血管内皮生长因子165、神经营养因子3、血管生成素1基因转染诱导骨髓间充质干细胞向神经元及血管内皮细胞分化

Transfection with vascular endothelial growth factor 165,neurotrophin 3 and angiopoietin 1 induces the differentiation of bone marrow mesenchymal stem cells into neurons and vascular endothelial cells

背景:骨髓间充质干细胞是一种多功能间充质干细胞,具有多向分化潜能,可通过腺病毒转染等方法对其进行基因修饰,使其能够产生多种生长因子,如血管内皮生长因子(vascular endothelial growth factor,VEGF)、神经营养因子3(neurotrophin 3,NT-3)、血管生成素1(angiopoietin 1,Ang-1)等,从而应用于组织工程研究中。目的:探讨VEGF165、NT-3、Ang-1基因转染大鼠骨髓间充质干细胞向神经元及血管内皮细胞诱导分化的可能性。方法:采用差速贴壁法分离培养骨髓间充质干细胞,进行流式细胞术和成骨、成脂诱导分化能力鉴定。以巨细胞病毒(CMV)作为启动子,构建2个腺病毒载体,其一为双顺反子载体,携带h VEGF165及Ang-1基因(Adv-Bic);其二为携带NT-3基因载体,即Adv NT-3。以不同感染复数值将Adv-Bic(绿色荧光)及Adv NT-3(红色荧光)同时转染大鼠骨髓间充质干细胞,转染2 d后根据荧光显微镜下绿色及红色荧光蛋白表达情况确定最佳感染复数值。将生长良好的第3代骨髓间充质干细胞分为2组,实验组以最适感染复数值转染Adv-Bic(无荧光标记)及Adv NT-3(无荧光标记);对照组以相同感染复数值转染空白对照病毒。两组分别于体外培养7 d后进行免疫荧光及q PCR检测。结果与结论:(1)间质干细胞表面分子CD29、CD44呈阳性表达,造血干细胞表面分子CD34、CD45呈阴性表达。骨髓间充质干细胞能够定向分化为成骨细胞及脂肪细胞;(2)病毒转染骨髓间充质干细胞48 h后荧光显微镜下观察有绿色及红色荧光蛋白表达,随着感染复数值增大荧光表达量逐渐增强,感染复数值为100时荧光表达最强烈;(3)免疫荧光及q PCR结果显示,实验组VEGF165、NT-3、Ang-1表达水平高于对照组,且血管内皮特异性标记物CD31、CD34表达水平高于对照组,神经元特异性标记物NSE、Nestin表达水平高于对照组;(4)结果提示,VEGF165、NT-3、Ang-1基因转染骨髓间充质干细胞,使其过表达VEGF165、NT-3、Ang-1生长因子,能够诱导骨髓间充质干细胞定向分化为神经元及血管内皮细胞。

BACKGROUND:Bone marrow mesenchymal stem cells(BMSCs)are a kind of multifunctional mesenchymal stem cells that have multi-directional differentiation potential.These cells can be genetically modified by methods such as adenovirus transfection,to produce a variety of growth factors such as neurotrophin-3(NT-3),angiopoietin-1(Ang-1),and vascular endothelial growth factor(VEGF).BMSCs have been applied as seed cells in tissue engineering research.OBJECTIVE:To transfect VEGF165,NT-3 and Ang-1 gene into rat BMSCs and to induce them to differentiate into neuronal cells and vascular endothelial cells.METHODS:BMSCs were isolated using differentia velocity adherent method and identified by flow cytometry.Differentiation potential of BMSCs into osteogenesis and adipogenesis was also identified.We constructed two adenoviral vectors with cytomegalovirus(CMV)as a promoter.One of the two vectors was a bicistronic vector(Adv-Bic),carrying hVEGF165 and Ang-1 gene;the other vector was an AdvNT-3 vector,carrying NT-3 gene.Adv-Bic(green fluorescence)and AdvNT-3(red fluorescence)were simultaneously transferred into rat BMSCs at various multiplicities of infection(MOIs).Two days after the transfection,the optimal MOI value was determined based on the expression of green and red fluorescent proteins under a fluorescence microscope.Then,well-grown BMSCs at passage 3 were divided into two groups.The experimental group was transfected with Adv-Bic(without fluorescence)and AdvNT-3(without fluorescence)at the optimal MOI.The control group was transfected with blank control virus at the same MOI.The two groups were cultured in vitro for 7 days and then were detected with immunofluorescence and qPCR.RESULTS AND CONCLUSION:The expression of CD29 and CD44,which are identifications of BMSCs,was positive,while the expression of CD34 and CD45,which are identifications of hematopoietic stem cells,was negative.BMSCs could be differentiated into osteoblasts and adipocytes.After BMSCs were transfected with adenovirus for 48 hours,the expression of green and red fluorescent proteins was observed under a fluorescence microscope.With the increase of MOIs,the intensity of fluorescence gradually increased,and peaked when MOI was equal to 100.Immunofluorescence and qPCR results showed that the expression of VEGF165,NT-3 and Ang-1 in the experimental group was higher than that in the control group.The expression level of CD31 and CD34,the markers of vascular endothelium,in the experimental group was higher than that in the control group.The expression level of neuron-specific markers NSE and Nestin in the experimental group was higher than that in the control group.These results suggested that VEGF165,NT-3 and Ang-1 genes were successfully transfected into BMSCs.This transfection made BMSCs overexpress growth factors of VEGF165,NT-3 and Ang-1,which could induce BMSCs to differentiate into neuronal cells and vascular endothelial cells.