重组腺病毒介导的肝细胞生长因子基因体外转染骨髓间充质干细胞源性脂肪细胞  

Transfection of bone marrow mesenchymal stem cells-derived adipose cells with recombinant adenovirus carrying human hepatocyte growth factor gene in vitro

作　　者：刘婕婷[1] 刘毅[2] 哈小琴[3] 王有虎[1] 蔡黔[2] 

机构地区：[1]兰州大学临床医学院,甘肃省兰州市730000 [2]解放军兰州军区兰州总医院烧伤整形种,甘肃省兰州市730000 [3]解放军兰州军区兰州总医院实验中心,甘肃省兰州市730000

出　　处：《中国组织工程研究与临床康复》2008年第25期4847-4850,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：全军“十一五”医学科学技术研究面上项目(06MA079);甘肃省自然科学基金(3ZS061-A25-099)~~

摘　　要：背景：肝细胞生长因子可促进血管内皮细胞增生及新生血管形成，已广泛应用于病理性瘢痕、心肌缺血等的实验性治疗。目的：鉴于重组腺病毒的宿主范围广，探讨重组腺病毒介导的人肝细胞生长因子对骨髓间充质干细胞源性脂肪细胞转染效率、目的蛋白表达及细胞活性的影响。设计、时间及地点；2006—08／2007-07在解放军兰州军区兰州总医院完成的细胞对比实验。材料：清洁级2月龄Wistar大鼠25只用于制备骨髓间充质干细胞。携带绿色荧光蛋白基因的重组腺病毒毒株、重组腺病毒介导的人肝细胞生长因子由解放军兰州军区兰州总医院博士后科研工作站哈小琴博士惠赠。方法：向体外培养至第3代的骨髓间充质干细胞中加入含地塞米松、IBMX、牛胰岛素、吲哚美辛、胎牛血清的L-DMEM进行成脂诱导。以不同感染强度的携带绿色荧光蛋白基因重组腺病毒转染诱导后的脂肪细胞。重组腺病毒介导人肝细胞生长因子以最佳感染强度转染诱导后的脂肪细胞。主要观察指标：油红。染色检测成脂情况。流式细胞仪计数绿色荧光蛋白阳性表达，计算转染效率。ELISA法检测转染上清中肝细胞生长因子的表达，MTT法检测转染脂肪细胞的活性。结果：①骨髓间充质干细胞成脂诱导后胞质内富含橙红色的脂滴，成脂率（97-31±0．46）％。②以100pfu／cell携带绿色荧光蛋白基因的重组腺病毒转染脂肪细胞72h后，绿色荧光蛋白表达量达高峰，此时转染效率为65．39％。③以100pfu／cell作为最佳感染强度转染脂肪细胞72h后，肝细胞生长因子的表达量为峰值。与未转染空白对照比较，转染后24，48，72，96，168h脂肪细胞活性均无明显变化（t=-0．024～0．092，P〉0．05）。结论：重组腺病毒可介导肝细胞生长因子基因转染骨髓间充质干细胞来源的脂肪细胞，有效表达目的蛋白�BACKGROUND： Hepatocyte growth factor （HGF） can encourage hyperplasy of vascular endothelial cells and angiogenesis. It has been widely applied on healing pathological cicatricle and myocardial ischemia. OBJECTIVE： Respecting recombinant adenovirus owns extensive host, this experiment discussed transfer efficiency, target gene expression and bioactivity of bone marrow mesenchymal stem cells （MSCs）-derived adipose cells via a recombinant adenovirus carrying human HGF gene. DESIGN, TIME AND SETTING： The comparison study based on cells was conducted in the Lanzhou General Hospital of Lanzhou Military Area Command of Chinese PLA between August 2006 and July 2007. MATERIALS： Two-month-old Wistar rats （n=25） were used to prepare MSCs. The recombinant adenovirus carrying green fluorescence protein gene （Ad-GFP） and the recombinant adenovirus carrying human HGF gene （Ad-HGF） were provided by Doctor Ha Xiao-qin in the Postdoctoral Station, Lanzhou General Hospital of Lanzhou Military Area Command of Chinese PLA. METHODS： The MSCs, cultivated till the 3^nd passage in vitro, were induced to differentiate into adipose cells by L-DMEM adding dexamethasone, IBMX, bovine insulin, indomethacin and fetal calf serum. The adipose cells were transfected with Ad-GFP at the different multiplicity of infection （MOI）. The adipose cells were transfected with Ad-HGF at the best MOI. MAIN OUTCOME MEASURES： The ratio of adipose cells was calculated by oil red O staining. Expression of GFP was evaluated by flow cytometry, and the best MOI would be calculated. HGF expression and the bioactivity of transfected adipose cells were evaluated respectively by ELISA and MTT. RESULTS： MSCs-induced adipose cells included lipid vacuoles, which were stained into salmon. The ratio of adipose cells was （97.31 ±0.46）%. At 72 hours after transfecting adipose cells with Ad-GFP at 100 MOI, the expression of GFP reached the maximal value and the transfer efficiency was 65.39%.At 72 hours after transfecting

关 键 词：骨髓间充质干细胞 脂肪细胞 重组腺病毒 肝细胞生长因子 转染 

分 类 号：R394.2[医药卫生—医学遗传学]