中华显微外科杂志
中華顯微外科雜誌
중화현미외과잡지
Chinese Journal of Microsurgery
2012年
6期
475-478,后插7
,共5页
杨绍安%蔡进奎%肖晓桃%周初松%蔡宝塔
楊紹安%蔡進奎%肖曉桃%週初鬆%蔡寶塔
양소안%채진규%초효도%주초송%채보탑
酸性成纤维细胞生长因子%肌卫星细胞%基因转染%细胞工程
痠性成纖維細胞生長因子%肌衛星細胞%基因轉染%細胞工程
산성성섬유세포생장인자%기위성세포%기인전염%세포공정
aFGF gene%Muscle satellite cells%Gene transfection%Cell engineering
目的 探讨通过构建酸性成纤维细胞生长因子(aFGF)基因真核表达载体转染大鼠肌卫星细胞(MSCs)建立细胞工程种子细胞可行性.方法 首先提取人类肌肉组织总RNA,RT-PCR法调取aFGF基因,然后用直接化学合成法合成人类白细胞介素2信号肽序列(SPS),通过基因融合得到SPS-aFGF,再通过定向克隆到真核表达载体pEGFP-N1,最终得到重组质粒PEGFP-N1-SPS-aFGF,对重组质粒做测序和酶切鉴定;差速贴壁法获取大鼠MSCs,观察细胞形态,做免疫荧光鉴定;经鉴定正确的细胞随机分为3组:目的基因组(aFGF加N1)、空载质粒组(N1)、空白对照组(blank),前两组分别加入质粒PEGFP-N 1-SPS-aFGF与pEGFP-N1质粒,空白对照不加入任何质粒,均在脂质体Lipofectamine 2000TMReagent介导下转染,转染后荧光显微镜统计发绿色荧光的细胞占各组细胞比例,计算转染效率;转染72h后利用实时荧光定量PCR与Western blot检测aFGF基因在MSCs中表达情况.结果 ①重组质粒测序结果与GenBank公布的序列一致,酶切鉴定条带与实际大小相符.②荧光显微镜下观察到转染细胞有荧光表达,并在转染72 h达到最高峰.③转染后72 h,实时荧光定量PCR结果显示目的基因组表达量平均相对比值(aFGF加N1)组(1464.96)显著高于N1组(1.016)、Blank组(1.000),差异有统计学意义(P＜0.05).Western blot检测显示aFGF表达呈极强阳性,而N1组及Blank组aFGF表达极弱.结论 成功构建aFGF基因真核表达载体并转染MSCs,aFGF在MSCs内高表达,此种方法有希望获取具备特殊生物学功能细胞.
目的 探討通過構建痠性成纖維細胞生長因子(aFGF)基因真覈錶達載體轉染大鼠肌衛星細胞(MSCs)建立細胞工程種子細胞可行性.方法 首先提取人類肌肉組織總RNA,RT-PCR法調取aFGF基因,然後用直接化學閤成法閤成人類白細胞介素2信號肽序列(SPS),通過基因融閤得到SPS-aFGF,再通過定嚮剋隆到真覈錶達載體pEGFP-N1,最終得到重組質粒PEGFP-N1-SPS-aFGF,對重組質粒做測序和酶切鑒定;差速貼壁法穫取大鼠MSCs,觀察細胞形態,做免疫熒光鑒定;經鑒定正確的細胞隨機分為3組:目的基因組(aFGF加N1)、空載質粒組(N1)、空白對照組(blank),前兩組分彆加入質粒PEGFP-N 1-SPS-aFGF與pEGFP-N1質粒,空白對照不加入任何質粒,均在脂質體Lipofectamine 2000TMReagent介導下轉染,轉染後熒光顯微鏡統計髮綠色熒光的細胞佔各組細胞比例,計算轉染效率;轉染72h後利用實時熒光定量PCR與Western blot檢測aFGF基因在MSCs中錶達情況.結果 ①重組質粒測序結果與GenBank公佈的序列一緻,酶切鑒定條帶與實際大小相符.②熒光顯微鏡下觀察到轉染細胞有熒光錶達,併在轉染72 h達到最高峰.③轉染後72 h,實時熒光定量PCR結果顯示目的基因組錶達量平均相對比值(aFGF加N1)組(1464.96)顯著高于N1組(1.016)、Blank組(1.000),差異有統計學意義(P＜0.05).Western blot檢測顯示aFGF錶達呈極彊暘性,而N1組及Blank組aFGF錶達極弱.結論 成功構建aFGF基因真覈錶達載體併轉染MSCs,aFGF在MSCs內高錶達,此種方法有希望穫取具備特殊生物學功能細胞.
