中华医学杂志
中華醫學雜誌
중화의학잡지
National Medical Journal of China
2010年
15期
1057-1061
,共5页
胡硕%熊玲静%余剑英%兰晓莉%张永学
鬍碩%熊玲靜%餘劍英%蘭曉莉%張永學
호석%웅령정%여검영%란효리%장영학
干细胞%转染%腺病毒%大鼠
榦細胞%轉染%腺病毒%大鼠
간세포%전염%선병독%대서
Stem cells%Transfectiom%Adenoviruses,rat
目的 对比研究两种腺病毒载体Ad5/EGFP和AdF35/EGFP转染SD大鼠骨髓间充质干细胞(rBMSC)的效率及其机制.方法 用密度梯度法从大鼠骨髓中分离出rBMSC,流式细胞仪检测其表面相关标志抗原(CD90、Cd29、CD44、CD34、CD11b和CD45)以鉴定rBMSC.用不同梯度感染复数(MOI)的Ad5/EGFP和AdF35/EGFP分别转染rBMSC,MTT检测二者对rBMSC生长的影响.转染后第1～9天,荧光显微镜下观察转染情况,同时流式细胞仪检测转染效率及平均荧光强度,实时PCR检测rBMSC表面CAR及CD46的表达水平.结果 rBMSC表达CD90、Cd29、CD44阳性,而CD34、CD11b、CD45表达为阴性.MOI≤1600时,Ad5/EGFP对rBMSC的生长无明显抑制作用(t=3.12,P=0.052);MOI≤1000时,AdF35/EGFP对rBMSC的生长无明显抑制作用(t=2.20,P=0.15)).荧光显微镜下,Ad5/EGFP转染rBMSC 1 d后即可见大量EGFP表达阳性的细胞,第2～3天阳性细胞数最多,随后减少,第9天仍可见少量的阳性细胞.AdF35/EGFP转染rBMSC后1～9 d EGFP阳性细胞数始终很少.Ad5/EGFP的转染效率显著性高于AdF35/EGFP的转染效率(t=2.45,P=0.008).经实时PCR检测,rBMSC高度表达CAR,而极低表达CD46,二者之间差异有统计学意义(t=2.57,P＜0.01).结论 Ad5/EGFP对rBMSC的转染效率明显高于AdF35/EGFP,这可能与rBMSC表面高表达CAR而极低表达CD46有关.AdF35不合适作为目的 基因转染rBMSC的载体,Ad5可作为目的 基因有效转染rBMSC的载体,这为后续目的 基因转染rBMSC治疗各种疾病的实验研究奠定了基础.
目的 對比研究兩種腺病毒載體Ad5/EGFP和AdF35/EGFP轉染SD大鼠骨髓間充質榦細胞(rBMSC)的效率及其機製.方法 用密度梯度法從大鼠骨髓中分離齣rBMSC,流式細胞儀檢測其錶麵相關標誌抗原(CD90、Cd29、CD44、CD34、CD11b和CD45)以鑒定rBMSC.用不同梯度感染複數(MOI)的Ad5/EGFP和AdF35/EGFP分彆轉染rBMSC,MTT檢測二者對rBMSC生長的影響.轉染後第1～9天,熒光顯微鏡下觀察轉染情況,同時流式細胞儀檢測轉染效率及平均熒光彊度,實時PCR檢測rBMSC錶麵CAR及CD46的錶達水平.結果 rBMSC錶達CD90、Cd29、CD44暘性,而CD34、CD11b、CD45錶達為陰性.MOI≤1600時,Ad5/EGFP對rBMSC的生長無明顯抑製作用(t=3.12,P=0.052);MOI≤1000時,AdF35/EGFP對rBMSC的生長無明顯抑製作用(t=2.20,P=0.15)).熒光顯微鏡下,Ad5/EGFP轉染rBMSC 1 d後即可見大量EGFP錶達暘性的細胞,第2～3天暘性細胞數最多,隨後減少,第9天仍可見少量的暘性細胞.AdF35/EGFP轉染rBMSC後1～9 d EGFP暘性細胞數始終很少.Ad5/EGFP的轉染效率顯著性高于AdF35/EGFP的轉染效率(t=2.45,P=0.008).經實時PCR檢測,rBMSC高度錶達CAR,而極低錶達CD46,二者之間差異有統計學意義(t=2.57,P＜0.01).結論 Ad5/EGFP對rBMSC的轉染效率明顯高于AdF35/EGFP,這可能與rBMSC錶麵高錶達CAR而極低錶達CD46有關.AdF35不閤適作為目的 基因轉染rBMSC的載體,Ad5可作為目的 基因有效轉染rBMSC的載體,這為後續目的 基因轉染rBMSC治療各種疾病的實驗研究奠定瞭基礎.
