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提高Lipofectamine2000對PC12細(xì)胞轉(zhuǎn)染效率的研究

2014-02-21 03:38:50邱燁
中國醫(yī)藥導(dǎo)報 2014年3期
關(guān)鍵詞:精胺氯喹脂質(zhì)體

邱燁

[摘要] 目的 探討提高PC12細(xì)胞轉(zhuǎn)染效率的方法。 方法 細(xì)胞培養(yǎng)板用10 μg/mLⅠ型牛膠原蛋白包被,通過在轉(zhuǎn)染過程中加入9 μmol/L趨溶酶體試劑氯喹及8 μmol/L聚胺類試劑亞精胺,同時調(diào)整Lipofectamine2000與DNA用量的比例和轉(zhuǎn)染時間??疾霥NA與Lipofectamine2000的比例對PC12細(xì)胞轉(zhuǎn)染效率的影響,轉(zhuǎn)染時間對PC12細(xì)胞轉(zhuǎn)染效率的影響,氯喹、亞精胺用量對PC12細(xì)胞轉(zhuǎn)染效率的影響,氯喹、亞精胺對PC12細(xì)胞活性的影響,氯喹、亞精胺對PC12細(xì)胞神經(jīng)軸突生長的影響及與4種常用脂質(zhì)體轉(zhuǎn)染試劑對PC12細(xì)胞轉(zhuǎn)染效率的比較。結(jié)果 ①對PC12細(xì)胞轉(zhuǎn)染,DNA∶Lipofectamine2000用量比例應(yīng)控制在1∶4。②當(dāng)轉(zhuǎn)染時間分別為1、2、4、8、24 h時,PC12細(xì)胞轉(zhuǎn)染率分別為35.5%、37.9%、40.5%、40.3%及38.6%,4 h達(dá)到最大轉(zhuǎn)染效率。③氯喹、亞精胺加入濃度的增加,PC12細(xì)胞轉(zhuǎn)染效率隨之增加,當(dāng)氯喹、亞精胺加入終濃度分別增至9 μmol/L和8 μmol/L時,PC12細(xì)胞的轉(zhuǎn)染效率最高,轉(zhuǎn)染效率為40.5%。④加入終濃度為9 μmol/L氯喹與8 μmol/L亞精胺前、后MTT試驗(yàn)吸光度(590 nm)分別為(0.466±0.042)與(0.451±0.038),差異無統(tǒng)計(jì)學(xué)意義(P > 0.05),對PC12細(xì)胞的活性無影響。⑤加入氯喹、亞精胺前、后,PC12細(xì)胞的神經(jīng)軸突生長數(shù)量與長度差異無統(tǒng)計(jì)學(xué)意義(P > 0.05)。⑥FuGENE、PolyJet、Lipofectamine LTX and Plus、Lipofectamine2000 4種脂質(zhì)體轉(zhuǎn)染試劑對PC12細(xì)胞轉(zhuǎn)染效率分別為10.5%、8.6%、11.8%及15.3%,本法PC12細(xì)胞轉(zhuǎn)染率為40.5%,差異有高度統(tǒng)計(jì)學(xué)意義(P < 0.01)。 結(jié)論 加入氯喹及亞精胺,實(shí)現(xiàn)陽離子脂質(zhì)體Lipofectamine2000對PC12細(xì)胞的高效轉(zhuǎn)染,為PC12細(xì)胞進(jìn)行神經(jīng)細(xì)胞基因功能及開發(fā)遺傳病治療方案等生物學(xué)研究提供了一種安全、廉價的新方法。

[關(guān)鍵詞] PC12細(xì)胞;Lipofectamine2000;陽離子脂質(zhì)體/DNA復(fù)合物;氯喹;亞精胺

[中圖分類號] R735.7 [文獻(xiàn)標(biāo)識碼] A [文章編號] 1673-7210(2014)01(c)-0020-04

Study of enhancement of the transfection efficiency in PC12 cells transfected with Lipofectamine 2000

