摘要
目的构建人β干扰素（hIFN—β）基因重组腺病毒，观察重组腺病毒介导的hIFN-β转染大鼠骨髓间充质干细胞（MSCs）后，细胞内hIFN—β表达情况以及重组腺病毒对MSCs生长的影响。方法利用细菌内同源重组技术快速构建Ad—hIFN-β腺病毒重组质粒，经酶切及测序鉴定正确后转染人胚肾细胞HEK293包装成为重组Ad—hIFN—β腺病毒，并进行滴度测定。转染的MSCs行逆转录-聚合酶链反应（RT—PCR）检测细胞内hIFN—βmRNA的表达；酶联免疫吸附试验（ELISA）法检测培养液上清hIFN—β的分泌情况；噻唑蓝（MTT）比色法检测细胞活力并绘制转染后MSCs生长曲线。结果经限制性内切酶检测、基因测序及和绿色荧光观察证实成功的构建了携带hIFN—β基因的重组腺病毒，且重组腺病毒滴度高达1×10^9pfu／ml。Ad—hIFN—β转染MSCs后在荧光显微镜下证实有绿色荧光；RT—PCR证明转染的MSCs内有hIFN-βmRNA的表达；ELISA检测转染组1、3、5、7、10、15、20d上清液中hlFN—β蛋白分泌量分别为192、273、436、957、605、472、279ng／L，明显高于对照组（P〈0．01）；Ad-hIFN-β转染的MSCs生长曲线与正常培养MSCs差异无统计学意义（P〉0．05）。结论成功构建了携带hIFN-β基因的重组腺病毒载体，重组腺病毒转染对MSCs的增殖能力无明显影响，而且Ad—hIFN-β转染大鼠MSCs能够表达并分泌hIFN—β蛋白。
Objective To construct the adenoviruse vector containing human beta-interieron gene （hIFN-β） and investigate the property of the transfected bone mesenchymal stem cells （MSCs） in vitro. Methods The recombinant plasmid pAdEasy-hIFN-β DNA was homologously recombinated in bacterial ceils. After amphfied in HEK293 cells ,the obtained adenovirus was transfected into HEK293 cells again, and GFP expression was detected. Ad-hIFN-β was transfected into MSCs cultured in vitro. The expression of hIFN-β in MSCs after transfection was detected by reverse transcription polymerase chain reaction （ RT- PCR）. ELISA method was applied to assay the secretion of hIFN-β. MTF method was applied to draw cells growth curve. Results Recombinant adenoviral hIFN-β was constructed successfully, which was confirmed by restriction enzyme digestion, gene sequence and GFP expression. GFP expression could be observed under fluorescent microscopy 24 h after transfection, and the expression of hIFN-β was confirmed by RT- PCR. ELISA analysis showed the expression levels of hIFN-β in transfection group were 192,273,436, 957,605,472,279 ng/L respectively after transfection for 1,3,5,7,10,15, and 20 days, which were higher than in control group （P 〈 0. O1 ）. There was no significant difference in the MSCs growth curve between transfection group and control group （ P 〉 0.05）. Conclusion The recombinant adenoviral vector carrying hIFN-β was successfully constructed. Exogenous hIFNA3 can be expressed in MSCs, which may offer the oossibility of a novel approach to local gene therapy of glioma.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2009年第2期216-218,共3页
Chinese Journal of Experimental Surgery
基金
基金项目：国家自然科学基金资助项目（30772223）
湖北省科技厅资助项目（2006ABA211）