목적 대비연구량충선병독재체Ad5/EGFP화AdF35/EGFP전염SD대서골수간충질간세포(rBMSC)적효솔급기궤제.방법 용밀도제도법종대서골수중분리출rBMSC,류식세포의검측기표면상관표지항원(CD90、Cd29、CD44、CD34、CD11b화CD45)이감정rBMSC.용불동제도감염복수(MOI)적Ad5/EGFP화AdF35/EGFP분별전염rBMSC,MTT검측이자대rBMSC생장적영향.전염후제1～9천,형광현미경하관찰전염정황,동시류식세포의검측전염효솔급평균형광강도,실시PCR검측rBMSC표면CAR급CD46적표체수평.결과 rBMSC표체CD90、Cd29、CD44양성,이CD34、CD11b、CD45표체위음성.MOI≤1600시,Ad5/EGFP대rBMSC적생장무명현억제작용(t=3.12,P=0.052);MOI≤1000시,AdF35/EGFP대rBMSC적생장무명현억제작용(t=2.20,P=0.15)).형광현미경하,Ad5/EGFP전염rBMSC 1 d후즉가견대량EGFP표체양성적세포,제2～3천양성세포수최다,수후감소,제9천잉가견소량적양성세포.AdF35/EGFP전염rBMSC후1～9 d EGFP양성세포수시종흔소.Ad5/EGFP적전염효솔현저성고우AdF35/EGFP적전염효솔(t=2.45,P=0.008).경실시PCR검측,rBMSC고도표체CAR,이겁저표체CD46,이자지간차이유통계학의의(t=2.57,P＜0.01).결론 Ad5/EGFP대rBMSC적전염효솔명현고우AdF35/EGFP,저가능여rBMSC표면고표체CAR이겁저표체CD46유관.AdF35불합괄작위목적 기인전염rBMSC적재체,Ad5가작위목적 기인유효전염rBMSC적재체,저위후속목적 기인전염rBMSC치료각충질병적실험연구전정료기출.
Objective To investigate the infective efficiency of Ad5/EGFP versus AdF35/EGFP to rat bone marrow mesenchymal stem cells(rBMSC)and understand its mechanism.Methods rBMSC were isolated from rat bone marrow by density gradient method.The expressions of CD90,Cd29,CD44,CD34,CD11b and CD45 on the surface of rBMSC were measured by flow cytometry.The EGFP-carrying Ad5 and AdF35 were infected into rBMSC respectively.The effect of Ad5/EGFP and AdF35/EGFP with different MOIs to rBMSC was tested by MTT.Infected rBMSC were observed by fluorescence microscopy.The infective efficiency and mean fluorescence intensity were determined by flow cytometry after a cellular infection for 1-9 days.The expressions of CAR and CD46 on rBMSC were measured by real-time PCR.Results The expressions of CD90,Cd29 and CD44 on rBMSC were positive while those of CD34,CD11b and CD45 were negative.When MOI ≤1600 and ≤1000,Ad5/EGFP and AdF35/EGFP could not inhibit the growth of rBMSC respectively(t=3.12,P=0.052;t=2.20,P=0.15).RBMSC with EGFP expression could be observed under fluorescence microscope after a 1-day infection by Ad5/EGFP.The number of observed cells peaked after an infection for 2-3 days and then decreased gradually.And a few cells could be still observed for 9 days.Fewer cells could be observed after an infection by AdF35/EGFP for 1-9 days.The infective efficiency of Ad5/EGFP was significantly higher than that of AdF35/EGFP(t=2.45,P=0.008).There was a high expression of CAR on rBMSC.The expression of CD46 was quite low.There was significant difference between them(t=2.57,P＜0.01).Conclusion The infective efficiency of Ad5/EGFP is significantly higher than that of AdF35/EGFP.It may explain the expression patterns of CAR and CD46 on rBMSC.AdF35 iS not a suitable gene-carrying vector to infect rBMSC.Yet Ad5 can be an efficient vector carrying target gene to infect rBMSC for curing diseases.
