重组腺病毒介导血小板衍生生长因子感染人类脐带间质干细胞的实验研究

Experimental study of recombinant adenoviral-mediated PDGF gene delivery of human umbilical cord derived mesenchymal stem cells

目的检测重组腺病毒方法介导血小板衍生生长因子（PDGF）对人类脐带间质干细胞（MSC）的感染能力，为将其用于基因治疗奠定基础。方法反转录．聚合酶链反应（RT—PCR）法扩增人类PDGF的cDNA序列，构建重组病毒载体AdPDGF。分离培养人类脐带MSC。体外以不同感染复数感染MSC后，流式细胞术检测感染效率，荧光显微镜观察绿色荧光蛋白（GFP）表达。锥虫蓝染色及四甲基偶氮唑蓝（MTY）法检测感染后细胞生存活力及增殖能力。酶联免疫吸附（ELISA）法检测细胞上清液中PDGF分泌水平。结果成功构建病毒载体AdPDGF。其对MSC的感染效率随感染复数增加而增高，当感染复数为50时，达到最高，为87．36％。未感染的MSC、感染AdPDGF和对照病毒的MSC活力分别为（97．8±2．3）％、（91．9±4．0）％和（92．8±4．0）％，增殖能力分别为（100±16．8）％，（95．9±12．0）％和（87．5±9．7）％，感染AdPDGF的MSC与两个对照比较差异均无统计学意义（P〉0．05）。感染48h后MSC能有效分泌PDGF，感染复数为10、30、50时，PDGF分泌水平分别为（1．53±0．37）、（3．03±0．68）和（5．25±0．92）ng／ml，MSC—GFP及MSC组未检测到有PDGF分泌。结论成功构建AdPDGF重组腺病毒并高效感染人类脐带MSC。

Objective To investigate the infect capability of recombinant adenovirus mediated PDGF to human umbilical cord derived mesenchymal stem cells （MSCs）. Methods The PDGF cDNA sequence was amplified with RT-PCR and constructed recombinant adenovirus vector AdPDGF. The human umbilical cord derived MSCs were isolated and cultured. In vitro AdPDGF infected MSCs with various MOI, and determine the efficiency using FACS and fluorescence microscope. Trypan blue and MTT assay was used to detect cell viability and proliferation. ELISA was used to quantify PDGF secretion. Results Recombinant adenovirus AdPDGF was successfully and constructed, in which infection efficiency to MSCs was dose dependent. The highest efficiency was around 87.36 % when MOI=50, at which cell viability and proliferation wasn＇t impaired. Cell viability of uninfected, AdPDGF infected and control virus infected MSCs was （97.8±2.3） %, （91.9±4.0） % and （92.8±4.0） %, separately. The cell proliferation was （100±16.8） %, （95.9±12.0） % and （87.5±9.7） %, separately. There was no statistic significance among AdPDGF infected,control virus infected and uninfected MSCs. 48 hrs post infection, PDGF secretion can be measured from infectious MSCs＇ supernatant. The secretion level was （1.53±0.37）, （3.03±0.68） and （5.25±0.92） ng/ml, separately, when MOI= 10, 30, 50. No PDGF can be detected from MSC-GFP and control MSC group. Conclusion Recombinant adenovirus AdPDGF was constructed and can infect human umbilical cord derived MSC at high efficiency.