。實(shí)驗(yàn)前在無(wú)病源菌實(shí)驗(yàn)室自由飲食飼養(yǎng)1周。

        2 主要試劑和儀器

        髓鞘少突膠質(zhì)細(xì)胞糖蛋白35-55多肽(myelin oligodendrocyte glycoprotein 35-55, MOG35-55;西安聯(lián)美生物科技有限公司);完全弗氏佐劑(complete Freund’s adjuvant, CFA; Sigma);百日咳毒素(pertussis toxin, PTX; Enzo);抗巢蛋白(nestin)、神經(jīng)生長(zhǎng)因子(nerve growth factor, NGF)、睫狀神經(jīng)營(yíng)養(yǎng)因子(ciliary neurotrophic factor, CNTF)、神經(jīng)營(yíng)養(yǎng)因子3(neurotrophin-3, NT-3)和腦源性神經(jīng)營(yíng)養(yǎng)因子(brain-derived neurotrophic factor, BDNF)抗體(Abcam);抗膠質(zhì)纖維酸性蛋白(glial fibrillary acidic protein, GFAP)抗體(Epitomics);抗膠質(zhì)細(xì)胞源性神經(jīng)營(yíng)養(yǎng)因子(glial cell-derived neurotrophic factor, GDNF)抗體(Santa Cruz);抗Tuj1抗體、抗2’,3’-環(huán)核苷酸-3’-磷酸二酯酶(2’,3’-cyclic-nucleotide 3’-phosphosiesterase,CNPase)抗體及Alexa Fluor?555標(biāo)記的山羊抗兔、抗小鼠 II 抗(Cell Signaling Technology);fasudil注射液(天津紅日藥業(yè)股份有限公司);胎牛血清(fetal bovine serum,F(xiàn)BS)、牛血清白蛋白(bovine serum albumin,BSA)、無(wú)血清培養(yǎng)添加劑B27、青霉素、鏈霉素和DMEM/F12細(xì)胞基礎(chǔ)培養(yǎng)液(Gibco);重組小鼠堿性成纖維細(xì)胞生長(zhǎng)因子2(recombinant mouse basic fibroblast growth factor 2,mFGF2)和重組小鼠表皮生長(zhǎng)因子(recombinant mouse epidermal growth factor,mEGF)購(gòu)自PeproTech。CO2培養(yǎng)箱(Thermo);正置熒光顯微鏡和倒置相差熒光顯微鏡(Olympus);冰凍切片機(jī)(Leica)。

        3 主要方法

        3.1BM-NSCs的獲得與鑒定 參考前人的實(shí)驗(yàn)方法[4, 12],將4周齡的小鼠頸椎脫臼處死,分離股骨和脛骨,用含雙抗的PBS清洗2~3次。無(wú)菌注射器反復(fù)沖洗骨髓腔,收集骨髓基質(zhì)細(xì)胞于15 mL離心管中,調(diào)整細(xì)胞數(shù)為1×1010/L,置于37 ℃、5% CO2、飽和濕度培養(yǎng)箱培養(yǎng)。48 h后更換新鮮培養(yǎng)液,除去未貼壁的懸浮細(xì)胞,之后每3 d半量換液1次。待細(xì)胞克隆球出現(xiàn)后,收集細(xì)胞球,100×g離心3 min,用AccutaseTM細(xì)胞消化液消化,調(diào)整細(xì)胞數(shù)為1×1010/L,每隔4 d傳代1次,第5代細(xì)胞用于后期實(shí)驗(yàn)。將細(xì)胞懸浮于分化培養(yǎng)液中培養(yǎng)2周,進(jìn)行免疫熒光染色鑒定。BM-NSCs增殖培養(yǎng)液(DMEM/F12,2% B27,20 μg/L mEGF,20 μg/L mFGF2,1% MEM非必需氨基酸,55 μmol/L β-巰基乙醇)。神經(jīng)元分化培養(yǎng)液(DMEM/F12,2% B27,2 mmol/L GlutaMAX-1,0.5 mmol/L cAMP)。星形膠質(zhì)細(xì)胞分化培養(yǎng)液(DMEM/F12,1% N-2,2 mmol/L GlutaMAX-1,1% FBS)。少突膠質(zhì)細(xì)胞分化培養(yǎng)液(DMEM/F12,2% B27,2 mmol/L GlutaMAX-1,20 μg/L T3)。

