目的探討電轉(zhuǎn)方法用于modRNA轉(zhuǎn)染細(xì)胞的可行性,并篩選出不同細(xì)胞電轉(zhuǎn)所需的最適電壓和脈沖時間。方法 （1）篩選電轉(zhuǎn)EGFP modRNA進(jìn)入人成纖維細(xì)胞的最佳電壓和脈沖時間參數(shù);（2）在此基礎(chǔ)上將人成纖維細(xì)胞轉(zhuǎn)染n GFP/EGFP+mCherry modRNA;（3）將Hela、293T、3T3三種常見貼壁細(xì)胞電轉(zhuǎn)EGFP modRNA。結(jié)果 （1）人成纖維細(xì)胞電轉(zhuǎn)modRNA的最適電壓為440 V,最適脈沖時間為30 ms;（2）nGFP可在核內(nèi)表達(dá),EGFP和mCherry兩種蛋白可同時在胞質(zhì)中表達(dá);（3）Hela、293T、3T3細(xì)胞電轉(zhuǎn)最適電壓分別為425 V、400 V和440 V,最適脈沖時間均為30 ms。結(jié)論本實(shí)驗(yàn)證明（1）電轉(zhuǎn)法轉(zhuǎn)染modRNA進(jìn)入細(xì)胞質(zhì)和細(xì)胞核的可行性;（2）可同時將兩種modRNA導(dǎo)入同一個細(xì)胞內(nèi)并表達(dá);（3）最適電壓和最適脈沖時間需根據(jù)不同的細(xì)胞種類分別篩選。 

【文章來源】：組織工程與重建外科雜志. 2019,15(04)

【文章頁數(shù)】：7 頁

【部分圖文】：

流式檢測表達(dá)效率和熒光強(qiáng)度Fig.4Detectionofexpressioneffi-ciencyandfluorescenceintensitybyflowcytometry

化圖A:12hoursafterelectroporation;B:1dayafterelectroporation;C:2daysafterelec-troporation;D:3daysafterelectroporation;E:4daysafterelectroporation;F:5daysafterelectroporation;G:6daysafterelec-troporation;H:7daysafterelectropora-tion;I:ChangesofexpressionefficiencyofEGFPmodRNAinhumanfibroblasts;J:ChangesoffluorescenceintensityofEGFPmodRNAinhumanfibroblasts圖4流式檢測表達(dá)效率和熒光強(qiáng)度Fig.4Detectionofexpressioneffi-ciencyandfluorescenceintensitybyflowcytometryA：光鏡圖；B：nGFP熒光圖；C：DAPI染色；D：Merge圖A:Underlightmicroscopy;B:nGFPfluorescentstaining;C:DAPIstaining;D:Mergediagram圖5電轉(zhuǎn)nGFPmodRNA后熒光圖Fig.5FluorescencediagramafterelectroporationofnGFPmodRNAA：mCherry熒光圖；B：EGFP熒光圖；C：DAPI染色；D：Merge圖A:mCherryfluorescentstaining;B:EGFPfluorescentstaining;C:DAPIstaining;D:Mergediagram圖6電轉(zhuǎn)EGFP+mCherrymodRNA后熒光圖Fig.6FluorescencediagramafterelectroporationofEGFP+mCherrymodRNAA：EGFP熒光圖；B：DAPI染色；C：Merge圖；D：流式分析A:EGFPfluorescentstaining;B:DAPIstaining;C:Mergediagram;D:Flowanalysis圖7Hela細(xì)胞電轉(zhuǎn)EGFPmodRNA后熒光圖Fig.7FluorescenceofHelacellsafterelectroporationwithEGFPmodRNA·219·

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