GFAP啟動(dòng)子與骨髓基質(zhì)干細(xì)胞神經(jīng)膠質(zhì)分化的篩選(

1、GFAP啟動(dòng)子與骨髓基質(zhì)干細(xì)胞神經(jīng)膠質(zhì)分化的篩選 作者單位 100044 北京大學(xué)人民醫(yī)院創(chuàng)傷骨科通訊作者 姜保國 本課題受國家863計(jì)劃（2002AA205071），國家973重大基礎(chǔ)研究項(xiàng)目（2001cca01300）基金資助張培訓(xùn) 姜保國 何湘君 趙富強(qiáng) 魏光如 張殿英 傅中國 張宏波摘要 目的 研究骨髓基質(zhì)干細(xì)胞體外誘導(dǎo)分化，結(jié)合特異性GFAP啟動(dòng)子控制的EGFP的表達(dá)來篩選誘導(dǎo)的骨髓基質(zhì)干細(xì)胞。方法 構(gòu)建報(bào)告載體pGFAP-EGFP, 原代培養(yǎng)的骨髓基質(zhì)干細(xì)胞，在-ME, RA, forskolin, bFGF, PDGF-AA, HRG-等復(fù)合物的誘導(dǎo)7天后，pGFAP-EGFP載

2、體轉(zhuǎn)染，G418抗生素篩選。熒光顯微鏡下觀察存活細(xì)胞體內(nèi)的EGFP表達(dá)情況，流式細(xì)胞儀測(cè)定熒光細(xì)胞的比率。 結(jié)果 報(bào)告載體pGFAP-EGFP構(gòu)建成功，轉(zhuǎn)染骨髓基質(zhì)干細(xì)胞后部分細(xì)胞存活，流式細(xì)胞儀測(cè)定82.74%的細(xì)胞表達(dá)EGFP。篩選出的細(xì)胞GFAP免疫細(xì)胞化學(xué)鑒定陽性。體外擴(kuò)增后可以得到大量的目的細(xì)胞。 結(jié)論 特異性GFAP 啟動(dòng)子控制的EGFP的表達(dá)可以在體外有效的進(jìn)行骨髓基質(zhì)干細(xì)胞的篩選和擴(kuò)增。篩選后的細(xì)胞具有膠質(zhì)細(xì)胞表型，可提供足夠的膠質(zhì)細(xì)胞進(jìn)行神經(jīng)系統(tǒng)的細(xì)胞替代治療。關(guān)鍵詞 骨髓基質(zhì)干細(xì)胞; GFAP啟動(dòng)子; 增強(qiáng)型綠色熒光蛋白（EGFP）; 細(xì)胞標(biāo)記The differentia

3、tion selection of bone marrow stromal cells into neuralglial cells with GFAP promoter Zhang peixun, Jiang baoguo, He xiangjun, et al. Traumatic orthopaedics of People Hospital, Peking University, Beijing, 100044, China.Abstract Objective To study the bone marrow stromal cells differentiation in vitr

4、o and to select the differentiated bone marrow stromal cells with the GFAP promoter-controled EGFP expression. Methods Reconstruct the report vector pGFAP-EGFP, culture bone marrow stromal cells primarily and induce with composite of -ME, RA, forskolin, bFGF, PDGF-AA, HRG- for 7 days. . Transfected

5、bone marrow stromal cells with pGFAP-EGFP and selected with G418 antibiotic. The survived bone marrow stromal cells were observed under fluorescence microscopy and flow cytometry. Results The report vector pGFAP-EGFP was reconstructed successfully. Transfected bone marrow stromal cells surviced and

6、82.74% expressed EGFP. Survived cells were positive to GFAP immunocytochemistry staining. The survived bone marrow stromal cells can be enriched in vitro. Conclusions Bone marrow stromal cells can be selected and enriched in vitro under the GFAP-controled EGFP expression. The selected cells possesse

7、d the phenotypes of neuralglial cells and can provide plenty of cells quantities for cell substitution therapy.Key Words: Bone marrow stromal cells; GFAP promoter; EGFP; cell labeling 骨髓基質(zhì)干細(xì)胞（bone marrow stromal cells,MSCs）是成年動(dòng)物骨髓中保持分裂和分化潛能的細(xì)胞群體19 11。具有多能性分化的骨髓基質(zhì)干細(xì)胞是細(xì)胞替代療法的可能來源。MSC在體外預(yù)誘導(dǎo)后注射入周圍神經(jīng)損傷處（