        3.2EAE模型制備與分組 將300 μg MOG35-55溶解于生理鹽水中,4 g/L PTX溶于弗氏佐劑,制成油包水樣乳白色混懸液。在小鼠脊柱背側(cè)中線兩側(cè),分4點(diǎn)皮下注射抗原乳劑,每只0.1 mL,各組小鼠在免疫當(dāng)天和免疫48 h后,分別腹腔注射PTX,每只500 ng。32只EAE小鼠隨機(jī)分為ddH2O組、fasudil組、BM-NSCs組和fasudil+BM-NSCs組。免疫后第12天,乙醚麻醉小鼠,BM-NSCs組和fasudil+BM-NSCs組小鼠每個(gè)鼻孔滴入3 μL透明質(zhì)酸酶溶液(PBS溶解,100 U),30 min后滴鼻給予12 μL BM-NSCs液(1.5×105);鼻腔給藥后,保持小鼠體位不變,使溶液充分吸入鼻腔上部[13]。第14~27天,fasudil組和fasudil+BM-NSCs組小鼠隔天腹腔注射fasudil溶液(每只400 μg);腹腔注射及鼻腔滴入等體積ddH2O為對(duì)照。

        3.3臨床評(píng)分 自免疫當(dāng)日開(kāi)始,隔天觀察記錄臨床評(píng)分及體重變化。采用國(guó)際通用的5分法對(duì)小鼠進(jìn)行臨床評(píng)分:無(wú)任何臨床癥狀者為0分;尾部張力消失,可見(jiàn)輕度步態(tài)笨拙者為1分;單側(cè)后下肢無(wú)力,被動(dòng)翻身可以恢復(fù)者為2分;雙后肢癱瘓,被動(dòng)翻身不能恢復(fù),但給予刺激可以挪動(dòng)者為3分;雙后肢癱瘓伴前肢癱瘓者為4分;瀕死狀態(tài)或死亡者為5分。癥狀介于兩標(biāo)準(zhǔn)之間者以±0.5分計(jì)。

        3.4標(biāo)本采集 免疫后第28天,腹腔注射5%的水合氯醛(每只0.2 mL)。麻醉后,剝離小鼠大腦,用生理鹽水灌注,4%多聚甲醛灌注固定。OCT包埋,制備冰凍切片(Bregma -1.34到-1.70 mm, 10 μm),-80 ℃保存。

        3.5免疫熒光染色 將上述制備的冰凍切片用PBS洗3次,每次5 min; 1% BSA室溫封閉1 h;I 抗(稀釋比例1∶1 000)4 ℃孵育過(guò)夜;次日,用PBS洗3次,每次5 min;Alexa Fluor?555熒光標(biāo)記的II 抗室溫孵育2 h;PBS洗3次,每次5 min,DAPI染核5 min,50%甘油封片,鏡檢。陰性對(duì)照為只加II抗,省略I抗。免疫熒光染色腦片用Image-Pro Plus 6.0軟件測(cè)量工具中的計(jì)數(shù)功能進(jìn)行雙盲檢測(cè)。

        4 統(tǒng)計(jì)學(xué)分析

        采用GraphPad Prism 5軟件進(jìn)行統(tǒng)計(jì)分析。數(shù)據(jù)用均數(shù)±標(biāo)準(zhǔn)差(mean±SD)表示,組間比較采用單因素方差分析,以P<0.05 為差異有統(tǒng)計(jì)學(xué)意義。

        結(jié) 果

        1 BM-NSCs的鑒定與分化

        從小鼠骨髓中分離的細(xì)胞,取傳代到第5代的細(xì)胞球行免疫熒光染色,結(jié)果顯示細(xì)胞球?yàn)閚estin陽(yáng)性表達(dá),見(jiàn)圖1A。用Accutase消化細(xì)胞球?yàn)閱渭?xì)胞,懸浮于分化培養(yǎng)液中培養(yǎng)2周,行免疫熒光染色,結(jié)果顯示細(xì)胞可以分化為T(mén)uj1+的神經(jīng)元、GFAP+的星形膠質(zhì)細(xì)胞和CNPase+的少突膠質(zhì)細(xì)胞,見(jiàn)圖1B。上述結(jié)果表明分離培養(yǎng)的細(xì)胞球?yàn)镹SCs克隆球。

        Figure 1. Identification and differentiation of BM-NSCs. A: neurospheres were stained with nestin antibody (×10); B: BM-NSCs were induced to differentiated into neurons (Tju1+), astrocytes (GFAP+) and oligodendrocytes (CNPase+) in specific differentiation media for 2 weeks.

