腺病毒载体介导SCL基因转染Cajal间质细胞及其表达  

Adenovirus vector mediating SCL gene expression and transfecting Cajal interstitial cells and expression of adenovirus vector

作　　者：张林[1] 童卫东[1] 刘宝华[1] 李春穴[1] 

机构地区：[1]第三军医大学大坪医院野战外科研究所普通外科,重庆400042

出　　处：《中华消化外科杂志》2007年第2期132-135,共4页Chinese Journal of Digestive Surgery

基　　金：国家自然科学基金资助项目（No.30300156）

摘　　要：目的构建含有人干细胞白血病（stemcellleukemia，SCL）基因的重组腺病毒载体，并观察其对Cajal间质细胞的转染及其介导的SCL基因的表达。为下一步在体转染Cajal间质细胞，观察其对Cajal间质细胞表型逆转的影响奠定基础。方法利用Ad—Easy系统、细菌内同源重组法快速构建重组腺病毒Ad-GFP／SCL。通过观察绿色荧光蛋白的表达评估重组腺病毒对培养的Cajal间质细胞的转染效率。通过RT—PCR法分析转染Cajal间质细胞后SCLmRNA的表达。结果酶切鉴定及PCR结果证明成功构建了含有人SCL基因的重组腺病毒，病毒滴度为1．6×10^11pfu／ml；对cajal间质细胞的转染率高达100％；RT—PCR法检测转染Cajal间质细胞后SCLmRNA有表达。结论成功构建的含人SCL基因的重组腺病毒对cajal间质细胞有很强的转染能力，可介导SCL基因在Cajal间质细胞中表达。Objective To construct recombinant adenovirus vector containing human stem cell leukemia （hSCL） gene to observe its ability in transfecting Cajal interstitial cells and mediating SCL gene expression, which may lay basis for further in vivo transfection of Cajal interstitial cells to observe the effect of the recombinant adenovirus vector on changes of monotype of Cajal interstitial cells. Methods The recombinant adenovirus Ad-GFP/ SCL was rapidly constructed by using Ad-Easy system based on the homologous recombination in bacteria. Then, Cajal interstitial cells cultured in vitro were infected with 1.6 × 10^9 pfu of recombinant adenovirus. The distribution and efficiency of recombinant adenovirus mediated hSCL was observed by expression of green fluorescence protein （GFP） under the fluorescent microscope. The expression of hSCL mRNA transfected with Cajal interstitial cells was measured by RT-PCR method. Results Restriction endonuclease and PCR analyses confirmed that the hSCL gene was successfully inserted into the adenovirus vector, with titer of the recombinant adenovirus for 1.6 × 10^11 pfu/ml. The adenovirus had a high transfection efficiency up to 100%. RT-PCR analysis showed expression of hSCL mRNA in Cajal interstitial cells transfected by hSCL. Conclusions The recombinant adenovirus containing human hSCL gene is successfully constructed by homogenous recombination in bacteria and it has a high transfection efficiency and can mediate expression of Cajal interstitial cells.

关 键 词：腺病毒载体 干细胞白血病基因 同源重组 转染 CAJAL间质细胞 

分 类 号：R686[医药卫生—骨科学]