SPIO、EGFP雙標(biāo)NGF基因修飾中腦神經(jīng)干細(xì)胞的建立

1、    SPIO、EGFP雙標(biāo)NGF基因修飾中腦神經(jīng)干細(xì)胞的建立鄧興力1，龍江1，蔡山1，任仲坤1，劉如恩2，楊智勇1*(1.昆明醫(yī)學(xué)院第一附屬醫(yī)院神經(jīng)外科，昆明 650032;2.中日友好醫(yī)院神經(jīng)外科，北京 100029)摘要 目的：建立超順磁氧化鐵(SPIO)、增強(qiáng)型綠色熒光蛋白(EGFP)雙標(biāo)神經(jīng)生長(zhǎng)因子(NGF)基因修飾中腦神經(jīng)干細(xì)胞。方法：以質(zhì)粒pcDNA3-hNGF、pEGFPN1共轉(zhuǎn)染第三代大鼠胚胎中腦神經(jīng)干細(xì)胞并用SPIO標(biāo)記。熒光顯微鏡檢測(cè)EGFP的表達(dá);免疫細(xì)胞化學(xué)、western blot鑒定NGF的表達(dá);普魯士藍(lán)染色、透射電鏡鑒定

2、SPIO標(biāo)記。結(jié)果：EGFP在基因轉(zhuǎn)染12 h后開(kāi)始表達(dá);免疫細(xì)胞化學(xué)、western blot表明細(xì)胞能正確表達(dá)NGF;普魯士藍(lán)染色顯示細(xì)胞標(biāo)記率達(dá)100%，透射電鏡顯示SPIO顆粒位于吞飲小泡和胞漿內(nèi)。結(jié)論：建立了SPIO、EGFP雙標(biāo)記NGF基因修飾中腦神經(jīng)干細(xì)胞，為進(jìn)一步開(kāi)展細(xì)胞移植治療帕金森病的研究奠定基礎(chǔ)。關(guān)鍵詞 超順磁氧化鐵;增強(qiáng)型綠色熒光蛋白;神經(jīng)生長(zhǎng)因子;中腦神經(jīng)干細(xì)胞Establishment of superparamagnetic iron oxide and enhanced fluorescence protein double labeled nerve grow

3、th factor gene modified midbrain derived neural stem cellsDeng Xing-li1, Long Jiang1, Cai Shan1, Ren Zhong-kun1, Liu Ru-en2, Yang Zhi-yong1(1. Department of Neurosurgery, First Affiliated Hospital of Kunming Medical College, Kunming 650032; 2. Department of Neurosurgery, China-Japan Friendship Hospi

4、tal, Beijing, 100029, China)Abstract Objective: To establish superparamagnetic iron oxide (SPIO) and enhanced green fluorescence protein (EGFP) double labeled nerve growth factor (NGF) gene modified neural stem cells. Methods: The rat embryonic midbrain derived neural stem cells of the third passage

5、 were co-transfected with plasmid pcDNA3-hNGF and pEGFPN1 and labeled with SPIO. The expression of EGFP was observed with fluorescence microscope and the expression of NGF was analyzed by immunocytochemistery as well as western blot. Prussian blue stain and transmission electron microscopy was used

6、to identify the SPIO particles in cells. Results: The expression of EGFP was initially found 12 hours after transfection. The immunocytochemistery and western blot showed the NGF was expressed successfully in cells. Prussian blue stain showed the percentage of labeled cells was 100%. Transmission el

7、ectron microscopy showed vacuolar structures of different sizes under the cytoplasma within and outside of which there were highly density particles. Conclusion: The SPIO and EGFP double labeled NGF gene modified midbrain derived neural stem cells were successfully established, which will provide th

8、e foundation for the further research about cell therapy of Parkinson disease.Key words superparamagnetic iron oxide; enhanced green fluorescent protein; nerve growth factor; midbrain derived neural stem cells神經(jīng)生長(zhǎng)因子(nerve growth factor，NGF)在神經(jīng)再生的基礎(chǔ)及臨床研究中具有重要的理論意義和實(shí)用價(jià)值1-4。然而，NGF屬生物大分子，不能直接通過(guò)血腦屏障，極大地限