        圖1BM-NSCs的鑒定與分化

        2 Fasudil聯(lián)合BM-NSCs更加顯著緩解EAE臨床癥狀

        ddH2O組小鼠于免疫后第10天相繼開(kāi)始發(fā)病,平均起病時(shí)間(10.7±1.0) d,最高臨床評(píng)分達(dá)到4.5分,發(fā)病率為100%,無(wú)死亡,平均最高臨床評(píng)分為3.92±0.92。單用fasudil和BM-NSCs均能有效延遲平均起病時(shí)間[(17.5±2.5) d和(16.3±3.5) d,P<0.05]并降低最高臨床評(píng)分(1.88±0.75和1.67±0.93,P<0.01)。Fasudil+BM-NSCs更顯著延遲平均起病時(shí)間[(21.6±5.2) d,P<0.01],降低臨床評(píng)分(1.30±0.45,P<0.01)。另外,EAE臨床癥狀加重伴隨體重減輕,fasudil組、BM-NSCs組和fasudil+BM-NSCs組小鼠體重減輕狀況明顯緩解,fasudil+BM-NSCs減少體重丟失最明顯(P<0.05或P<0.01),見(jiàn)圖2。

        3 Fasudil聯(lián)合BM-NSCs促進(jìn)BDNF表達(dá)

        免疫熒光染色結(jié)果顯示,與ddH2O組比較,BM-NSCs組和fasudil+BM-NSCs組的BDNF陽(yáng)性細(xì)胞數(shù)均明顯增加,fasudil+BM-NSCs組增加更為明顯(P<0.05或P<0.01),見(jiàn)圖3。

        Figure 2. Fasudil combined with BM-NSCs markedly reduced EAE clinical score and decreased body weight loss. Mean±SD.n=8.*P<0.05,**P<0.01vsddH2O group.

        圖2Fasudil聯(lián)合BM-NSCs明顯降低EAE臨床評(píng)分并減少體重丟失

        Figure 3. Fasudil combined with BM-NSCs promoted the expression of BDNF. Mean±SD.n=5.*P<0.05,**P<0.01vsddH2O group.

        圖3Fasudil聯(lián)合BM-NSCs促進(jìn)BDNF表達(dá)

        4 Fasudil聯(lián)合BM-NSCs促進(jìn)GDNF表達(dá)

        免疫熒光染色結(jié)果顯示,與ddH2O組比較,fasudil組、BM-NSCs組和fasudil+BM-NSCs組的GDNF陽(yáng)性細(xì)胞數(shù)均明顯增加,fasudil+BM-NSCs組增加最明顯(P<0.05或P<0.01),見(jiàn)圖4。

        5 Fasudil聯(lián)合BM-NSCs促進(jìn)NGF表達(dá)

        免疫熒光染色結(jié)果顯示,與ddH2O組比較,BM-NSCs組和fasudil+BM-NSCs組的NGF陽(yáng)性細(xì)胞數(shù)均明顯增加, fasudil+BM-NSCs組增加更為明顯(P<0.05或P<0.01),見(jiàn)圖5。

        6 Fasudil聯(lián)合BM-NSCs促進(jìn)NT-3表達(dá)

        免疫熒光染色結(jié)果顯示,與ddH2O組比較,BM-NSCs組和fasudil+BM-NSCs組的NT-3陽(yáng)性細(xì)胞數(shù)均明顯增加, fasudil+BM-NSCs組增加更為明顯(P<0.01),見(jiàn)圖6。

        7 Fasudil聯(lián)合BM-NSCs促進(jìn)CNTF表達(dá)

        免疫熒光染色結(jié)果顯示,與ddH2O組比較,fasudil組、BM-NSCs組和fasudil+BM-NSCs組的CNTF陽(yáng)性細(xì)胞數(shù)均明顯增加,fasudil+BM-NSCs組增加最顯著(P<0.05或P<0.01),見(jiàn)圖7。

        Figure 4. Fasudil combined with BM-NSCs promoted the expression of GDNF. Mean±SD.n=5.*P<0.05,**P<0.01vsddH2O group.