8、Mari Dezawa et al 2001）或直接將MSC注射入神經(jīng)損傷處（Pedro Cuevas et al 2002）能夠促進(jìn)神經(jīng)再生過程，但只有極小一部分MSC分化為了雪旺細(xì)胞。因此建立體外篩選、擴(kuò)增、富集已分化的神經(jīng)膠質(zhì)細(xì)胞或前體細(xì)胞的方法將是一大突破。Michael Brenner發(fā)現(xiàn)了在膠質(zhì)細(xì)胞特異表達(dá)的膠質(zhì)纖維酸性蛋白GFAP的基因序列。1994年又成功克隆了在星形細(xì)胞中允許轉(zhuǎn)基因表達(dá)的特異性GFAP啟動(dòng)子，可以隨著膠質(zhì)細(xì)胞的成熟而特異性開啟并表達(dá)下游的目的基因5。本實(shí)驗(yàn)以特異性GFAP 啟動(dòng)子控制的增強(qiáng)型綠色熒光蛋白的表達(dá)來監(jiān)測(cè)MSC的分化和擴(kuò)增，旨在探索一種標(biāo)記誘導(dǎo)分化的

9、MSC并定向篩選神經(jīng)膠質(zhì)細(xì)胞,為臨床細(xì)胞替代療法提供目的細(xì)胞的方法。材料和方法1載體構(gòu)建pGfa2-cLacZ載體(gift of Micheal Brenner, National Institute of Health, Betheda,MD), 轉(zhuǎn)化感受態(tài)大腸桿菌DH5，搖菌擴(kuò)增后大提質(zhì)粒（Qiagen plasmid maxi kit）。BglII-SalI酶切pGfa2-cLacZ載體后將回收得到的小片段2.2kb的GFAP啟動(dòng)子克隆至經(jīng)VspI-SalI酶切后的pEGFP-N3載體(Clontech, gift of Tang fouchou, Beijing university

10、)中以取代原來的CMV啟動(dòng)子，得到重構(gòu)的pGFAP-EGFP-N3載體。割膠回收后轉(zhuǎn)化感受態(tài)大腸桿菌DH5，搖菌擴(kuò)增大提（Qiagen plasmid maxi kit）。重構(gòu)載體的酶切位點(diǎn)圖及酶切鑒定結(jié)果如圖A、B所示。各限制性內(nèi)切酶均購自華美公司。2 MSCs培養(yǎng)根據(jù)Friedenstein12 13和Azizi SA14的方法進(jìn)行成年SD大鼠MSC的分離。過量的苯巴比妥殺死大鼠（200g）后以-MEM (Gibco)灌注脛骨和股骨骨髓，灌注液在含10%的胎牛血清(Gibco)、200 mg/ml卡那霉素的-MEM中培養(yǎng)。同時(shí)加入10ng/ml Leukemia inhibitory fa

11、ctor (LIF, Sigma ,US)以維持MSC的分化能力。48小時(shí)后更換培養(yǎng)基，利用MSC的貼壁生長(zhǎng)特性反復(fù)更換培養(yǎng)基來去除血源性細(xì)胞。3 MSC的誘導(dǎo)分化 經(jīng)傳代篩選培養(yǎng)的MSCs在含-mercaptoethanol (-ME，1mmol/L)的-MEM培養(yǎng)基中培養(yǎng)24小時(shí)。更換培養(yǎng)基后添加all-trans-retinoic acid (RA, 35ng/ml， Sigma)培養(yǎng)3天。移去培養(yǎng)基PBS沖洗后，-MEM培養(yǎng)基培養(yǎng)7天，同時(shí)添加forskolin (FSK, 5mmol/L，Sigma)、recombinant human basic-fibroblast growth

12、 factor (bFGF, 10ng/mL，Peprotech, London, UK)、recombinant human platelet derived growth facor-AA (PDGF-AA, 5ng/mL，Peprotech, London, UK)、recombinant human heregulin-(HRG-, 200ng/mL，R&D System, Minneapolis, MN)和10%的胎牛血清。復(fù)合物誘導(dǎo)期間細(xì)胞不傳代，僅更換一次培養(yǎng)基。4誘導(dǎo)后的MSCs免疫熒光鑒定誘導(dǎo)后的MSCs進(jìn)行免疫熒光鑒定，應(yīng)用的一抗為兔抗大鼠S100, P75 (Si