9、制了其應(yīng)用?；蛑委熓悄壳敖鉀QNGF給藥途徑問(wèn)題最有希望的方案。神經(jīng)干細(xì)胞(neural stem cells，NSCs)被認(rèn)為是目前中樞神經(jīng)系統(tǒng)基因治療的理想載體5-6。胚胎腹側(cè)中腦來(lái)源的NSCs(midbrain derived neural stem cells，mNSCs)是細(xì)胞移植治療PD的理想細(xì)胞源7-8。綠色熒光蛋白(green fluorescent protein，GFP)基因標(biāo)記技術(shù)具有高效、穩(wěn)定、無(wú)毒、易于檢測(cè)等特性，能在活細(xì)胞中直接觀察其表達(dá)9。MRI可顯示磁性納米材料超順磁氧化鐵(Superparamagnetic Iron Oxide，SPIO)標(biāo)記的細(xì)胞，實(shí)現(xiàn)對(duì)移

10、植入體內(nèi)細(xì)胞存活、遷徙情況的活體動(dòng)態(tài)觀察10。本研究將建立SPIO、EGFP雙標(biāo)記NGF基因修飾中腦神經(jīng)干細(xì)胞，為進(jìn)一步應(yīng)用其移植治療帕金森病的研究奠定基礎(chǔ)。材料和方法1 材料質(zhì)粒pcDNA3-hNGFb，pEGFPN1;E. coli DH5;E14 SD孕鼠;DL2000 DNA marker，-EcoT14 I digestion DNA marker(Takara);Hind III，xho I(MBI);無(wú)內(nèi)毒素質(zhì)粒提取試劑盒(Qiagen);DMEM/F12(Hyclone);Opti-MEM I低血清培養(yǎng)基，G418，N2，胎牛血清(Invitrogen);EGF，bFGF(R&

11、amp;D Systems);FuGENE HD轉(zhuǎn)染試劑(Roche);Feridex(Advanced Magnetic); 小鼠抗巢蛋白(nestin)單克隆抗體(abcam);兔抗NGF多克隆抗體(SANTA CRUZ);TRITC標(biāo)記山羊抗兔/小鼠IgG，HRP標(biāo)記山羊抗兔IgG，BSA(北京中山金橋);LumiGLO化學(xué)發(fā)光底物(KPL);Pro-PREPTM蛋白提取試劑(北京賽柏盛)。2 方法2.1 質(zhì)粒的純化與鑒定 質(zhì)粒pcDNA3-hNGF、pEGFPN1轉(zhuǎn)化E. coli DH5，分別接種LB/Amp、LB/Kana平板37 培養(yǎng)過(guò)夜，挑取單菌落，搖菌擴(kuò)增后用Endo-Fre

12、e Plasmid Maxi Kit純化質(zhì)粒，紫外分光光度法測(cè)定其濃度和純度，限制酶Hind III、xho I雙酶切鑒定質(zhì)粒pcDNA3- hNGF。2.2 大鼠胚胎mNSCs的分離培養(yǎng)與鑒定 參考我們的方法11，常規(guī)分離培養(yǎng)大鼠胚胎mNSCs。2.3 NGF/EGFP基因修飾 (1)基因轉(zhuǎn)染: 轉(zhuǎn)染前1 d離心收集神經(jīng)球，機(jī)械分離制備單細(xì)胞懸液，106個(gè)/孔(2 ml)接種6孔板，37 5% CO2培養(yǎng)1824 h。取質(zhì)粒pcDNA3-hNGF與pEGFPN1各1 g、FuGENE HD轉(zhuǎn)染試劑6 l加入100 l Opti-MEM I低血清培養(yǎng)基形成轉(zhuǎn)染復(fù)合物，將其均勻滴加于神經(jīng)干細(xì)胞培

13、養(yǎng)液，輕輕晃動(dòng)培養(yǎng)板使之混合均勻。37 50 ml/L CO2培養(yǎng)72 h后按1×105/ml的密度傳代，更換含有G418(100 g/ml)的神經(jīng)干細(xì)胞培養(yǎng)液進(jìn)行篩選。同時(shí)設(shè)置轉(zhuǎn)染pcDNA3和不轉(zhuǎn)染基因的細(xì)胞為陰性對(duì)照?；蜣D(zhuǎn)染后，熒光倒置顯微鏡下觀察細(xì)胞產(chǎn)生綠色熒光的情況。(2)免疫細(xì)胞化學(xué): 基因轉(zhuǎn)染48 h，離心收集細(xì)胞，以神經(jīng)干細(xì)胞分化培養(yǎng)液(DMEM/F12、1% N2、10%胎牛血清)重懸，接種于預(yù)先放置多聚賴氨酸包被蓋玻片的培養(yǎng)板中，37 5% CO2 培養(yǎng)1 h后進(jìn)行nestin、NGF免疫細(xì)胞化學(xué)鑒定。(3)Western Blot： 基因轉(zhuǎn)染48 h，離心收集

