摘要
目的构建慢病毒-血管内皮生长因子。(VEGF165)载体并转染脂肪组织来源干细胞(ADSCs)以得到用于基因治疗的种子细胞。方法通过聚合酶链反应(PCR)方法将EHSl001—68950485313912克隆突变成VEGF165(NM_001025368)转录本后采用三质粒系统(pGC—FU、pHelpl.0和pHelp2.0)进行包装慢病毒。通过定时定量PCR测定滴度后使用慢病毒-VEGFl65转染ADSCs。免疫荧光、Westernblot鉴定VEGF165在ADSCs中的表达。结果PCR(535bp)、DNA测序、Westernblot(49×10^3)检测证明成功构建的载体为VEGF165与绿色荧光蛋白(GFP)融合表达载体。实时定量PCR确定病毒滴度达到2×10^8TU/ml。慢病毒-VEGF165贷载体转染ADSCs后经免疫荧光验证约95%ADSCs可表达VEGF165,同时Westernblot也证明基因稳定表达。结论通过获得稳定表达VEGF165的ADSCs,为血管性疾病特别是糖尿病性勃起障碍的治疗奠定了基础。
Objective Many studies found that stem cells can act as gene therapeutic vector. But there is few related report focus on the Adipose tissue-derived stem ceils (ADSCs). Therefore we intend to construct a lentiviral vascular endothelial growth factor165 ( VEGF165 ) over expression vector and infect the ADSCs to produce therapeutic seed cells. Methods VEGF165 transcript (NM_001025368) fragment was origin from the EHS1001-6895048 5313912 clone and mutated to the purpose transcript by polymerase chain reaction (PCR) method. The Lentivirus was enveloped with 3 plasmids (pGC-FU, pHelper 1.0 and pHelper 2. 0). And then use the successful constructed vectors infected the ADSCs (MOI = 20) after titer determination. Stable expression of VEGF16s in ADSCs was confirmed by immunofluorescence (95% affected) staining and western blotting analysis. Results VEGFI65 was linked to the green fluorescent protein (GFP) fused vector which was verified by the PCR (535 bp), DNA sequencing, Western blotting (49 × 10^3). Quantitative PCR determined that the virus titer is up to 2 × 10^8 which. VEGF165 transduced cells could show green fluorescence which was confirmed by immunofluorescence. And the Western blotting analyses confirmed VEGF165 expression in VEGF165-transduced cells. Conclusion We obtained the VEGF165 over-expression ADSCs and these cells could use as a gene therapeutic tool and may be applied for vascular regeneration, especially in treatment of erectile dysfunction.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2011年第6期893-895,共3页
Chinese Journal of Experimental Surgery
基金
基金项目:国家自然科学基金资助项目(30872571)
国家青年自然科学基金资助项目(30901487)
2010教育部博士点基金资助项目(20100171110060)
广东省科技计划资助项目(2008GBCBC01250014149)
广东省自然科学基金-博士启动基金资助项目(8451008901000774)