重组腺病毒介导BMP-9及促红细胞生成素双基因共转染促进脂肪来源干细胞体外成骨的研究

RECOMBINANT ADENOVIRUS-MEDIATED BONE MORPHOGENETIC PROTEIN 9 AND ERYTHROPOIETIN GENES CO-TRANSFECTION IN PROMOTING OSTEOGENIC DIFFERENTIATION OF ADIPOSE-DERIVED STEM CELLS IN VITRO

目的探讨重组腺病毒介导BMP-9及促红细胞生成素（erythropoietin,EPO）双基因共转染对脂肪来源干细胞（adipose-derived stem cells,ADSCs）体外成骨分化的影响。方法取4月龄新西兰兔腹股沟脂肪组织,采用酶消化法与贴壁法分离培养ADSCs并鉴定其多向分化能力。将第3代ADSCs分为5组：A组为正常细胞,B组为空质粒转染细胞作为对照,C、D组分别采用BMP-9、EPO重组腺病毒转染细胞,E组采用BMP-9、EPO重组腺病毒共转染细胞。转染培养7 d后倒置相差显微镜观察各组细胞生长变化;计算48 h时病毒转染效率,14 d荧光显微镜下观察各组细胞荧光表达情况;14 d时Western blot检测各组细胞BMP-9及EPO蛋白表达。取转染培养14 d的5组细胞进行成骨诱导培养,于诱导培养3、7、14 d检测各组细胞ALP活性;3周时茜素红染色观察钙结节形成情况,实时荧光定量PCR检测骨桥蛋白（osteopontin,OPN）、骨钙素（osteocalcin,OCN）基因表达。结果转染培养7 d后倒置相差显微镜下见A、B组部分细胞变为椭圆形、圆形和不规则形;C、D组可见少量长梭形细胞;E组长梭形细胞明显增多,仅见少量圆形细胞。荧光显微镜下观察BMP-9、EPO及BMP-9/EPO重组腺病毒均能稳定转染ADSCs,转染效率为80%-93%。Western blot检测示E组BMP-9及EPO蛋白相对表达量显著高于其他组,差异有统计学意义（P〈0.05）。成骨诱导培养3、7、14 d时各组细胞ALP活性明显升高,E组优于其余各组（P〈0.05）;3周时茜素红染色示E组钙结节数亦显著高于其余各组（P〈0.05）,实时荧光定量PCR示E组OPN和OCN基因相对表达量明显高于其他组,C、D组高于A、B组,比较差异有统计学意义（P〈0.05）。结论重组腺病毒介导BMP-9及EPO基因均可转染ADSCs并稳定表达,双基因共转染后能显著促进成骨相关基因和蛋白的表达。

Objective To investigate the effect of recombinant adenovirus-mediated bone morphogenetic protein 9（BMP-9） and erythropoietin（EPO） genes co-transfection on osteogenic differentiation of adipose-derived stem cells（ADSCs） in vitro. Methods The inguinal adipose tissue was harvested from 4-month-old New Zealand rabbits, ADSCs were isolated with enzyme digestion and adherence method, and multipotent differentiation capacity was identified. The 3rd generation ADSCs were divided into 5 groups： normal cells（group A）, empty plasmid control group（group B）, BMP-9 or EPO recombinant adenovirus transfected cells（groups C and D）, BMP-9 and EPO recombinant adenovirus co-transfected cells（group E）. The inverted phase contrast microscope was used to observe the cell growth at 7 days; the expression of cell fluorescence was observed under a fluorescence microscope at 14 days, and viral transfection efficiency was calculated at 48 hours; Western blot was used to detect the expressions of BMP-9 and EPO proteins at 14 days. The expression of alkaline phosphatase（ALP） activity was detected at 3, 7, and 14 days after osteogenic induction, and alizarin red staining was used to detect calcium nodules formation and real-time fluorescence quantitative PCR to detect the expressions of osteopontin（OPN） and osteocalcin（OCN） at 3 weeks. Results At 7 days after transfected, some cells showed oval, round, and irregular shape under the inverted phase contrast microscope in groups A and B; a few fusiform cells were observed in groups C and D; oval cells increased obviously, and there were only few round cells in group E. The fluorescence microscope observation showed that BMP-9 and EPO, BMP-9/EPO recombinant adenovirus could stably transfected ADSCs, with transfection efficiency of 80%-93%. The expressions of BMP-9 and EPO proteins significantly higher in group E than the other groups by Western blot（P〈0.05）. The ALP activity significantly increased in group E when compared with that in the other groups at 3, 7, and 14 days after osteogenic induction（P〈0.05）; the number of calcium nodules in group E was significantly more than that in the other groups（P〈0.05）. Real-time fluorescence quantitative PCR showed that OPN and OCN genes expressions were significantly higher in group E than other groups（P〈0.05）, and in groups C and D than groups A and B（P〈0.05）. Conclusion Recombinant adenovirus-mediated BMP-9 and EPO genes can transfect ADSCs, which can stably express in ADSCs, BMP-9/EPO genes co-transfection can more promote the expressions of osteoblast-related genes and protein than non-transfected and single gene transfection.