摘要
[目的]研究人血管内皮生长因子(vascular endothelial growth factor,VEGF)转染人脂肪组织来源干细胞(adipose tissue-derived stem cells,ADSCs)后对该细胞成骨分化能力的影响。[方法]采用Trizol法获取编码VEGF成熟肽的基因序列,将获得的目的基因与线性载体pGEM-T Easy连接,测序后与含绿色荧光蛋白的双顺反子表达载体双酶切连接成重组质粒pSELECT-GFP zeo-VEGF,并转染第2代ADSCs,荧光显微镜下观察转染效率。对转染后的ADSCs行成骨培养,Western Blot及Realtime PCR检测细胞对碱性磷酸酶(alkaline phosphatase,ALP)和骨钙蛋白(osteocalcin,OC)的分泌,分别对细胞行Von Kossa及ALP染色。[结果]荧光显微镜下可见与细胞形态相似的绿色荧光。成骨条件培养下,Western Blot及Realtime PCR检测结果示VEGF转染后的ADSCs组对ALP和OC的分泌量明显高于空载体转染组和空白细胞组,结果有统计学意义(P<0.01)。VEGF转染组细胞经Von Kossa及ALP染色可见不透光的钙结节沉着。[结论]经脂质体介导将人VEGF目的基因片段转染后的ADSCs成骨分化能力明显增强,转染后的ADSCs可以有效促进成骨,从而为基因治疗促进骨组织工程发展提供了必要的试验基础。
[ Objective] To study the effect of the osteogenic differentiation capability of adipose tissue-derived stem cells （ADSCs） after transfection of human Vascular Endothelial Growth Factor （VEGF）. [ Method ] Gene sequence encoding human VEGF mature peptide was obtained using Trizol reagent method. Target gene VEGF was connected with linearity vector of pGEM-T Easy. After sequencing, the pGEM-T Easy was connected with bicistronic expression vector containing green fluorescent protein to form pSELECT-GFP zeo-VEGF, which transfected second generation ADSCs. Transfection resuhs were verified under fluorescence microscope. Second generation transfected ADSCs were cultured under osteogenic conditions. Alkaline phosphatase （ALP） and osteocalcin （OC） secretion in the supernatant liquid were detected. Van Kossa and ALP staining were performed. [ Result ] There was green fluorescence similar to the cell shape under fluorescence microscope. After osteogenic culture, reahime PCR and Western Blot detection showed that secretion of ALP and OC of VEGF transfected group was significantly higher than empty vector transfected group and blank cell group. The results had a statistical difference （P 〈 0.01 ）. The lightproof calcium clumping was showed in the VEGF transfected group by Van Kossa and ALP staining under the microscope. [ Conclusion] After transfected by liposome-mediated VEGF target gene fragment, the directional osteogenic differentiation capability of transfected ADSCs was significantly enhanced. This study laid necessary experimental basis for gene therapy to promote the development of bone tissue engineering.
出处
《中国矫形外科杂志》
CAS
CSCD
北大核心
2013年第24期2515-2521,共7页
Orthopedic Journal of China
关键词
血管内皮生长因子
脂肪组织
干细胞
转染
成骨细胞
vascular endothelial growth factor, adipose tissue, stem cells, transfection, osteoblasts