        圖4Fasudil聯(lián)合BM-NSCs促進(jìn)GDNF表達(dá)

        Figure 5. Fasudil combined with BM-NSCs promoted the expression of NGF. Mean±SD.n=5.*P<0.05,**P<0.01vsddH2O group.

        圖5Fasudil聯(lián)合BM-NSCs促進(jìn)NGF表達(dá)

        Figure 6. Fasudil combined with BM-NSCs promoted the expression of NT-3. Mean±SD.n=5.**P<0.01vsddH2O group.

        圖6Fasudil聯(lián)合BM-NSCs促進(jìn)NT-3表達(dá)

        Figure 7. Fasudil combined with BM-NSCs BM-NSCs promoted the expression of CNTF. Mean±SD.n=5.*P<0.05,**P<0.01vsddH2O group.

        圖7Fasudil聯(lián)合BM-NSCs促進(jìn)CNTF表達(dá)

        討 論

        近年來(lái), NSCs移植在神經(jīng)系統(tǒng)疾病的治療方面已經(jīng)顯示出有效性。大量關(guān)于CNS損傷機(jī)制及神經(jīng)保護(hù)再生的研究發(fā)現(xiàn),通過(guò)干細(xì)胞移植治療可以改善CNS微環(huán)境,促進(jìn)髓鞘或神經(jīng)再生,從而恢復(fù)信息傳導(dǎo)[14]。NSCs存在于CNS多個(gè)部位,具有自我更新和多向分化潛能,能夠分泌多種神經(jīng)營(yíng)養(yǎng)因子,使殘存脫髓鞘的神經(jīng)纖維和新生的神經(jīng)纖維形成髓鞘而起到改善CNS微環(huán)境、促進(jìn)神經(jīng)再生和神經(jīng)保護(hù)的作用[15]。在所有NSCs來(lái)源中,BM-NSCs因取材方便、無(wú)倫理道德及免疫排斥問(wèn)題及最小的致瘤性,在治療神經(jīng)系統(tǒng)退行性疾病方面顯示出巨大的潛力。

        在干細(xì)胞治療中,影響神經(jīng)形成的一個(gè)關(guān)鍵因素是細(xì)胞的移行路線。目前,常見(jiàn)的細(xì)胞移行路線包括靜脈注射、腹腔注射、顱腦注射、腦室注射及鼻腔給藥。因?yàn)榫哂刑厥獾倪B接結(jié)構(gòu),鼻腔給藥能提供一個(gè)有效的、非侵害的、快速的傳遞細(xì)胞到腦的路線,這也是本研究選擇鼻腔給藥的原因。