QIU Ye

Department of Laboratory, Nanxun Hospital of Integrated Traditional Chinese and Western Medicine of Huzhou City, Zhejiang Province, Huzhou 313009, China

[Abstract] Objective To study the method of enhancement of the transfection efficiency of PC-12 cells. Methods Cell culture plates were coated by 10 μg/mL collagen typeⅠ, with the help of the simultaneous treatment with the lysosomotropic agent chloroquine at the concentration of 9 μmol/L combined with the polyamine spermidine at the concentration of 8 μmol/L, and adjustment of the ratio of NDA∶Lipofectamine2000 transfection reagents and the transfecting time. The influence of ratio of NDA∶Lipofectamine2000 on PC12 cell transfection efficiency, the influence of transfection time on PC12 cell transfection efficiency, the influence of chloroquine, spermine dosage on PC12 cell transfection efficiency, the influence of chloroquine, spermine on PC12 cell activity, the influence of chloroquine, spermine on PC12 cell neural axon growth were investigated; PC12 cell transfection efficiency among the improving method and 4 commonly used liposome transfection reagents were compared. Results ①It was right to control the ratio of NDA∶Lipofectamine2000 at 1∶4. ②PC12 cells transfection rate were 35.5%, 37.9%, 40.5%, 40.3%, 38.6% respectively at the time point of 1, 2, 4, 8, 24 h; the biggest transfection efficiency was appeared at the time point of 4 h. ③PC12 cells transfection rate was increased as the concentration increase of chloroquine and spermine; the biggest transfection efficiency of 40.5% was appeared at the 9 μmol/L concentration of chloroquine and 8 μmol/L concentration of spermine. ④Absorbancy (590 nm) of MTT before and after the 9 μmol/L chloroquine and 8 μmol/L spermine were (0.466±0.042) and (0.451±0.038) respectively, the differences were not statistically significant (P > 0.05). ⑤The differences of neural axon growth number and length before and after the chloroquine and spermine was not statistically significant (P > 0.05). ⑥PC12 cell transfection efficiency of FuGENE, PolyJet, Lipofectamine LTX and Plus, Lipofectamine2000 were 10.5%, 8.6%, 11.8%, 15.3% respectively; PC12 cell transfection efficiency of this method was 40.5%, the differences were statistically significant (P < 0.01). Conclusion Increased transfection efficiency of PC-12 cells was achieved with the help of adding chloroquine and spermidine, provides a novel, safe and inexpensive way to study gene functions of neuronal cells and develop gene therapies for inherited diseases.

[Key words] PC-12 cells; Lipofectamine2000; Cationic liposome/DNA compound; Chloroquine; Spermidine

PC12細(xì)胞誘導(dǎo)產(chǎn)生的神經(jīng)軸突能作為研究神經(jīng)退行性疾病及脊柱損傷的研究模型[1-2],也是研究神經(jīng)內(nèi)分泌試驗(yàn)的模型[3]。細(xì)胞轉(zhuǎn)染技術(shù)是分子生物學(xué)及基因工程關(guān)鍵步驟之一[4],也是體外研究神經(jīng)細(xì)胞特定基因表達(dá)調(diào)控及遺傳病基因治療的重要技術(shù)之一[5],但常用的陽離子脂質(zhì)體轉(zhuǎn)染試劑對PC12細(xì)胞轉(zhuǎn)染效率低[1,6-8]。本研究參照相關(guān)文獻(xiàn)[8-10],通過在陽離子脂質(zhì)體轉(zhuǎn)染過程中加入氯喹及亞精胺,同時調(diào)整陽離子脂質(zhì)體轉(zhuǎn)染試劑與DNA用量的比例和轉(zhuǎn)染時間,實(shí)現(xiàn)提高PC12細(xì)胞轉(zhuǎn)染率,且未影響細(xì)胞的正常功能,為PC12細(xì)胞進(jìn)行神經(jīng)細(xì)胞基因功能及開發(fā)遺傳病治療方案等生物學(xué)研究提供了一種安全、廉價的新方法。