14、細(xì)胞，提取總蛋白，Brandford法定量?？偟鞍?0 g進(jìn)行15% SDS-PAGE，電泳結(jié)束取出凝膠，用電轉(zhuǎn)印儀將蛋白轉(zhuǎn)移在硝化纖維膜上。轉(zhuǎn)移膜經(jīng)5%脫脂奶粉封閉后，分別與兔抗NGF多抗(1200，SANTA CRUZ，sc-548)、HRP標(biāo)記的山羊抗兔IgG孵育(150，KPL)，然后與LumiGLO化學(xué)發(fā)光底物孵育后X光片直接曝光。2.4 SPIO標(biāo)記與鑒定 (1)SPIO標(biāo)記：標(biāo)記前1 d離心收集神經(jīng)球，機(jī)械分離制備單細(xì)胞懸液，106個(gè)/孔(2ml)接種6孔板，37 5% CO2培養(yǎng)1824 h。取Feridex(11.2 mg Fe/ml)4 l、FuGENE HD轉(zhuǎn)染試劑2 l

15、加入100 l Opti-MEM I低血清培養(yǎng)基形成Feridex-FuGENE復(fù)合物，將其均勻滴加于神經(jīng)干細(xì)胞培養(yǎng)液，輕輕晃動(dòng)培養(yǎng)板使之混合均勻。37 5% CO2培養(yǎng)24 h后按1×105個(gè)/ml的密度傳代繼續(xù)培養(yǎng)供進(jìn)一步實(shí)驗(yàn)用。(2)普魯士藍(lán)染色： 離心收集細(xì)胞，以神經(jīng)干細(xì)胞分化培養(yǎng)液重懸，接種于預(yù)先放置多聚賴氨酸包被蓋玻片的6孔板中，37 5% CO2培養(yǎng)1h使細(xì)胞充分貼壁，D-Hanks液漂洗2遍，4%多聚甲醛固定30 min，蒸餾水洗滌3遍，Perls反應(yīng)液(2% 亞鐵氰化鉀、6%鹽酸，新鮮配制)作用30 min，蒸餾水洗滌2遍，核固紅復(fù)染12 min，蒸餾水洗去多余的核

16、固紅，顯微鏡下觀察。(3)透射電鏡觀察：離心收集細(xì)胞，戊二醛固定，四氧化鋨后固定，經(jīng)脫水、包埋后制成超薄切片，鉛鈾染色后透射電鏡下觀察細(xì)胞的超微結(jié)構(gòu)并照相。結(jié) 果1 質(zhì)粒的純化與鑒定質(zhì)粒經(jīng)擴(kuò)增純化后，紫外分光光度法測(cè)定其OD260/OD280為1.98、濃度為0.5 g/l。重組質(zhì)粒pcDNA3- hNGF經(jīng)Hind III和xho I酶切后，可見(jiàn)750bp和5.2kb兩條帶，與預(yù)期結(jié)果一致(Fig.1)。2 NGF /EGFP基因修飾基因轉(zhuǎn)染12 h可觀察到少量EGFP陽(yáng)性細(xì)胞，24 h EGFP陽(yáng)性細(xì)胞明顯增加并于48 h 達(dá)到頂峰(轉(zhuǎn)染效率為8%10%)。經(jīng)G418篩選3 周后EGFP陽(yáng)

17、性細(xì)胞形成細(xì)胞克隆，4 周后細(xì)胞克隆增大形成神經(jīng)球。免疫細(xì)胞化學(xué)染色顯示細(xì)胞呈nestin、NGF免疫陽(yáng)性反應(yīng)(Fig.2)。此外，Western Blot可檢測(cè)到細(xì)胞內(nèi)NGF的表達(dá)(Fig.3)。3 SPIO標(biāo)記mNSCs的鑒定普魯士藍(lán)染色結(jié)果顯示，SPIO標(biāo)記mNSCs胞漿內(nèi)有大量藍(lán)染鐵顆粒，標(biāo)記率為100%;而對(duì)照組胞漿內(nèi)未見(jiàn)著色顆粒(Fig.4)。透射電鏡顯示，高電子致密度的鐵顆粒位于mNSCs吞飲小泡和胞漿內(nèi)(Fig.5)。Fig. 1 Analyze plasmid pcDNA3-hNGF by enzyme digestion. Lane 1: -EcoT14 I digest