        研究發(fā)現(xiàn),損傷的CNS微環(huán)境不利于NSCs的移植和存活,故單獨(dú)移植NSCs對(duì)神經(jīng)再生和神經(jīng)功能恢復(fù)是有限的。與單獨(dú)移植NSCs比較,聯(lián)合干預(yù)能夠更好地促進(jìn)干細(xì)胞生存[16-18]。ROCK信號(hào)通路作為機(jī)體普遍存在的信號(hào)通路,與細(xì)胞的生長(zhǎng)、增殖、分化、收縮和移行等許多細(xì)胞功能密切相關(guān),廣泛參與包括抑制軸突再生及神經(jīng)元死亡等多個(gè)病理生理過(guò)程,被認(rèn)為是治療神經(jīng)系統(tǒng)變性疾病的潛在藥物靶點(diǎn)[19-20]。大量證據(jù)顯示,許多CNS變性疾病均存在ROCK信號(hào)通路的異常激活[21-22]。ROCK抑制劑具有促進(jìn)干細(xì)胞增殖和維持干細(xì)胞分化、促進(jìn)干細(xì)胞存活、直接抑制Rho/ROCK信號(hào)通路、促進(jìn)軸突再生和保護(hù)神經(jīng)元等重要作用,已逐漸成為神經(jīng)系統(tǒng)疾病重要的治療藥物[12, 23-24]。Fasudil作為唯一在臨床上使用的ROCK抑制劑,能有效地減緩EAE發(fā)病,減輕EAE的臨床癥狀,減少CNS和外周的炎癥反應(yīng),改善CNS微環(huán)境。Chen等[25]研究表明,fasudil作為ROCK抑制劑,具有促進(jìn)神經(jīng)軸突生長(zhǎng)、抑制髓鞘脫失、減少炎癥細(xì)胞聚集及炎癥因子的釋放和改善CNS微環(huán)境等作用。Chen等[26]報(bào)道fasudil能通過(guò)Notch信號(hào)通路刺激C17.2神經(jīng)干細(xì)胞分化和軸突生長(zhǎng)。Li等[12]報(bào)道fasudil能有效提高被移植到大腦的BM-NSCs的存活率。鑒于上述分析,本研究評(píng)估fasudil聯(lián)合BM-NSCs是否能協(xié)同促進(jìn)CNS分泌神經(jīng)營(yíng)養(yǎng)因子,改善CNS微環(huán)境,達(dá)到神經(jīng)保護(hù)和促進(jìn)神經(jīng)再生的作用。

        神經(jīng)營(yíng)養(yǎng)因子(neurotrophic factors,NTFs)是一類由機(jī)體產(chǎn)生的具有促進(jìn)和維持神經(jīng)元生長(zhǎng)、發(fā)育、分化、成熟和存活功能的特異性蛋白質(zhì)或多肽類分子,其不僅參與調(diào)節(jié)神經(jīng)細(xì)胞的生長(zhǎng)發(fā)育過(guò)程,而且能夠促進(jìn)神經(jīng)元修復(fù)、軸突再生、阻止受損神經(jīng)元死亡,對(duì)神經(jīng)退行性疾病等具有顯著的神經(jīng)營(yíng)養(yǎng)及神經(jīng)保護(hù)作用。NTFs成員包括NGF、BDNF、NT-3、GDNF和CNTF等[27]。研究顯示,神經(jīng)營(yíng)養(yǎng)因子分泌不足導(dǎo)致神經(jīng)元死亡是影響神經(jīng)元再生的重要因素[28]。Yu等[29]報(bào)道,fasudil通過(guò)增加EAE小鼠CNS神經(jīng)營(yíng)養(yǎng)因子和減少CNS炎性微環(huán)境來(lái)阻止突觸損傷和促進(jìn)突觸形成。Selim等[30]報(bào)道,靜脈給予人胚胎源間充質(zhì)干細(xì)胞,在EAE大鼠腦中顯著表達(dá)BDNF、NGF和NT-3等神經(jīng)營(yíng)養(yǎng)因子,通過(guò)提供直接的神經(jīng)營(yíng)養(yǎng)支持獲得良好的治療效果。Li等[31]報(bào)道,新型ROCK抑制劑WAR-5通過(guò)上調(diào)神經(jīng)營(yíng)養(yǎng)因子BDNF和NT-3表達(dá),表現(xiàn)出良好的EAE治療潛力。以上研究表明,神經(jīng)營(yíng)養(yǎng)因子分泌增加有助于改善CNS微環(huán)境,促進(jìn)神經(jīng)細(xì)胞的生長(zhǎng)和功能恢復(fù),起到神經(jīng)保護(hù)的作用。