1 材料與方法

1.1 試劑與儀器

主要試劑:PC12細(xì)胞(American Culture Collection),RPMI1640培養(yǎng)基(Sigma公司),Lipofectamine2000(Invitrogen公司),F(xiàn)uGENE HD(Roche公司),PolyJet(Signagen Laboratoies公司),Lipofectamine LTX and Plus(Invitrogen公司),pEGFP-N1(BD Biosciences公司),氯喹(Sigma公司),亞精胺(Sigma公司),Ⅰ型牛膠原蛋白(BD Biosciences公司),神經(jīng)生長因子(NGF)(Peprotech公司),噻唑藍(lán)(MTT)(Sigma公司)。Axiovert 100 M倒置熒光顯微鏡(Carl Zeiss公司),MetaMorph圖像分析處理軟件(Molecular Devices公司),EL808酶標(biāo)儀(Bio-Tek Instrument公司)。

1.2 方法

1.2.1 PC12細(xì)胞培養(yǎng) PC12細(xì)胞培養(yǎng)參照相關(guān)文獻(xiàn)[11-12],細(xì)胞密度至70%左右時進(jìn)行傳代,傳至2~3代的PC12細(xì)胞備用。

1.2.2 PC12細(xì)胞轉(zhuǎn)染 PC12細(xì)胞培養(yǎng)參照文獻(xiàn)[11]的方法,轉(zhuǎn)染的基本步驟[8]:將4 μL Lipofectamine2000轉(zhuǎn)染試劑加入75 μL不含血清的新鮮RPMI 1640培養(yǎng)基中混勻,同時將1 μg質(zhì)粒pEGFP-N1加入150 μL不含血清的新鮮RPMI 1640培養(yǎng)基中孵育5 min后將兩溶液混勻,室溫靜置30 min,另將含(或不含)36 mmol/L氯喹、32 μmol/L亞精胺的無血清RPMI 1640培養(yǎng)基75 μL加入上述陽離子脂質(zhì)體/DNA復(fù)合物,移去細(xì)胞培養(yǎng)液后加入以上陽離子脂質(zhì)體/DNA復(fù)合物,培養(yǎng)4 h后再棄去上述含陽離子脂質(zhì)體/DNA復(fù)合物的細(xì)胞培養(yǎng)液,換成新鮮含5%胎牛血清、10%小牛血清的RPMI 1640培養(yǎng)基,每室300 μL。再培養(yǎng)48 h,然后在Axiovert 100 M倒置熒光顯微鏡,物鏡40×和目鏡10×放大倍數(shù)下拍照及計(jì)數(shù)分析。見圖1(封三)。

1.2.3 四甲基偶氮唑鹽(MTT)試驗(yàn) 參照文獻(xiàn)[1]方法,用MTT試驗(yàn)分別測定對照孔、在轉(zhuǎn)染過程中加(或不加)氯喹與亞精胺孔的PC12細(xì)胞活性。

1.2.4 PC12細(xì)胞神經(jīng)軸突生長試驗(yàn) 按文獻(xiàn)方法[11],PC12細(xì)胞轉(zhuǎn)染48 h后,將細(xì)胞培養(yǎng)基換成含1%胎牛血清、含(或不含)100 ng/mL 神經(jīng)生長因子(NGF)的新鮮RPMI 1640培養(yǎng)基,培養(yǎng)5 d后,在倒置熒光顯微鏡物鏡40×和目鏡10×放大倍數(shù)下拍照及計(jì)數(shù)分析見圖2(封三)。