13、gma, US),。二抗為羅丹明標(biāo)記的山羊抗兔IgG(H+L) (Jackson immunoResearch company,US).5. 誘導(dǎo)后的MSCs轉(zhuǎn)染應(yīng)用Lipofectamine2000和Plus試劑（Invitrogen）介導(dǎo)pGFAP-EGFP轉(zhuǎn)染誘導(dǎo)后的MSCs細(xì)胞，轉(zhuǎn)染后的MSC在含400ug/mlG418(Clontech)的培養(yǎng)基中篩選培養(yǎng)10天。轉(zhuǎn)染的各項(xiàng)操作均按照試劑說明書進(jìn)行。6篩選后存活的細(xì)胞克隆培養(yǎng)擴(kuò)增，GFAP免疫熒光鑒定 篩選后存活的細(xì)胞經(jīng)過培養(yǎng)擴(kuò)增后形成較大的克隆，對(duì)細(xì)胞克隆進(jìn)行兔抗大鼠GFAP(Sigma, US)免疫熒光鑒定。7. 篩選擴(kuò)增后的細(xì)胞

14、流式細(xì)胞儀測(cè)定表達(dá)EGFP的細(xì)胞比率 篩選擴(kuò)增以后的細(xì)胞培養(yǎng)傳代，0.25%胰蛋白酶消化，離心收集后重懸，流式細(xì)胞儀檢測(cè)。結(jié)果1報(bào)告載體pGFAP-EGFP構(gòu)建成功，限制性內(nèi)切酶切鑒定符合預(yù)計(jì)設(shè)計(jì)（見圖A）。載體的具體酶切位點(diǎn)如圖B所示。 圖1 報(bào)告載體pGFAP-EGFP的酶切鑒定圖1 為DNA/Hind III maker, 2為Xho I 和Sal I消化片段，3為XbalI消化片段。4 為EcoR I消化片段。重構(gòu)載體的酶切鑒定中Xbal I具有三個(gè)酶切位點(diǎn),其中在EGFP下游的位點(diǎn)被甲基化，不參與酶切，因此3中切出的片段只有兩條。圖2 重構(gòu)質(zhì)粒載體各個(gè)酶切位點(diǎn)Xho I 和Sal I

15、各有一個(gè)位點(diǎn)，Xbal I有三個(gè)酶切位點(diǎn)，EcoR I沒有酶切位點(diǎn)。2誘導(dǎo)后的MSCs細(xì)胞免疫熒光鑒定。S100和P75均呈現(xiàn)陽性。如圖3 圖4 所示圖 3 誘導(dǎo)的MSCs細(xì)胞S100免疫熒光染色紅色的為陽性的熒光染色； 藍(lán)色的為DAPI復(fù)染的細(xì)胞核，二者為同一視野（100x）圖 4 誘導(dǎo)的MSCs細(xì)胞P75免疫熒光染色紅色的為陽性的熒光染色； 藍(lán)色的為DAPI復(fù)染的細(xì)胞核，二者為同一視野（100x）3. 篩選后存活的細(xì)胞克隆GFAP免疫熒光鑒定結(jié)果：篩選后表達(dá)綠色熒光蛋白的細(xì)胞GFAP染色出現(xiàn)紅色的熒光。圖 5 篩選后的MSCs細(xì)胞GFAP免疫熒光染色紅色的為陽性的熒光染色； 綠色的為細(xì)胞表

16、達(dá)的綠色熒光蛋白，二者為同一視野（100x）4. 篩選后存活細(xì)胞的流式細(xì)胞儀測(cè)定熒光細(xì)胞的比率圖6 流式細(xì)胞儀測(cè)定表達(dá)EGFP的細(xì)胞比率上圖為陰性對(duì)照組，下圖為轉(zhuǎn)染篩選后的細(xì)胞，顯示82.74%的細(xì)胞表達(dá)EGFP。討論pGfa2-cLacZ載體中2163到47為h-GFAP啟動(dòng)子序列，其中+15位的密碼子ATG突變?yōu)門TG。人，小鼠和大鼠的GFAP基因具有高度同源性18。重構(gòu)載體的酶切鑒定中Xbal I具有三個(gè)酶切位點(diǎn),其中在EGFP下游的位點(diǎn)甲基化，因此切出的片段只有兩條，4道的三條帶提示重構(gòu)的質(zhì)粒載體具有三種分子構(gòu)象.酶切后得到的片段數(shù)量和大小恰好符合，提示載體重構(gòu)正確。GFAP啟動(dòng)子正確

