CAJ | 학술논문

背景:利用基因工程细胞进行替代治疗和基因治疗中合适的供体细胞、靶细胞以及相应载体是细胞和基因治疗最为困难的部分.目的:检测重组腺病毒Ad5和Ad5F35、腺相关病毒rAAV1/2和rAAV2、慢病毒LV对大鼠骨髓间充质干细胞的感染效率和外源基因表达水平,拟筛选能够高效转导骨髓间充质干细胞的载体.设计、时间及地点:细胞基因工程对照观察,于2006-10/2007-03在上海市第一人民医院中心实验室完成.材料:清洁级SD大鼠10只用于制备骨髓间充质干细胞.所有重组病毒均携带增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)报告基因,AdS由本实验室制备,Ad5F35由解放军第二军医大学钱其军教授惠赠,rAAV2及rAAV1/2为本元正阳基因技术有限公司产品,LV由中科院上海生化与细胞生物学研究所郭礼和教授惠赠.方法:采用淋巴细胞分离液密度梯度离心法体外分离培养大鼠骨髓间充质干细胞,传至第4代用于实验.按1×105/孔接种于24孔细胞培养板中,1 d后细胞贴壁,设立5组:第1组向细胞培养液中加入10,100,1 000 MOI Ad5-EGFP,第2组加入10,100,1 000 MOIAdSF35-EGFP,第3组加入1×104,1×105vg rAAV1/2-EGFP,第4组加入1×104,1×105vg rAAV2-EGFP,第5组加入30 TU LV-EGFP.Ad病毒感染2d,rAAV及LV病毒感染6d.主要观察指标:EGFP阳性表达及荧光强度.结果:Ad3-EGFP感染24 h后镜下可见EGFP阳性细胞,随着病毒用量的增加EGFP阳性细胞数目增多,荧光亮度增强,12d后阳性细胞开始逐渐减少,荧光亮度减弱.AdSF35-EGFP感染情况与Ad5-EGFP基本相似,但EGFP阳性细胞数和荧光亮度均增加.rAAV1/2-EGFP或rAAV2-EGFP感染6 d后,EGFP阳性细胞数和荧光亮度均较弱.LV-EGFP感染第2天即可见少量EGFP阳性细胞,随着时间延长EGFP阳性细胞数目逐渐增多,至第6天表达EGFP荧光的细胞数量及亮度不再有明显变化.结论:腺病毒Ad5、AdSF35与慢病毒LV能够有效感染体外培养的骨髓间充质干细胞,并表达外源基因,感染效率与病毒用量之间存在量效关系.腺相关病毒rAAV1/2和rAAV2体外感染效果不佳. 揹景:利用基因工程細胞進行替代治療和基因治療中閤適的供體細胞、靶細胞以及相應載體是細胞和基因治療最為睏難的部分.目的:檢測重組腺病毒Ad5和Ad5F35、腺相關病毒rAAV1/2和rAAV2、慢病毒LV對大鼠骨髓間充質榦細胞的感染效率和外源基因錶達水平,擬篩選能夠高效轉導骨髓間充質榦細胞的載體.設計、時間及地點:細胞基因工程對照觀察,于2006-10/2007-03在上海市第一人民醫院中心實驗室完成.材料:清潔級SD大鼠10隻用于製備骨髓間充質榦細胞.所有重組病毒均攜帶增彊型綠色熒光蛋白(enhanced green fluorescent protein,EGFP)報告基因,AdS由本實驗室製備,Ad5F35由解放軍第二軍醫大學錢其軍教授惠贈,rAAV2及rAAV1/2為本元正暘基因技術有限公司產品,LV由中科院上海生化與細胞生物學研究所郭禮和教授惠贈.方法:採用淋巴細胞分離液密度梯度離心法體外分離培養大鼠骨髓間充質榦細胞,傳至第4代用于實驗.按1×105/孔接種于24孔細胞培養闆中,1 d後細胞貼壁,設立5組:第1組嚮細胞培養液中加入10,100,1 000 MOI Ad5-EGFP,第2組加入10,100,1 000 MOIAdSF35-EGFP,第3組加入1×104,1×105vg rAAV1/2-EGFP,第4組加入1×104,1×105vg rAAV2-EGFP,第5組加入30 TU LV-EGFP.Ad病毒感染2d,rAAV及LV病毒感染6d.主要觀察指標:EGFP暘性錶達及熒光彊度.結果:Ad3-EGFP感染24 h後鏡下可見EGFP暘性細胞,隨著病毒用量的增加EGFP暘性細胞數目增多,熒光亮度增彊,12d後暘性細胞開始逐漸減少,熒光亮度減弱.AdSF35-EGFP感染情況與Ad5-EGFP基本相似,但EGFP暘性細胞數和熒光亮度均增加.rAAV1/2-EGFP或rAAV2-EGFP感染6 d後,EGFP暘性細胞數和熒光亮度均較弱.LV-EGFP感染第2天即可見少量EGFP暘性細胞,隨著時間延長EGFP暘性細胞數目逐漸增多,至第6天錶達EGFP熒光的細胞數量及亮度不再有明顯變化.結論:腺病毒Ad5、AdSF35與慢病毒LV能夠有效感染體外培養的骨髓間充質榦細胞,併錶達外源基因,感染效率與病毒用量之間存在量效關繫.腺相關病毒rAAV1/2和rAAV2體外感染效果不佳.