1.2.5 轉(zhuǎn)染效率與神經(jīng)軸突判定及統(tǒng)計(jì) PC12細(xì)胞轉(zhuǎn)染48 h后,計(jì)數(shù)綠色熒光蛋白陽性的細(xì)胞占細(xì)胞總數(shù)的百分比即為轉(zhuǎn)染效率。參照文獻(xiàn)方法[11],用Meta Morph圖像分析處理軟件計(jì)數(shù)并測量PC12細(xì)胞神經(jīng)軸突的長度。

1.3 統(tǒng)計(jì)學(xué)方法

采用統(tǒng)計(jì)軟件SPSS 15.0對數(shù)據(jù)進(jìn)行分析,正態(tài)分布計(jì)量資料以均數(shù)±標(biāo)準(zhǔn)差(x±s)表示,兩獨(dú)立樣本的計(jì)量資料采用t檢驗(yàn)。以P < 0.05為差異有統(tǒng)計(jì)學(xué)意義。

2 結(jié)果

2.1 DNA與Lipofectamine 2000的比例對PC12細(xì)胞轉(zhuǎn)染效率的影響

當(dāng)質(zhì)粒pEGFP-N1的用量為1 μg時,Lipofectam ine2000用量相對較低時,PC12細(xì)胞轉(zhuǎn)染效率與Lipofectamine2000用量呈正相關(guān),但隨著Lipofectam ine2000用量超過4 μL,其毒副作用逐漸明顯,導(dǎo)致PC12細(xì)胞轉(zhuǎn)染效率逐步降低,且綠色熒光蛋白陽性細(xì)胞的死亡率明顯增加,本研究結(jié)果表明,對PC12細(xì)胞轉(zhuǎn)染,DNA∶Lipofectamine2000用量比例應(yīng)控制在1∶4。

2.2 轉(zhuǎn)染時間對PC12細(xì)胞轉(zhuǎn)染效率的影響

當(dāng)轉(zhuǎn)染時間分別為1、2、4、8、24 h時,PC12細(xì)胞轉(zhuǎn)染率分別為35.5%、37.9%、40.5%、40.3%及38.6%。實(shí)驗(yàn)結(jié)果表明,在未達(dá)到最佳轉(zhuǎn)染時間前,轉(zhuǎn)染效率隨轉(zhuǎn)染時間的延長而增加,4 h達(dá)到最大轉(zhuǎn)染效率,但在超過4 h后轉(zhuǎn)染時間后,轉(zhuǎn)染效率則會緩慢降低。

2.3 氯喹、亞精胺用量對PC12細(xì)胞轉(zhuǎn)染效率的影響

隨著氯喹、亞精胺加入濃度的增加,PC12細(xì)胞轉(zhuǎn)染效率也隨之增加,當(dāng)氯喹、亞精胺加入終濃度分別增至9 μmol/L和8 μmol/L時,PC12細(xì)胞的轉(zhuǎn)染效率最高,轉(zhuǎn)染效率為40.5%。

2.4 氯喹、亞精胺對PC12細(xì)胞活性的影響

PC12細(xì)胞在轉(zhuǎn)染過程中加入終濃度為9 μmol/L氯喹與8 μmol/L亞精胺前、后MTT試驗(yàn)吸光度(590 nm)分別為(0.466±0.042)與(0.451±0.038),差異無統(tǒng)計(jì)學(xué)意義(P > 0.05),表明PC12細(xì)胞的活性無影響。

2.5 氯喹、亞精胺對PC12細(xì)胞神經(jīng)軸突生長的影響

轉(zhuǎn)染試劑Lipofectamine2000對轉(zhuǎn)染前后的PC12細(xì)胞及經(jīng)NGF誘導(dǎo)后產(chǎn)生的神經(jīng)軸突長度無影響。氯喹、亞精胺對PC12細(xì)胞的神經(jīng)軸突生長數(shù)量與長度無影響;也間接表明在轉(zhuǎn)染過程中加入9 μmol/L氯喹及8 μmol/L亞精胺對PC12細(xì)胞的功能無影響。見表1。