목적 탐토통과구건산성성섬유세포생장인자(aFGF)기인진핵표체재체전염대서기위성세포(MSCs)건립세포공정충자세포가행성.방법 수선제취인류기육조직총RNA,RT-PCR법조취aFGF기인,연후용직접화학합성법합성인류백세포개소2신호태서렬(SPS),통과기인융합득도SPS-aFGF,재통과정향극륭도진핵표체재체pEGFP-N1,최종득도중조질립PEGFP-N1-SPS-aFGF,대중조질립주측서화매절감정;차속첩벽법획취대서MSCs,관찰세포형태,주면역형광감정;경감정정학적세포수궤분위3조:목적기인조(aFGF가N1)、공재질립조(N1)、공백대조조(blank),전량조분별가입질립PEGFP-N 1-SPS-aFGF여pEGFP-N1질립,공백대조불가입임하질립,균재지질체Lipofectamine 2000TMReagent개도하전염,전염후형광현미경통계발록색형광적세포점각조세포비례,계산전염효솔;전염72h후이용실시형광정량PCR여Western blot검측aFGF기인재MSCs중표체정황.결과 ①중조질립측서결과여GenBank공포적서렬일치,매절감정조대여실제대소상부.②형광현미경하관찰도전염세포유형광표체,병재전염72 h체도최고봉.③전염후72 h,실시형광정량PCR결과현시목적기인조표체량평균상대비치(aFGF가N1)조(1464.96)현저고우N1조(1.016)、Blank조(1.000),차이유통계학의의(P＜0.05).Western blot검측현시aFGF표체정겁강양성,이N1조급Blank조aFGF표체겁약.결론 성공구건aFGF기인진핵표체재체병전염MSCs,aFGF재MSCs내고표체,차충방법유희망획취구비특수생물학공능세포.
Objective To construct human acidic fibroblast growth factor (aFGF) recombinant eu karyotic expression vector and transfect it into muscle satellite cells(MSCs) of rat,in purpose of further study the method to set up cell bank.Methods The aFGF gene was cloned from human total RNA which was obtained from human skeletal muscle tissue by RT-PCR method.Human interleukin 2 (IL-2) signal peptide sequence (SPS) was obtained by direct chemosynthesis method.Then aFGF and SPS were fused to obtain SPS-aFGF.Finally,directional cloning SPS-aFGF into pEGFP-N1,the recombinant (pEGFP-N1-SPS-aFGF) was obtained.The recombinant was confirmed by endonuclease digestion and DNA sequencing.MSCs were purified by difference-speed adherence method and were ideontified by immunofluorescence assay.The correct cells were divided into 3 groups:Experimental group (aFGF +N 1),control group (N 1),blank group (blank).All the groups were transfected by Lipofectamine 2000TM Reagent,and pEGFP-N1-SPS-aFGF,pEGFP-N1 were respectively added in experimental group and control group while blank group was added none plasmid.Fluorescence microscope was employed to detect transfection efficiency tendency along with time changes.The expression of target gene was detected by fluorescent quantitation PCR and Western blot.Results (1) The sequencing of pEGFP-N1-SPS-aFGF was completely correct and the outcome of endonuclease was equal to actual ban s-ize.(2)The expression of GFP in transfected cells were observed by fluorescencemicroscope and transfection efficiency reached the peak at 72 h.(3)Real-time fluorescent quantitation PCR proved strong aFGF mRNA expression in transfected cells (the average relative expression of experimental group was 1464.95)with aFGF gene,while it was detected a little in the other groups (the average relative expression of control group was 1.016 and blank group was 1.000) (P ＜ 0.05).Western blot also proved strong expression in Experimental group then the other two groups.Conclusion aFGF eukaryotic expression vector was successfully constructed and transfected into MSCs.This study may be expected to obtain some specific functions cells.
