摘要
背景:近些年来应用相关干细胞及生长因子促进肺再生修复肺气肿病变的研究逐渐成为热点,那么通过干细胞携带相关外源生长因子基因能否更好的修复肺气肿病变值得关注。目的:观察携带绿色荧光蛋白基因的腺病毒(adenovirus vecto rmediated green fluorescence protein,Ad-GFP)转染骨髓间充质干细胞的效率、对细胞增殖的影响,及携带目的基因的间充质干细胞尾静脉移植在肺气肿大鼠肺部的定居情况。方法:采用密度梯度离心结合贴壁培养法分离、培养间充质干细胞,流式细胞仪鉴定;在不同感染复数时,激光共聚焦显微镜检测转染效果及转染效率,MTT法检测转染48h的细胞活性;Wistar大鼠16只,随机分为模型组和对照组,每组8只,采用单纯被动烟熏法制备肺气肿大鼠模型。通过尾静脉移植Ad-GFP修饰的间充质干细胞,于24h后取大鼠肺组织做快速冰冻切片,在荧光显微镜下观察间充质干细胞在肺部的分布情况。结果与结论:实验成功获得大鼠间充质干细胞,Ad-GFP成功转染间充质干细胞,且在MOI=200时,转染效率达到88.42%。在不同MOI下,Ad-GFP转染间充质干细胞对细胞增殖无影响(P>0.05)。移植24h后,模型组大鼠及对照组大鼠肺部可见到发绿色荧光的间充质干细胞,且模型组荧光强度明显强于对照组(P<0.05)。提示导入目的基因可能不会影响骨髓间充质干细胞增殖和归巢特性。
BACKGROUND：Recently,application of stem cells and growth factor to promoting lung regeneration in repair of emphysema lesion has been a hot focus in study.Thus,it is worth to pay attention on whether stem cells carrying relevant foreign growth factor gene can repair emphysema lesion.OBJECTIVE：To evaluate the efficiency of adenovirus vector mediated green fluorescence protein（Ad-GFP） transfecting bone marrow mesenchymal stem cells（BMSCs） and its effect on the cell proliferation,to explore oriented migration of intravenously administrated BMSCs transfected with Ad-GFP in the lung tissues of pulmonary emphysema rats.METHODS：MSCs were separated and purified from the bone marrow of rats by density gradient centrifugation and by adherence.At different multiplicity of infection（MOI）,transfection efficiency was observed by laser confocal microscopy.At 48 hours of transfection,MTT method was used to evaluate the proliferation of MSCs.A total of 16 Wistar rats were randomly divided into emphysema model group and control group（n = 8）.Model rats were established by exposure to cigarette smoke.MSCs,transfected with Ad-GFP,were grafted into the body of rats via tail vein.Lungs derived at 24 hours after implantation,and frozen sections were made.Migration and survival of MSCs in the lung tissues were observed by fluorescence microscopy.RESULTS AND CONCLUSION：MSCs from Wistar rats were successfully cultured,grew well and infected by Ad-GFP.The highest transfection effincincy（88.42 %） could be achieved at MOI of 200.Green fluorescent protein labeling had little effect on proliferation of MSCs by different MOI（P〉 0.05）.At 24 hours posttransplantation,the green fluorescence-positive tissue was found in the lung tissues of emphysema model group and control group.Compared with control group,the expression of GFP in lung tissues was higher in emphysema model group（P 〈0.05）.These suggested that introduction of target gene cannot affect proliferation and homing property of BMSCs.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2010年第14期2528-2532,共5页
Journal of Clinical Rehabilitative Tissue Engineering Research