表1 氯喹、亞精胺對PC12細(xì)胞神經(jīng)軸突生長的影響(x±s)

2.6 本法與4種常用脂質(zhì)體轉(zhuǎn)染試劑對PC12細(xì)胞轉(zhuǎn)染效率的比較

本文以pEGFP-N1為報告基因,用本法與常用的FuGENE、PolyJet、Lipofectamine LTX and Plus、Lipofectamine2000等4種脂質(zhì)體轉(zhuǎn)染試劑對PC12細(xì)胞轉(zhuǎn)染效率進(jìn)行了比較,結(jié)果:本法的PC12細(xì)胞轉(zhuǎn)染率為40.5%,其它四種脂質(zhì)體轉(zhuǎn)染試劑對PC12細(xì)胞轉(zhuǎn)染效率分別為10.5%、8.6%、11.8%及15.3%。本法對PC12細(xì)胞的轉(zhuǎn)染效率明顯高于其他4種脂質(zhì)體轉(zhuǎn)染法,差異有高度統(tǒng)計(jì)學(xué)意義(P < 0.01)。見圖3。

圖3 本法與4種常用脂質(zhì)體轉(zhuǎn)染試劑對PC12細(xì)胞轉(zhuǎn)染效率的比

3 討論

PC12細(xì)胞具有類似神經(jīng)細(xì)胞的形態(tài)學(xué)改變及生理學(xué)特征而作為神經(jīng)研究的模式細(xì)胞[1]。氯喹、亞精胺能明顯提高PC12細(xì)胞的轉(zhuǎn)染效率,且不影響PC12細(xì)胞的活性與正常功能,與文獻(xiàn)報道一致[1,8]。

氯喹是一種疏水性的弱堿,在PC12細(xì)胞轉(zhuǎn)染過程中能進(jìn)入細(xì)胞吞噬溶酶體,在酸性環(huán)境下被質(zhì)子化,隨著質(zhì)子化的氯喹在吞噬溶酶體的累積,吞噬溶酶體開始腫脹,其包膜穩(wěn)定性破壞,導(dǎo)致陽離子脂質(zhì)體/DNA復(fù)合物釋放入細(xì)胞漿,同時,氯喹抑制吞噬溶酶體酸化與成熟,阻止陽離子脂質(zhì)體/DNA復(fù)合物在溶酶體中的降解[8-9],轉(zhuǎn)染過程中氯喹能提高PC12細(xì)胞轉(zhuǎn)染效率與上述氯喹的作用機(jī)制有關(guān)。亞精胺是一種多胺類制劑能通過增強(qiáng)DNA的復(fù)制與轉(zhuǎn)錄作用來提高PC12細(xì)胞轉(zhuǎn)染效率,同時能增強(qiáng)報告基因的表達(dá)強(qiáng)度[8]。

當(dāng)DNA:Lipofectamine2000比小于1∶4時,Lipofectamine2000用量相對過多,N∶P比增加,陽離子脂質(zhì)體/DNA復(fù)合物表面所帶的凈正電荷增多,PC12轉(zhuǎn)染效率也隨之增加[6],但對PC12細(xì)胞毒性作用也隨之增強(qiáng),這與部分結(jié)合的Lipofectamine從陽離子脂質(zhì)體/DNA復(fù)合物中分離,引起Lipofectamine內(nèi)化而損傷細(xì)胞或破壞帶負(fù)電荷的細(xì)胞膜而影響PC12細(xì)胞的狀態(tài),PC12細(xì)胞轉(zhuǎn)染效率與細(xì)胞活性也會降低[1],及時更換RPMI1640培養(yǎng)基可以減少其毒性作用。

本文得到美國伊利諾斯州立大學(xué)芝加哥分校醫(yī)學(xué)院Beatrice Y.J.T. Yue教授指導(dǎo),特此致謝!

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(收稿日期:2013-10-17 本文編輯:李繼翔)

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