배경:이용기인공정세포진행체대치료화기인치료중합괄적공체세포、파세포이급상응재체시세포화기인치료최위곤난적부분.목적:검측중조선병독Ad5화Ad5F35、선상관병독rAAV1/2화rAAV2、만병독LV대대서골수간충질간세포적감염효솔화외원기인표체수평,의사선능구고효전도골수간충질간세포적재체.설계、시간급지점:세포기인공정대조관찰,우2006-10/2007-03재상해시제일인민의원중심실험실완성.재료:청길급SD대서10지용우제비골수간충질간세포.소유중조병독균휴대증강형록색형광단백(enhanced green fluorescent protein,EGFP)보고기인,AdS유본실험실제비,Ad5F35유해방군제이군의대학전기군교수혜증,rAAV2급rAAV1/2위본원정양기인기술유한공사산품,LV유중과원상해생화여세포생물학연구소곽례화교수혜증.방법:채용림파세포분리액밀도제도리심법체외분리배양대서골수간충질간세포,전지제4대용우실험.안1×105/공접충우24공세포배양판중,1 d후세포첩벽,설립5조:제1조향세포배양액중가입10,100,1 000 MOI Ad5-EGFP,제2조가입10,100,1 000 MOIAdSF35-EGFP,제3조가입1×104,1×105vg rAAV1/2-EGFP,제4조가입1×104,1×105vg rAAV2-EGFP,제5조가입30 TU LV-EGFP.Ad병독감염2d,rAAV급LV병독감염6d.주요관찰지표:EGFP양성표체급형광강도.결과:Ad3-EGFP감염24 h후경하가견EGFP양성세포,수착병독용량적증가EGFP양성세포수목증다,형광량도증강,12d후양성세포개시축점감소,형광량도감약.AdSF35-EGFP감염정황여Ad5-EGFP기본상사,단EGFP양성세포수화형광량도균증가.rAAV1/2-EGFP혹rAAV2-EGFP감염6 d후,EGFP양성세포수화형광량도균교약.LV-EGFP감염제2천즉가견소량EGFP양성세포,수착시간연장EGFP양성세포수목축점증다,지제6천표체EGFP형광적세포수량급량도불재유명현변화.결론:선병독Ad5、AdSF35여만병독LV능구유효감염체외배양적골수간충질간세포,병표체외원기인,감염효솔여병독용량지간존재량효관계.선상관병독rAAV1/2화rAAV2체외감염효과불가.
BACKGROUND: Genetically engineered cells have been used in the replacement therapy and gene therapy. However, how to select proper donor cells, target cells, and corresponding viral vectors is the most difficult in the therapy.OBJECTIVE: To compare the transduction efficiency of recombinant adenovirus Ad5 and AdSF35, adeno-associated virus rAAV1/2 and rAAV2, and lentivirus LV in bone marrow mesenchymal stem cells (BMSCs) and exogenous gene expression level so as to select the vectors, which can efficiently transduce BMSCs.DESIGN, TIME AND SETTING: Gene engineering controlled observation, performed in the Central Laboratory, Shanghai First People's Hospital between October 2006 and March 2007.MATERIALS: Ten Sprague Dawley rats of clean grade were used to prepare BMSCs. All recombinant viral vectors carded enhanced green fluorescent protein (EGFP) report gene. Ad5 was prepared by the Central Laboratory, Shanghai First People's Hospital. Ad5F35 was gifted by professor Qian Qi-jun from the Second Military Medical University of Chinese PLA. rAAV2 and rAAVI/2 were the products of Benyuan Zhengyang Gene Technique Co.,Ltd. LV was gifted by professor Cuo Li-be from Shanghai Institute of Biochemistry and Cytobiology, Chinese Academy of Sciences.METHODS: Rat BMSCs were in vitro isolated and cultured by density gradient centrifugation. BMSCs of passage 4 were inoculated into 24-well plate at lxlO5/well. One day later, ceils adhered to the wall and allocated to 5 groups. Ad5-EGFP [10, 100,1 000 multiplicity of infection (MOI)], Ad5F35-EGFP (10,10, 1 000 MOI), rAAVI/2-EGFP (1×104,1x10× vg), rAAV2-EGFP(1×104, 1x105vg), and LV-EGFP (30 TU) were respectively added into the 5 groups. BMSCs were transduced for 2 days with Ad virus and separately for 6 days with rAAV and LV virus.MAIN OUTCOME MEASURES: EGFP-positive expression and fluorescence intensity.RESULTS: After twenty-four hours of Ad5-EGFP transduction, EGFP-positive cells were visible under the microscope. With virus dose increasing, EGFP-positive cells increased and fluorescence intensity strengthened. Twelve days later, EGFP-positive cells gradually reduced and fluorescence intensity weakened. For Ad5F35-EGFP, its transduction was basically similar to Ad5-EGFP, but EGFP-positive cell number and fluorescence intensity were increased. After 6 days of rAAV1/2-EGFP or rAAV2-EGFP transduction, EGFP-posidve cell number and fluorescence intensity were decreased. For LV-EGFP transduction, a small amount of EGFP-positive cells could be visible on the second day, and then EGFP-positive ceils and fluorescence intensity were gradually increased until the sixth day.CONCLUSION: Ad5, Ad5F35 and LV could effectively transduce BMSCs cultured in vitro and express exogenous gene.Furthermore, transduction efficiency was correlated with virus dose in dose-dependent manner, rAAVI/2 and rAAV2 had poor/in vitro transduction efficiency.