摘要
目的:探讨人血管内皮细胞生长因子(VEGF165)基因转染大鼠脂肪干细胞(rADSCs)的稳定表达系统对皮瓣成活的影响。方法:①培养和鉴定大鼠ADSCs,脂质体介导pcDNA3.1-VEGF165质粒转染大鼠(rADSCs),经G418筛选抗性rADSCs并继续培养4周,用ELISA法检测转染后细胞培养上清中VEGF浓度。②Brdu标记被转染rADSCs后,在大鼠背部制备8.0cm×2.5cm的随意型皮瓣模型,实验组(10只大鼠)为注射pcDNA3.1-VEGF165质粒转染的rADSCs悬液到大鼠腹腔,对照组(8只大鼠)为注射2ml被空载体pcDNA3.1(-)转染的rADSCs悬液,术后7天用透明纸描记法记录皮瓣成活范围,记数坐标纸格子并换算为成活面积百分率。分别于术后3天、5天、7天在成活皮瓣的远端切取皮瓣组织标本行免疫组化染色。结果:①ELISA检测4株转染和经G418筛选抗性并继续培养2周的第二代rADSCs上清中VEGF浓度多达(4.21±0.35)pmol VEGF/1×106细胞/48h(x±s,n=4),有2株细胞培养液上清分泌VEGF较强,其余2株细胞相对较弱,均大于对照的空载体转染组和未转染组rADSCs培养液上清中的(0.49±0.15)pmol VEGF/1×106细胞/48h(x±s,n=3)和(0.62±0.86)pmol VEGF/1×106细胞/48h(x±s,n=3),均P<0.01,差异有统计学意义。分泌VEGF较强的rADSCs经Brdu标记后阳性率达到70%以上。②将获得的稳定表达VEGF165的rADSCs移植到随意型皮瓣动物模型体内,7天后实验组皮瓣的成活面积为(59.6±0.52)%,大于对照组的皮瓣成活面积(40.5±0.27)(%P<0.01)。成活皮瓣的远端组织标本切片在显微镜下可见皮下脂肪中有大量核仁深染棕黄色的细胞。结论:ADSCs可以作为载体细胞运送VEGF基因到达皮瓣远端并通过分泌VEGF提高皮瓣的成活面积。
Objective To explore if adipose derived stem cells（ADSCs） can act as carrier to transporting VEGF165 gene to the distal part of rat flap model and improve the viability of distal part of skin flap. Methods ①Rat adipose derived stern cells （rADSCs）isolation and culture and identification,and then the plasmid of pcDNA3.1- VEGF165 was transfected into rADSCs by using Lipofectin method. The positive cell clones were selected with G418 and cultured for 4 weeks.The expression of VEGF in the supernatant of the positive cells was determined by ELISA analysis. ②A 8.0cm×2.5 cm random flap model was dissected in the back of SD rat, and then 2ml transfected rADSCs was injected into abdominal cavity of rat in experimnetal group （10 rats）, and the 2ml pcDNA3.1 （-）vector control tranfectants rADSCs was also injected to abdominal cavity in control group （8 rats）, respectively. ③The survival area of skin flap was calculited by tracing cellophane paper 7d postoperaton, respectively. And The specimen of flap was cutted from the distal survival part of flap aftrer 3d, 5d, 7d, respectively, and was examed by the immunocytochemical staining. Results ①After G418 selection, ELISA analysis, 2 out of 4 cell lines transfected with pcDNA3.1- VEGF165 expressed with very high level of VEGF, and the transductced cells release as much as （4.21±0.35） pmol VEGF/1×10^6 cells/48h （^-x±s,n=4）, much higher than （0.62±0.86）pmol VEGF/1×10^6 cells/48h （^-x±s, n=3） in the un-transfected control group, （0.49±0.15） pmol VEGF/1×10^6 cells /48h （^-x±s, n=3） in the vector control tranfectants group, P〈0.01. The immunocytochemical staining of Brdu of rADSCs revealed that more than70% of rADSCs was marked by displying intranucleus Brdu stained with an anti- Brdu antibody and a HRPconjugated secondary antibody.②The survival area of skin flap was （59.6±0.52）%, much greater than （40.5±0.27）% in the control group, P〈0.01. ③The immunocytochemical staining showed that there are great of rADSCs staining yellowed occupied in the subcutanoeous, but there was no rADSCs ocuupied in skin epiderm. Conclusion rADSCs can act as carrier to transport VEGF gene and to migrate to the distal part of skin flap, and can be used to improve the skin flap viability.
出处
《中国美容医学》
CAS
2007年第1期7-10,共4页
Chinese Journal of Aesthetic Medicine
基金
国家重点基础研究发展规划资助项目(2005CB522603)
全军十一五课题(069116)
国家自然科学基金资助项目(30400172)