18、DNA Marker; lane 2: pcDNA3-hNGF digested by Hind III; lane 3: pcDNA3-hNGF digested by Hind III and xho I , which was digested into 750 bp and 5200 bp fragments; lane 4: DNA Marker-C.Fig. 2 Establishment of NGF-EGFP gene modified rat mNSCs. A: 12 h after transfection, the GFP positive cells could be

19、observed under fluorescence microscope, which peaked at 48 hours (×200); B: 72 h after transfection, the cells were screened by G418. 3 wk later, the GFP positive cells formed cell colonies (×200); C and E: cell colonies increased in size and formed neurospheres after screen for 4 wk (

20、5;200); D: immunofluorescence photomicrograph showed all neurospheres were immunoreactive for the NSCs marker nestin (×200); F: immunofluorescence photomicrograph showed the neurospheres of plasmid pcDNA3-hNGF, pEGFPN1 co-transfection group were strongly immunopositive for NGF (×200).Fig.

21、3 Western Blot identification of NGF expression in rat mNSCs. Lane 1: plasmid pcDNA3-hNGF, pEGFPN1 co-transfection group; lane 2: plasmid pcDNA3-hNGF transfection group; lane 3: plasmid pcDNA3 transfection group; lane 4: non-transfection group.Fig. 4 Prussian blue stain identification of SPIO labele

22、d rat mNSCs. A: There are numerous blue stained particles in the cytoplasma of SPIO labeled cells (×200); B: there is not any blue stained particle in cells of control group (×200).Fig. 5 Transmission electron microscopy identification of SPIO labeled rat mNSCs. A: There are numerous high

23、density particles in the cytoplasma of SPIO labeled cells (×10000); B: there is not any high density particle in cells of control group (×10000).討 論NGF對(duì)多種神經(jīng)元的發(fā)育分化與生長(zhǎng)再生具有維持和促進(jìn)作用1-4。由于組織中NGF含量極微，不足以滿足神經(jīng)再生的需要，加之其屬于生物大分子，不能通過(guò)血腦屏障，故如何有效地將NGF導(dǎo)入體內(nèi)成為近年神經(jīng)科學(xué)研究的熱點(diǎn)?；蛑委熓悄壳敖鉀QNGF給藥途徑問(wèn)題最有希望的方案。NSCs具有來(lái)源豐富

24、，增殖、分化能力強(qiáng)，能整合于宿主細(xì)胞，易于基因修飾，極低或無(wú)免疫源性，無(wú)致瘤性等特點(diǎn)，是中樞神經(jīng)系統(tǒng)基因治療的理想載體5-6。最近研究表明，源于胚胎中樞神經(jīng)系統(tǒng)各區(qū)域的NSCs具有不同的分化潛能， mNSCs更易于分化為TH免疫陽(yáng)性的多巴胺能神經(jīng)元7,8。GFP基因標(biāo)記技術(shù)是近年來(lái)迅速發(fā)展起來(lái)的一種新型細(xì)胞示蹤技術(shù)，無(wú)須反應(yīng)底物即可產(chǎn)生綠色熒光，敏感性強(qiáng)且性質(zhì)穩(wěn)定，不干擾細(xì)胞功能，易于檢測(cè)，可實(shí)現(xiàn)活細(xì)胞的實(shí)時(shí)定位觀察。EGFP是野生型GFP的突變型，熒光強(qiáng)度增加了35倍，大大提高了檢測(cè)靈敏度，已成為細(xì)胞生物學(xué)研究廣泛應(yīng)用的標(biāo)志分子9。本研究應(yīng)用FuGene HD轉(zhuǎn)染試劑將NGF表達(dá)質(zhì)粒pcDN

