四川大学学报（医学版）
四川大學學報（醫學版）
사천대학학보（의학판）
JOURNAL OF SICHUAN UNIVERSITY(MEDICAL SCIENCE EDITION)
2010年
2期
203-207
,共5页
蒋星%杨昌永%毛棉%刘强强%王莉
蔣星%楊昌永%毛棉%劉彊彊%王莉
장성%양창영%모면%류강강%왕리
重组腺相关病毒%增强型绿色荧光蛋白%成骨细胞%转染
重組腺相關病毒%增彊型綠色熒光蛋白%成骨細胞%轉染
중조선상관병독%증강형록색형광단백%성골세포%전염
Recombinant adeno-associated virus%Enhanced green fluorescent protein%Osteoblast%Transfection
目的 比较2种不同重组腺相关病毒(rAAV)介导的增强型绿色荧光蛋白(EGFP)对大鼠成骨细胞的转染效率,评价其作为成骨细胞病变基因治疗载体的可行性.方法 采用Ⅰ型胶原酶阶段消化法分离培养大鼠成骨细胞并鉴定,rAAV-EGFP按转染复数(MOI) 1×10~3、1×10~4、1×10~5、5×10~5分为只加rAAV和rAAV与腺病毒(ADV)共同转染组转染成骨细胞,倒置荧光显微镜观察转染后荧光强度随转染时间的变化,流式细胞仪检测rAAV2/6-EGFP和rAAV2/9-EGFP对成骨细胞的转染效率及荧光强度,确定转染的较佳MOI值,以此值用MTT法描绘细胞生长曲线,观察rAAV对细胞的毒性.结果 分离培养的细胞具有体内成骨细胞的生物学行为,rAAV对成骨细胞的转染效率随MOI值的增加而提高,ADV(-)组荧光强度在第5 d达到高峰,当MOI为1×10~5时,rAAV2/6-EGFP和rAAV2/9-EGFP的转染效率分别为90.2%、66.1%,MOI值增到5×10~5时转染效率无显著提高;ADV(+)组荧光强度在第3 d即达高峰,MOI值为5×10~5时,rAAV2/6-EGFP和rAAV2/9-EGFP的转染效率为47.6%、30.5%.细胞生长正常,rAAV对细胞活性影响小.结论 两种病毒载体对成骨细胞转染效率均较高,其中rAAV2/6高于rAAV2/9,是一种理想的基因治疗载体.
目的 比較2種不同重組腺相關病毒(rAAV)介導的增彊型綠色熒光蛋白(EGFP)對大鼠成骨細胞的轉染效率,評價其作為成骨細胞病變基因治療載體的可行性.方法 採用Ⅰ型膠原酶階段消化法分離培養大鼠成骨細胞併鑒定,rAAV-EGFP按轉染複數(MOI) 1×10~3、1×10~4、1×10~5、5×10~5分為隻加rAAV和rAAV與腺病毒(ADV)共同轉染組轉染成骨細胞,倒置熒光顯微鏡觀察轉染後熒光彊度隨轉染時間的變化,流式細胞儀檢測rAAV2/6-EGFP和rAAV2/9-EGFP對成骨細胞的轉染效率及熒光彊度,確定轉染的較佳MOI值,以此值用MTT法描繪細胞生長麯線,觀察rAAV對細胞的毒性.結果 分離培養的細胞具有體內成骨細胞的生物學行為,rAAV對成骨細胞的轉染效率隨MOI值的增加而提高,ADV(-)組熒光彊度在第5 d達到高峰,噹MOI為1×10~5時,rAAV2/6-EGFP和rAAV2/9-EGFP的轉染效率分彆為90.2%、66.1%,MOI值增到5×10~5時轉染效率無顯著提高;ADV(+)組熒光彊度在第3 d即達高峰,MOI值為5×10~5時,rAAV2/6-EGFP和rAAV2/9-EGFP的轉染效率為47.6%、30.5%.細胞生長正常,rAAV對細胞活性影響小.結論 兩種病毒載體對成骨細胞轉染效率均較高,其中rAAV2/6高于rAAV2/9,是一種理想的基因治療載體.
목적 비교2충불동중조선상관병독(rAAV)개도적증강형록색형광단백(EGFP)대대서성골세포적전염효솔,평개기작위성골세포병변기인치료재체적가행성.방법 채용Ⅰ형효원매계단소화법분리배양대서성골세포병감정,rAAV-EGFP안전염복수(MOI) 1×10~3、1×10~4、1×10~5、5×10~5분위지가rAAV화rAAV여선병독(ADV)공동전염조전염성골세포,도치형광현미경관찰전염후형광강도수전염시간적변화,류식세포의검측rAAV2/6-EGFP화rAAV2/9-EGFP대성골세포적전염효솔급형광강도,학정전염적교가MOI치,이차치용MTT법묘회세포생장곡선,관찰rAAV대세포적독성.결과 분리배양적세포구유체내성골세포적생물학행위,rAAV대성골세포적전염효솔수MOI치적증가이제고,ADV(-)조형광강도재제5 d체도고봉,당MOI위1×10~5시,rAAV2/6-EGFP화rAAV2/9-EGFP적전염효솔분별위90.2%、66.1%,MOI치증도5×10~5시전염효솔무현저제고;ADV(+)조형광강도재제3 d즉체고봉,MOI치위5×10~5시,rAAV2/6-EGFP화rAAV2/9-EGFP적전염효솔위47.6%、30.5%.세포생장정상,rAAV대세포활성영향소.결론 량충병독재체대성골세포전염효솔균교고,기중rAAV2/6고우rAAV2/9,시일충이상적기인치료재체.
Objective To compare the transfection efficiency of two kind of recombinant adeno-associated virus-mediated transfection to rats osteoblasts with enhanced green fluorescent protein and assess the feasibility of it as a vector for gene therapy of osteoblast lesions. Methods The osteoblasts of rats were isolated, cultured and identified with typeⅠcollagen staged digestion method. According to different multiplicity of infection (MOI) (MOI=1×10~3,1×10~4,1×10~5,5×10~5), rAAV-EGFP was transfected into osteoblasts with rAAV only and rAAV-ADV co-transfection respectively. The expression of EGFP along with the transfection time was observed under inverted fluorescence microscope. The transfecion efficiency and fluorescence intensity was evaluated by flow cytometry. The best MOI value was analysed and the cell growth curves were obtained according to the best MOI value to evaluate the toxical effects of rAAV-EGFP. Results The cultured cells possessed the biological behaviors of osteoblasts. The transfecion efficiency of the rAAV was increased with the increasing of MOI. The EGFP expression reached the maximum on day 5 in ADV(-) group, the transfection efficiency of rAAV2/6-EGFP and rAAV2/9-EGFP was 90.2% and 66.1% respectively when MOI was 1×10~5 and no significant increasewas observedwhen MOI was 5×10~5. In ADV(+) group, EGFP expression reached its maximum on day 3, the transfection efficiency of rAAV2/6-EGFP and rAAV2/9-EGFP was 47.6% and 30.5% respectively when MOI was 5×10~5. And no significant biologic effects on the cyto-activity was observed. Conclusion The transfection efficiency of two kind of virus vectors was both very high and rAAV2/6's is higher than that of rAAV2/9. This suggested the potential of rAAV-EGFP as a safe and efficient vector for gene therapy.
