非病毒載體負載增強型綠色熒光蛋白轉(zhuǎn)染永生化神經(jīng)前體細胞

1、非病毒載體負載增強型綠色熒光蛋白轉(zhuǎn)染永生化神經(jīng)前體細胞            非病毒載體負載增強型綠色熒光蛋白轉(zhuǎn)染永生化神經(jīng)前體細胞    2008-5-9 12:23:44                      &

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3、                                    作者：桂伶俐，劉志恒，張傳漢，高峰    【關(guān)鍵詞】  綠色熒光蛋白質(zhì)類；,基因表達；,轉(zhuǎn)染；,神經(jīng)前體細胞 

4、0;  Transfection of enhanced green fluorescent protein into immortalized neural progenitor cells by nonviral vectors    【Abstract】 AIM: To investigate an effective transfection method of nonviral vectors and the expression of enhanced green fluorescent protein (EGFP) in

5、 the transfected immortalized neural progenitor cells (INPC). METHODS: The plasmid EGFPC1 was transfected into INPC by three different nonviral vectors respectively, including Lipofectamine 2000, TRANSfection and Sofast. The expression of EGFP and the transfection efficiency of the 3 vectors were me

6、asured at 24 h after transfection. The transfection efficiency of the vector which was the most efficient, was detected 12, 24, 48 and 72 h after transfection to determine the expression peak of EGFP. The viability of transfected and nontransfected cells was measured by trypan blue rejection test. A

7、fter the plasmid EGFPC1 was transfected into INPC, the stable cell clone designated INPC/EGFP was isolated by G418 selection. The positive rate of EGFP was observed. And the specific molecular marker of neural progenitor cells, nestin, was detected using immunocytochemistry. After INPC/EGFP was indu

8、ced by 50 mL/L fetal bovine serum, the cell morphology and the expression of EGFP were observed in the differentiated cells. RESULTS: The transfection efficiency of Lipofectamine 2000, TRANSfection and Sofast was (25.5±2.9)%, (4.0±1.7)%, (7.9±1.4)% respectively, at 24 h after transfec

9、tion. And Lipofectamine 2000 was the most efficient. Its transfection efficiency at 12, 24, 48 and 72 h after transfection was (17.1±0.7)%, (25.5±2.9)%, (19.4±0.9)%, (15.6±1.4)%, respectively. The expression of EGFP was the highest at 24 h after transfection. The positive rate of

10、 EGFP was 95% in INPC/EGFP. And INPC/EGFP was still nestin positive. The differentiated cells presented as neurons or astrocytes, and EGFP was still found in their somas and processes. CONCLUSION: Extrinsic genes can be effectively transfected into INPC by Lipofectamine 2000. And EGFP is one of the

11、valuable tracking tools for INPC.    【Keywords】 green fluorescent protein; gene expression; transfection; neural progenitor cell    【摘要】 目的: 尋找能高效轉(zhuǎn)染永生化神經(jīng)前體細胞(INPC)的非病毒載體，觀察轉(zhuǎn)染后增強型綠色熒光蛋白(EGFP)的表達. 方法: 用陽離子脂質(zhì)體Lipofectamine 2000，TRANSfection或陽離子聚合物Sofast分別負載質(zhì)粒EGFP

12、C1轉(zhuǎn)染INPC，利用熒光顯微鏡檢測轉(zhuǎn)染后24 h的轉(zhuǎn)染效率. 對轉(zhuǎn)染效率最高的一種載體，觀察其轉(zhuǎn)染后12，24，48和72 h轉(zhuǎn)染效率，確定EGFP表達最高峰. 用臺盼藍排斥試驗檢測轉(zhuǎn)染和未轉(zhuǎn)染細胞的活力. 質(zhì)粒EGFPC1轉(zhuǎn)染INPC后，經(jīng)G418篩選，挑選細胞克隆，命名為INPC/EGFP,觀察其EGFP陽性率. 應用巢蛋白抗體鑒定INPC/EGFP. 50 mL/L胎牛血清誘導INPC/EGFP分化，觀察分化后細胞的形態(tài)及EGFP表達. 結(jié)果: Lipofectamine 2000，TRANSfection和Sofast轉(zhuǎn)染INPC后24 h，轉(zhuǎn)染效率分別為(25.5±2.9