        本實(shí)驗(yàn)研究結(jié)果表明,fasudil、BM-NSCs及fasudil聯(lián)合BM-NSCs均能促進(jìn)EAE小鼠腦組織神經(jīng)營(yíng)養(yǎng)因子BDNF、GDNF、NGF、NT-3和CNTF的分泌,其中fasudil聯(lián)合BM-NSCs上調(diào)神經(jīng)營(yíng)養(yǎng)因子效果最佳。綜上所述,我們推測(cè)fasudil聯(lián)合BM-NSCs發(fā)揮神經(jīng)保護(hù)作用的可能機(jī)制為:fasudil通過(guò)減少炎性細(xì)胞聚集和炎性因子釋放,減輕炎癥反應(yīng);分泌更多的神經(jīng)營(yíng)養(yǎng)因子,改善CNS微環(huán)境,有利于更多的BM-NSCs存活,二者相互協(xié)作,起到神經(jīng)保護(hù)的作用。本研究以神經(jīng)營(yíng)養(yǎng)因子的分泌作為主要觀測(cè)對(duì)象,對(duì)fasudil聯(lián)合BM-NSCs干預(yù)EAE的神經(jīng)保護(hù)作用作前期的探索,后續(xù)研究將進(jìn)一步探討fasudil聯(lián)合BM-NSCs干預(yù)EAE后,小鼠腦內(nèi)炎性因子的變化,及星形膠質(zhì)細(xì)胞和少突膠質(zhì)細(xì)胞等變化,為臨床治療MS提供基礎(chǔ)數(shù)據(jù)和參考。

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        Neuroprotective effect of fasudil combined with bone marrow-derived neural stem cells on mice with experimental autoimmune encephalomyelitis

        SONG Guo-bin1, XI Guo-ping1, LI Yan-hua1, LI Jia-shan1, LIU Jian-chun2, CHAI Zhi2, XIAO Bao-guo3, ZHANG Guang-xian1, 4, MA Cun-gen1, 2

        (1InstituteofBrainScience,DatongUniversity,Datong037009,China;2NeurobiologyResearchCenter,ShanxiUniversityofTraditionalChineseMedicine,Taiyuan030024,China;3InstituteofNeurology,HuashanHospital,FudanUniversity,Shanghai200025,China;4DepartmentofNeurology,ThomasJeffersonUniversity,Philadelphia,PA19107,USA.E-mail:macungen2001@163.com;guang-xian.zhang@jefferson.edu)

        AIM: To explore the neuroprotective effect of fasudil combined with bone marrow-derived neural stem cells (BM-NSCs) on the mice with experimental autoimmune encephalomyelitis (EAE).METHODSFemale C57BL/6 mice (8~10 weeks old,n=32) were immunized with myelin oligodendrocyte glycoprotein 35-55 (MOG35-55) to establish chronic EAE model. The mice were randomly divided into control (ddH2O ) group, fasudil group, BM-NSCs group, and fasudil+BM-NSCs group. The clinical score and body weight were recorded every other day. The expression of neurotrophic factors was determined by immunofluorescence staining.RESULTSIn comparison with ddH2O group, fasudil combined with BM-NSCs delayed onset and ameliorated severity of EAE. The numbers of brain-derived neurotrophic factor, glial cell-derived neurotrophic factor, nerve growth factor, neurotrophin-3 and ciliary neurotrophic factor positive cells in fasudil group, BM-NSCs group and fasudil+BM-NSCs group were all increased in various extents. In particularly, the expression of these neurotrophic factors in fasudil+BM-NSCs group was significantly higher than that in the mice treated with fasudil or BM-NSCs alone (P<0.01).CONCLUSIONFasudil combined with BM-NSCs promotes the expression of neurotrophic factors and improves microenvironment of central nervous system, thus playing a positive role in neural restoration and regeneration through a synergistic and superimposed effect.

        Fasudil; Bone marrow-derived neural stem cells; Experimental autoimmune encephalomyelitis; Neurotrophic factors

        1000- 4718(2017)12- 2113- 08

        2017- 04- 17

        2017- 09- 28

        國(guó)家自然科學(xué)基金資助項(xiàng)目(No.81501198; No. 81272163); 山西大同大學(xué)青年科研基金資助項(xiàng)目(No.2013Q11);山西省回國(guó)留學(xué)人員重點(diǎn)科研資助項(xiàng)目(No. 2014-重點(diǎn)7);山西中醫(yī)學(xué)院“2011”培育計(jì)劃項(xiàng)目(No.2011PY-1)

        △通訊作者 馬存根 Tel: 18203515288; E-mail: macungen2001@163.com; 張光先 Tel: (215)-9558935; E-mail:guang-xian.zhang@jefferson.edu

        R593.1; R329.5

        A

        10.3969/j.issn.1000- 4718.2017.12.001

        (責(zé)任編輯: 陳妙玲, 宋延君)

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