25、A3-hNGF與EGFP表達(dá)質(zhì)粒pEGFPN1共轉(zhuǎn)染大鼠胚胎mNSCs，經(jīng)G418篩選獲得NGF/EGFP穩(wěn)定表達(dá)mNSCs，熒光顯微鏡下可檢測(cè)到綠色熒光，同時(shí)免疫細(xì)胞化學(xué)染色示NGF免疫反應(yīng)陽(yáng)性、western blot可檢測(cè)到NGF表達(dá)，說(shuō)明NGF和EGFP能在mNSCs中正確表達(dá)。新霉素抗性基因(Neor)-G418選擇系統(tǒng)為常用的基因轉(zhuǎn)染篩選系統(tǒng)。G418通過(guò)抑制核糖體功能而阻止哺乳動(dòng)物細(xì)胞蛋白質(zhì)合成。Neor基因編碼一種磷酸轉(zhuǎn)移酶，其能使G418失活12。質(zhì)粒pcDNA3-hNGF和pEGFPN1均含有Neor基因，故成功轉(zhuǎn)染的mNSCs能在含有G418的選擇性培養(yǎng)液中正常生長(zhǎng)，而未

26、被轉(zhuǎn)染的mNSCs則逐漸死亡。Feridex是一種新型MR對(duì)比劑，核心為Fe2O3，外包葡聚糖，直徑(50±29)nm。由于其具有非常小的晶體結(jié)構(gòu)，F(xiàn)eridex在外加磁場(chǎng)中，呈單一磁矩順磁場(chǎng)排列，即使在較弱的磁場(chǎng)中也能產(chǎn)生較大的磁性，而外加磁場(chǎng)撤銷后磁性也迅速消失，即具有所謂的超順磁性。Feridex不均勻分布于組織后，造成局部磁場(chǎng)不均勻，從而縮短了組織的橫向弛豫時(shí)間(T2)和縱向弛豫時(shí)間(T1)，以T2值縮短更為明顯，表現(xiàn)為T2 WI信號(hào)降低13。MR具有較高敏感度和分辨率，可提供細(xì)胞級(jí)分辨率的圖像，故通過(guò)MRI示蹤Feridex標(biāo)記的細(xì)胞可以了解其在宿主體內(nèi)的存活和遷徙情況9。

27、由于細(xì)胞膜與Feridex均帶有負(fù)電荷，故本研究應(yīng)用帶有正電荷的FuGene HD轉(zhuǎn)染試劑通過(guò)靜電作用包裹Feridex，從而使其通過(guò)靜電作用與細(xì)胞表面受體結(jié)合，進(jìn)而經(jīng)內(nèi)吞作用進(jìn)入細(xì)胞內(nèi)。普魯士藍(lán)染色證實(shí)Feridex進(jìn)入細(xì)胞內(nèi)，細(xì)胞標(biāo)記率達(dá)100%，透射電鏡檢查結(jié)果顯示標(biāo)記細(xì)胞吞飲小泡和胞漿中含有高電子致密度的Feridex顆粒，證實(shí)應(yīng)用FuGene HD轉(zhuǎn)染試劑介導(dǎo)的標(biāo)記方法標(biāo)記率高，簡(jiǎn)單易行。研究表明，較高的細(xì)胞密度、較長(zhǎng)的標(biāo)記時(shí)間及較高濃度的SPIO有助于提高SPIO的細(xì)胞標(biāo)記率，然而延長(zhǎng)標(biāo)記時(shí)間及提高SPIO的濃度將導(dǎo)致細(xì)胞的活性降低14-16。本研究采用5×105/ml的

28、細(xì)胞密度、25 g Fe/ml Feridex標(biāo)記24h，標(biāo)記效果良好。巢蛋白(nestin)是NSCs特征性的生物學(xué)標(biāo)志17。本研究建立的SPIO、EGFP雙標(biāo)NGF基因修飾大鼠胚胎mNSCs呈nestin免疫陽(yáng)性且具有克隆形成能力并在連續(xù)傳代培養(yǎng)中依舊維持這種克隆能力，說(shuō)明其具有自我更新能力。因此，NGF/EGFP基因修飾及SPIO標(biāo)記對(duì)大鼠胚胎mNSCs的增殖能力無(wú)明顯影響。綜上所述，本研究建立了SPIO、EGFP雙標(biāo)NGF基因修飾大鼠胚胎mNSCs，為進(jìn)一步應(yīng)用其移植治療帕金森病的研究奠定基礎(chǔ)。參考文獻(xiàn)1 Sofroniew MV, Howe CL, Mobley WC. Nerve

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