13、)%，(4.0±1.7)%，(7.9±1.4)%. Lipofectamine 2000的轉(zhuǎn)染效率最高. 其轉(zhuǎn)染后12，24，48和72 h的轉(zhuǎn)染效率分別為(17.1±0.7)%，(25.5±2.9)%，(19.4±0.9)%，(15.6±1.4)%，EGFP在轉(zhuǎn)染后24 h表達最高. INPC/EGFP中，EGFP陽性率約為95%. INPC/EGFP巢蛋白表達陽性，其分化后呈神經(jīng)元或星形膠質(zhì)細胞樣，且胞體及突起中仍可見綠色熒光.  結(jié)論: Lipofectamine 2000可高效轉(zhuǎn)染INPC. EGFP是INPC理想的

14、示蹤方法之一.    【關(guān)鍵詞】 綠色熒光蛋白質(zhì)類；基因表達；轉(zhuǎn)染；神經(jīng)前體細胞    0引言    永生化神經(jīng)前體細胞（immortalized neural progenitor cell, INPC）具有自我更新、無限增殖及多向分化的潛能，且較原代神經(jīng)前體細胞易行基因操作，成為神經(jīng)系統(tǒng)損傷細胞替代及基因治療的理想靶細胞. 而選擇高效的外源基因?qū)胂到y(tǒng)對基因治療的效果至關(guān)重要. 盡管病毒介導的基因轉(zhuǎn)染具有更佳的轉(zhuǎn)染效率，但由于其安全性和技術(shù)方面的原因,探討高效率的非病毒載

15、體基因轉(zhuǎn)染方法更具意義. 本實驗擬以增強型綠色熒光蛋白(enhanced green fluorescent protein, EGFP)為報告基因， 采用不同種類陽離子脂質(zhì)體或陽離子聚合物轉(zhuǎn)染大鼠INPC, 檢測其轉(zhuǎn)染效率， 并觀察EGFP的表達， 以期尋找轉(zhuǎn)染效率較高的非病毒載體及理想的INPC示蹤方法.    1材料和方法    1.1材料    Neurobasal，B27添加劑，胎牛血清購自美國GIBCO公司；堿性成纖維生長因子（basic fibroblast g

16、rowth factor, bFGF）、表皮生長因子（epidermal growth factor, EGF）購自英國PeproTech公司；多聚鳥氨酸、明膠購自美國SIGMA公司； Lipofectamine 2000購自美國Invitrogen公司；TRANSfection購自北京天為時代公司；Sofast購自北京天來公司；巢蛋白抗體購自美國NeoMarkers公司；免疫組化試劑盒購自北京中山公司；質(zhì)粒提取試劑盒購自上海華舜公司；攜帶質(zhì)粒EGFPC1（美國BD公司）的菌種由同濟醫(yī)院麻醉學教研室保存.    1.2方法  

17、0;     原代大鼠神經(jīng)前體細胞后建立的INPC，由同濟醫(yī)院麻醉學教研室構(gòu)建并保存. INPC應用含20 mL/L B27，20 g/L bFGF，20 g/L EGF的無血清Neurobasal培養(yǎng)基，在預先采用10 mg/L多聚鳥氨酸和2 g/L明膠包被的六孔板中貼壁培養(yǎng).        將攜帶EGFPC1質(zhì)粒的菌種接種于含氨芐青霉素的LB培養(yǎng)基中，振搖12 h，按試劑盒操作說明提取質(zhì)粒，采用瓊脂糖凝膠電泳及紫外分光光度法確定質(zhì)粒的純度與濃度. 分別參照陽離子

18、脂質(zhì)體Lipofectamine 2000，TRANSfection及陽離子聚合物Sofast的說明書進行轉(zhuǎn)染（轉(zhuǎn)染試劑與質(zhì)粒按說明書推薦量）. 將兩者混合物輕輕滴加于匯合率為80%90%的INPC中，繼續(xù)培養(yǎng)5 h后換液. 每組設(shè)3 個復孔. 轉(zhuǎn)染后24 h，倒置熒光顯微鏡觀察EGFP的表達. 分別記錄每個轉(zhuǎn)染細胞培養(yǎng)孔隨機5個視野內(nèi)明視野和暗視野所觀察到的細胞數(shù). 轉(zhuǎn)染效率（%）暗視野發(fā)綠色熒光細胞數(shù)/明視野細胞數(shù)×100%. 對轉(zhuǎn)染效率最高的一種載體，在轉(zhuǎn)染后12，24，48和72 h觀察轉(zhuǎn)染效率.        細胞活力取出同期培養(yǎng)的未轉(zhuǎn)染和轉(zhuǎn)染剛結(jié)束的細胞培養(yǎng)板，室溫下用0.4 g/L臺盼藍溶