17、插入載體替代了原來的CMV啟動(dòng)子，其下游具有EGFP的基因序列。EGFP已經(jīng)被廣泛應(yīng)用于細(xì)胞的追蹤檢測(cè)3。Marius Wernig等利用Tau基因中插入的EGFP來篩選胚胎干細(xì)胞分化來的神經(jīng)元4。Christian Andressen等利用Nestin在神經(jīng)前體細(xì)胞內(nèi)表達(dá)的特性，采用了Nestin特異性啟動(dòng)子控制的EGFP表達(dá)來篩選胚胎干細(xì)胞來源的神經(jīng)前體細(xì)胞8。Yamaguchi M等利用Nestin啟動(dòng)子控制的GFP表達(dá)來監(jiān)測(cè)中樞神經(jīng)系統(tǒng)內(nèi)的神經(jīng)形成15。我們以Micheal Brenner的GFAP啟動(dòng)子為基礎(chǔ)，重構(gòu)質(zhì)粒載體pGFAP-EGFP, 轉(zhuǎn)染MSC后經(jīng)G418篩選后，利用GF

18、AP啟動(dòng)子在神經(jīng)膠質(zhì)細(xì)胞內(nèi)特異表達(dá)來篩選來源于MSCs的神經(jīng)膠質(zhì)細(xì)胞,為臨床實(shí)驗(yàn)提供足量的細(xì)胞來源。應(yīng)用細(xì)胞替代療法的前提是獲得較高純度的細(xì)胞，通常有兩種方法4：其一是定向誘導(dǎo),這需要探索嚴(yán)格的分化外環(huán)境和特定的細(xì)胞因子作用。即使探索的條件很好也面臨分化的細(xì)胞多樣性的問題。其二是細(xì)胞系篩選，這需要相對(duì)特異的細(xì)胞系標(biāo)志。我們把兩者結(jié)合在一起,在體外進(jìn)行相對(duì)定向誘導(dǎo)后,采用特異性GFAP啟動(dòng)子篩選分化的神經(jīng)膠質(zhì)細(xì)胞,擴(kuò)增后可以進(jìn)行體內(nèi)的細(xì)胞功能替代。神經(jīng)膠質(zhì)細(xì)胞包括Schwann細(xì)胞,星形細(xì)胞,少突細(xì)胞等。Schwann細(xì)胞也表達(dá)膠質(zhì)細(xì)胞的特異蛋白GFAP。當(dāng)誘導(dǎo)后的MSCs表達(dá)GFAP，經(jīng)重構(gòu)的

19、質(zhì)粒載體轉(zhuǎn)染后，GFAP啟動(dòng)子開啟下游的綠色熒光蛋白得以表達(dá)，分化的神經(jīng)膠質(zhì)細(xì)胞中綠色熒光蛋白的表達(dá)與GFAP的表達(dá)和細(xì)胞形態(tài)的變化密切相關(guān)。表達(dá)綠色熒光蛋白的膠質(zhì)細(xì)胞可以通過熒光顯微鏡觀察，通過流式細(xì)胞儀得到分離純化。此方法可以用來標(biāo)記MSC來源的神經(jīng)膠質(zhì)細(xì)胞并監(jiān)測(cè)其分化過程和去向，甚至從MSC分化來的多種細(xì)胞中分離用于體內(nèi)移植神經(jīng)膠質(zhì)細(xì)胞。在本實(shí)驗(yàn)室培養(yǎng)MSC的過程中發(fā)現(xiàn)，大鼠MSC的體外培養(yǎng)需要LIF因子的作用，早期未加LIF的MSC在培養(yǎng)中出現(xiàn)細(xì)胞形態(tài)變大，端粒酶檢測(cè)發(fā)現(xiàn)MSC細(xì)胞衰老，分化能力減弱（未發(fā)表資料），這與Yuehua Jiang等17的發(fā)現(xiàn)一致。神經(jīng)再生微環(huán)境中增加一定數(shù)

20、量的Schwann細(xì)胞可以有效促進(jìn)神經(jīng)軸突的生長(zhǎng)6 16。獲得足量Schwann細(xì)胞以及避免免疫排斥反應(yīng)仍是一大難題。MSCs臨床獲得容易，用MSCs分化成的神經(jīng)膠質(zhì)細(xì)胞（Schwann細(xì)胞）來促進(jìn)神經(jīng)軸突再生無疑會(huì)給神經(jīng)再生帶來希望。本研究為MSC的體內(nèi)移植促進(jìn)周圍神經(jīng)再生做了前期的細(xì)胞標(biāo)記工作。致謝Thanks Professor Michael Brenner for his kindly presentation of pGfa2-cLacZ plasmid and technical support, presently Associate Professor of Neurobio

21、logy with a joint appointment in the Department of Physical Medicine & Rehabilitation, Ulabama,USA. Thanks Professor Mari Dezawa, Department of Anatomy, Yokohama City University School of Medicine, Japan, for his kindly theoritical and technical support and encouragement. 參考文獻(xiàn) 1 Devine SM, Cobbs

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